Second Edition

0
 ‫لجنةىاردادىالمنكاج‬

‫ى‬
‫د‪.‬ركودىربدىالدتارىربدىالجبار‬ ‫د‪.‬ىمحمدىرباسىالكرخي‬
‫دكتوراهىاحواءىمجكروة ى‬ ‫دكتوراهىاحواءىمجكروةى ى‬
‫ى‬

‫ى‬
‫د‪.‬رليىفاضلىكاظمى ى‬ ‫نورىربدىالردولىمحمد‬
‫دكتوراهىاحواءىمجكروة ى‬ ‫ماجدتورىاحواءىمجكروة ى‬
‫ى ى‬

‫أحمدىكفاحىربد ى‬
‫تقنيىتحلوالتىمرضوةىأختصاصى ى‬

‫اذراف ى‬
‫ىىالدكتورىحازمىهاديىالخفاجي ى‬
‫ىىمدورىقدمىالمختبرات ى‬

‫ىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىالتنضودىوالتصمومى ى‬
‫ىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىىى رليىفاروقىوحوى ى‬
‫رقمىاالوداعىفيىدارىالكتبىوالوثائق‪/‬بغدادى(‪)5863‬ى‪ 4243‬ى‬

‫‪1‬‬
 CHAPTER
1
INVESTIGATIONS AND BIOCHEMICAL
REACTIONS
The laboratory diagnosis of infectious diseases involves two main approaches: one is
the bacteriologic approach, in which the organism is identified by staining and culturing the
organism, and the other is the immunologic (serologic) approach, in which the organism is
identified by detection of antibodies against the organism in the patient‟s serum.In the
bacteriologic approach to the diagnosis of infectious diseases, several important steps
precede the actual laboratory work:
(1) Choosing the appropriate specimen to examine, which requires an understanding of the
pathogenesis of the infection;
(2) Obtaining the specimen properly to avoid contamination from the normal flora;
(3) Transporting the specimen promptly to the laboratory or storing it correctly; and (4)
providing essential information to guide the laboratory personnel.
In general, there are three approaches to the bacteriologic laboratory work:
1. Observing the organism in the microscope after staining.
2. Obtaining a pure culture of the organism by inoculating it onto a bacteriologic medium.
3. Identifying the organism by using biochemical reactions, growth on selective media,
DNA probes, or specific antibody reactions. Which of these approaches are used and in
what sequence depend on the type of specimen and organism. After the organism is
grown in pure culture, its sensitivity to various antibiotics is determined by test of
sensitivity.

STAINING TECHNIQUES
1. Staining of bacteria
Bacterial staining is the process of coloring of colorless bacterial structural components
using stains (dyes). The principle of staining is to identify microorganisms selectively by
using dyes.
Uses
1. To observe the morphology, size, and arrangement of bacteria.
2. To differentiate one group of bacteria from the other group.
A. Gram staining method
Developed by Christian Gram.Most bacteria are differentiated by their gram reaction due to
differences in their cell wall structure.Gram-positive bacteria are bacteria that stain purple
with crystal violet after decolorizing with acetone-alcohol.Gram-negative bacteria are
bacteria that stain pink with the counter stain (safranin) after losing the primary stain (crystal
violet) when treated with acetone-alcohol.

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Procedure:
1. Prepare the smear from the culture or from the specimen (drop of normal salin+colony
from culture media).
2. Allow the smear to air-dry completely.
3. Rapidly pass the slide (smear upper most) three times through the flame.
4. Cover the fixed smear with crystal violet for 1 minute and wash with distilled water.
5. Tip off the water and cover the smear with gram‟s iodine for 1minute.
6. Wash off the iodine with clean water.
7. Decolorize rapidly with acetone-alcohol for 30 seconds.
8. Wash off the acetone-alcohol with clean water.
9. Cover the smear with safranin for 1 minute.
10. Wash off the stain wipe the back of the slide. Let the smear to air-dry.
11. Examine the smear with oil immersion objective to look for bacteria.

Figure (1 -1): Gram staining method

Interpretation:
Gram-positive bacterium )Purple(
Gram-negative bacterium )Pink(

Figure (1-2): GM positive cocci Figure (1-3): GM negative rod

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 Table 1–1: Shape of bacteria and their Gram staining property
Gram-positive cocci arranged in
Cluster Staphylococcus
Chain Streptococcus
Pairs, lanceolate shaped Pneumococcus
Tetrads Micrococcus
Octate Sarcina
Pair or in short chain, spectacle eyed shaped Enterococcus
Gram-positive bacilli arranged in
Chain (bamboo stick appearance) Bacillus anthracis
Chinese letter arrangement C.diphtheriae
Pallisade arrangement Other
Corynebacterium
Branching GPB (Gram-positive bacilli ) species
Actinomycetes

B. Ziehl-Neelsen staining method

Developed by Paul Ehrlichin1882, and modified by Ziehl and Neelson , Ziehl-Neelson stain
(Acid-fast stain) is used for staining Mycobacteria which are hardly stained by gram staining
method.Once the Mycobacteria is stained with primary stain it cannot be decolorized with
acid, so named as acid-fast bacteria.

Procedure
1. Prepare the smear from the primary specimen and fix it by passing through the flame and
label clearly.
2. Place fixed slide on a staining rack and cover each slide with concentrated carbol fuchsin
solution.
3. Heat the slide from underneath with sprit lamp until vapor rises (do not boil it) and wait
for 3-5 minutes.
4. Wash off the stain with clean water.
5. Cover the smear with 3% acid-alcohol solution until all color is removed (15–30 seconds).
6. Wash off the stain and cover the slide with 1% methylene blue for 30 seconds.
7. Wash off the stain with clean water and let it air-dry.
8. Examine the smear under the oil immersion objective to look for acid fast bacilli.

Interpretation
Acid fast bacilli (Red)
Back ground (Blue)

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Figure (1-4): Ziehl Neelsen staining procedure

Figure (1-5): Mycobacterium tuberculosis

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C. Spore staining method
Procedure
1. Prepare smear of the spore-forming bacteria and fix inflame.
2. Cover the smear with 5% malachite green solution and heat over steaming for 5minutes.
3. Wash with clean water.
4. Apply 1% safranin for 30 seconds.
5. Wash with clean water.
6. Dry and examine under the oil immersion objective.

Figure (1-6): Spore staining procedure

Figure (1-7): Endospores staining with spore stain

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D. Capsule staining (Welch method)
Capsule stain is a type of differential stain which involves the use of two stains; primary
stain and the counterstain.Two of the most commonly used methods used in capsule staining
include:(India ink method & Anthony's stain method).

India ink method

Requirements
1. India ink
2. Crystal violet
3. Glass slide
4. Inoculating loop
Procedure
1. Place a drop of India ink onto a clean glass slide.
2. Using a sterile loop place the sample on the glass slide.
3. Obtain another sterile glass slide and having laid it at an angle on one end of the first
slide, spread out the drop into a film to have a thin layer of the smear-ink mixture.
4. Allow the slide to stand for about 5 minutes (air- dry).
5. Saturate the slide with crystal violet for a minute and then tilt the slide to drain excess
dye.
6. Allow the slide to dry (air-dry).
7. View the slide using 100x.
India ink is often used as the negative stain. However, some of the other stains that can also
be used include: (Congo red, Nigrosin , Eosin).
This is a negative staining technique that is essentially used to identify the presence of
capsules. Because of its acidic nature, India ink (or Congo red, nigrosin) stains the
background dark.

Crystal violet is used for number of reasons including:

1. To act as a fixative.
2. Increase penetration power.
3. Stain the cells (being a basic dye).
4. Decrease pH of smear.
When viewed under the microscope, the background will be dark as a result of India ink,
bacteria cells will be purple having taken the crystal violet dye while the capsule will be
clear against a dark background given that it takes no stain.Nigrosin is recommended to India
ink because it gives a more even background and spreads more easily.

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Anthony's Stain Method
Some of the requirements include:
A sample (36-48 hour culture of capsulated bacteria), Glass slide, Inoculating loop.

Procedure
1. Introduce a drop of crystal violet on to a clean glass slide.
2. Using a sterile loop, place the sample on to the glass slide.
3. Obtain another glass slide and at an angle, spread the drop and sample to form a thin film.
4. Allow the film to dry (air dry) for about 6 minutes.
5. Title the slide and rinse with 20% copper sulfate solution.
6. Allow the side to air dry for about 3 minutes.
7. Place the slide on to the microscope stage and observe using oil immersion.

This is a positive staining method used in capsule staining. After the slide is air dried, it
becomes possible to observe the stain that remained on the capsular layer. Here, one will see
a dark violet color of the cell and a light violet color of the capsule.

Figure (1-8): (A) Capsule (B) Capsule staining

Staining India ink Method ANTHONY‟s method

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CULTIVATION OF BACTERIA IN CULTURE MEDIA
AND BIOCHEMICAL TESTS
Classification of culture media:
1. Based on their consistency
A. Solid medium
B. Liquid medium
C. Semi solid medium
2. Based on the constituents /ingredients
A. Simple medium
B. Complex medium
C. Special medium :
(i) Enrichment media
(ii) Enriched Media
(iii) Selective media
(iv) Indicator media
(v) Differential media
(vi) Sugar media
(vii) Transport media
(viii) Media for biochemical reaction
3. Based on oxygen requirement
A. Aerobic media
B. Anaerobic media

Culture media
The basic constituents of culture media are:
1. Peptone: Mixture of partially digested proteins
2. Agar: It is used for solidifying the culture media. It has no nutritional property.
• It is prepared from seaweeds (red algae of species Gelidium and Gracilaria ).
• Agar is preferred over gelatine, as it is bacteriologically inert, and it melts at 98°C and
usually solidifies at 42°C.
3. Others: Meat extract, Yeast extract, Blood and serum, Water and Electrolytes (NaCl).

Concentration of agar:
1. For solid agar: 1–2% (Japanese agar 2% or New Zealand agar 1.2 %(.
2. For semisolid agar: 0.5%.
3. For solid agar to inhibit Proteus swarming: 6%.

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Simple/Basal Media
They contain minimum ingredients that support the growth of non-fastidious bacteria.
Examples include:
1. Peptone water: Is a microbial growth medium composed of peptic digest of animal
tissue and sodium chloride and is rich in tryptophan (an α-amino acid that is used in
the biosynthesis of proteins) . Peptone water is also a non-selective broth medium which
can be used as a primary enrichment medium for the growth of bacteria. It contains
peptone (1%) + NaCl (0.5%) + water. The pH of the medium is 7.2±0.2 at 25 °C.

Figure (1-9 ): Peptone water medium

2. Nutrient broth: It is made-up of peptone water + meat extract (1%).

Figure (1-10): Nutrient broth media

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3. Nutrient agar: Is a general purpose liquid medium supporting growth of a wide range of
non-fastidious organisms. It typically contains (mass/volume):

 0.5% peptone - this provides organic nitrogen.

 0.3% beef extract/yeast extract - the water-soluble content of these contribute vitamins,
carbohydrates, nitrogen, and salts.
 1.5% agar - this gives the mixture solidity.
 0.5% sodium chloride - this gives the mixture proportions similar to those found in
the cytoplasm of most organisms.
 distilled water - water serves as a transport medium for the agar's various substances
 PH adjusted to neutral (6.8) at 25 °C.

Figure: (1-11) A: Escherichia coli ,B: Pseudomonas aeruginosa ,C: Staphylococcus aureus,
D: Streptococcus agalactiae.

The basal media are used for:

1. Testing the non-fastidiousness of bacteria.
2. They serve as the base for the preparation of many other media.
3. Nutrient broth is used for studying the bacterial growth curve.
4. Nutrient agar is the preferred medium for:
 Performing the biochemical tests such as oxidase, catalase and slide agglutination
 To study the colony character and Pigment demonstration.

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Enriched Media
When a basal medium is added with additional nutrients such as blood, serum or egg, it is
called enriched medium. They also support the growth of fastidious bacteria, e.g.:

1. Blood agar: Prepared by adding 5–10% of sheep blood to the molten nutrient agar at
45°C. It is used to test the hemolytic property of the bacteria.
Composition of Blood Agar
 0.5% Peptone
 0.3% beef extract/yeast extract
 1.5% agar
 0.5% NaCl
 Distilled water
(Since Blood Agar is made from Nutrient Agar, above is the composition of Nutrient
Agar)
 5%-10% Sheep Blood
 PH should be from 7.2 to 7.6 (7.4)

Preparation of Blood Agar

1. Suspend 28 g of nutrient agar powder in 1 liter of distilled water.
2. Heat this mixture while stirring to fully dissolve all components.
3. Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.
4. Once the nutrient agar has been autoclaved, allow it to cool but not solidify.
5. When the agar has cooled to 45-50 °C, Add 5% (vol/vol) sterile defibrinated blood
that has been warmed to room temperature and mix gently but well.
6. Avoid Air bubbles.
7. Dispense into sterile plates while liquid.

Uses of Blood Agar

1. Blood Agar is a general purpose enriched medium often used to grow fastidious
organisms
2. To differentiate bacteria based on their hemolytic properties (β-hemolysis, α-
hemolysis and γ-hemolysis (or non-hemolytic)).
Hemolysis is the breakdown of red blood cells (RBC). A substance that causes hemolysis is
a hemolysin. Brown (1919) introduced three terms alpha, beta and gamma to indicate three
types of streptococci based on haemolytic reactions observed on blood agar plates.

Beta-hemolytic Streptococci
Beta-hemolysis (β-hemolysis) is associated with complete lysis of red cells surrounding the
colony. Beta hemolysis is caused by two hemolysins O and S; the former is inactive in the
presence of oxygen. Thus, stabbing of the plate increases the intensity of the hemolysis
reaction. S is an oxygen-stable cytotoxin.
It exhibits a wide zone (2-4 mm wide). Beta hemolysis is more marked when the plate has
been incubated anaerobically. They are generally commensals of throat and causes
opportunistic infections.
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Examples: Streptococcus pyogenes, or Group A beta-hemolytic Strep (GAS).

Weakly beta-hemolytic
species: Streptococcus agalactiae, Clostridium perfringens, Listeria monocytogenes

Alpha-hemolytic Streptococci
Alpha-hemolysis (α-hemolysis) is a partial or “green” hemolysis associated with reduction
of red cell hemoglobin. Alpha hemolysis is caused by hydrogen peroxide produced by the
bacterium, oxidizing hemoglobin to green methemoglobin.
It exhibit incomplete haemolysis with 1-2 mm wide. Persistence of some unhaemolysed
RBC‟s can be seen microscopically.
Examples: Streptococcus pneumoniae and a group of oral streptococci (Streptococcus
viridans or viridans streptococci)

Gamma-hemolytic (Non-haemolytic) Streptococci

Colonies show neither typical alpha nor beta haemolysis. There may be, however, slight
discoloration in the medium. The streptococci included in this group are usually not
pathogenic.
Examples: Enterococcus faecalis (formerly called “Group D Strep”)

Figure (1-12) : Types of Hemolysis on blood agar

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 Figure (1-13): Hemolysis of Streptococci on blood agar

Figure (1-14) : Streptococcus pyogenes showing beta-hemolysis on blood agar

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2. Chocolate agar: Chocolate Agar (CAP or CHOC) is a nonselective, enriched medium
used for the isolation and identification of fastidious pathogens.
Chocolate agar is modified blood agar which is heated to lyse the RBC. The lysis of RBC
releases intracellular nutrients such as hemoglobin, hemin (X factor), and the coenzyme
nicotinamide adenine dinucleotide (NAD or V factor) into the agar for utilization by
fastidious bacteria. Peptone provides the organism with nitrogen, amino acids, and other
elements essential for growth, sodium chloride maintains the osmotic balance, and agar acts
as a solidifying agent. The name is derived from the fact that the lysis of RBC gives the
medium a chocolate-brown color.

Preparation of Chocolate Agar

1. Prepare the blood agar base as instructed by the manufacturer.
2. Sterilize by autoclaving at 121°C for 15 minutes.
3. Add 5-7% v/v of defibrinated blood (horse or sheep blood) and place the media in a
water bath of 75 -80°C, and keep swirling gently until the color changes to dark
brown.
4. Pour into sterile Petri plates under aseptic conditions after the media has cooled to 50-
55°C.
5. Label the plates with the name of the media, and date of preparation, and store them
invertedly at 2-8°C until use.

Uses of Chocolate Agar

1. To isolate fastidious organisms such as H. influenzae, N. gonorrhoeae, and N.
menigitidis from various clinical specimens, that aid in the diagnosis of disease.
2. Chocolate agar with bacitracin acts as a selective medium for screening H.
influenzae from specimens e.g. sputum containing a mixed flora of microorganisms

Limitations of Chocolate Agar

1. Since chocolate agar is an enriched medium, it gets contaminated very easily.
2. Use of chocolate agar only, makes it difficult to differentiate non-pathogenic
organisms from pathogenic ones as the former overgrow pathogenic bacteria.
3. Chocolate agar has a reduced concentration of agar, so the surface of the plate is
fragile and prone to scratches during streaking.

Modifications of Chocolate Agar

1. Thayer-Martin Media: Thayer martin medium is a modification of chocolate agar
supplemented with vancomycin, nystatin, and colistin to inhibit the normal flora,
including nonpathogenic Neisseria for the selective isolation of N. gonorrhoeae and N.
meningitidis.
2. Chocolate Agar with bacitracin: This modification is used as a selective medium to
improve the primary isolation of H. influenzae from specimens such as sputum that
contains a mixed flora of bacteria and/or fungi.
3. Chocolate agar supplemented with GC base and growth supplement: This variant
of chocolate agar is used to support the special growth requirements (hemin and NAD)
needed for the isolation of Haemophilus spp. when incubated at 35-37°C in a 5% CO2
atmosphere.
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 4. Chocolate agar with TSA and growth supplements: It is a modification of
chocolate agar that supports the special growth requirements (hemin and NAD)
needed for the isolation of fastidious organisms such as H. influenzae when incubated
at 35-37°C in a 5% CO2 atmosphere.

Colony Morphology
Almost all organisms grow on chocolate agar giving grey colonies of various sizes.
However, since it is used specifically to isolate fastidious pathogens such
as Neisseria and Haemophilus, their colony morphologies are described below.

Table 1–2: Colony Morphology on chocolate agar.

Organism Colony morphology

pinkish-brown and translucent, exhibit smooth consistency and

Neisseria gonorrhoeae defined margins, and are typically 0.5-1 mm in diameter

grayish, non-hemolytic, round, convex, smooth, moist, glistening

colonies with a clearly defined edge, larger in size as compared to N.
N. meningitidis gonorrhoeae

Nonhemolytic, colorless, moist colonies with a characteristic

Haemophilus influenzae “mousy” odor.

3. Loeffler’s serum slope is used for isolation of Corynebacterium diphtheriae.

4. Blood culture media: Used for blood culture. They are of two types:
 Monophasic medium is made-up of brain heart infusion (BHI) broth
 Biphasic medium has a liquid phase (BHI broth) and a solid agar slope (BHI agar).

Enrichment Broth
Liquid media that allow certain organism (pathogens) to grow and inhibit others (normal
flora)
• Selenite F and Tetrathionate broth used for Salmonella and Shigella
• Alkaline peptone water (APW) used for Vibrio cholerae.

Selective Media
Solid media that allow certain organism (pathogens) to grow and inhibit others (normal
flora):
1. Lowenstein Jensen (LJ) medium is used for isolation of Mycobacterium tuberculosis
2. Thiosulphate Citrate Bilesalt Sucrose (TCBS) agar used for isolation of Vibrio species.
3. For the isolation of enteric pathogens such as Salmonella and Shigella from stool :
a) DCA (Deoxycholate Citrate Agar).
b) XLD (Xylose Lysine Deoxycholate) agar.

16
4. Potassium tellurite agar (PTA) is used for isolation of Corynebacterium diphtheriae.
5. Wilson Blair bismuth sulphite medium: It is used for isolation of Salmonella Typhi.
6. MacConkey agar: is a selective and differential culture medium for bacteria. It is designed
to selectively isolate Gram-negative and enteric bacteria and differentiate them based on
lactose fermentation. Lactose fermenters turn red or pink on McConkey agar, and
nonfermenters do not change color.
Using neutral red pH indicator, the agar distinguishes those Gram-negative bacteria that can
ferment the sugar lactose (Lac+) from those that cannot (Lac-). This medium is also known
as an "indicator medium" and a "low selective medium". Presence of bile salts inhibits
swarming by Proteus species.

Transport Media
They are used for the transport of specimens containing delicate organism or when the delay
is expected. Bacteria do not multiply in the transport media; they only remain viable.

Table 1–3: Transport media used for common bacteria

Organism Transport media
Streptococcus Pike’s medium
Neisseria Amies medium, Stuart’s medium
Vibrio cholerae VR (Venkatraman-Ramakrishnan) medium, Cary Blair medium
and Autoclaved sea water
Shigella, Buffered glycerol saline, and Cary Blair medium
Salmonella

Enteric pathogen Carry Blair medium

Bordetella Modified Stuart’s (with casamino acid) ,Mischulow’s charcoal agar

Dacron or calcium alginate swab used

Differential Media
These media differentiate between two groups of bacteria by using an indicator.
1. MacConkey agar: It differentiates organisms into LF or lactose fermenters (produce pink
colonies, e.g. E. coli and Klebsiella) and NLF (produce colorless colonies, e.g. Shigella
and Salmonella).
2. CLED agar (Cysteine lactose electrolyte-deficient agar): This is similar to MacConkey
agar, differentiates between LF and NLF. It is used as an alternative to combination of
blood agar and MacConkey agar, for the processing of urine specimens:
• Advantages over MacConkey agar: It is less inhibitory than MacConkey agar, supports
gram-positive bacteria (except β hemolytic Streptococcus) and Candida.
• Advantage over blood agar: It can prevent the swarming of Proteus.

17
Table 1–4: Diagram of some microbiological culture media

18
Anaerobic Culture Media
Anaerobic media contain reducing substances which take-up oxygen and create lower redox
potential and thus permit the growth of obligate anaerobes such as Clostridium. Examples
are:
1. Robertson’s cooked meat (RCM) broth: It contains chopped meat particles (beef heart),
which provide glutathione and unsaturated fatty acids.
2. Other anaerobic media include:
• BHIS agar: Brain heart infusion agar with supplements (vitamin K and hemin)
• Thioglycollate broth, Anaerobic blood agar
• Neomycin blood agar, Egg yolk agar and Phenylethyl agar.

CULTURE METHODS
Various Aerobic Culture Methods:
A. Streak culture by using loop with intermittent heating: It is the routinely used
method, is a technique used to isolate a pure strain from a single species of bacteria.
Samples can then be taken from the resulting colonies and a microbiological culture can
be grown on a new plate so that the organism can be identified, studied, or tested.

Figure (1-15): Streak culture methods

19
 Figure (1-16): A plate which has been streaked showing the colonies thinning as the
streaking moves clockwise

Objectives of Streak Plate Method

1. To obtain a pure culture of bacteria from a mixed culture
2. To obtain well-isolated colonies
3. To propagate bacteria

Principle of Streak Plate Method

The streak plate method is based on dilution during the process of mechanical spreading of
inoculum over the surface of solidified culture media in order to obtain well-isolated
colonies of the sample at the terminal streaks. Sample can be either colony on solid media or
suspension in broth. The sample is picked by using different tools, mostly using a sterile
inoculating loop or swab. The sample is placed over a surface of sterile solid media at one
edge of the petri dish and a smear is prepared.
Using the tool, the smear is successively streaked over the agar medium on different patterns.
As the streaking proceeds, the inoculum is gradually diluted to the point where bacterial cells
are separated as individual cells or as a colony-forming unit (CFU) at a gap of a few
millimeters. When these inoculated plates are incubated, the isolated bacterium or a CFU
will give rise to a well-isolated colony. This will allow us to get a pure culture as well as
describe the colony morphology of the organism.

Types of Streak Plate Method

Based on the pattern of streaking, the streak plate method can be classified into 4 types :
Quadrant Streaking, T-Streaking, Continuous Streaking, and Radiant Streaking.

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1. Quadrant Streaking
It is the most commonly used and the most preferred method where four equal-sized sections
of the agar plate are streaked. It is also referred to as the “four-quadrant streak” or “four
sectors” or “four-way streak” method.
In this method, each plate is divided into four equal sectors and each adjacent sector is
streaked sequentially. The sector which is streaked first is called the first sector or the first
quadrant, and it has the highest concentration of inoculum. Gradually the second, third, and
fourth quadrants will have diluted inoculum. By the time the fourth quadrant is streaked, the
inoculum is highly diluted giving rise to isolated colonies following the incubation.
Mostly, a discontinuous fashion of streaking is followed where the loop is sterilized at the
end of each quadrant prior to streaking over the next quadrant. However, if the bacterial load
is too small (or highly diluted), continuous fashion can also be used. In the latter, the loop
needs not be sterilized at the end of every quadrant.
Although being the most popular method, it limits us to use only one specimen per plate. If
we try two or more specimens in a single 10 cm plate, this method is not suitable.

Figure (1-17) : Four Quadrant Streak Method Steps

21
2. T-Streaking
It is another method of streaking where the agar Petri plate is divided into three sections and
each section is streaked. Hence, this method is also known as the “three-sector streak”
method. The media is divided into three sections by drawing a letter (T) and each adjacent
section is streaked sequentially.
By the time the final section is being streaked, the inoculum is diluted to the point to give
rise to isolated colonies following the incubation. Mostly discontinuous fashion of streaking
is followed; however, a continuous fashion can also be used in the very dilute specimen.
As in quadrant streaking, it is difficult to culture two or more samples in a single 10 cm plate
using this method.

Figure (1-18) : Steps of T- Streak Pattern

22
 Figure (1-19) : T streak (three-sector method)

3. Continuous Streaking
It is another commonly followed method where an inoculum is evenly distributed in a single
continuous movement from starting point to the center of the plate. There is no need to
divide the plate and sterilize the loop during the process. It is easy and quick; however, the
problem is that we can use it only if the inoculum is either very diluted or we just have to
propagate pure culture rather than isolate one.
We can divide the 10 cm Petri plate into different sections (mostly 2 to 6), and in each
section, we can streak different specimens following this method. Hence, it is used in the
clinical laboratory to culture urine, sputum, pus, etc. if multiple samples have arrived at a
single time. This will allow us to save media and get maximum output using a minimum
resource.

4. Radiant Streaking
It is another method of streaking where the inoculum is first streaked at one edge and spread
in vertical lines above the edge. Finally, the vertical lines are cross streaked diagonally. This
method is suitable to propagate pure culture, and also in the case of a dilute specimen.
There are other modified forms of streaking like:
5. Semi-quantitative Streaking
It is routinely followed in urine culture. It is a modified form of continuous streaking. In this
method, a calibrated loop (usually a loop of 1 or 2μl) is used to streak a certain volume of the
liquid specimen. A loopful of the specimen is streaked in a horizontal line in the middle of
the Petri plate, and the specimen is spread all over the plate in a single continuous back and
forth movement.

23
This method allows us to approximately quantify the viable load (in a range, not an exact
number) as well as get the pure culture in a single go.
6. Zigzag Streaking
It is another form of continuous streaking where a loopful of the specimen is streaked all
over the plate in a zigzag pattern in a single continuous movement. It is commonly done to
propagate the pure culture and culture them in large quantities.

Figure (1-20) : Zigzag,Radiant and Continuous Streaking Methods

Requirements of Streak Plate Method

1. Streaking tool
This is a sterile tool used to streak the specimen over the surface of culture media. The tools
used for streaking are cotton swabs, inoculating loop (both metal and plastic), toothpicks,
and wooden or metal or plastic sticks/wires. The most commonly used one is inoculating
loop (nichrome wire loop).
(In this whole article, we will talk about inoculating loop.)

2. Sample culture
Sample bacteria may be in the form of suspension, liquid broth, or colonies over solid media.
The sample is picked by using an inoculating loop and transferred over the surface of fresh
culture media to perform streaking.

3. Solid culture media

Specific culture media is used for the isolation and differentiation of suspected (or specific)
bacteria. The culture medium is a solid agar medium that is pre-solidified before use.

24
4. Bunsen burner and other laboratory facilities
A Bunsen burner is used to sterilize the loop and also to create a sterile zone around the
flame. Besides, other chemicals, sterilizing materials, and laboratory apparatus are also
required.

Procedure or Protocol of Streak Plate Method

The general procedure of the streak plate method can be summarized as:
1. Arrange all the requirements, put on the PPE, sterilize the work surface, and allow all
the samples and media to come to room temperature if were refrigerated.
2. If the sample is very concentrated then dilution can be helpful to get the isolated
colonies. (But it is not compulsory as the sample will be diluted during the streaking
process.)
3. Sterilize the inoculating loop by flaming and allow it to cool. Pick a small portion of
the isolated colony. (if the sample is in the suspension then take a loopful of the
sample)

Figure (1-21) : Procedure or Protocol of Streak Plate Method

25
The inoculating procedure is different according to the method of streaking :

Quadrant Streaking Procedure

1. Lift the Petri plate in your left hand and hold it at an angle of 60°.
(if you are left-handed, hold the plate in your right hand)
2. The sample is spread over about 1/4th of the media in the Petri plate from the rim to
the center of the plate using a rapid, gentle, back and forth motion.
(For ease, a beginner can draw two diameters intersecting each other diagonally at the back
of the petri dish to divide the media into 4 equal sections)
3. Re-flame the loop and allow it to cool. Turn the Petri plate by 90° anticlockwise, and
place the loop to the last streaks of the first quadrant. Move the loop back and forth to
spread the inoculum over the last half of the streaks in the first quadrant into the empty
second quadrant.
(Be sure not to move the loop to the streaks in the first half of the first quadrant.)
4. Repeat the process (3) for streaking the third quadrant and the fourth quadrant.
5. For the fourth quadrant similar step can be followed. However, many people prefer to
draw a few (6 to 7 streaks) well-separated streaks by touching the second half of
streaks in the third quadrant. Also, some prefer to make the final streak in a zigzag
fashion making a tail.
(In a discontinuous fashion, the loop is sterilized after streaking each quadrant. In a
continuous fashion, there is no need to flame the loop after streaking each quadrant. But, this
is preferred only if the sample is very dilute.)

T-Streaking Procedure
1. Lift the Petri plate in your left hand and hold it at an angle of 60 0. (if you are left-
handed, hold the plate in your right hand).
2. The sample is spread over about 1/3rd of the media in the Petri plate from the rim to
the center of the plate using a rapid, gentle, back and forth motion.
(For ease, a beginner can draw a letter (T) at the back of the petri dish to divide the media
into 3 sections).
3. Re-flame the loop and allow it to cool. Turn the Petri plate by 90 0 anticlockwise, and
place the loop to the last streaks of the first quadrant. Move the loop back and forth to
spread the inoculum over the last half of the streaks in the first quadrant into the empty
second quadrant.
(Be sure not to move the loop to the streaks in the first half of the first quadrant.)
4. Repeat the process (iii) for streaking the third quadrant. As in quadrant streaking, you
can follow any one of the streaking patterns at the 3rd quadrant.

Continuous Streaking Procedure

1. Lift the Petri plate in your left hand and hold it at an angle of 60 0. (if you are left-
handed, hold the plate in your right hand)
2. Place the loop at one end of the plate and start streaking the inoculum from that point
in a continuous movement to the center of the plate.
3. Rotate the plate by 1800 and without sterilizing the loop, follow the step (ii) to streak
the remaining half of the plate.

26
Radiant Streaking Procedure
1. Lift the Petri plate in your left hand and hold it at an angle of 60 0. (if you are left-
handed, hold the plate in your right hand)
2. Spread the inoculum over the near edge of the agar plate using a gentle zigzag motion.
3. Sterilize the loop and allow it to cool. Then, the streak from the point of primary
spread in a radial direction up to the far edge of the Petri plate. (Be sure to streak the
lines far away from each other.)
4. Re-flame the loop and allow it to cool. Then draw horizontal lines crossing the radial
streaks.

Semi-quantitative Streaking Procedure

1. Lift the Petri plate in your left hand and hold it at an angle of 60 0. (if you are left-
handed, hold the plate in your right hand)
2. Using a calibrated loop take a loopful of the sample (urine).
3. Draw the sample into a vertical or horizontal streak (primary streak) at the center of
the plate.
4. Using the same loop spread the inoculum by continuously moving the loop in back
and forth (zigzag) motion crossing the primary streak.
5. Label at the edge of the bottom of the plate with the date, name, sample ID, and other
required information.
6. Incubate the plate in an inverted position under suitable incubation conditions (mostly
for 24 hours at 370C).

Result Interpretation of Streak Plate Method

 Results can be interpreted after the incubation period (mostly 24 hours at 37 0C).
Following incubation, the terminal streaks are observed for isolated colonies.
Morphologies of the isolated colonies are tabulated. If the culture media used is
indicator type, then changes in the medium are also tabulated (E.g. lactose
fermentation in MacConkey Agar).
 In the first area of streaking, there is heavy growth with fused colonies, and gradually
there are fewer colonies in subsequent streaks giving a few well-isolated colonies in
the final streak.
 Colonies with similar appearances are expected in pure culture.
 If the sample contained single species then colonies with similar morphologies are
obtained. But, in the case of mixed culture, colonies with different morphologies are
obtained.
 If there are different types of colonies, each colony must be streaked again in another
plate to get a pure culture of each species.

[Exception: in some cases where colony characters of two or more bacterial species are the
same, all the colonies may look alike even if they are of a different individual.]

27
Precautions during Streak Plate Method
1. Media should be properly solidified before use. If it is refrigerated, allow it to come to
room temperature.
2. Check for the presence of water droplets and/or any contamination or foreign
substance in media prior to streaking.
3. Follow proper safety protocols. Treat every unknown or clinical specimen as
hazardous and follow safety accordingly.
4. If using a toothpick for streaking, use the blunt end by holding the pointed end
between your thumb and ring finger at an angle of 10 to 20 0 to the medium. Dispose it
after streaking each quadrant and take a new one to streak the second quadrant.
5. If the sample is in suspension, properly mix the suspension before taking inoculum.
Follow the aseptic technique during the process. Flame the rim of the test tube or
bottle before and after taking the inoculum. If using a micropipette, don‟t touch the
wall of the tube or bottle with the pipette barrel.
6. If the sample is a colony, gently touch the colony with a sterile and cool loop. Don‟t
take the entire colony or large portion, just touch the colony and it will be enough.
7. Always work in a sterile area (between flames of a Bunsen burner or in a biosafety
cabinet). Properly sterilize the inoculating loop before and after use. If flame
sterilization is followed, make sure that the loop is cooled before using.
8. Appropriate media should be selected.
9. Use only a small amount of sample. Heavy inoculum doesn‟t produce isolated
colonies.
10.Streak gently without applying high pressure.
11.Flame the loop after streaking each quadrant.
12.Rotate the plate anticlockwise after streaking each quadrant. Do not streak from the
first half of the previous quadrant.
13.Follow the suitable streaking pattern. If multiple samples are streaked in the same
plate, ensure that there is at least a 20 – 30 mm gap between the streaking zones of
each sample.
14.Label properly and incubate under suitable conditions.

Applications of Streak Plate Method

1. Used to obtain a pure culture from the mixed culture in order to perform
morphological, biochemical, and molecular tests to identify and for other applications.
2. Used to define the specimen as pure or mixed species.
3. Used to study colony characters of bacteria.
4. Used to produce a colony of genetically identical individuals
5. Used in inoculation of clinical specimens in diagnostic laboratories to grow isolated
colonies of pathogen
6. Used in urine culture to isolate pathogens and semi-quantify the uropathogens to
determine the significance of the infection. A calibrated loop is used for this purpose.

28
Advantages of Streak Plate Method
1. It is a simple, reliable, convenient, and easy-to-perform method of inoculation.
2. It results in well-isolated colonies, each of genetically identical individuals; hence, we
can perform further tests and applications on the isolates. Hence, it is followed in
clinical diagnosis.
3. Dilution is done along with the process of inoculation (or streaking), hence, no need to
perform separate dilution of the sample.
4. Allow manually to control the sample and sample size and the inoculating area in a
petri dish.
5. Different patterns of streaking give flexibility in selecting the appropriate method
based on sample size, availability of Petri dishes, and other requirements.
6. It is a suitable and less-time consuming method to culture aerobic organisms.
7. We can use a sample in both states; from the broth or suspension, as well as colonies
from solid media.

B. Lawn or carpet culture: Useful for Carrying out antimicrobial susceptibility

testing by disk diffusion method, Bacteriophage typing and producing large amount of
bacterial growth for preparation of bacterial antigens and vaccines.

Figure (1-22): A bacterial lawn used in antibiotic resistance testing

C. Stroke culture: Stroke culture provides a pure growth of bacteria for carrying out
slide agglutination and other diagnostic tests. It is carried out in tubes usually containing
nutrient agar slopes.

29
 Figure (1-23): Stroke culture media

D. Stab culture: Stabbing the semisolid agar butt by a straight wire. It is used for:
(i) maintaining stock cultures, (ii) OF test (iii) Mannitol motility medium, (iv) Nutrient agar
semisolid butts, (v) TSI (here both stroke and stab cultures are made).

Figure (1-24): Stab culture media

E. Liquid culture:
Liquid culture is prepared in a liquid media enclosed in tubes, flasks, or bottles. The medium
is inoculated by touching with a charged loop or by adding the inoculum with pipettes or
syringes and incubating at 37°C, followed by subculture on to solid media for final
identification.

F. Pour-plate culture: Quantitative culture method, used to estimate viable bacterial count.

30
G. Anaerobic Culture: Obligate anaerobes are bacteria that can live only in the absence of
oxygen. These anaerobes are killed when exposed to the atmosphere for as briefly as 10
minutes. Some anaerobes are tolerant to small amounts of oxygen. Facultative anaerobes are
those anaerobes that grow with or without oxygen.

Anaerobic bacterial culture is a method used to grow anaerobes from a clinical specimen.
Culture and identification of anaerobes is essential for initiating appropriate treatment.

Figure (1-25): Anaerobic Culture jar

Incubatory Conditions
1. Most of the pathogenic organisms grow best at 37°C.
2. Candle jar: It provides capnophilic atmosphere (3–5% CO2). This is useful for
Brucella abortus, Streptococcus, pneumococcus and gonococcus.
3. Microaerophilic bacteria such as Campylobacter and Helicobacter require 5% oxygen.

Anaerobic Culture Methods

1. Production of vacuum.
2. By displacement and combustion of oxygen: This principle is used in:
• McIntosh and Filde‟s anaerobic jar (It involves evacuation of air and replacement with
hydrogen gas manually)
• Anoxomat System (principle is same, but done by automated instrument)
3. Gas pak System (Absorption of oxygen chemically, e.g. using alkaline pyrogallol).
Indicators of anaerobiosis are:
• Chemical indicator: Reduced methylene blue
• Biological indicator: Pseudomonas
4. Anaerobic Glove Box (or anaerobic chamber)
5. By reducing agents: Such as glucose, thioglycollate, meat, cysteine and ascorbic acid.
6. PRAS media (Prereduced, Anaerobically Sterilized).

31
Aerobic culture methods
1. Streak culture
2. Lawn or carpet culture
3. Stroke culture
4. Stab culture
5. Liquid culture
6. Pour-plate culture

Anaerobic Culture Methods

1. Production of vacuum
2. By displacement and combustion of oxygen:
• McIntosh and Filde's an- aerobic jar
• Anoxomat System
3. Gas pak System
4. Anaerobic Glove Box
5. By reducing agents
6. PRAS media

Preservation of Microorganisms
1. Short-term methods: (i) Sub-culturing, (ii) Immersing the culture in glycerol, or
sterile distilled water, (iii) Freezing at –20°C and (iv) Drying (for moulds and spores).
2. Long-term methods: By Ultra temperature freezing and Lyophilization (freeze-
drying).

IDENTIFICATION OF BACTERIA
Identification of bacteria can be done by: (i) conventional (culture and identification by
biochemical reactions), (ii) automated culture techniques (iii) molecular methods.

Automated Culture Techniques

Conventional culture methods often yield poor results because of low bacterial load.
Therefore, various automated blood culture techniques have been in use since last decade.
Advantages: The major advantages of automated blood culture techniques are:
• Continuous automated monitoring (once in every 15–20 min by the instrument).
• Other advantages: More sensitive,↑ yield, rapid, less labor intensive.
Disadvantages: (i) high cost (ii) inability to observe the colony morphology as liquid
medium is used, (iii) no separate detection in mixed cultures, (iv) radioactive hazards for
BACTEC.

32
MOLECULAR METHODS
Nucleic acid amplification techniques (NAATs) include:
1. Polymerase chain reaction (PCR) and its modification including Real time PCR.
2. Ligase chain reaction (LCR) and Transcription-mediated amplification (TMA).

Table 1–5: Automated culture systems in diagnostic bacteriology

For bacterial culture Principle used

1. BACTEC In was radiometry based, but later changed to fluorescent based

detection technique
2. BacT/Alert CO2 liberated from bacteria, causes a pH change, detected by
colorimeter
3. ESP culture CO2 liberated from bacteria, causes a pressure change, detected
system by manometer
For bacterial •Phoenix bacterial identification system
identification • MALDI –TOF (Matrix-assisted laser desorption/ionization time-

of-flight), e.g. VITEK MS

• VITEK 2 bacterial identification and antimicrobial sensitivity

system
• Microscan Walkaway system

For M. Tuberculosis MGIT (Mycobacterial Growth Indicator Tube)

• Nucleic acid sequence based amplification (NASBA)

• Strand displacement amplification (SDA).

33
Table 1 – 6: Examples of Special Media

Organism Medium
Enteric pathogens—for Hektoen enteric agar (S) Xylose-lysin-deoxycholate agar (S)
Salmonella, Shigella Deoxycholate citrate agar (S) Eosin methylene blue agar (S)
MacConkey (D and S) Salmonella Shigella agar (S)
Wilson blair for Salmonella (S)Selenite F broth (En), Tetrathionate
broth (En)
Blood culture—for Castaneda’s biphasic media (E): Brain-heart infusion agar slope and
blood-borne pathogens broth

Vibrio cholerae (likes TCBS (Thiosulfate Citrate Bile Sucrose agar) (S) Mansour’s Gelatin
alkaline growth Taurocholate Trypticase agar (S) Alkaline bile salt agar (S)
medium) APW: Alkaline Peptone Water (En)

S.aureus Mannitol salt agar (S) Milk salt agar (S) Ludlam’s medium (S)
Streptococcus Crystal violet blood agar (S)
Neisseria Chocolate agar (E), Thayer-Martin media (S),
Modified New York medium (S)
Corynebacterium Loeffler’s serum medium (E)
Potassium tellurite agar (S)
Bacillus anthracis PLET: Polymyxin Lithium EDTA Thallous acetate (S)
Bacillus cereus MYPA: Mannitol egg yolk phenol red polymyxin agar (S)
Anaerobes Thioglycollate (En)
Robertson cooked meat broth (En)
Listeria PALCAM agar (S)
Pseudomonas Cetrimide agar (S), King’s media (for pigment)
Burkholderia Ashdown’s medium
Haemophilus Blood agar with staph streak (E) Chocolate agar (E)
Levinthal’s medium (E), Fildes agar (E)
Bordetella Regan low media (E)
Bordet Gengou Glycerin potato blood agar (E) Lacey’s DFP media (S)
Mycobacterium Lowenstein Jensen, Dorset egg (S)
Leptospira EMJH (E), Fletcher’s (E), Korthof’s (E)
Campylobacter Skirrow’s, Butzler, Campy BAP (S)
Legionella BCYE (Buffered charcoal yeast extract) (E)
Reiter’s treponema Smith Noguchi media
Urinary pathogen MacConkey agar (D and S)
Cystein Lactose Electrolyte-deficient agar (CLED agar) (D and S)

34
Polymerase Chain Reaction (PCR)
PCR is a technology in molecular biology used to amplify a single or few copies of a piece
of DNA to generate millions of copies of DNA. It was developed by Kary B Mullis.

Principle: PCR involves three basic steps:

1. DNA extraction from the organism.
2. Amplification of extracted DNA: The extracted DNA is subjected to repeated cycles (30–
35 numbers) of amplification in a thermocycler which takes about 3–4 hours. Each
amplification cycle has three steps:
• Denaturation at 94°C: This involves separation the dsDNA into two separate ssDNA.
• Primer annealing (54°C): Primer is a short oligonucleotide complementary to a small
sequence of the target DNA. It anneals to the complementary site on ssDNA.
• Extension of the primer (72°C): This step is catalyzed by Taq Polymerase enzyme.

3. Gel electrophoresis of amplified product: The amplified DNA is electrophoretically

migrated according to their molecular size to form bands; seen under UV rays.

COMPONENTS OF PCR
1.DNA template:
DNA template is DNA target sequence. DNA template is the DNA molecule that contains
the DNA region (segment) to be amplified, the segment we are concerned which is the target
sequence.
2.DNA polymerase:
DNA polymerase sequentially adds nucleotides complimentary to template strand at 3‟-OH
of the bound primers and synthesizes new strands of DNA complementary to the target
sequence. The most commonly used DNA polymerase is Taq DNA polymerase (from
Thermus aquaticus, a thermophillic bacterium) because of high temperature stability. Pfu
DNA polymerase (from Pyrococcus furiosus) is also used widely because of its higher
2+
fidelity (accuracy of adding complimentary nucleotide). Mg ions in the buffer act as co-
factor for DNA polymerase enzyme and hence are required for the reaction.
3.Primers:
To achieve selective amplification of nucleotide sequences from a DNA extract by PCR, it is
essential to have least one pair of oligonucleotides. These oligonucleotides, which will serve
as primers for replication, are synthesized chemically and must be the best possible
complementarity with both ends of the sequence of interest that one wishes to amplify. One
of the primers is designed to recognize complementarily a sequence located upstream of the
fragment 5′–3′ strand DNA of interest; the other to recognize, always by
complementarity, a sequence located upstream complementary strand (3′–5′) of the same
fragment DNA.
Primers are single-stranded DNAs whose hybridization on sequences flanking the sequence
of interest will allow its replication so selective. The size of the primers is usually between
10 and 30 nucleotides in order to guarantee a sufficiently specific hybridization on the
sequences of interest of the matrix DNA.

35
Two primers must be designed for PCR; the forward primer and the reverse primer. The
forward primer is complimentary to the 3‟ end of antisense strand (3‟-5‟) and the reverse
primer is complimentary to the 3‟ end of sense strand (5‟-3‟). If we consider the sense strand
(5‟-3‟) of a gene, for designing primers, then forward primer is the beginning of the gene and
the reverse primer is the reverse-compliment of the 3‟ end of the gene.

4.Nucleotides (dNTPs or deoxynucleotide triphosphates):

All types of nucleotides are "building blocks" for new DNA strands and essential for
reaction. It includes Adenine(A), Guanine(G), Cytosine(C), Thymine(T) or Uracil(U).

5. Magnesium
Magnesium affects primer annealing and template denaturation, as well as enzyme activity.
An excess of magnesium gives non-specific amplification products, while low magnesium
yields lesser amount of desired product.

Procedure
There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on
an automated cycler, which can heat and cool the tubes with the reaction mixture in a very
short time.

Denaturation at 94°C :
During the heating step (denaturation), the reaction mixture is heated to 94°C for 1 min,
which causes separation of DNA double stranded. Now, each strand acts as template for
synthesis of complimentary strand.

36
Annealing at 54°C:
This step consist of cooling of reaction mixture after denaturation step to 54°C, which causes
hybridization (annealing) of primers to separated strand of DNA )template). The length and
GC-content (guanine-cytosine content) of the primer should be sufficient for stable binding
with template.
Guanine pairs with cytosine with three hydrogen bonding adenine binds with thymine with
two hydrogen bonds. Thus, higher GC content results in stronger binding. In case GC
content is less, length may be increased to have stronger binding (more number of H bonding
between primer and template).

Extension at 72°C :
The reaction mixture is heated to 72°C which is the ideal working temperature for the Taq
polymerase. The polymerase adds nucleotide (dNTP's) complimentary to template on 3‟ –
OH of primers thereby extending the new strand.

Final hold:
First three steps are repeated 35-40 times to produce millions of exact copies of the target
DNA. Once several cycles are completed, during the hold step, 4–15 °C temperature is
maintained for short-term storage of the amplified DNA sample.

37
Advantages
PCR has the following advantages compared to the conventional culture:
1. More sensitive: It can amplify very few copies of a specific DNA, so it is more
sensitive.
2. More specific: By use of primers targeting specific DNA sequence of the organism
3. Detects the organism: (i) either from sample, (ii) to confirm culture isolate.
4. Detect the organisms that are highly fastidiousor noncultivable by conventional culture
methods.
5. Detect genes coding drug resistance (e.g. MecA gene detection in Staphylococcus
aureus)
6. Detects genetic diseases such as sickle cell anemia, phenylketonuria, etc.

Disadvantages
1. Conventional PCR detects only the DNA, but not the RNA (detected by RT-PCR).
2. Qualitative, not quantitative (Quantitation is done by real time PCR).
3. Viability: PCR cannot differentiate between viable or nonviable organisms.
4. False positive amplification may occur due to contamination with environmental
DNA.
5. False negative: by PCR inhibitors present in some specimens such as blood, feces, etc.

Modification of PCR
1. Reverse transcriptase PCR (RT-PCR): For amplifying RNA, RT-PCR is done.
• After RNA extraction, the first step is addition of reverse transcriptase enzyme that
coverts RNA into DNA. Then, the amplification of DNA and gel documentation steps is
similar to that described for conventional PCR.
• Useful for detection of RNA viruses or 16SrRNA genes of the organisms.
2. Nested PCR: The amplified products of the first round PCR is subjected to another
round of amplification using a second primer targeting a different gene of same
organism.
• More sensitive: Double round of amplification yields high quantity of DNA.

38
 • More specific: Use of two primers targeting same organisms makes more specific.
• Disadvantage: There is more chance of contamination of the PCR tubes, which may lead
to false positive results.

3. Multiplex PCR: It uses more than one primer which can detect many DNA sequences
of several organisms in one reaction.
• Syndromic approach: To diagnose infectious syndrome caused by more than one
organism.
• Contamination chances of reaction tubes with environmental DNA.

Real-time PCR (rt-PCR)

Real time PCR, though expensive, but has many advantages over a conventional PCR:
1. Quantitative, hence can be used for monitoring treatment response, e.g. in HIV or
HBV.
2. Takes less time: As the amplification can be visualized even when the amplification
cycle is going on.
3. Contamination rate is extremely less.
4. Sensitivity and specificity of rt-PCR assays are extremely higher.

Detection of amplification products of real time PCR

1. Nonspecific methods use SYBR green dye that stains any nucleic acid
nonspecifically.
2. Specific methods use fluorescent labeled DNA probe such as (i) TaqMan or
hydrolysis probe, (ii) Molecular beacon and (iii) FRET (Fluorescence Resonance
Energy Transfer) probe.

MICROBIAL TYPING
Microbial typing refers to characterization of an organism beyond its species level. It is used
to determine the relatedness between different microbial strains of the same species and
thereby helps to (i) investigate outbreaks, (ii) determine the source and routes of infections.
Genotypic methods are more reliable and have better reproducibility and discriminative
power than phenotypic methods, however they are expensive.

Bacteriophage typing is done for:

• Staphylococcus aureus

• SalmonellaTyphi
• Vibrio cholerae

• Brucella

• Corynebacterium diphtheriae

Bacteriocin typing is done for:

• Shigella sonnei (colicin typing)

• Klebsiella (klebocin typing),

• E.coli (colicin typing)

• Proteus (proticin typing),

• Pseudomonas (pyocin typing)

39
Biotyping is done for:
• C. diphtheriae
• Vibrio cholerae

Table 1–7: Typing methods

Phenotypic methods Genotyping methods
Bacteriophage typing: Based on their susceptibility to Non Amplification based
bacteriophages. It is done for methods
• Staphylococcus aureus and SalmonellaTyphi 1. Plasmid profile analysis
• Vibrio cholerae, Brucella and Corynebacterium diphtheriae 2. Chromosomal DNA analysis
3. RFLP (Restricted fragment
Bacteriocin typing: Based on the ability of a strain to produce length polymorphism)
particular bacteriocin which inhibits the growth of a set of 4. Ribotyping (RFLP analysis of
selected indicator strains. It is done for: ribosomal DNA)
• Shigella sonnei (colicin typing) 5. Pulse field gel electrophoresis
• Klebsiella (klebocin typing), E.coli (colicin typing) (PFGE)-Gold standard method
• Proteus (proticin typing),Pseudomonas (pyocin typing) Amplification based methods
1. PCR-RFLP
Biotyping: Based on different biochemical properties of the
2. Amplified Fragment Length
organism. It is done for
Polymorphism (AFLP)
• C.diphtheriae (gravis, intermedius and mitis
3. Sequencing-based methods
• Vibrio choleraeO1(classical and El Tor) and Yersinia pestis
4. Microarrays
Antibiogram typing: Based on their resistance pattern to different
antimicrobials. It is the most commonly used typing method.
Auxotyping: Based on nutritional requirement of the organism
(e.g. Gonococcus)
Morphotyping: Based on different type of colonies in culture (e.g.
Pseudomonas)

Serotyping: Based on the antigenic property of an organism.

• Streptococcus (Lancefield grouping)
• Based on capsular antigen, e.g. pneumococcus,
meningococcus and H. influenzae
• Based on somatic antigen- E. coli, Shigella, Salmonella and
Vibrio cholerae.

40
COMMON BIOCHEMICAL TESTS
1. CATALASE TEST
This test is used to differentiate those bacteria that produce the enzyme catalase, such as
staphylococci, from non-catalase producing bacteria such as streptococci.

Principle:
2H2O2→catalase→ 2H2O + O2 (bubbles)
Water free oxygen

Results
Active bubbling ----------------- Positive test (Catalase produced)
No release of bubbles ---------- Negative test (No catalase produced)

Controls
Positive catalase control: Staphylococcus species.
Negative catalase control: Streptococcus species.

Figure (1-26): Positive catalase test

2.COAGULASE TEST
This test is used to differentiate Staphylococcus aureus which produces the enzyme
coagulase(CPS), from S.epidermidis and S.saprophyticus which do not produce
coagulase.(CNS).

Principle
Coagulase causes plasma to clot by converting fibrinogen to fibrin. Two types of coagulase
are produced by most strains of S.aureus:
Free coagulase which converts fibrinogen to fibrin by activating a coagulase – reacting
factor present in plasma. Free coagulaseis detected by the appearance of a fibrin clot in the
tube test.
Bound coagulase (clumping factor) which converts fibrinogen directly to fibrin without
requiring a coagulase – reacting factor. It can be detected by the clumping of bacterial cells
in the rapid slide test.

41
Method for slide test (to detect bound coagulase)
Place a drop of physiological saline on each end of a slide, or on two separate slides.
Emulsiy a colony of the test organism from blood agar in each of the drops to make two
thick suspensions. Add a drop of plasma to one of the suspensions, and mix gently.Look for
clumping of the organisms within 10 seconds. No plasma is added to the second suspension.
This is used to differentiate any granular appearance of the organism form true coagulase
clumping.

Results
Clamping within 10 secs ------------------------ S.aureus(CPS)
No clumping within 10 secs-----------No bound coagulase produced.(CNS)

Controls
Positive coagulase control: Staphylococcus aureus
Negative coagulase control: Staphylococcus epidermidis

Figure (1-27): Coagulase test of staphylococcus spp.

Method for tube test (detect free coagulase)

1. Dilute the plasma 1 in 10 in physiological saline (mix 0.2 ml of plasma with 1.8ml of
saline).
2. Take three small test tubes and label:
T = Test organism (18-24h broth culture)
Pos = Positive control (18-24h Staph. aureus broth culture)
Neg = Negative control (sterile broth).
42
3. A suitable broth is brain heart infusion. Pipette 0.5ml of the diluted plasma into each tube.
4. Add 5 drops (about 0.1ml) of the test organism culture to the tube labeled „T‟. Add 5
drops of the Staph. aureus culture to the tube labeled „Pos‟.
5. Add 5 drops of sterile broth to the tube labeled „Neg‟.
6. After mixing gently, incubate the three tubes at 35-37Cₒ.
7. Examine for clotting after 1 hour. If no clotting has occurred,examine at 30minute
intervals for up to 6 hours.When looking for clotting, gently tilt each tube.Most Staph,
aureus strains produce a fibrin clot within 1hour of incubation. There should be no fibrin clot
in the negative control tube.

Results
Fibrin clot ----------------------------- S. aureus
No fibrin clot ------------------------- No free coagulase produced.

3.Mannitol salt agar (MSA)

Is both a selective and differential media used for the isolation of Staphylococci spp.

Principle
On MSA, only pathogenic Staphylococcus aureus produces small colonies surrounded by
yellow zones. The reason for this color change is that S. aureus have the ability to ferment
the mannitol, producing an acid, which changes the Indicator color from red to yellow. The
growth of other types of bacteria is usually inhibited. This growth differentiates S. aureus
from S.epidermidis.

Results
On MSA, pathogenic Staphylococcus aureus ferments mannitol, thereby changing the
colour of the medium from red to yellow.
Staphylococcus epidermidis grows on MSA, but does not ferment mannitol (media remains
light pink in color, colonies are colorless).

Figure (1-28): Staphylococcus aureus on mannitol salt agar

43
4.Bacitracin test
Principle
This test is commonly used to distinguish between the b-hemolytic streptococci:
Streptococcus agalactiae (bacitracin resistant) and Streptococcus pyogenes (bacitracin
sensitive).
Method:
1.Streak a blood agar plate with the isolated organism.
2.Place bacitracin disc in the streaked area.
3.Incubate the plate for 24 hours at 37 cₒ. Examine the plate for a zone of no-growth around
the disc.
Results
No-growth around the disc. (Sensitive)..…………… S.pyogenes
Growth around the disc (Resist)………………… Other streptococci.

Figure (1-29): Bacitracin test of Streptococcus pyogenes

5.Optochin test
Principle:
Streptococcus pneumoniae is sensitive to optochin disc unlike other alpha-hemolytic
streptococci. The antibiotic bacitracin inhibits the synthesis of bacterial cell walls by
interfering the peptidoglycan synthesis of bacteria.

Method:
1. Streak a blood agar plate with the isolated organism.
2. Place optochin disc in the streaked area.
3. Incubate the plate for 24 hours at 37 c0 in 5% to 10% carbon dioxide (CO2).
4. Examine the plate for a zone of no-growth around the disc.
No-growth around the disc ..…S.pneumoniae
Growth around the disc … Other Alpha hemolytic Streptococci.

44
 Figure (1-30): Optochin test of Streptococcus pneumonia

6.BILE SOLUBILITY TEST

This helps to differentate S.pneumoniae, which is soluble in bile and bile salts, from
viridans streptococci which are insoluble.

Principle
A heavy inoculum of the test organism is emulsified in physiological saline to give a turbid
suspension. The bile salt sodium deoxycholate is then added. The test can also be performed
by adding the bile salt to a broth culture of the organism. The bile salt dissolves
S.pneumoniae as shown by a clearing of the turbidity within 10-15 minutes.
Viridans streptococci are not dissolved and therefore there is no clearing of the turbidity.
An amidase is an intracellular autolytic enzyme possessed by pneumococcus that is
responsible for the rapid autolysis of the organism, when cultivated on artificial medium.
The bile salts lower the surface tension of the medium and cause cell membrane disruption.
The working mechanism of the test is not clearly known; however, one theory states the bile
salts accelerates lysis of pneumococcal cells by activating the autolytic enzyme.

Method
1. Emulsify several colonies of the test organism in a tube containing 2ml of sterile
physiological saline, to give a turbid suspension.
2. Divide the organism suspension between two tubes.
3. To one tube, add 5 ml of 10% sodium deoxycholate reagent and mix. , To the other tube,
add sterile distilled water and mix.
4. Leave both tubes for 10-15 minutes.
5. Look for a clearing of turbidity in the tube containing the sodium deoxycholate.

Results
Clearing of turbidity -------------- Probably S.pneumoniae
No clearing of turbidity -----------organism is probably Not S.pneumoniae

45
 Control
Bile solubility positive control :Streptococcus pneumoniae.
Bile solubility negative control: Streptococcus faecalis

Figure (1-31): Bile solubility test of Streptococcus spp.

7. CAMP test (Christie, Atkins , Munich Paterson )

Principle:
Certain organisms (including group B streptococci) like S.agalaciae produce a diffusible
extracellular hemolytic heat-stable protein (CAMP factor) that acts synergistically with the
beta-lysin of Staphylococcus aureus to cause enhanced lysis of red blood cells. The group B
streptococci are streaked perpendicular to a streak of S. aureus on sheep blood agar. A
positive reaction appears as an arrowhead zone of hemolysis adjacent to the place where the
two streak lines come into proximity.
The hemolytic activity of the beta-hemolysin produced by most strains of Staphylococcus
aureus is enhanced by extracellular protein produced by group B streptococci. Interaction of
the beta-hemolysin with this factor causes “synergistic hemolysis,” which is easily observed
on a blood agar plate. This phenomenon is seen with both hemolytic and non-hemolytic
isolates of group B streptococci.

Method:
1. Streak S.aureus isolate across sheep blood agar plate.
2. Inoculate the test bacteria at right angle to staphylococci without touching it.
3. Incubate over night at 35-37 c0.
4. Formation of an arrow-head shaped area of hemolysis indicates interaction of camp
factor with staphylococci hemolysin.

Results
Positive: Enhanced hemolysis is indicated by an arrow head-shaped zone of beta-hemolysis
at the junction of the two organisms.
Negative: No enhancement of hemolysis.

46
Control
Positive: Streptococcus agalactiae enhanced arrowhead hemolysis.
Negative:Streptococcus pyogenes beta-hemolysis without enhanced arrowhead formation.

Figure (1-32): CAMP test of Streptococcus agalactiae

8. OXIDASE TEST (Cytochrome c Oxidase)

The oxidase test is used to assist in the identification of Pseudomonas, Neisseria, Vibrio,
Pasteurella, Compylobacter and Helicobacter species,all of which produce oxidase
enzymes.

Principle
Cytochrome containing organisms produce an intracellular oxidase enzyme. This oxidase
enzyme catalyzes the oxidation of cytochrome c. Organisms which contain cytochrome c as
part of their respiratory chain are oxidase-positive and turn the reagent blue/purple.
Organisms lacking cytochrome c as part of their respiratory chain do not oxidize the reagent,
leaving it colorless within the limits of the test, and are oxidase-negative.
Oxidase positive bacteria possess cytochrome oxidase or indophenol oxidase (an iron
containing haemoprotein). Both of these catalyse the transport of electrons from donor
compounds (NADH) to electron acceptors (usually oxygen).
The test reagent, N, N, N‟, N‟-tetramethyl-p-phenylenediamine dihydrochloride acts as an
artificial electron acceptor for the enzyme oxidase. The oxidised reagent forms the coloured
compound indophenol blue.The cytochrome system is usually only present in aerobic
organisms which are capable of utilising oxygen as the final hydrogen receptor.
The end product of this metabolism is either water or hydrogen peroxide (broken down by
catalase).
A piece of filter paper is soaked with a few drops of oxidase reagent.A colony of the test
organism is then smeared on the filter paper. If the organism is oxidase - producing, the
phenylenediamine in the reagent will be oxidized to a deep purple colour.

47
Method
Place a piece of filter paper in a clean petri dish and add 2 or 3 drops of freshly prepared
oxidase reagent.Using a piece of stick or glass rod (not an oxidized wire loop), remove a
colony of the test organism, and smear it on the filter paper.Look for the development of a
blue – purple colour within a few Seconds.

Results
Blue-purple colour ------------------------------------- Positive test
(within 10 seconds) Oxidase produced
No blue – purple colour ------------------------------- Negative test
(within 10 seconds) No oxidase produced.
Positive oxidase control: Pseudomonas ,Neisseria, Vibrio, Compylobacter and Helicobacr.

Note: It is used to differentiate Pseudomonas from other non lactose enterobecteriaceae.

Figure (1-33): Oxidase test

9.INDOLE TEST
Testing for indole production is important in the identification of enterobacteria. Most strains
of E.coli, P.vulgaris, P.rettgeri , M.morganll, and providencia species break down the
amino acid tryptophan with the release of indole.

Principle
The indole test can be performed by culturing the organism in tryptone water or peptone
water containing tryptophan, and detecting indole production by adding Kovac‟s or Ehrlich‟s
reagent to an 18-24h culture. Tryptophan, an essential amino acid, is oxidized by some
bacteria by the action of trytophanase enzyme. Bacteria that produce the enzyme
tryptophanase are able to degrade the amino acid tryptophan into pyruvic acid, ammonia,
and indole.

Results
Reddening of strip -----------------------------Positive test ) Indoloe produced(
No red colour ---------------------------------- Negative test (No Indole produced)
48
Control
Positive indole control: Escherichia coli
Negative indoloe control: Enterobacter aerogenes

Figure (1-34) : E.coli shows Positive Indole test

10. CITRATE UTILIZATION TEST

This test is one of several techniques used to assist in the identification of enterobacteria. The
test is based on the ability of an organism to use citrate as its only source of carbon and
ammonia as its only source of nitrogen.

Principle
The test organism is cultured in a medium which contains sodium citrate, an ammonium salt,
and the indicator bromo – thymol blue.
Bacteria that can grow on this medium produce an enzyme, citrate-permease, capable of
converting citrate to pyruvate.When the bacteria metabolize citrate, the ammonium
salts are broken down to ammonia, which increases alkalinity. The shift in pH turns
the bromthymol blue indicator in the medium from green to blue above pH 7.6.

Results
Turbidity and blue colour ------------------------------------ Positive test Citrate utilized
No growth ----------------------------------------------Negative test Citrate not utilized

Controls
Positive citrate control: Klebslella pneumonlae
Negative citrate control: Escherichia coli

49
 Figure (1-35): Klebsiella pneumonia shows positive citrate test

11. Motility Test

This is shown by a spreading turbidity from the stab line or a turbidity throughout the
medium (compare with an un inoculated tube).

Figure (1-36): Motility test

12. UREASE TEST

Testing for urease enzyme activity is important in differentiating entrobacteria. Proteus
strains are strong urease producers.Y.enterocolitica also shows urease activity (Weakly a 35-
37 cₒ) Salmonella and shigella do not produce urease.

50
Principle
The test organism is cultured in a medium which contains urea and the indicator phenol
red. If the strain is urease-producing, the enzyme will break down the urea (by hydrolysis) to
give ammonia and carbon dioxide. With the release of ammonia, the medium becomes
alkaline as shown by a change in color of the indicator to red-pink.
Results
Red-pink medium …… Positive test.
Urease produced
No red-pink color …… Negative test No urease produced.
Controls
Positive urease control: Proteus vulgaris.
Negative urease control:Shigella spp

Figure (1-37): Shows positive test of urease test In Proteus spp.

13. Triple Sugar Iron (TSI) test

Triple Sugar Iron Agar„ it‟s a test that has three sugar (lactose, sucrose, and glucose) and also
iron; and it contains agar as solidifying agent (TSI is a semi-solid media having slant and
butt).
Is a microbiological test named for its ability to test a microorganism‟s ability to ferment
sugars and to produce hydrogen sulfide. An agar slant of a special medium with multiple
sugars constituting a pH-sensitive dye (phenol red), 1% lactose, 1% sucrose,
0.1% glucose, as well as sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate
is used for carrying out the test. All of these ingredients when mixed together and allowed
solidification at an angle result in a agar test tube at a slanted angle.

51
 Table (1-8): Composition Of TSI Agar
Ingredients
Grams/liter

Beef extract
3.0 g

3.0 g
Yeast extract

Peptone 20.0 g

Glucose 1.0 g

Lactose 10.0 g

Sucrose 10.0 g

Ferrous sulfate or ferrous ammonium sulfate 0.2 g

NaCl 5.0 g

Sodium thiosulfate 0.3 g

Phenol red 0.024 g

13.0 g
Agar

Distilled water 1,000 mL

Lactose, sucrose and glucose are in the concentration of 10:10:1 (i.e. 10 part lactose (1%), 10
part sucrose (1%) and 1 part glucose (0.1%)). TSI is similar to Kligler’s iron agar
(KIA), except that Kligler‟s iron agar contains only two carbohydrates: glucose (0.1%) and
lactose (1%).

 0.1% glucose: If only glucose is fermented, only enough acid is produced to turn the butt
yellow. The slant will remain red.
 1.0 % lactose/1.0% sucrose: If lactose or sucrose or both sugars are fermented, a large
amount of acid will produce which turns both butt and slant yellow. So the appearance of
yellow color in both slant and butt indicates that the isolate has the ability to ferment
lactose or sucrose or both.
 Iron (ferrous sulfate): Indicator of H2S formation
 Phenol red: Indicator of acidification (It is yellow in acidic conditions and red under
alkaline conditions).
 It also contains peptone which acts as a source of nitrogen (remember that whenever
peptone is utilized under aerobic condition, ammonia is produced).

52
Why Sucrose is added to TSI Agar?
Addition of sucrose in TSI agar permits earlier detection of coliform bacteria that ferment
sucrose more rapidly than lactose. Adding sucrose also aids the identification of certain
gram-negative bacteria that could ferment sucrose but not lactose. Another basic
understanding is TSI tube contains a butt-poorly oxygenated area on the bottom and a slant-
angled well-oxygenated area on the top.

Figure (1-38) : Inoculation in TSI agar

Preparation of TSI Agar

 Combine the ingredients, and adjust the pH to 7.3
 Boil to dissolve the agar
 Dispense it into tubes
 Sterilize by autoclaving at 121°C for 15 minutes
 Cool in a slanted position to give a 2.5 cm butt and a 3.8 cm slant.
TSI agar is also available commercially.

Procedure for TSI Agar Test

1. With a sterilized straight inoculation needle touch the top of a well-isolated colony
2. Inoculate TSI agar by first stabbing through the center of the medium to the bottom of the
tube and then streaking on the surface of the agar slant.
3. Leave the cap on loosely and incubate the tube at 35°C in ambient air for 18 to 24 hours.

53
Principle of TSI Agar Test
1. If lactose (or sucrose) is fermented, a large amount of acid is produced, which turns the
phenol red indicator yellow both in the butt and in the slant. Some organisms generate
gases, which produce bubbles/cracks in the medium.
2. If lactose is not fermented but the small amount of glucose is, the oxygen-deficient butt
will be yellow (remember that butt has comparatively more glucose than slant i.e. more
media and more glucose), but on the slant, the acid produced (less acid produces in slant
as media in slant is less) will be oxidized to carbon dioxide and water by the organism and
the slant will be red (alkaline or neutral pH).
3. If neither lactose/sucrose nor glucose is fermented, both the butt and the slant will be
red. The slant can become a deeper red-purple (more alkaline) as a result of the production
of ammonia from the oxidative deamination of amino acids (remember peptone is a major
constituent of TSI agar).
4. if H2S is produced, the black color of ferrous sulfide is seen.

Due to the building of acid during fermentation of sugars(glucose,sucrose,lactose), the pH

falls. The acid-base indicator Phenol red is incorporated for detecting carbohydrate
fermentation that is indicated by the change in color of the carbohydrate medium from
orange-red to yellow in the presence of acids.Sodium thiosulfate and ferrous ammonium
sulfate present in the medium detects the production of hydrogen sulfide and is indicated by
the black color in the butt of the tube.

Uses:
The test is used primarily to differentiate members of the Enterobacteriaceae family from
other gram-negative rods.It is also used in the differentiation among Enterobacteriaceae
on the basis of their sugar fermentation patterns.

54
 Figure (1-39): Shows TSI media ,

1. Alkaline slant/no change in butt (K/NC) i.e Red/Red = glucose, lactose, and sucrose non-
fermenter.
2. Alkaline slant/alkaline butt (K/K) i.e Red/Red = glucose, lactose, and sucrose non-fermenter
3. Alkaline slant/acidic butt (K/A); Red/Yellow = glucose fermentation only, gas (+ or -), H2S (+
or -).
4. Acidic slant/acidic butt (A/A); Yellow/Yellow = glucose, lactose and/or sucrose fermenter gas (+
or -), H2S (+ or -).

55
 Table(1-9): Results of Triple Sugar Iron (TSI) Agar Tests
Name of the organism Slant Butt Gas H2S

Escherichia, Klebsiella, Acid (A) Acid (A) Pos (+) Neg (-)
Enterobacter

Alkaline (K) Neg (-) Neg (-)

Shigella, Serratia Acid (A)

Alkaline (K)
Salmonella, Proteus Acid (A) Pos (+) Pos (+)

Pseudomonas
Alkaline (K) Alkaline (K) Neg (-) Neg (-)

Table (1 - 10): Interpretation of results of TSI

Organisms Growth

Shigella sonnei Growth; red slant(Alkaline), yellow butt(Acid) AK/A , no gas, no

H2S produced

Pseudomonas aeruginosa Growth; red slant(Alkaline), red butt (Alkaline)AK/AK, no gas, no

H2S produced

Control(Un inoculated) Growth; red slant, red butt, no gas , no H2S produce
(No change/no change)

Proteus vulgaris
Red slant (Alkaline), yellow butt(Acid)..ALK/A, no gas production;
H2S produced

Enterobacter aerogenes Yellow slant (Acid), yellow butt (Acid) A/A gas production; no H2S
E.coli produce
Klebsiella pneumonia

56
 Figure (1 -40): Shows biochemical reaction of Pseudomonas aeruginosa :
1. INDOLE TEST …. NEGATIVE (NO RED RING)
2. MOTILITY TEST ... MOTILE (Turbidity throughout the Medium)
3. CITRAETE ...... POSITIVE (Turbidity and Blue Color)
4. UREASE TEST .... NEGATIVE (No Red-Pink Colour)
5. TSI TEST ... ALK/ALK (NO GAS,NO H2S)

14. MacConkey agar

MacConkey agar is a selective and differentiating agar that only grows gram-negative
bacterial species; it can further differentiate the gram-negative organisms based on their
lactose metabolism. Key components of the MacConkey medium include crystal violet dye,
bile salts, lactose, and neutral red (pH indicator). Crystal violet dye and bile salts inhibit the
growth of gram-positive bacteria. This allows only gram-negative species to form colonies
on MAC agar.

Principle
Lactose (Lac) positive (pink colonies):
Lactose fermenting species will grow pink colonies. Lactose fermentation will produce
acidic byproducts that lower the pH, and this turns the pH indicator to pink. Example of Lac
positive species: Escherichia coli, Enterobacteria, Klebsiella .

Lactose (Lac) negative (white colonies):

Gram-negative bacterial species will still form colonies, but colonies will have a white
appearance as there will be no change in pH in the absence of lactose fermentation. Example
of Lac negative species: Salmonella, Proteus, Yersinia, Pseudomonas.
Weak lactose fermenters will form colonies slower than the rest. Example of slow lac
fermenters: Serratia, Citrobacter .

57
 Figure (1 -41): Shows Bacterial colonies culture lactose fermenter on
MacConkey agar media

Figure (1 -42): Shows Bacterial colonies culture non lactose fermenter on MacConkey
agar media

58
15. X AND V FACTOR DISCS FOR DIFFERENTIATION OF
HAEMOPHILUS SPECIES

Hemophilus influenzae requires two accessory growth factors:

Principle
Members of the genus Haemophilus require accessory growth factors in vitro.
Some Haemophilus spp. require X factor (hemin) alone, V factor (nicotinamide adenine
dinucleotide [NAD]) alone, or a combination of the two. Consequently, this significant
difference in growth factor requirements of Haemophilus spp. allows for their
differentiation.
In the laboratory, a lawn of the test organism is streaked onto heart infusion agar, tryptic soy
agar, Haemophilus agar, or nutrient agar. The impregnated disks or strips (X, V, or XV) are
placed directly on the confluent inoculation, allowing diffusion of the accessory growth
factor into the medium. The organisms will grow only around the disk that provides the
appropriate factor for growth of the organism ie. the presence of growth around the disc but
not elsewhere on the plate indicates a requirement for that particular factor. Differentiation is
hence based on the presence or absence of growth around and/or between disks impregnated
with factors X, V and XV.

Procedure
1. Make a very light suspension (MacFarland 0.5) of the organism in sterile saline.
2. Dip a sterile swab into the organism suspension. Roll the swab over the entire surface
of a trypticase soy agar plate.
3. Place the X, V, and XV factor disks on the agar surface. If using separate disks, place
them at least 4 to 5 cm apart.
4. Incubate the plate overnight at 35°-37°C in ambient air.
5. Following incubation examine the plate for growth around the disk.
Positive test
 Growth around the XV disk only shows a requirement for both factors.
 Growth around the V disk, no growth around the X disk, and light growth around the
XV disk shows a V factor requirement.
Negative test
Growth over the entire surface of the agar indicates no requirement for either X or V factor.

Quality Control
1. Haemophilus influenza : halo of growth around the XV disk, no growth on the rest
of the agar surface.
2. Haemophilus parainfluenzae : halo of growth around the XV and V disks
Haemophilus ducreyi: halo of growth around the XV and X disks.

59
 Figure (1-43): Shows X and V factor test of
(Haemophilus influenzae)

16. String Test

Purpose
1. To differentiate Vibrio cholerae ( positive) from Aeromonas spp (negative). Both are
isolated from diarrheal stool, show same biochemical properties on culture media and
are oxidase positive.
2. To differentiate Vibrio cholerae (positive) from other Vibrio spp (negative).

Principle
When an isolated colony (18-24 hour growth) of a suspected bacterium is emulsified in
Sodium deoxycholate or Sodium taurocholate (commonly known as bile salt), it lyses the
cell wall of the bacterium releasing the DNA. The suspension loses turbidity and the mixture
becomes viscous. A mucoid “string” is formed when an inoculating loop is drawn slowly
away from the suspension.
Requirements: Freshly prepared 0.5% bile salt, glass slide, inoculating loop.
Procedure
1. Take a clean grease free slide and put a drop of 0.5% bile salt.
2. Emulsify an isolated colony of the bacterium using an inoculating loop.
3. Keep on rubbing the loop vigorously for 2-3 mins until it appears viscous.
4. Gently, pull the loop upwards from the slide.

Result and interpretation

Formation of a thread like mucoid string indicates positive test.
Note: Based on colony morphology, positive oxidase test and positive string test, the isolate
can be confirmed as Vibrio cholerae.

60
 Figure (1-44): Shows string test of Vibrio cholera

17. Analytical profile index or API

The analytical profile index or API is a classification of bacteria based on biochemical
tests, allowing fast identification. This system is developed for quick identification of
clinically relevant bacteria. Because of this, only known bacteria can be identified. The API
range introduced a standardized, miniaturized version of existing techniques, which up until
then were complicated to perform and difficult to read.
One of the API systems is specific for differentiating between members of the Gram negative
bacterial Family Enterobacteriaceae and is called API-20E. The other API system is specific
for Gram positive bacteria, including Staphylococcus species, Micrococcus, species, and
related organisms, and is called API-Staph.
API test strips consists of wells containing dehydrated substrates to detect enzymatic
activity, usually related to fermentation of carbohydrate or catabolism of proteins or amino
acids by the inoculated organisms. A bacterial suspension is used to rehydrate each of the
wells and the strips are incubated.
During incubation, metabolism produces color changes that are either spontaneous or
revealed by the addition of reagents. For example, when carbohydrates are fermented, the pH
within the well decreases and that change is indicated by a change in the color of the pH
indicator. All positive and negative test results are compiled to obtain a profile number,
which is then compared with profile numbers in a commercial codebook (or online) to
determine the identification of the bacterial species.

61
 Figure (1-45) : API System

The different tests in API 20E :

1- ONPG test : The ONPG test is used to detect the enzyme β-galactosidase
2- ADH : Decarboxylation of the amino acid arginine by arginine dihydrolase
3- LDC: Decarboxylation of the amino acid lysine by lysine decarboxylase
4- ODC: Decarboxylation of the amino acid ornithine by ornithine decarboxylase
5- CIT : Use of citrate as sole carbon source
6- H2S : Hydrogen sulfide production
7- URE : Urease enzyme test
8- TDA (Tryptophan deaminase) : Detection of the tryptophan deaminase enzyme
(detected by adding Ferric chloride).
9- IND : Indole test : production of indole from tryptophan by the enzyme tryptophanase
(Indole is detected by adding Kovac's reagent).
10- VP : The Voges-Proskauer test for the detection of acetoin (acetylmethylcarbinol)
produced by fermentation of glucose by bacteria using the butylene glycol
pathway
11- GEL : Production test of the enzyme gelatinase which liquefies gelatin
12- GLU : Glucose fermentation (hexose sugar)
13- MAN : Fermentation of mannose (sugar hexose)
14- INO : Fermentation of inositol (cyclic polyalcohol)
15- SOR : Fermentation of sorbitol (sugar alcohol)
16- RHA : Rhamnose fermentation (methyl pentose sugar)
17- SAC : Fermentation of sucrose (disaccharide)
18- MEL : Fermentation of melibiose (disaccharide)
19- AMY : Fermentation of amygdalin (glycoside)
20- ARA Fermentation of arabinose (pentose sugar)

62
  A bacterial suspension is used to rehydrate each of the wells and the strips are incubated.
During incubation, bacterial metabolism produces color changes that are either
spontaneous or revealed by the addition of reagents.

Procedure of Api 20 E
1. Confirm the culture is of an Enterobacteriaceae. To test this, a quick oxidase
test for cytochrome c oxidase may be performed.
2. Pick a single isolated colony (from a pure culture) and make a suspension of it in
sterile distilled water.
3. Take the API20E Biochemical Test Strip which contains dehydrated bacterial
media/bio-chemical reagents in 20 separate compartments.
4. Using a pasteur pipette, fill up (up to the brim) the compartments with the bacterial
suspension.
5. Add sterile oil into the ADH, LDC, ODC, H2S and URE compartments.
6. Put some drops of water in the tray and put the API Test strip and close the tray.
7. Mark the tray with identification number (Patient ID or Organism ID), date and your
initials.
8. Incubate the tray at 37oC for 18 to 24 hours.

Result Interpretation
1. For some of the compartments, the color change can be read straightway after 24
hours but for some reagents must be added to them before interpretation.
2. Add following reagents to these specific compartments:
 TDA: Put one drop of Ferric Chloride
 IND: Put one drop of Kovacs reagent
 VP: Put one drop of 40 % KOH (VP reagent 1) & One drop of VP Reagent 2 (α-
Naphthol)
3. Get the API Reading Scale (color chart) by marking each test as positive or negative
on the lid of the tray. The wells are marked off into triplets by black triangles, for
which scores are allocated.
4. Add up the scores for the positive wells only in each triplet. Identify the organism by
using API catalog or apiweb (online)

63
Advantages of API Test
1. It is a fast and efficient method of identification and differentiation of organisms (18-
24 hour identification of Enterobacteriaceae and other non-fastidious gram-negative
bacteria).
2. It is also useful for fungal identifications ( yeasts).
3. It is an easy-to-run, user-friendly, and standardized method of identification and
differentiation of microorganisms.
4. API strips have a long shelf life, enabling every laboratory to keep the test kits on
hand, and are very useful in medium-level set-up laboratories where identification of
organisms is difficult using conventional tests and lack of sources like MALDI-TOF-
MS and molecular testing ( absence of thermocyclers).
5. This allows accurate identifications based on extensive databases.

Figure (1-46): Shows results of Enterobacteriaceae

By API 20E

64
18. Germ Tube Test
Is a screening test which is used to differentiate Candida albicans from other yeast. Candida
can be isolated by culturing specimen on sabouraud dextrose agar (SDA).

Principle of SDA
Peptone (Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue) provide the
nitrogen and vitamin source required for organism growth in SDA. Dextrose is added as the
energy and carbon source.Chloramphenicol and/or tetracycline may be added as broad
spectrum antimicrobials to inhibit the growth of a wide range of gram-positive and gram-
negative bacteria. Gentamicin is added to further inhibit the growth of gram-negative
bacteria.

65
 Figure (1-47): Shows Candida albicans (Gm stain)

Principle of germ tube test

Formation of germ tube is associated with increased synthesis of protein and ribonucleic
acid. Germ Tube solutions contains tryptic soy broth and fetal bovine serum, essential
nutrients for protein synthesis.It is lyophilized for stability.Germ tube is one of the virulence
factors of Candida albicans.
This is a rapid test for the presumptive identification of C. albicans.

Procedure of Germ Tube Test

1. Put 0.5 ml of sheep or human serum into a small tube.
Note: Fetal bovine serum can also be used instead of human serum.
2. Using a Pasteur pipette, touch a colony of yeast and gently emulsify it in the serum.
Note: Too large of an inoculum will inhibit germ tube formation.
3. Incubated the tube at 37°C for 2 to 4 hours.
4. Transfer a drop of the serum to a slide for examination.
5. Coverslip and examine microscopically under low and high power objectives.

Results
Positive Test a short hyphal (filamentous) extension arising laterally from a yeast cell, with
no constriction at the point of origin. Germ tube is half the width and 3 to 4 times the length
of the yeast cell and there is no presence of nucleus.
Examples: Candida albicans

Negative Test : No hyphal (filamentous) extension arising from a yeast cell or a short
hyphal extension constricted at the point of origin.
Examples: C. tropicalis, C. glabrata and other yeasts.

Control
Positive Control : C. albicans
Negative Control : C. tropicalis , C. glabrata

66
 Figure (1-48): Shows germ tube of Candida albicans

19. Antibiotic Sensitivity test

Purpose
The purpose of test antibiotic Sensitivity test is to determine the sensitivity or resistance of
pathogenic aerobic and facultative anaerobic bacteria to various antimicrobial compounds in
order to assist a physician in selecting treatment options for his or her patients. The
pathogenic organism is grown on Mueller-Hinton agar in the presence of various
antimicrobial impregnated filter paper disks. The presence or absence of growth around the
disks is an indirect measure of the ability of that compound to inhibit that organism.

Kirby-Bauer Test to Measure Antibiotic Sensitivity

The basics are easy: The bacterium is swabbed on the agar and the antibiotic discs are placed
on top. The antibiotic diffuses from the disc into the agar in decreasing amounts the further it
is away from the disc. If the organism is killed or inhibited by the concentration of the
antibiotic, there will be NO growth in the immediate area around the disc: This is called
the zone of inhibition . The zone sizes are looked up on a standardized chart to give a result
of sensitive, resistant,or intermediate.Many charts have a corresponding column that also
gives the MIC (minimal inhibitory concentration) for that drug. The MIC is currently the
standard test run for antibiotic sensitivity testing because it produces more pertinent
information on minimal dosages .The Mueller-Hinton medium being used for the Kirby-
Bauer test is very high in protein.

67
Procedure
1. Swab a Mueller-Hinton plate with the bacteria. Dip a sterile swab into the broth and
express any excess moisture by pressing the swab against the side of the tube.
2. Swab the surface of the agar completely (do not leave any unswabbed agar areas at
all).
3. After completely swabbing the plate, turn it 90 degrees and repeat the swabbing
process. (It is not necessary to re-moisten the swab.) Run the swab around the circumference
of the plate before discarding it in the discard bag.
4. Allow the surface to dry for about 5 minutes before placing antibiotic disks on the agar.

THE ANTIBIOTIC DISKS

1. You are using individual antibiotic dispensers.
2. You will probably have to use a pair of forceps to remove an antibiotic disc from the
dispenser: the forceps have to be sterile. Place the forceps in alcohol, flame the forceps
until they catch on fire, let the flame go out----sterile forceps.
3. Lightly touch each disc with your sterile inoculating loop to make sure that it is
in good contact with the agar surface. Incubate upside down and incubate at 37o C.

Interpretation
1. Place the metric ruler across the zone of inhibition, at the widest diameter, and
measure from one edge of the zone to the other edge. Holding the plate up to the light
might help.
2. Use millimeter measurements. The disc diameter will actually be part of that number.
3. If there is NO zone at all, report it as 0---even though the disc itself is around 7 mm.
4. Zone diameter is reported in millimeters, looked up on the chart, and result reported
as sensitive, resistant, or intermediate.
5. Record the results for your table, as well as other tables, in the table.

68
Figure (1- 49): Shows measurement of inhibition zone

Figure (1- 50): Shows table of antibiotic discs standards

69
Figure (1-51): Shows Sensitivity test of streoptococcus pneumonia
On blood agar

Figure (1-52): Shows Sensitivity test of of Haemophilus influenzae

In Chocolate Agar

70
 CHAPTER
2

LABORATORY COLLECTION, TRANSPORT AND

EXAMINATION OF SPECIMEN

SPECIMEN COLLECTION
Learning Objectives
1. List types of specimens for microbiology lab.
2. Understand the concept of proper specimen collection.
3. Handle the collected specimen.
4. Know the proper transportation of specimen.
5. Value the rejection criteria for mishandled specimen.
6. If pathogens are to be isolated successfully, the type of specimen, its collection, time
and method of its dispatch to the laboratory must be correct.
7. Adequate information about the patient‟s condition and antimicrobial treatment must
also be sent in the Specimen.

Specimens
1. Some of the specimens that have been collected for microbiology laboratory.
2. Throat swabs: ex Streptococcal sore throat.
3. Eye swab: ex Conjunctivitis.
4. Ear swab: otitis media.
5. Urine: UTI.
6. Blood: Septicemia.
7. Biopsy (Surgical specimen) ex. Leprosy.

Figure (2-1): Shows sore throat and conjunctivitis

71
 Figure (2-2): Shows Otitis media

Table ( 2-1 ): Procedural Guidelines for Specimen Collection

PRINCIPLE JUSTIFICATION
Place specimens and swabs in appropriate to To facilitate laboratory identification of
accurately identify the correctly labeled microorganisms and safe transportation.
containers.
When collecting specimens staff must To reduce the risks of cross infection and
submit with the trust hand hygiene policy. cross contamination.
Patients should be aware of why a specimen is To ensure consent and reassure.
being collected and when results will be
available.
Any specimens collected should be To recorded that samples have been
documented in the patient's record. collected.

When to Collect the Earliest Specimen

 Start collection of specimens for all cultures before starting an Antibiotic.
 The advice is ideal but may not be possible, as many prescribe Antibiotics before
considers the microbiological diagnostic options.
What containers to use
1. Containers must be leaking proof.
2. Unbreakable.
3. For cultures sterile containers a Must.
4. Microbiology specimen should never be sent in formalin.

When to Request transport medium

Transport medium:
1. Allows organisms (pathogens and contaminants) to survive.
2. Non-nutritive - does not allow organisms to proliferate.
3. When facilities are not available to perform the desired tests at the place of collection or
laboratory located far away, request the diagnostic laboratories to advice on transportation
72
 of specimens, and consider how to preserve and transport in ideal medium before it is
processed.

Popularly used transport medium are:

1. Amie's transport Medium.
2. Stuart's Transport Medium.
3. Venkatrman Ramakrishnan (VR) Medium.

Handling of Specimen
1. Label the container(s) of sample with the patient‟s name, ID number, specimen
type(s), and date collected.
2. Every specimen collected should be considered as a potentially hazardous.
3. Those delivering, receiving and examining specimens must be informed when a
specimen is likely to contain highly infectious.
4. Such specimen should be labeled HIGH RISK.

Label High risk Specimens

1. Sputum with suspected (Tuberculosis).
2. Fecal samples suspected with Cholera, Typhoid, Anthrax
3. Serum when suspected with HIV/ HBV/HCV,infections .

Figure (2-3): Shows symbol of biohazard

Specimen Rejection Criteria

1. Unlabeled specimen.
2. Insufficient patient information.
3. Haemolysed specimen.
4. Wrong tube drawn.
5. Wrong specimen submitted.
6. Inadequate volume for the amount of preservative.
7. Insufficient quantity.
8. Prolonged transport.

73
1. Collection, Transport and examination of Urine
Urinary tract infections (UTIs) include infections of the urethra, bladder, and kidneys, and
are common causes of urethritis, cystitis, pyelonephritis, and glomerulonephritis. Bacteria
are the most common causes of UTIs, especially in the urethra and bladder.

Possible pathogens
Gram-negative bacteria such as Escherichia coli (most commonly), Proteus
vulgaris, Pseudomonas aeruginosa, and Klebsiella pneumoniae cause most bladder
infections. Gram-positive pathogens associated with cystitis include the coagulase-
negative Staphylococcus saprophyticus, Enterococcus faecalis, and Streptococcus
agalactiae.
Urine specimens for culture should be collected in the morning. It is advisable to ask the
patient the night before to refrain from urinating until the specimen is to be collected.
midstream urine specimen, particularly in females and children should be collected in sterile,
wide-mouthed,glass or plastic jars.
Since urine itself is a good culture medium, all specimens should be processed by the
laboratory within 2 hours of collection, or be kept refrigerated at 4C0 until delivery to the
laboratory and processed no longer than 18 hours after collection.

Urinary Tract Infection (UTI)

E.coli (uropathogenic E.coli or UPEC) is the single most common pathogen, accounting for
85–95% of all cases of UTI. Uropathogenic E. coli (UPEC) serotypes O1, O2, O4, O6, O7
and O75 are responsible for most UTIs.

Route of Spread
1. Ascending route: After colonizing the periurethral area, E.coli ascends the urinary
tract to reach bladder and later to kidney.
2. Descending route: Hematogenous seeding of E.coli into kidneys result in
pyelonephritis.

74
Types of UTI
Depending on the site involved, there are two types of UTI: Lower and upper UTI
Table (2-2): Types of UTI
Lower UTI Upper UTI
Syndromes Cystitis and Pyelonephritis
urethritis

Symptoms Local Systemic manifestations:

manifestations: Fever, vomiting, abdominal pain
dysuria, urgency,
frequency.

Route of spread Ascending route Both ascending (common) and descending route

Occurrence More common Less common

Virulence factors Fimbriae (e.g. P Capsular K antigen
fimbriae)

Common virulence factors responsible are:

• Cytotoxins (CNF 1-cytotoxic necrotizing factor 1 and SAT-Secreted auto transporter toxin)
• Hemolysins

Predisposing Factors that Promote UTI

1. Females: Due to short urethra and close proximity to anus.
2. Pregnancy: Physiological obstruction in urinary tract due to growing fetus may lead
to prolonged stasis of urine and asymtpomatic bacteriuria.
3. Others: Presence of urinary catheters, urinary stones or prostate enlargement.

Laboratory Diagnosis of UPEC

Specimen collection
1. Clean voided midstream urine: It is the most common specimen for UTI.
2. Suprapubic aspiration is the most ideal specimen (for coma or infants).
3. In catheterized patients: Collected from the catheter tube and not from the bag.
• Transport: Stored in refrigerator or stored by adding boric acid
• Direct examination: Screening tests done are as follows:
1. Wet mount examination is done to demonstrate the pus cells in urine. Pyuria of > 8
pus cells/mm3 or 4 lakh pus cells excreted in urine/hour is taken as significant.
2. Others: Leukocyte esterase test, Nitrate reduction test (Griess test), Catalase test
3. Gram staining of urine is not a reliable indicator as (i) low bacterial count in urine,
pus cells rapidly deteriorate and may not be seen well. It may be limited to
pyelonephritis and invasive UTI cases and a count of ≥ 1 bacteria/oil immersion filed
is taken as significant.

75
Culture
Culture Media: Urine is inoculated onto:
▪ MacConkey agar and blood agar combination or
▪ CLED (cysteine lactose electrolyte deficient) agar.

Kass concept of significant bacteriuria:

1. A count of ≥ 105 colony forming units (CFU)/ml of urine is considered as sig- nificant –
indicates infection (referred as „significant bacteriuria‟ developed by Kass)
2. Low count of ≤ 104 CFU/ml is due to commensal bacteria (due to contamination during
voiding) and is of no significance. However, low counts may be significant in following
conditions:
a. Patient on antibiotic treatment or on diuretic drugs
b. Infection with some gram-positive bacteria like S.aureus, and Candida.
c. Pyelonephritis and acute urethral syndrome
d. Sample taken by suprapubic aspiration.

Quantitative culture is done to count the number of colonies. This is done by:
1. Semi quantitative method, such as standardized loop technique
2. Quantitative method, such as pour plate method.
3. Antibody coated bacteria test is used to differentiate upper and lower UTI.

Laboratory examination of urine

Routine manual urinalysis
Urine: is a liquid waste product of the body secreted by the kidneys by process of filtration
from blood. The average amount of urine excreted in 24 hours is about 1,200 cubic cm and
normally, it contains about 960 parts of water to 40 parts of solid matter. A complete
urinalysis consists of three distinct testing phases:

A. Physical Examination Includes

1. Volume. 2. Color. 3. Odor. 4. Reaction (pH). 5. Specific gravity.

B. Biochemical Examination Includes

(1) Proteins (2) Sugars (3) Ketone bodies (4) Bile salts (5) Bile Pigments (6) Blood.

C. Microscopic Tests Include

(1) Cells (2) Crystals (3) Casts (4) Microorganism (5) Parasites (6) Contamination.

76
Table (2-3): Types of urine sample
Sample Type Sampling Purpose
Random specimen No specific time most common, Routine screening
taken any time of day
Morning sample First urine in the morning, most Pregnancy test, microscopic test
concentrated
Clean catch midstream Discard first few ml, collect the rest
Used for quantitative and
qualitative analysis of substances
24 hours All the urine passed during the day Used for quantitative and
and night and next day 1st sample is qualitative analysis of substances
collected
Postprandial 2 hours after meal Determine glucose in diabetic
monitoring
Supra-pubic aspired Needle aspiration Obtaining sterile urine

Urine Physical examination

1 – Urine volume:
Urine volume measurements are part of the assessment for fluid balance and kidney function.
The normal volume of urine voided by the average adult in a 24-hour period ranges from 600
to 2500 ml; the typical amount is about 1200 ml.

Urine volume has many conditions

1 - Polyuria : is a condition characterized by the passage of large volumes of urine (at least
2.5 L over 24 hours in adults).
2 - Oliguria : are the decreased production of urine.
3 - Anuria : It non passage of urine.

2 - Urine color: Normal urine color ranges from pale yellow to deep amber.
Very pale yellow or colorless urine
 Large fluid intake.
 Diabetes Mellitus.
 Diabetes Insipidus.
 Alcohol ingestion (inhibit ADH release).
 Caffeine ingestion (increase GFR by dilating afferent arterioles).
 Diuretic therapy.
Deep yellow urine
 Dehydration or drinking too few fluids can concentrate urochrome (or Urobilin is the
chemical primarily responsible for the yellow color of urine) .
 Concentrated urine caused by fever, sweating, reduced fluid intake.
Orange urine
 Certain medications such as the antibiotic Rifampicin.
 Large amounts of carotene.

77
Red or pink urine
 The presence of red blood cells is the main reason.
 Hemoglobinuria turns urine translucent red.
 Beets, and blackberries can turn urine red.
 Methyldopa in alkaline urine , antipsychotics drugs such as chlorpromazine.
Black or dark-colored urine
 Melanuria caused by a melanoma (Melanoma, also known as malignant melanoma,
is a type of skin cancer that develops from the pigment-producing cells known as
melanocytes).
 Urine that darkens on standing may indicate antiparkinsonian agents such as
levodopa.

Greenish-yellow urine
 May indicate bilirubin in the urine.
 Greenish urine may be caused by dietary supplemental vitamins, especially the B
vitamins.

Blue to green urine

 A number of medications produce blue urine, including the anti-nausea drug
phenergan and several multivitamins.
 Pseudomonas infection Urine.

3 – Urine Specific gravity: is defined as the ratio of the density of a given solid or liquid
substance to the density of water at a specific temperature and pressure.

Figure (2-4): Shows range of urine Specific gravity

Normal : The range of urine specific gravity depends on the state of hydration and usually
between 1.015 and 1.025 .

Urine Chemical examination

1 – Urine reaction: is a measure of the acidity or basicity of a solution. The kidneys
maintain normal acid-base balance primarily through reabsorption of sodium and tubular
secretion of hydrogen and ammonium ions.

Figure (2-5): Shows range of PH in urine

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Normal: The pH of normal urine can vary widely, from 4.6 to 8.0 (The average pH value is
about 6.0 (acid).

Acidic urine ( PH < 7.0 ) occurs in :

 Prolonged diarrhea
 Starvation
 Uremia
 UTIs caused by Escherichia coli
 Gout
 A diet high in meat and protein keeps the urine acid

Alkaline urine (pH >7.0) occurs in:

 UTIs caused by urea-splitting bacteria
 Renal tubular acidosis
 Chronic renal failure
 Metabolic alkalosis (as in prolonged vomiting due to lose of hydrogen ion)
 Age of specimen (loss of CO2 from the sample to the air raises the pH)
 Drugs that increase urine pH include potassium citrate, and sodium bicarbonate.

2 – (A).Urine Albumin: In a healthy renal and urinary tract system, the urine contains no
protein or only traces amounts. These consist of albumin.

(A). Dipstick test

Figure (2-6): Shows Albumin test in urine

Normal: - Only trace amounts (about 20 mg/dL/24h) half of this amount is Tamm-hors fall
protein (Is high molecular weight glycoprotein Also Known as Uromodulin).

(B). Sulfosalicylic acid Test: The chemical added causes the precipitation of dissolved
proteins, which is measured from the degree of turbidity.

79
 Figure (2-7): Shows bottle of Sulfosalicylic acid

Figure (2-8): Shows results of Albumin in urine

Physiological proteinuria
1- Transient proteinuria: it is usually transient and due to a temporary increase in
intraglomerular capillary pressure.
Causes:
 Excessive ingestion of proteins
 Emotional stress
 High fever
 Dehydration

80
2 - Intermittent proteinuria
It is frequently associated with postural changes (protein excretion is normal when the
patient is lying down but is increased when a person is sitting or standing).

Pathological proteinuria
1 - Pre-renal proteinuria: This proteinuria is caused by conditions unrelated to the kidney
(protein loss occurs in the normal kidneys) and will disappear when those underlying
conditions are resolved.

2 - Renal proteinuria:
A - Glomerular proteinuria (albuminuria): occurs when there is disruption of the
filtration barrier (due to Loss of fixed negative charge on the glomerular capillary wall)
Causes:
 Idiopathic glomerulonephritis
 Post-streptococcal glomerulonephritis
 Nephrotic syndrome.

B - Tubular proteinuria: Develop if the tubules fail to reabsorb proteins that are normally
filtered in small amounts by the glomeruli.
C - Mixed glomerular-tubular proteinuria: This condition may be seen in advanced renal
disease that involves the entire nephron, such as chronic renal failure and chronic
pyelonephritis.
3 - Post-renal proteinuria
Post-renal proteinuria occurs when protein enters the urine because of haemorrhage or
inflammation within the lower urinary tract.

Causes: Inflammation,Hemorrhage .

3 – Urine sugar: Glycosuria or glucosuria,, Under normal circumstances glucose in not

excreted in urine. Glucose is freely filtered then reabsorbed in the proximal tubule, but
resorptive capacity is limited. Glucosuria occurs when : blood glucose exceeds this renal
threshold, for example : Diabetes mellitus Glucosuria in the absence of hyperglycaemia
reflects: a tubular resorption defect eg: Fanconi syndrome (is a rare disorder of kidney
tubule function that results in excess amounts of glucose, bicarbonate, phosphates, uric acid,
potassium, and certain amino acids being excreted in the urine ).

Chemical strip testing

Figure (2-9): Shows results of glucose in urine

81
False positive reactions
 The presence of bacterial peroxidases (e.g. cystitis).
 Drugs: Nalidixic acid, cephalosporins, Chloramphenicol.
 Stress, excitement, testing after a heavy meal.

False negative reactions

 High concentrations of ascorbic acid.
 Drugs: salicylates, tetracyclines.
 High pH inhibit the reaction.

4 – Urine Ketone: Ketone bodies are three water-soluble compounds that are produced as
by-products when fatty acids are broken down for energy in the liver and kidney.

Chemical strip testing

Figure (2-10): Shows results of ketone in urine

Interfering Factors
False-positive results: caused by drugs such as Levodopa , Insulin , Penicillamine.
False-negative results: occur if urine stands too long, due to loss of ketones into the air.

Metabolic conditions
 Diabetes mellitus (diabetic acidosis).
 Glycogen storage disease (von Gierke's disease).

Dietary conditions
 Starvation, fasting
 High-fat diets
 Prolonged vomiting, diarrhea (cause dehydration)
 Anorexia (poor appetite whatever the cause)
 Low-carbohydrate diet

5 – Urine Nitrite: Whereas NITRATES are normal in urine (mainly coming from food
additives and from food protein,), the presence of NITRITES is not normal Bacteria that
cause a urinary tract infection.
Chemical test strip

Figure (2-11): Shows results of Nitrite in urine

Note : A positive nitrate test is a reliable indicator of bacteria (especially E. Coli).

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6 – Urine Bilirubin: Bilirubin is the yellow breakdown product of normal heme catabolism.
The constituents are:
• Bilirubin (bile pigments)
• Bile salts
• Urobilin and Urobilinogen
• Bilirubin appears in JAUNDICE
• Increased bilirubinuria may be caused by liver diseses, cholestasis or haemolytic
anaemia
• Bilirubin in urine is in the form of conjugated bilirubin

Chemical strip testing

Normal: Normally, a tiny amount of bilirubin is excreted in the urine, accounting for the
light yellow color.

7 - Urine Urobilinogen : Urobilinogen is a colorless product of bilirubin reduction. It is

formed in the intestines by bacterial action.

Chemical strip testing

Figure (2-12): Shows results of Urobilinogen in urine in urine

Microscopic examination
Classification of Sediment
A .Cells
• Erythrocytes-Hematuria
• Leucocytes-Infective etiology
• Epithelial cells

B. Casts
• Hyaline cast
• Red cell cast
• Granular cast
• Epithelial cast
• Waxy cast
• Fatty cast

83
C. Crystals
Some are common in Acidic urine; some are common in Alkaline urine.

D. Bacteria
Normal urine is free from bacteria.

E. Yeast.
F Ova.

CELLS
Erythrocytes
Usually appear as hourglass appearance, presence of red cells 1-2RBCs/HPF is not
considered abnormal. Increased red cells in urine above normal level is termed hematuria.

Figure (2-13): Shows morphology of Erythrocytes in urine under microscope

Leucocytes
Normal up to 1-2 WBCs/HPF.
Larger than red cells and smaller than renal epithelial cells.
Presence of many white cells in clumps is strongly suggestive of acute urinary tract
infection.
An increase in urinary WBCs is called pyuria and indicates the presence of an infection or
inflammation in the genitourinary system.
Microscopic examination and chemical testing are used to determine the presence of
leukocytes in the urine.

84
 Figure (2-14): Shows morphology of leukocytesin urine under microscope

Epithelial cells
Any site in genitourinary tract from PCT to the urethra or from the vagina. Normally a few
cells from these sites can be found, A marked increase indicates inflammation of that
proportion of urinary tract from which the cell is derived.

TYPES
Renal tubular ,Transitional , Squamous.

Figure (2-15): Shows morphology of epithelial cells in urine under microscope

85
Crystals
Usually not found in fresh urine but appear when urine strands for a while. Many of crystal
found in urine have little clinical significance except in case of metabolic disorders. Crystals
are identified by their appearance and their solubility characteristics.

TYPES
Acidic & Alkaline crystals.

Crystals In Acidic Urine

1. Uric acid.
2. Amorphous urates
3. Calcium oxalate
4. Cystine
5. Leucine
6. Tyrosine
7. Sulpha

Uric acid crystals

Presence of uric acid crystals in urine is a normal appearance. Increase in-gout,Chronic
nephritis , high purine Metabolism .

86
 Figure (2-16): Shows morphology of Uric acid crystals in urine under microscope

Calcium oxalate crystals

Octahedral or envelope shaped crystal. Can be present in normal urine after ingestion of
various oxalate rich foods (Beans.Beer.Beets.Chocolate.Coffee.Dark green vegetables, such
as spinach.Nuts.Oranges.Soda (cola).Soy beans. Sweet potatoes. Tea (black). Pathological-
DM -Liver disease- chronic renal disease.

87
 Figure (2-17): Shows morphology of Calcium oxalate Crystals in urine under microscope

Amorphous urates
Urates salts of sodium, potassium and calcium, having a granular appearance. Present in
urine as non crystalline amorphous forms. No clinical significance. The precipitate of
amorphous urate is pink (known as brick dust).

88
Figure (2-18): Shows morphology of Amorphous urates under microscope and in test tube

Hippuric acid crystals: Can also occur in healthy urine.

Figure (2-19): Shows morphology of Hippuric acid Crystals in urine under microscope

89
Cystine crystals
Cystine stones are caused by a rare disorder called “cystinuria.” The disorder causes a
natural substance called “cystine”. When there is too much cystine in the urine, kidney
stones can form. These stones can get stuck in the kidneys, bladder, or anywhere in the
urinary tract.

Figure (2-20): Shows morphology of Cystine Crystals in urine under microscope

Leucine crystals
Clinically very significant.
Maple syrup disease (is a rare but serious inherited condition. It means the body cannot
process certain amino acids , causing a harmful build-up of substances in the blood and
urine. Also in serious liver disease.

Figure (2-21): Shows morphology of Leucine Crystals in urine under microscope

90
Tyrosine crystals
Clinically significance, (Severe liver disease & tyrosinosis) .

Figure (2-22): Shows morphology of Tyrosine Crystals in urine under microscope

Cholesterol crystals
Presence of excessive in urine indicates tissue breakdown.

Figure (3-23): Shows morphology of Cholesterol Crystals in urine under microscope

91
Sulfa drugs crystals
May be history of sulfa drugs medication.

Figure (2-24) : Shows morphology of Sulfa drugs Crystals in urine under microscope

Crystals In Alkaline Urine

1. Triple phosphate
2. Calcium carbonate
3. Cholesterol
4. Amorphous phosphates
5. Ammonium biurate

Figure (2-25): Shows morphology of triple phosphates Crystals in urine under

microscope

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Amorphous phosphates
No clinical significance.

Figure (2-26): Shows white precipitation of Amorphous phosphate

Figure (2-27): Shows morphology of Amorphus phosphates Crystals in urine under

microscope

Calcium carbonate
The formation of calcium carbonate crystals can be caused by a combination of factors
including decreased urine volume or a condition that alkalinizes (increases the pH of) urine,
such as a vegetarian diet, chronic diarrhea, urinary tract infections or certain medications.

93
 Figure (2-28): Shows morphology of Calcium carbonate Crystals in urine under
microscope

Corpora amylacea
Corpora amylacea, also known as amyloids, is the accumulation of dense amounts of
calcified materials that are protein based in nature. Benign prostatic hyperplasia is a
condition found in older men where the prostate naturally swells to an abnormal size and
shape. When this happens it can cause the prostate to press on the urethra and make it
painful, or difficult to pass urine.
The abnormal size is what is believed to cause the prostate stones (Corpora amylacea),
because the prostate is not its normal size the ducts cannot function properly and release the
fluids down the tubes because they become twisted or narrow from the swelling.

94
Figure (2-29): Shows a corpora amylacea from the urinary sediment

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Casts
Presence of casts is frequently associated with proteinuria. Always renal in origin and
indicates central renal disease.

Red cells cast

Meaning renal hematuria. Always pathological.

Figure (2-30): Shows morphology of Red cells cast in urine under microscope

White cell cast

White blood cell (WBC) casts are common with acute kidney infections and interstitial
nephritis.

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 Figure (2-31): Shows morphology of White cells cast in urine under microscope

Granular casts
Degeneration of cellular casts or direct aggregation of serum proteins. Almost always
indicate significant renal disease. May be fine granular or coarse granular casts.

Figure (2-32): Shows morphology of Granular cast in urine under microscope

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Epithelial cells casts
Result as statis and desquamation of renal tubular epithelial cells, indicates tubular injury.

Figure (2-33): Shows morphology of epithelial cells cast in urine under microscope

Waxy casts
Results from degeneration of granular casts, found in acute and chronic renal disease.

Figure (2-34): Shows morphology of Waxy cast in urine under microscope

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Fatty casts
Found in fatty degeneration of tubular epithelial.

Figure (2-35): Shows morphology of Fatty cast in urine under microscope

Hyaline cast
Damage to glomerular capillary membrane, fever, orthostatic proteinuria (also known as
postural proteinuria), is a condition where an abnormally large amount of protein is
excreted in the urine when the patient is in an upright position and normal protein excretion
in the supine position, may be due to Chronic renal disease - eg, diabetic kidney disease,
glomerulonephritis,SLE and emotional stress or active exercise.

Figure (2-36): Shows morphology of Hyaline cast in urine under microscope

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Bacteria
When accompanied with white cells usually indicates UTI. Occurs as rod or chains or cocci.

Figure (2-37): Shows morphology of Bacteria in urine under microscope

Artifacts
Starch crystals

Figure (2-38): Shows morphology of starch crystals in urine under microscope

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 Fibres

Figure (2-39): Shows morphology of fibers in urine under microscope

Types of fibers
A. Hair
B. Pollen grain
C. Talcum powder
D. Air bubble
E. Fat droplets
F. Cloth fiber

Figure (2-40): Shows morphology different types of fibers in urine under microscope

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Urine parasites
Trichomonas vaginalis

Figure (2-41): Shows morphology Trichomonas vaginalis in urine under microscope

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 Schistosoma haematobium

Figure (2-42): Shows morphology of Schistosoma haematobium in urine under

microscope

Urine Collection and Culture

To isolate, quantify and permit presumptive identification and differentiation of the major
microorganisms causing urinary tract infections (UTIs). Specimen collection Urine specimens
may be collected in several ways:
1. Mid-stream specimen
2. Clean catch specimen Bag specimen
3. Catheter specimen (either from an in-out catheter or an indwelling catheter)
4. Supra-pubic aspirate (needle directly into the bladder).

Wherever possible a specimen of urine should be collected aseptically directly into a sterile
universal container following cleaning of the perineal area. Specimen transport and storage
Specimens should ideally be stored and transported in sealed plastic bags. Laboratory
processing should occur as soon as possible after specimen collection. Specimens should be
refrigerated if delays in processing over two hours are unavoidable.

Culture
 Tip over the container to re-mix the urine sample
 Describe the appearance of the urine (clear or cloudy) and colour
 Blood agar and macconkey agar. Inoculate the urine on plates of culture media.
 Routine urine cultures should be plated using calibrated loops for the semi quantitative
method.
 This method has the advantage of providing information regarding the number of
cfu/mL, as well as providing isolated colonies for identification and susceptibility
testing.

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  Spread the entire volume over the surface of Blood agar and MaCconkey agar or
CLED agar (cysteine lactose electrolyte-deficient agar) by making a single streak
across the centre. Spread the inoculum evenly at right angles to the primary streak .

CLED agar (Cystine Lactose Electrolyte Deficient )

Is differential, non selective culture medium used for the isolation, enumeration and
differentiation of urinary microorganisms. It promotes the growth of urinary pathogens, but
prevents excessive swarming of Proteus species due to its lack of electrolytes.
The medium allows quantitative determination of urinary pathogens including Proteus when
calibrated loops are used for inoculation.

Principle of CLED Agar

The medium contains cystine, lactose, bromothymol blue and is electrolyte deficient.

 Cystine is added for the benefit of organisms that have a specific need for cystine. It
promotes the growth of coliforms.
 Lactose is included to provide an energy source for organisms capable of utilizing it
through a fermentation mechanism.
 Bromothymol blue is the indicator used in the agar, it turns yellow in case of acid
production during lactose fermentation or turns dark blue in case of alkalinization.
Lactose-positive bacteria form yellow colonies. Bacteria that decarboxylate L-cystine
cause an alkaline reaction and form dark blue colonies
 Electrolyte deficiency reduces the invasion of the environment by Proteus.

Taple (2-4) : Interpretation of CLED Agar

Bacteria Growth
Escherichia coli, Citrobacte Yellow colonies, opaque; yellow medium
Enterobacter, Klebsiella Yellow to whitish-blue colonies, often mucoid; yellowish
medium
Proteus Translucent blue colonies; blue-green to blue medium
Pseudomonas Green colonies with typical matted surface and rough
periphery; blue medium
Streptococci Small, opaque, pale yellow colonies
Staphylococci Very small, opaque, yellow colonies
Enterococci Small yellow colonies, about 0.5 mm in diameter; yellow
medium

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Figure(2-43):Urine culture on blood agar Figure(2-44):Urine culture on macconley agar

Figure (2-45) : CLED agar

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 Figure (2-46) : E.coli on CLED agar

Figure (2-47) : Serratia marcescens on CLED medium

Q : Is CLED selective?

A : CLED agar is a non-selective medium, It allows the growth of a wide variety of non-
fastidious germs.
Q : Does Salmonella grow on CLED?

A : Yes, salmonella can grow on CLED, salmonella doesn't utilize lactose (blue-green
colonies on the surface of CLED agar).

106
Q : Why does E coli grow as yellow colonies on CLED?

A : Organisms capable of fermenting lactose, like E coli, will lower the pH and change the
color of the medium to yellow (colonies appear yellowish because the agar will turn
yellowish).

Figure (2-48) : Lactose fermenting (yellow colonies) and Lactose Non-fermenting colonies in
CLED

Figure (2-49): Shows procedure of urine culture

If many samples are being processed use half a plate per sample. -Incubate the plate
aerobically at 35-37°C for at 18-24 hours. -After inoculation, estimate the number of bacteria
by counting the number of colonies on the surface of the media two type of standard loop.
one loop 10μl(0.01ml) and other equal 1 μl(0.001ml) if used standard loop 0.01ml the number
of colonies on the surface of the media ×100= cfu/mL but used standard loop 0.001ml in this
case the number of colonies on the surface of the media ×1000 = cfu/mL.
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 Figure (2-50) : Urine culture using the calibrated loop/surface streak method

Interpretation
Examine and report the cultures
 Examine the blood agar for Gram-positive pathogens associated with cystitis include
the coagulase-negative Staphylococcus saprophyticus, Enterococcus faecalis, and
Streptococcus agalactiae.
 Examine MacConkey agar for Enterobacteriaceae , Pseudomonas aeruginosa
 Report wet amount as an initial report.
 Report the isolated pathogen and its sensitivity pattern as a final report.

Culture results: Results are categorised on the basis of quantity of growth. Below a
recognised threshold (105 cfu/mL) the likelihood is that the organisms grown are
contaminants, particularly if more than one type of organism is present. Above the threshold
it is more probable that a true urinary tract infection is occurring.

Colony count Number organisms per mL of urine

<10 <104 CFU/mL

10-100 104–105 CFU/mL

>100 >105 CFU/mL

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If there is a pure growth of 10-100 or over 100 colonies, sub culture the isolate for
identification and antimicrobial susceptibility testing. For cultures that contain two organisms,
one in low numbers (<100 colonies) and the other over 100 colonies, then only sub-culture
the predominant organism because the organism of lower numbers is unlikely to be causing
disease.

If both are present at over 100 colonies, sub-culture both organisms.If more than two
organisms are isolated, then do not sub-culture / identify any of them since this is highly likely
to be a contaminated specimen. Must be repeated the culture with mid-stream urine sample.

Table (2-5) : Sites of Urinary Tract Infections

Lower UTI Upper UTI
Sites involved Urethra and bladder Kidney and ureter
Symptoms Local manifestations- dysuria, urgency, Local and systemic manifestations (fever, vomiting,
frequency abdominal pain)

Route of Ascending route Both ascending (common) and descending route

spread

Occurrence More common Less common

Table (2-6) : Microorganisms causing UTI

Bacterial agents: Other agents:

Gram-negative bacilli: Fungus:

• Escherichia coli- Commonest agent of UTI Candida albicans
• Proteus mirabilis
• Klebsiella pneumoniae
Parasites:
• Schistosoma haematobium
• Pseudomonas aeruginosa
• Trichomonas vaginalis
• Acinetobacter species
• Enterobacter species
• Serratia species

Gram-positive cocci: Viruses:

• Staphylococcus saprophyticus • Herpes simplex virus
• Staphylococcus aureus • Adenovirus
• Staphylococcus epidermidis • JC and BK virus
• Enterococcus spp. • Cytomegalovirus

Note: Most organisms are acquired by ascending route. Organisms acquired by

descending route include (Staphylococcus aureus, Salmonella, Mycobacterium
tuberculosis, Leptospira and Candida) .

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Collection, transport and examination of stool specimens
DEFINITION
• Human feces is called as stool.
• faeces / feces is plural of latin term faex meaning RESIDUE.
• Meconium is newborn‟s first feces.

Common pathogens
Helicobacter pylori ,Salmonella spp. , E. coli O157:H7 , Staphylococcus aureus ,
Campylobacter spp. ,Vibrio cholerae ,Yersinia enterocolitica ,Shigella spp.,Clostridium
difficile .

ADVANTAGES OF STOOL EXAMINATION

Examination of stools aids in investigation of GIT diseases.
Detection of parasites : Detection of worms (adult worms, tapeworms, larvae, ova),
detection of protozoa (trophozoites or cysts).
Evaluation of chronic diarrhea
• Random diarrheal sample tested for occult blood, fat, pH, white blood cells, culture, or
microscopy.
• A 48- or 72-hour sample is examined for weight, fat content, carbohydrate, osmolality
or chymotrypsin activity.
• Examination of stools aids in investigation of GIT diseases.

Evaluation of dysentery
Identification of causative organism is definitive in amebic V/S bacillary dysentery.

Bacteriologic examination
Infection by bacteria such as Salmonella, Shigella, Vibrio, Yersinia, or Clostridium difficile
can be identified by stool culture. Bacterial toxins released by Clostridium botulinum or
Clostridium difficile can also be identified.

Chemical examination
1. To detect occult blood (in ulcerated lesions of gastrointestinal tract, especially occult
carcinoma of colon).
2. Excess fat excretion (malabsorption syndrome).
3. Presence or absence of urobilinogen (obstructive jaundice).

Differentiating infection by invasive bacteria (like Salmonella or Shigella ) from that

due to toxin producing bacteria (like Escherichia coli or Vibrio cholerae):
Increased numbers of polymorphonuclear neutrophils (identified by methylene blue stain)
are seen in the former, Examination of stools aids in investigation of GIT diseases.

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Identification of Rotavirus
Common cause of diarrhea in infants & young children. It can be identified by examination
of stool by electron microscopy, latex agglutination, immunofluorescence or ELISA.

COLLECTION OF STOOL SAMPLES

• A random specimen of stool (at least 4 ml or 4 cm3) is collected in a clean, dry, container
with a tightly fitting lid & transported immediately to the laboratory.
• About 20-40 grams of formed stool or 5-6 tablespoons of watery stool should be collected.
• Parasites are best detected in warm, freshly passed stools which should be examined as
early as possible (preferably within 1 hour of collection).
• Stool should not be contaminated with urine, water, soil, or menstrual blood.
• Sample may be refrigerated if delay in examination is anticipated.
• A fixative may be used if specimen is to be transported to a distant laboratory.
o 10% formalin for preservation of eggs, larvae & cysts.
o Polyvinyl alcohol for preservation of trophozoites & cysts & for permanent staining.
• 3 separate samples collected at 3-day intervals are recommended to detect all parasite
infections. One negative report for ova and parasites does not exclude the possibility of
infection.

Figure (2-51): Shows container of stool collection

MACROSCOPIC EXAMINATION
Findings
1. Consistency
2. Color
3. Odor
4. Presence of blood
5. Presence of mucus
6. Presence of adult worms or segments of tapeworms

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MACROSCOPIC EXAMINATION
Consistency of Stool samples
1. Formed
2. Loose
3. Watery
• Cysts are likely to be found in formed stools.
• Trophozoites are most likely to be found in loose or watery stools or in stools containing
blood and mucus.
• Trophozoites die soon after being passed and therefore such stools should be examined
within 1 hour of passing.
• Examination of formed stools can be delayed but should be completed on the same day.

Consistency of Stool samples

• Dry and hard stools are seen in chronic constipation.
• Pasty stools are due to fat contents
• Common bile obstruction
• Celiac disease
• Ribbon like stools are seen in
• Spastic bowel
• Rectal narrowing
• Stricture
• Partial GIT obstruction

Figure (2-52): Shows consistency of stool samples

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 Table (2-7): Color/Appearance of Stool samples

Color/Appearance Interpretation
Brown Normal/Stercobilinogen

Black and tarry Bleeding in upper GIT(proximal to cecum)

Drugs (iron salts, bismuth salts, charcoal)
Lower GIT bleeding/tumors, inflammatory process
Red
Anal fissure,hemmorhoids,tumors
Undigested tomatoes or beetroot.
Yellow or yellow green Diarrhoea
Clay-colored
(gray-white) Biliary obstruction

Silvery Carcinoma of ampulla of Vater (The ampulla of Vater is

a small opening that enters into the first portion of the
small intestine, known as the duodenum).
Watery Certain strains of Escherichia coli, Rotavirus enteritis,
Cryptosporidiosis
Rice water Cholera

Unformed with blood Bacillary dysentery, Ulcerative Colitis, Intestinal

mucus, and pus tuberculosis,Amoebiasis, Enteritis
Unformed, frothy, foul
smelling, which float on Steatorrhoea
Water
Pale color stool with Pancreatic deficiency due to malabsorption
greasy appearance

MACROSCOPIC EXAMINATION
Presence of mucus in stools
• Translucent gelatinous material clinging to surface of stool.
• Produced by colonic mucosa in response to parasympathetic stimulation.
• Seen in: (Severe constipation, mucous colitis).
Presence of mucus and blood in stools
Seen in:
• Bacillary dysentery
• Ulcerative Colitis
• Intestinal tuberculosis
• Amoebiasis

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Mucus with blood clinging to stool is seen in:
• Lower GIT malignancy.
• Inflammatory lesions of anal canal

PREPARATION OF SLIDES
1. A drop of normal saline is placed near one end of a glass slide and a drop of Lugol
iodine solution is placed near the other end.
2. A small amount of feces is mixed with a drop each of saline and iodine using a wire
loop, and a cover slip is placed over each preparation separately.
3. If the specimen contains blood or mucus, that portion should be included for
examination (trophozoites are more readily found in mucus).
4. If the stools are liquid, select the portion from the surface for examination.
5. Saline wet mount is used for demonstration of eggs and larvae of helminths, and
trophozoites and cysts of protozoa.
6. Saline wet mount can also detect red cells and white cells.
7. The iodine wet mount is useful for identification of protozoal cysts as iodine stains
glycogen and nuclei of the cysts.
8. Trophozoites become non-motile in iodine mounts.
9. A liquid, diarrheal stool can be examined directly without adding saline.

Figure (2-53): Shows Wet preparation of direct stool examination with normal saline and
Iodine

MICROSCOPIC EXAMINATION
Findings:
1. Leukocytes (WBCs)
2. Red Blood Cells (RBCs)
3. Macrophages
4. Epithelial cells
5. Bacteria
6. Ova/ Cysts/ Trophozoites of parasites
7. Meat/muscle fibres
8. Fat

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Leukocytes (WBCs)
Normal stool may contain occasional (0-1) WBCs, to look for WBCs; the smears should be
prepared from areas of mucous or watery stools.

Increased numbers of WBCs in stools are associated with:

• Bacillary dysentery
• Chronic ulcerative colitis
• Shigellosis
• Salmonella infections
• Invasive E-Coli infections
• Anal/Rectal Fistula
• Localised abscess
• Amoebiasis & typhoid

Figure (2-54): Shows Leukocytes (WBCs) in stool under microscope

Red Blood Cells (RBCs)

• Bright red stool is seen in cases of lower GIT bleeding.
• Black and tarry blood are seen in cases of:
a. Upper GIT bleeding.
b. Occult bleeding.
Present in:
• Dysentery
• Hemorrhoids
• GIT Malignancies

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 Figure (2-55): Shows Erythrocytes in stool under microscope

Macrophages
Seen in Bacillary dysentery, Ulcerative colitis

Figure (2-56): Shows Macrophages in stool under microscope

Epithelial cells
Seen in inflammatory conditions of the bowel

Figure (2-57): Shows Epithelial cell in stool under microscope

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Bacteria
Stool cultures are commonly done to identify bacteria associated with enteric infection. Stool
samples are examined to detect toxins & to rule out infection by:
a. Salmonella, Shigella, Campylobacter & predominating numbers of Staphylococcus
organisms.
b. Pseudomonas, Yersinia, Vibrio & Shiga toxin–producing E. coli.
c. Clostridium difficile causing antibiotic-associated colitis.
d. Yeast.
At least 3 stool cultures collected on separate days are recommended if the patient‟s clinical
picture suggests bacterial involvement, despite previous negative cultures.

Fat
Present in:
1. Malabsorption
2. Deficiency of pancreatic digestive enzyme
3. Deficiency of bile

Figure (2-58): Shows fat droplet in stool under microscope

Meat/muscle fibers in stool

Their presence show impaired intraluminal digestion. Increased amount of meat fibres are
found in:
1. Malabsorption syndrome.
2. Pancreatic functional defect like cystic fibrosis.

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 Figure (2-59): Shows muscle fibers in stool under microscope

Ova/ Cysts/ Trophozoites of parasites

1. Normally there are no parasites/eggs in the stool sample.
2. Multiple stool samples should be examined to rule out parasitic infestations.
3. At least 3 consecutive days‟ stool samples are examined to rule out parasitic infestations
4. Cytoplasm shows outer transparent ectoplasm and inner finely granular endoplasm.
5. Nucleus is visible in the iodine preparation.
6. Fine granules of peripheral nuclear chromatin are evenly distributed. Small, single
central karyosome. (Motility is lost in iodine mount).

Entamoeba histolytica
1. Demonstration of trophozoites of E. histolytica in stool samples is required for
diagnosis of amoebic dysentery.
2. For diagnosis, at least three fresh stool samples should be examined to increase
sensitivity.
3. Trophozoites vary from 15 to 40 μ in diameter.
4. In saline wet mounts,
5. Trophozoites show motility in one direction via pseudopodia, which form rapidly.
6. Cytoplasm shows outer transparent ectoplasm and inner finely granular endoplasm.
7. Nucleus is visible in the iodine preparation.
8. Fine granules of peripheral nuclear chromatin are evenly distributed. Small, single
9. Central karyosome. (Motility is lost in iodine mount).
10. Diagnostic feature of E. histolytica trophozoites is the presence of ingested red cells.

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 Figure (2-60): Shows trophozoite of Entamoeba histolytica in stool under microscope

Cysts of E. histolytica are:

1. Spherical and measure 10-15 μ in diameter.
2. Nuclei are 1, 2, 3, or 4 and are similar in morphology to trophozoite nucleus.
3. Mature cyst contains 4 nuclei.
4. Nuclear membrane is regular and thin with finely granular peripheral chromatin.
5. Karyosome is small and central.
6. Immature cysts may show chromatoid bodies (aggregates of ribosomes) that are
oblong structures with rounded ends and glycogen clumps – infective stage.

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 Figure (2-61): Shows cyst of Entamoeba histolytica in stool under microscope

Giardia intestinalis (lamblia)

1. Cysts are more likely to be found in formed or loose stools.
2. Cysts of G. lamblia are 8-12 μ in diameter, oval and contain 4 nuclei, axonemes,
median bodies and remains of flagella.

Figure (2-62): Shows cyst of Giardia lamblia in stool under microscope

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Giardia lamblia trophozoites
1. G. lamblia trophozoites are found as clusters in fresh liquid stools, particularly in
flakes of mucus.
2. Trophozoites of G. lamblia are pear-shaped,12-15 μ in diameter, have 4 pairs of
flagellae, 2 large and oval nuclei, 2 axonemes (axial filaments of flagella), and 1 or 2
curved median bodies.
3. Motility is likened to that of “falling leaf”.

Figure (2-63): Shows Trophozoite of Giardia lamblia in stool under microscope

MICROSCOPIC EXAMINATION – HELMINTHS

Ascaris lumbricoides (Roundworm)
1. Diagnosis of A. lumbricoides infection is made by demonstration of eggs on stool
examination.
2. Eggs can be demonstrated in direct wet mount of stools in moderate to heavy infections.
3. The recommended procedure is formol-ethyl acetate sedimentation technique for
concentration of eggs.
4. In stools, the types of eggs are found:
a. fertilized (double-shelled or decorticated)
b. Unfertilised (double-shelled or decorticated)

121
 Figure (2-64): Shows Ascaris lumbricoides (Egg&Larva) in stool under microscope

Unfertilized eggs:
• Single female worms discharge eggs which are slightly larger & elliptical in shape.
• Size : 80 μm x 55 μm.
• The egg has a thinner shell with an irregular coating of albumin.
• The egg is filled with a mass of large disorganized highly refractile granules of various
sizes (atrophied ovum).

Fertilized eggs:
• Eggs are round, oval, golden brown in color & always bile stained.
• Size : 70 μ × 50 μ in size.
• Surrounded by thick smooth translucent shell with an outer coarsely mammillated
albuminous coat, a thick transparent middle layer & the inner lipoidal vitelline membrane.
• The egg contains a single central granular mass (fertilized ovum).

122
 Figure (2-65): Shows Egg of Ascaris lumbricoides in stool under microscope

MICROSCOPIC EXAMINATION - HELMINTHS

Identification of adult worms
1. Occasionally adult worms are passed spontaneously in the feces and brought to the
laboratory for identification.
2. Adult ascaris worms are cylindrical or round, pinkish, and measure about 15 cm
(male) or 30 cm (female) in length.
3. 0.5 cm in diameter.
4. Tail is either curved (male) or straight (female).
5. There are three lips at the anterior end (mouth).

Hookworms
• Hookworms are:
Ancylostoma duodenale (old world hookworm).
Necator americanus (new world hookworm).
1. Diagnosis is based on identification of hookworm eggs on stool examination.
2. Alternatively, direct wet mount of stool sample can demonstrate eggs in moderate to
heavy infections.
3. Eggs of A. duodenale & N. americanus are morphologically similar.
4. Eggs of A. duodenale & N. americanus are 50-75 μ in length and 40 μ in width,
oval, colourless, and have a thin shell.
5. In fresh stools, eggs show 4-8 gray, granular cells.
6. If stool > 12 hrs old, eggs will show a rhabditiform larva folded upon itself, which
are called embryonated eggs.
7. If feces > 24 hrs old, free rhabditiform larvae will be seen.
8. Buccal cavity of hookworm larva is longer which distinguishes itself from
9. larvae of Strongyloides stercoralis test for occult blood in stools is positive.
10. Blood examination often shows eosinophilia.
11. Microcytic hypochromic anemia develops due to chronic blood loss.

123
 Figure (2-66): Shows Egg of Ancylostoma duodenale in stool under microscope

Trichuris trichiura (Whipworm)

1. Diagnosis depends on identification of typical eggs on stool examination.
2. Eggs measure 50 × 25 μ in size, are yellowbrown and barrel-shaped.
3. A rounded, transparent plug is present at both poles
4. Eggs contain central, uniformly granular mass.
5. Eggs are often quantitated to assess the severity of infection.

Figure (2-67): Shows male of Trichuris trichiura in stool under microscope

124
 Figure (2-68): Shows Ova of Trichuris trichiura in stool under microscope

Hymenolepis nana
1. Eggs are oval and measures 30-50 μ in size.
2. The inner membrane has 2 poles, from which 4-8 polar filaments spread out between the
two membranes.
3. The oncosphere has 6 Hooklets.

Figure (2-69): Shows Ova of Hymenolepis nana in stool under microscope

125
Enterobius vermicularis
Special technique for collection and demonstration of pinworm eggs:
1. Adult female pinworm migrates at night from the intestine (cecum) to the perianal
skin folds and deposits eggs which are often not found on routine stool examination.
2. Pinworm eggs can be collected either by a transparent adhesive tape (“cellophane
tape test”) or by anal swab.
3. Specimen should preferably be collected late into the night or early morning before
patient passes urine, feces or takes a bath.
4. A transparent adhesive tape is folded over the end of a glass slide, spoon handle or a
wooden tongue depressor (sticky surface outwards).
5. Patient‟s buttocks are separated & the slide or spoon handle covered with tape is
pressed over perianal skin at many sites.
6. The tape is then spread over a glass slide with adhesive side down & pressed flat onto
the slide surface.
7. Slide covered with tape is then examined under the microscope.
8. Eggs of E.vermicularis measure 60 μ × 30 μ in size, are oval & flattened on one side.
9. They are colorless, transparent with a double-lined smooth shell & contain a small
granular mass or a larva.
10. Adult pinworms may be recovered from perianal skin folds (by adhesive tape) or
may be found in children‟s feces.
11. They are white, motile & small in size (male: 0.5 cm; female: 1 cm).
12. Diagnosis depends on demonstration of eggs in samples collected from perianal skin
or demonstration of adult worms.

Figure (2-70): Shows Ova of Enterobius vermicularis in stool under microscope

126
Taenia solium
Identification of eggs:
1. Egg measures 30-40 μ in diameter, is round to oval, and has a thick, brown wall with
transverse lines.
2. The egg contains an embryo, which is a round granular mass containing 3 pairs of
hooklets and surrounded by a fine membrane.
3. Occasionally, the egg is enclosed in a clear sac.
4. Eggs are discharged intermittently by the tapeworm and therefore may not be detected
easily.
5. Repeated stool examinations and formol-ether concentration technique are often
required for their demonstration.

Figure (2-71): Shows Ova of Taenia solium in stool under microscope

Eggs of T. saginata can be identified in feces and perianal skin. They are morphologically
similar to those of T. solium

MICROSCOPIC EXAMINATION - ARTEFACTS

It is also important to identify artifacts during microscopic examination of stool samples
which could be confused with ova & cysts of various protozoa & helminthes. Fecal
specimens should be collected in the early stages of the diarrhoeal disease, when pathogens
are present in the highest number, and preferably before antimicrobial treatment is started, if
appropriate.
The specimen should be collected in the morning to reach the laboratory before noon, so that
it can be processed the same day. Provide the patient with two small wooden sticks and a
suitable container with a leak proof lid (e.g. a clean glass or plastic cup or a special container
with a spoon attached to the lid).

127
The specimen should contain at least 5 g of feces and, if present, those parts that contain
blood, mucus or pus. It should not be contaminated with urine.
Once the specimen has been placed in the specimen container, the lid should be sealed. The
patient should be asked to deliver the specimen to the clinic immediately after collection.
If it is not possible for the specimen to be delivered to the laboratory within 2 hours of its
collection, a small amount of the fecal specimen (together with mucus, blood and epithelial
threads, if present) should be collected on two or three swabs and placed in a container with
transport medium (Cary–Blair, Stuart or Amies) or 33 mmol/l of glycerol–phosphate buffer.

For cholera and other Vibrio spp., alkaline peptone water is an excellent transport (and
enrichment) medium. Pathogens may survive in such media for up to 1week, even at room
temperature, although refrigeration is preferable.

Figure (2-72): Shows Yeast & fungal elements, red arrow show Candida budding, and
blue arrow show Candida non-budding (40X).

A B
Figure (2-73): Shows Pollen grain (A) & Plant fibre (B) in stool under microscope

128
 Taple (2-8) : Possible pathogens in stool

Culture the specimen

1. MacConkey agar with crystal violet is recommended as ageneral purpose medium.
Various points which have to be noted are:
• Consistency: formed, unformed (soft), loose or watery. The cysts have been mostly
found in the formed stools, while trophozoites have been most abundantly
found in watery stools.
• The presence of blood,mucus or pus.
• The presence of worms, e.g. Enterobius Vermicularis, Ascaris, tapeworm segments,
e.g. Taenia species.
• Colour (white, yellow,brown or black).
• Normal faeces appear brown and formed or semiformed. Infant faeces are yellow-
green and semiformed.

2. Xylose–lysine–deoxycholate (XLD) agar is recommended as a medium of moderate or

high selectivity for the isolation of Shigella and Salmonella spp.

3. Deoxycholate citrate agar (DCA), Hektoen enteric agar (HEA) and Salmonella– Shigella
(SS) agar.

4. Thiosulfate citrate bile salts sucrose (TCBS) agar is selective for V. cholerae O1 and non-
O1 and for V. parahaemolyticus.
Inoculate all media above with stool specimen. After inoculation, incubate the agar plates.
Incubate the plates for the isolation of Salmonella, Shigella and Yersinia spp. and V.
cholerae at 35 - 37C0 in an aerobic incubator (without CO2) for 24 hr.

129
Examine and report the cultures
Examine MacConkey agar for Gram negative enterobacteriaceae. Examine other for
identification of bacteria mention ‫ه‬n above.

Biochemical reaction
• Indole, cimmon citrate, urease test , motility, triple sugar iron agar (TSI)
• String test for vibrio cholera and serology.
• Serological test of shigella spp.

Figure (2-74) : Salmonella spp on triple sugar iron agar

130
 Figure (2-75): This image depicts four test tubes each containing triple sugar iron agar (TSI)
medium. Test tube 1 only contained the agar medium, hence was uninoculated. Test tube 2,
contained medium that was inoculated with Shigella sp. bacteria. Test tube 3 contained
medium inoculated with Providencia sp. bacteria. Test tube 4 contained medium inoculated
with Pseudomonas sp. bacteria.

Table (2-9): Summary of morphology,Cultural characteristics,and biochemical reactions of

Enterobacteriaceae

TSI Indole MR VP Citrate Urease Motility

E.coli A/A/- +ve +ve -ve -ve -ve Motile
Citrobacter A/A/- +ve +ve -ve +ve -ve Motile
freundii
Klebsiella A/A/- -ve -ve +ve +ve +ve Non-motile
pneumonia
Enterobacter A/A/- -ve -ve +ve +ve +ve Motile
cloacae
Salmonella A/Alk/+ -ve +ve -ve +ve -ve Motile
typhi
Shigella A/Alk/- -ve +ve -ve -ve -ve Non-motile
boydii
Proteus A/Alk/+ -ve +ve -ve +ve +ve Motile
mirabilis Swarming

131
Table (2-10): Shows growth characteristics of Enterobacteriaceae

Species MacConkey XLD agar SS agar DCA agar Hekton agar

agar with
crystal
violet
Large, salmon-
Escherichia Pink to rose Large,flat,yello Pink to red, Pink,encircled pink to orange,
coli w opaque inhibited, by zone of encircled by zone
growth precipitate of precipitate

Shigella spp. Colourless Red Colourless Colourless to Green, moist and

tan Raised

Salmonella Colourless Red, with or Colourless, with Colourless to Blue-green, with

spp. without black or without tan, with or or without black
centre black centre without black centre
centre
Large, pale
Enterobacter Pink, mucoid Yellow, mucoid Pink,inhibitd mucoid with Large, salmon-
Klebsiella spp. growth pink centre Orange

Colourless, with Large,

Proteus Colourless, Red, some or without colourless
Providenci inhibited,sw Proteus spp. grey-black to tan, with or
spp. arming have black centre without black
centre centre

Yersinia Colourless Yellow, Colourless Colourless Salmon

enterocolitica irregular

Enterococci No No growth to No growth No growth to No growth to

Growth slight growth slight growth slight growth

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Table (2-11) : Infectious agents of acute diarrhea and the underlying mechanism
Mechanism Features Examples of pathogens involved

Non- Location: Bacteria: Viruses:

inflammatory Proximal small bowel (Mostly enterotoxin • Rotavirus
mediated) • Norovirus
Illness: • Vibrio cholerae • Enteric adenoviruses
Watery diarrhea • Escherichia coli Parasites:
○ Enteropathogenic • Giardia lamblia
Stool findings: ○ Enterotoxigenic • Cryptosporidium
○ Enteroaggregative
• No fecal leukocytes • Cyclospora species
• Clostridium perfringens
• Fecal lactoferrin-not • Microsporidia
increased • Bacillus cereus
• Staphylococcus aureus,
• Aeromonas hydrophila
• Plesiomonas shigelloides

Inflammatory Location: Predominantly dysentery: Parasite

(invasion or Colon or distal small bowel • Shigella species (predominantly dysentery):
cytotoxin) • Campylobacter jejuni • Entamoeba histolytica,
Illness: • Enterohemorrhagic E. coli Balantidium coli
• Dysentery or • Enteroinvasive E. coli
• Inflammatory diarrhea • Vibrio parahaemolyticus
Stool findings: Predominantly inflammatory
• Fecal pus cells diarrhea-
(polymorphonuclear • Salmonella species
leukocytes)– increased • Yersinia enterocolitica
Fecal lactoferrin–increased • Listeria monocytogenes
• Clostridium difficile
• Aeromonas hydrophila
• Plesiomonas shigelloides
Klebsiella oxytoca

Penetrating Location: Distal small Salmonella Typhi (enteric

bowel fever)
Illness: Enteric fever Yersinia enterocolitica
Stool findings:
Fecal mononuclear
leukocytes

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Table (2-12) : Infectious agents causing food poisoning
Organisms Symptoms Common food sources
1–6 h incubation period
Staphylococcus aureus Nausea, vomiting, diarrhea Ham, poultry, potato or egg salad, mayonnaise, cream pastries
Bacillus cereus Nausea, vomiting, diarrhea Fried rice
Clostridium botulinum Nausea, vomiting, diarrhea Canned food
8–16 h incubation period
Clostridium perfringens Abdominal cramps, diar- Beef, poultry, legumes, gravies
rhea (vomiting rare)
B. cereus Abdominal cramps, diar- Meats, vegetables, dried beans, cereals
rhea (vomiting rare)
> 16 h incubation period
Vibrio cholerae Watery diarrhea Shellfish, water
Enterotoxigenic E. coli Watery diarrhea Salads, cheese, meat, water
Enterohemorrhagic E. coli Bloody diarrhea Ground beef, roast beef, salami, raw milk, raw vegetables, apple
juice
Salmonella species Inflammatory diarrhea Beef, poultry, eggs, dairy products
Campylobacter jejuni Inflammatory diarrhea Poultry, raw milk
Shigella species Dysentery Potato or egg salad, lettuce, raw vegetables
Vibrio parahaemolyticus Dysentery Mollusks, crustaceans

Table (2-13): Agents of Traveller‟s diarrhea

Etiologic agent Comments
Bacteria (50–75%)
Enterotoxigenic Escherichia coli (10–45%) Single most important agent
Enteroaggregative E. coli (5–35%) Emerging enteric pathogen with worldwide distribution
Campylobacter jejuni (5–25%) More common in Asia
Shigella Major cause of dysentery
Salmonella Common in India
Others Aeromonas, Plesiomonas, and Vibrio cholerae
Viruses ( < 20%)
Norovirus (< 10%) Associated with cruise ships
Rotavirus (< 5%) Common among children
Parasites (0–10 %) Giardia lamblia,
Cryptosporidium, Entamoeba
histolytica, Cyclospora

Table (2-14) : Pathogenic mechanisms of diarrheal agents

Toxins Other mechanisms
Enterotoxins: Attachment within or close to mucosal cells:
• Cholera toxin • E. coli:
• Vibrio parahaemolyticus ○ Enteropathogenic E. coli (EPEC)
• E.coli: ○ Enterohemorrhagic E. coli (EHEC)
○ LT and ST of ETEC • Cryptosporidium species
○ EAST(enteroaggregative heat- • Cyclospora species
stable enterotoxin) of EAEC • Rotavirus
○ VT of EHEC • Norovirus
• Clostridium difficile (toxin A)
• Aeromonas
• Campylobacter jejuni

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 Cytotoxins Invasion of intestinal epithelium:
• Shigella dysenteriae type 1 • Shigella species
• Enterohemorrhagic E.coli (EHEC) • Enteroinvasive E. coli
• Clostridium difficile (toxin B) • Campylobacter jejuni
• Yersinia enterocolitica
Neurotoxins: • Plesiomonas shigelloides
• Staphylococcus aureus enterotoxin • Entamoeba histolytica
• Bacillus cereus toxin • Balantidium coli
• Clostridium botulinum toxin

Collection, Transport and examination of sputum

Possible pathogens
Gram positive: Streptococcus pnemoniae , Staphylococcus aureus , Streptococcus
pyogenes.
Gram negative: Haemophilus influenzae, Moraxella catarrhalis, Klebsiella pneumonia,
Pseudomonas aeruginosa, Proteus species,Yersina pestis.

Acid fast Bacilli (Mycobacterium tuberculosis)

In a hospital with a microbiology Laboratory

1. Give the patient a clean (need not be sterile), dry, wide necked,leak- proof container and
request him or her to cough deeply to produce a sputum specimen Note: The specimen
must be sputum, not saliva.
2. Sputum is best collected in the morning, soon after the patient wakes and before any
mouth-wash is used.
3. If the patient is young child and it is not possible to obtain sputum,gastric washings can
be used for the isolation of M.tuberculosis but not for other respiratory.
4. Label the container and fill the request form.
5. If pneumonia or bronchopneumonia is suspected, deliver the sputum to the laboratory
with as little delay as possible.
6. Because organisms such as S.pneumoniae and H.influenzae require culturing as soon as
possible. If the specimen is for the isolation of M.tuberculosis,ensure it is delivered to
the laboratory within 2 hoursor kept at 4 C0 until delivery is possible.
7. If pneumonia plague is suspected: Deliver the sputum to the laboratory as soon as
possible.

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 Figure (2-76): Shows sputum collection

Laboratory examination of sputum

Describe the appearance of the specimens:
1. Purulent: Green- looking with pus and mucus.
2. Muco purulent: Green- looking with pus and Mucous.
3. Mucoid: Mostly mucous.
4. Mucosalivary: Mucus with a small amount of saliva.
5. Bloody: should be reported.

Examine the specimen microscopically

Gram smear
Look for pus cells and bacteria
1. Gram positive diplococci that could be S. Pneumoniae.
2. Gram positive cocci that could be S. aureus.
3. Gram negative rods that could be H. Influenzae.
4. Gram negative capsulated rods that could be K.pneumoni

Ziehl-Neelsen stain (See page 4 )

1. Make a smear of the sputum on slides from the most purulent materials for
M.tuberculosis
2. Fix with alcohol.
3. Potassium hydroxide (KOH) preparation, if fungal infection is suspected (yeast
cells, Nocardia species, actinomycetes).

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Culture the specimen
Blood agar, chocolate agar in CO2 ,McConkey agar, Sabouraud agar if fungal infection
suspected.
1. Inoculate the sputum on all plates of culture media.
2. Incubate the blood agar plate in a CO2, chocolate agar also in a CO2 enriched
atmosphere at 35-37c0 for up to 48 hours, examining for growth after overnight
incubation.

Lowenstein Jensen medium

If pulmonary tubercles is suspected,Incubate at 35c0 -37c0.Examining twice a week for
growth.

Examine and report the cultures

Examine the blood agar ,macConkey agar and Chocolate agar culture for :
 S. pneumoniae
 S. aureus
 H. influezae
 K. pneumoniae
 Pseudomonas aeruginosa

Examine Lowenstein Jensen medium for Mycobacterium tuberculosis

Examine Sabouraud agar for fungi

Figure (2-77): Shows Growth characteristics of Mycobacterium tuberculosis on

Lowenstein Jensen medium

137
Figure (2-78): This is a close-up of a Mycobacterium tuberculosis culture revealing this
organism‟s colonial morphology. Note the colorless rough surface, which are typical
morphologic characteristics seen in Mycobacterium tuberculosis colonial growth

Collection, transport and examination of mouth and throat specimens

Possible pathogens :
Gram positive
Streptococcus pyogenes , Other beta-haemolytic streptococci, Corynebacterium
diphtheriae.

Gram negative
Vincent’s organisms
Viruses: Respiratory viruses, Enteroviruses and herpes simplex virus type-I .
Fungi: candida albicans.
Pathogens in the upper respiratory tract such as Bordetella,pertussis, streptoccus
pneumoniae, and Neisseria meningitidis, are usually more successfully isolated from
perinasal and nasopharyngeal specimens.

Dispatching to the microbiology laboratory

1. Using a sterile swab or silica gel collect a specimen from the infected area.
2. Taking care not to contaminate the swab, return it to its tube. Seal with adhesive tape and
label it.
3. Send the swab with its request form to the microbiology laboratory.

138
 Figure (2-79): Shows collection of throat swab

Culture the specimen

Blood agar
1. Inoculate the swab on a plate of blood agar.
2. If the swab is received in silica gel ( ex. from health center), moisten it first with sterile
nutrient broth and then inoculate the plate.
3. Add a bacitracin disc. This will help in the identification of S. pyogenes.
4. Incubate the plate preferably in a CO2 enriched atmosphere over night at 35-37C0
5. Beta-haemolytic streptococci produce larger Zones of haemolysis when incubated in
CO2.

Sabouraud agar : If thrush is suspected.

1. Inoculate the swab on sabouraud agar
2. Incubate at 35-37c0 for up to 48hours checking for growth after overnight incubation.

Examine the specimen microscopically,

Gram smear
Examine the smear for pus cells and Vincent‟s organisms:
• Vincent‟s organism are seen as Gram negative spirochaetes (B. vincenti) And fusiform
rods.
• If thrush is suspected, look for Gram positivee yeast-like cells.
• If diphtheria is suspected, look for Gram positive pleomorphic rods.
• Commensal diphtheroids, however, are strongly Gram positive and Unlike.

Examine and report the cultures

Blood agar culture
Look for beta-haemolytic colonies that could be group A streptococcu (S. pyogenes) or a
beta-haemolytic streptococcus belonging to another lancefield group such as group C or G.

139
Figure (2-80) : Two different large-zone, large colony, beta hemolytic Streptococcus isolates from
wounds. Group C (S. dysgalactiae subspecies equisimilus, which can also express Lancefield
antigen G) and Group A strep (S. pyogenes)

Bacitracin susceptibility test

Most group A strains show sensitivity to bacitracin. Group A strains can also be
distinguished from other groups by their sensitivity to bacitracin. A disc that contains 0.04U
of bacitracin inhibits the growth of more than 95% of group A strains, whereas 80-90% of
non–group A strains are resistant to this antibiotic.

Figure (2-81): Bacitracin test of Streptococcus pyogenes

140
Reporting of the throat swab cultures:
If a B-haemolytic streptococcus sensitive to bacitracin is isolated, report the culture as “S.
pyogenes presumptive group A isolated, Lancefield group to be confirmed.

Collection, Transport and examination of Ear Discharges

Possible pathogens:
Gram positive : S. aureus ,S.pyogenes ,Other beta-haemolytic streptococci , S. preumoniae

Gram negative: P. aeruginosa, H. influenzae Klebsiella species, proteus species

E.coli and other coliforms ,Bacteriodes species.

Fungi: Aspergillus species especially A. niger, candida species, and,occasionally various

species of dermatophyte or phycomycete.

External Ear infection is more commonly caused by:

S. aureus,S. pyogenes,P. aeruginosa.

Middle ear infection or otitis media are commonly caused by :

S. pneumoniae, S. pyogenes,and other B- haemolytic streptococci,H.influenzae (esp. in
children), Coliforms, Klebsiella SPP., S. aureus, P. aeruginosa.
Chronic ear infection is often caused by: Bacteroides species or fungi.

Collection and Transport of Ear Discharges

1. Collect a specimen of the discharge on a sterile cotton swab.
2. Place it in container of Amies transport medium, breaking off the swab stick to allow the
bottle top to be replaced tightly.
3. Label the specimens and send them with its request form to the laboratory.

Laboratory examination of Ear Discharges

Culture of specimen
Blood agar (CO2), MacConkey agar and Chocolate agar(CO2)
Inoculate the specimen on culture media .Incubate at 35-37c0 overnight. Inoculate the
specimen on chocolate (heated blood) agar for the isolation of H.influenza.Incubate the plate
in a carbon dioxide enriched atmosphere at 35-37c0 for up to 48 hours, examining for growth
after overnight incubation.

Sabouraud agar if a fungal infection is suspected

Inoculate the specimen on sabouraud agar, and incubate for up to 6 days.

141
Examine the specimen Microscopically
Gram smear
1. Gram positive cocci that could be S.aureus.
2. Gram positive streptococci or diplococi that could by streptococci pathogens.
3. Gram negative rods that could be H.influenzae,p. aeroginosa, klebsiella speceis, proteus
species, E.coli or others.
4. Gram positive yeast cells that could be candida species.
5. Small numbers of Gram positive cocci,streptococci, Rods and also Gram negative rods
may be seen in smears of ear discharges because these organisms form part of the
normal microbial flora of the external ear.

Additional:
Potassium hydroxide if a fungal infection is suspected.
Look for:
• Brnaching septate hyphae with small round spores, that could be Aspergillns speies
• Pseudohyphae with yeast cells, that could be candida specis (Gram positive).

Examine and Report the culture

Blood agar and MacConkey
S. aureus ,B-haemolytic streptococcus ,E.coli ,Proteus species ,P. Aeruginosa S. Pneumonia.
Examine chocolate agar: H. Influenza.
Examine Sabouroud agar for: fungal elements.

Collection, transport and examination of eye specimens

Possible pathogens

Gram positive Gram negative

Staphylococcus aureus Neisseria gonorrhoeae
Streptococcus pneumonae Haemophilus influenzae
Streptococcus agalactiae (Group – B) Pseudomonas aeroginosa
Streptococcus Pyogenes (Group – A) Moraxella lacunata
Other B- hemolytic streptococci

Chlamydiae: C. trachomatis
Viruses: Adenoviruse, herpesviruses and enteroviruses.

142
Collection and transport of eye specimen
1. Eye specimen should be collected by medical officer or experienced nurses.
Conjunctival scrapings to detect C.trachomatis must be collected by medical person.
2. Specimen from the eye must be cultured as soon as possible after collection because the
natural secretions of the eye contain antibacterial enzymes.

Laboratory examination of the eye specimen

Culture the specimen
Blood agar,McConkey agar and chocolate agar
1. Inoculate the eye discharge on blood , MacConkey and chocolate (heated blood) agar.
2. Incubate the blood agar in CO2 and MacConkey agar aerobically at 35-37C0 overnight.
3. Incubate the chocolate agar plate in a CO2 enriched atmosphere for 48 hours, checking
for growth after overnight incubation.
Additional
NYC selective medium if gonococcal conjunctivtis is suspected (infant less than 3 weeks
old).
• Inoculate the discharge on the plate
• Incubate at 35-37C0 in a CO2 enriched atmosphere overnight.
Loeffler serum slope if Moraxella infection is suspected:
• Inoculate the eye discharge on a loeffler serum slope.
• Incubate at 35-37C0 overnight.
Microscopically examination
Gram smear : Look for:-
1. Gram negative intracellular diplococci that could be N.Gonorrhoeae. If found, a
presumptive diagnosis of gonococcal conjunctioitis can be made A cervical swab from
the mother should also be cultured for the isolation of N.gonorrhoeae.
2. Gram positive streptococci or diplococci that could be streptococci pathogens.
3. Gram positive cocci that could be S.aureus
4. Gram negative rods that could be Haemophilus species.

Additional
Giemsa smear : Result
trachomatis inclusion bodies ---------- Blue-mauve to dark purple. Depending on the stage of
development; if the inclusion body is more mature, it will contain ---- red mauve staining
elementary particles.

Examine and Report the cultures

Blood agar and chocolate agar
Look for colonies that could be:
Haemophilus Influenzae,Staphylococcus aureus,Beta- helmolytic streptococci
Streptococcus Pneumoniae ,Pseudomonas aeruginosa.
NYC Medium culture
Look for N. gonorrheae, oxidase positive.

143
Collection, transport and examination of skin and wound specimen
Possible pathogens:

Gram positive Gram negative

S. aureus Escherichia coli
S. pyogenes Proteus spp.
Enterococci Pseudomonas aeruginosa
Bacillus anthracis yersinia pestis
Corynaebacterium ulcerans Vincent’s organisms
M. leprae
M. ulcerans
Virus: pox viruses and herpes viruses
Fungi: Ringworm
Parasite: Leishmania spp.

Collection of skin specimens and ulcer materials

1. Using a sterile dry cotton wool swab, collect a sample of discharge from the infected
tissue.
2. If there is no discharge, use swab moistened with sterile physiological saline to collect a
specimen.
3. Insert the swab in a sterile tube. If the tissue is deeply ulcerated and necrotic (full of
4. dead cells); Aspirate a sample of infected material from the side wall of the ulcer using
a sterile needle and syringe.
5. Fluid from pustules and blisters: Aspirates a specimen using a sterile needle and
syringe.
6. Brings immediately the specimen to the laboratory for examination.

Laboratory examination of skin specimens

Culture the specimen
Blood agar and MacConkey agar
Inoculate the specimen then incubate both plate aerobically at 35-37C0 overnight.

Additional:
Sabourand agar if a fungal infection is suspected
1. Inoculate to agar plate.
2. Modified Tinsdale Medium (MTM), if cutaneous diphitheria is suspected
3. Inoculate the sample for isolation of C. Ulcerans.
4. Incubate aerobically at 35-37C0 for up to 48hours,examining the growth after overnight
incubation.

144
 Figure (2-82) : Corynebacterium diphtheriae in Tinsdale Agar medium, Look for
black colonies with brown halo

Microscopical examination
Gram Smear Look for:
1. Gram positive cocci that could be S. aureus.
2. Gram positive streptococci that could be S. pyogenes or other streptococci.
3. Gram negative rods that could be p. aeruginosa, proteus speices, E.coli or other
coliforms.

If tropical ulcer is suspected, look for vincent‟s organisms.

The spirochetes and fusiform bacilli that are commonly grouped as Vincent's organisms are
frequently associated with infections in and about the tonsils, teeth, genital organs and
gastro-intestinal tract .
Acute necrotizing ulcerative gingivitis, otherwise known as Vincent's angina or trench
mouth, is caused by an imbalance in the normal flora of the gingival sulcus with
predominant presence of the spirochete Borrelia vincentii and the gram-negative bacillus
Fusiformis fusiform.

145
 Figure (2-83) : Vincent‟s organisms

If cutaneus anthrax is suspected, look for large Gram variable rods lying in chains that
could be B. anthracis.

Figure (2-84) : Gram-positive, rod-shaped, Bacillus anthracis bacteria

Additional:
Potassium hydroxide preparation, if ringworm or other superficial fungi infection is
suspected.

For detection of ringworm:

Giemsa techniques or wayson`s techniques,if bubonic plague is suspected. Polychrome
Loefler methylene blue smear if cutaneous anthrax is suspected.
Examine and report the culture
Blood agar and MacConkey agar cultures Look for: S. aureus, S. pyogenes, P.
aeruginosa , Enterococci ,Proteus species , Escherichia coli.

146
Aditional investigations
sabouraud agar -if fungal infection is suspected.
MTM - if cutaneous diphtheria is suspected .

Wound Examination
Specimen collection
Pus and wound swab Specimen collection :
1. No-touching technique: remove bandage with the forceps.
2. With the forceps take a sponge, dip it in the saline and wash the surface of the wound or
ulcers.
3. Remove the swab from its covering and extend the tip of the swab deep into the wound
taking care not to touch the adjacent skin margins.

Note: The laboratory is not responsible for taking the specimen

Wherever possible a specimen of pus/fluid collected aseptically into a sterile universal
container is preferred over a swab. However, a swab collected into a suitable transport
medium (e.g. Amies +/- charcoal) is acceptable if a pus/fluid specimen is not available. It is
important to accurately record the anatomical site of a swab specimen, since cultures from
swabs of a deep pus collection will require a different interpretation to those from a
superficial site.

Specimen transport and storage

Specimens should ideally be stored and transported in sealed plastic bags. Laboratory
processing should occur as soon as possible after specimen collection. Specimens should be
refrigerated if delays in processing over two hours are unavoidable.

Specimen processing
Specimen Processing (Laboratory Receiving) is the section of the laboratories where
specimens are received, sorted, entered into the Laboratory Information System, labelled
with barcoded labels and processed.

Microscopic examination
Gram stain
ZN staining (mandatory if TB is suspected).
If a single swab is received, prepare the slide after performing culture.

Culture
Inoculate and incubate culture media
Swab
 Blood agar 35 – 37 5 – 10% CO2 24 - 48h Daily β-haemolytic Streptococci
Pasteurella spp.(bite wounds) S. aureus.
 MacConkey agar 35 – 37 Air 24 h Daily Enterobacteriacea, Pseudomonads.
 Sabouraud agar 35 – 37 Air 24 - 48h Daily Fungi.
147
  Cooked meat medium Laboratory Subculture at 24 h, 48 h, and 72 h as indicated.
 blood agar when anaerobic infection is suspected Incubate anaerobically.
 Culture for M. tuberculosis requires facilities of a tuberculosis reference laboratory.

In superficial swabs, the following may be identified to a lower level: Enterobacteriaceae,

Pseudomonas: “pseudomonas” level (species level in burn swab)
Yeasts: “yeasts” level (species level for Cryptococcus sp.)

Examine Microscopically
 Gram smear: For pus cells and bacteria
 Ziehl-Neelsen smear: When tuberculosis or M. ulcerans disease is suspected.
 KOH preparation: When a fungal or actinomycete infection is suspected.

Anaerobic culture
When an anaerobic infection is suspected (specimen is often foul-smelling), or the Gram
smear shows an „anaerobic mixed flora‟, inoculate a second blood agar plate and incubate it
anaerobically for up to 48 hours. The anaerobic blood agar plate may be made selective by
adding neomycin to it .
Examine and report the cultures Blood agar and MacConkey agar cultures :
Look especially for colonies that could be: Staphylococcus aureus Streptococcus pyogenes
Pseudomonas aeruginosa Proteus species Escherichia coli Enterococcus species Klebsiella
species.

Anaerobic blood agar culture and cooked meat culture Look for growth that could be:
C. perfringens: Grows rapidly in cooked meat medium with hydrogen sulphide gas
production (gas bubbles in turbid medium) and reddening but no decomposition of the meat
(saccharolytic reaction). On anaerobic blood agar, colonies are usually seen after 48 h
incubation. Most strains produce a double zone of haemolysis (inner zone of clear
haemolysis, outer zone of partial haemolysis) C. perfringens .
B. fragilis : Grows in cooked meat medium producing decomposition with blackening of the
meat (foul-smelling proteolytic reaction). On anaerobic blood agar, non-haemolytic grey
colonies (Gram negative pleomorphic rods) are seen, usually within 48 hours.

Antimicrobial susceptibility testing :

Susceptibility testing may be required for S. aureus, enterobacteria and non-fermentative
Gram negative rods. Only routinely used antibiotics should be be tested on special request or
when the isolate is resistant to other drugs.
Anaerobic pathogens:
Susceptibility tests should not routinely be performed on anaerobic bacteria by the disc
diffusion technique. Most anaerobic infections are caused by penicillin-susceptible bacteria
with the exception of infections originating in the intestinal tract or vagina. Such infections
generally contain B. fragilis which produces beta-lactamase and is resistant to penicillins,
ampicillins and most cephalosporins. Such infections can be treated with clindamycin,

148
metronidazole or chloramphenicol. Aminoglycosides have no activity against anaerobes but
they are often used for the treatment of patients who have mixed infections.

Bacteroides Bile Esculin and Laked Kanamycin Vancomycin bi-plate –

BBE/LKV
BBE
Bacteroides Bile Esculin (BBE) agar is an enriched, selective, and differential medium used
for the isolation and presumptive identification of obligately anaerobic gram-negative bacilli
of the Bacteroides fragilis group and Bilophila wadsworthia.

Figure (2-85): B. fragilis Growing On BBE/LKV Bi-Plate

LKV
Laked Brucella Blood Agar with Kanamycin and Vancomycin (LKV) is an enriched,
selective, and differential medium for the isolation and partial identification of obligately
anaerobic gram-negative bacilli. LKV agar is useful for the rapid isolation
of Prevotella species.

149
 Figure (2-86) : C. perfringens on blood agar with double zone of hemolysis

Figure (2-87): Nagler‟s reaction of C. perfringens (lecithinase positive) and C. sporogenes

(lecithinase negative)

150
Colleciton, transport and examination of CerebroSpinal fluid
Cerebro spinal fluid The examination of cerebrospinal fluid (CSF) is an essential step in the
diagnosis of bacterial and fungal meningitis and CSF must always be considered as a priority
specimen that requires prompt attention by the laboratory staff.
Normal CSF is sterile and clear, and usually contains three leukocytes or fewer per mm3 and
no erythrocytes. The chemical and cytological composition of CSF is modified by meningeal
or cerebral inflammation, i.e. meningitis or encephalitis.

Common causes of bacterial and fungal meningitis

In neonates (from birth to 2 months)
 Escherichia coli
 Listeria monocytogenes
 Other Enterobacteriaceae: Salmonella spp., Citrobacter spp
 Streptococcus agalactiae (group B)

In all other age groups

 Haemophilus influenzae (capsular type B)
 Neisseria meningitidis
 Streptococcus pneumoniae
 Mycobacterium tuberculosis
 Listeria monocytogenes
 Cryptococcus neoformans
 Staphylococci
o Uncommon after the age of 5 years.
o In immuno compromised patients (including those with acquired immunodeficiency
syndrome (AIDS).
o Associated with neurosurgery and postoperative drains.

Viruses
Enteroviruses, especially echoviruses and coxsackie viruses,Rarely Polioviruses may also be
isolated from CSF.
Fungi: Cryptococcus neoformans
Parasites: Trypanosoma species, Naegleria fowleri,Acanthamoeba species .

Collection of specimens
It should be collected by medical officer in a septic procedure.The fluid is usually collected
from the arachnoid space. A sterile wide-bore needle is inserted between the 3th and 4th
lumbar vertebrate and CSF is allowed to drip into dry sterile containers for bacteriological
investigation, count and biochemical estimations
Approximately 5–10 ml of CSF should be collected in three sterile tubes by lumbar or
ventricular puncture performed by a physician.Part of the CSF specimen will be used for

151
cytological and chemical examination, and the remainder for the microbiological
examination.
The specimen should be delivered to the laboratory at once, and processed immediately,since
cells disintegrate rapidly also lead to a falsely low glucose value due to glycolysis.. Any
delay may produce a cell count that does not reflect the clinical situation of the patient.

Figure (2-88) : Shows collection of CSF

C.S.f should be cultured as soon as possible after collection, if a delay is una-voidable, the
fluid should be kept at 35-37C0or left at room temperature (never refrigerated).
Because the Haemophilus influenza and Neisseria meningitides may not survive in the cold
temperature. One exception to this rule involves CSF for viral studies. These specimens may
be refrigerated for as long as 23 hours after collection or frozen at -70°C if a longer delay is
anticipated and should never be frozen at temperatures above -70°C. If not processed
immediately, CSF specimen for hematology studies can be refrigerated, whereas the CSF for
chemistry and serologycan be frozen (-20°C).

Laboratory examination of C.S.F

It should be performed without delay and should be reported micro-organism especially
Gram smear. The fluid should be handled with special care because it is collected by lumbar
puncture and only a small amount can be withdrawn.

Describe the appearance of the specimens Report

The appearance of the CSF should be noted and recorded as: clear, hazy, turbid, purulent,
yellow, bloody (due to haemolysis or icterus), or blood- tinged, with fibrin web or pellicle.
Microscopic examination.

152
Perform a cell count
Test the specimen biochemically
Glucose estimation
A. Low Glucose →pyogenic meningitis
B. Normal →viral miningitis.

Microscopic examination
Preparation of specimen If, on gross examination, the CSF is purulent (very cloudy), it can
be examined immediately without centrifugation. In all other cases, the CSF should be
centrifuged in a sterile tube (preferably a 15-ml conical tube with screwcap) at 10000 g for
15–20 minutes. Remove the supernatant using a sterile Pasteur pipette fitted with a rubber
bulb, and transfer it to another tube for chemical and/or serological tests. Use the sediment
for further microbiological tests.

Direct microscopy Examine

One drop of the sediment microscopically :
 Leukocytes (polymorphonuclear neutrophils or lymphocytes)
 Erythrocytes
 Bacteria
 Yeasts.
 Trypanosomiasis

If the yeast-like fungus Cryptococcus neoformans is suspected, mix a loopful of the

sediment with a loopful of India ink on a slide place a coverslip on top, and examine
microscopically for the typical, encapsulated, spherical, budding yeast forms. (Naegleria
fowleri) they may be seen in the direct wet preparation as active motile amoebae about the
size of neutrophilic leukocytes.
Gram-stained smears As the causative agent of bacterial meningitis may often be observed in
a Gram-stained smear, this examination is extremely important. Air-dry the smear, fix with
gentle heat, and stain it by Gram‟s method. Examine at \1000 (oil-immersion) for at least 10
minutes, or until bacteria are found.
C.S.F should be cultured as soon as possible after collection.If a delay is unavoidable, the
fluid should be kept at 35-37C0 (never refrigerated).If the C.S.F appears only slightly cloudy,
centrifuge it in a sterile tube for 15-20minute and use the sediment for inoculating the plates.

Culture of C.S.F.
Culture
 If bacteria have been seen in the Gram stained smear, the appropriate culture media
should be inoculated.
 If no organisms have been seen, or if the interpretation of the Gram smear is unclear, it
is desirable to inoculate a full range of media, including blood agar with a streak of
Staphylococcus aureus to promote growth of H. influenzae.
 Blood agar and chocolate agar plates should be incubated at 35°C in an atmosphere
enriched with carbon dioxide.
 All media should be incubated for 3 days, with daily inspections .

153
Additional
Lowenstein Jensen medium if tuberculous meningitis is suspected.
Sabouraud agar if cryptococcal meningitis is suspected.If capsulated yeast cells are seen in
the microscopical preparations,inoculate a plate of sabouraud agar. Incubate at 35-37C0 for
up to72hours, checking for growth after overnight incubation.

Microscopy
Gram smear
1. Gram negative intracellular diplococci that could be N. meningitidis.
2. Gram positive diplococci or short streptococci that could be S. pneumoniae. It is often
possible to see the capsules as unstained are as around the bacteria.
3. Garm negative rods, possibly H. Influezae.
4. Gram negative rods, could also be E.coli or other coliforms, especially if the C.S.f is
from a newborn infant.
5. Gram positive cocci in groups and singly, possible S.aureus.
6. Gram positive streptococci, possibly S. agalacteae (G -.B).
7. Gram positive yeast cells, C. neoformans.

Figure (2-89): Gram‟s stain (Panel A) and India ink stain (Panel B) revealed abundant
encapsulated, round yeasts, with some budding forms

Additional
Ziehl-Neelsen – smear for M. Tuberculosis.
Indian ink preparation if cryptococcal meningitis is suspected:
Wet preparation to detect Amoebae or Trypanosome
Giemsa stain to detect morula cells or Burkett’s lymphoma cells these can be found when
trypanosomes have invaded the CNS.
They are larger than most lymphocytes, stain dark red mauve and contain vacuoles.

154
Preliminary identification
 Growth on MacConkey agar is suggestive of Enterobacteriaceae and should be further
identified using the methods and media recommended for enteric pathogens.
Enterobacteriaceae , Klebsiella spp. , Escherichia coli. , Acinetobacter baumannii
,Morganella morganii ,Serratia marcescenes ,Citrobacter freundii ,Enterobacter
cloacae ,Proteus mirabilis.
 Colonies of Gram-positive cocci with a narrow zone of b-haemolysis on blood agar
may be S. agalactiae (group B streptococci). This should be confirmed with the CAMP
test.
 Flat colonies with a concave centre and a slight green zone of alpha -haemolysis are
probably S. pneumoniae. For confirmation, a 6-mm optochin disc should be placed on
a blood agar plate heavily inoculated with a pure culture of the suspected.strain. After
overnight incubation, pneumococci will exhibit an inhibition zone of 14 mm or more
around the optochin disc. The best results are obtained after incubation on sheep blood
agar in a carbondioxide-enriched atmosphere.
 If the reading of this test on the primary blood agar plate is inconclusive,the test
should be repeated on a subculture. Colonies of H. influenzae will grow only on
chocolate agar, and as satellite colonies in the vicinity of the staphylococcal streak on
blood agar. Further identification may be accomplished using H. influenza type b
antiserum in the slide agglutination test.
 Gram-negative diplococci growing on blood and chocolate agar, and giving a rapidly
positive oxidase test, may be considered to be meningococci.
 Confirmation is accomplished by grouping with appropriate N. meningitidis antisera
(A, B, C) in the slide agglutination test.
 A negative agglutination test does not rule out meningococci as there are at least four
additional serogroups
 If the agglutination test is negative, carbohydrate utilization tests should be performed
and the culture sent to a central reference laboratory. A preliminary report should be
given to the physician at each stage of identification (Gram stain,growth,
agglutination, etc.), noting that a final report will follow.

Figure (2-90) : Hemophilus influenza on chocolate agar(left) , X&v FACTOR TEST (Right)

155
Colonies of Gram-positive rods with a b-haemolysis on blood agar may be Listeria
monocytogenes. The following confirmatory tests are suggested: positive catalase reaction,
motility in broth culture or in MIU and black discoloration on bile–aesculin agar.

Figure (2-91): Umbrella-like pattern (SIM) Tumbling motility of Listeria monocytogenes

Susceptibility testing
For Gram-negative rods and staphylococci, the standardized disc-diffusion method (Kirby–
Bauer) should be used.
 No susceptibility testing is needed for Listeria monocytogenes, S. agalactiae or N.
meningitidis since resistance to ampicillin and benzylpenicillin is extremelyrare.
 All strains of pneumococci should be tested on blood agar for susceptibility to
chloramphenicol and benzylpenicillin. For the latter, the oxacillin (1mg) disc is
recommended.
 Strains of H. influenzae should be tested for susceptibility to chloramphenicol using
chocolate agar or a supplemented blood agar.
Most ampicillin-resistant strains produce b-lactamase that can be demonstrated using one
of the rapid tests recommended for the screening of potential b-lactamase-producing
strains of gonococci.

156
Table (2-15) : Cytological and biochemical parameters in CSF of normal individuals and in
different types of meningitis
Character Normal Pyogenic meningitis Tuberculous Viral meningitis
individual meningitis

CSF pressure Normal (50–150) Highly elevated (>180 ) Moderately elevated Slightly
(mm of water) elevated/normal

Total leukocyte 0–5 100–10,000, 10–500, Neutrophils 25–500,

count (per mm3) Segmented PMN Mononuclear
neutrophils
Predominant cell Lymphocytes Neutrophils Lymphocytes Lymphocytes

Glucose (mg%) 40–70 (< 40 mg/dL) 20–40 mg/dL (Slightly Normal

(Decreased to absent) Decreased)

Total proteins 15–45 > 45 mg/dL 100–500 mg/dL 20–80 mg/dL

(mg%) (usually > 250) (moderate to markedly (Normal or
(markedly elevated) elevated) slightly elevated)

Table (2-16) : Causes of meningitis (pyogenic and aseptic)

Pyogenic meningitis Aseptic meningitis

Neonates or • Escherichia coli Viruses

infants of 0–2 • Group B streptococcus (S. agalactiae) Enteroviruses (Polioviruses, echoviruses,
months • Listeria monocytogenes Coxsackie viruses): The most common agents
• Other Gram-negative bacilli Herpes simplex virus 1 and 2
(like Klebsiella pneumoniae) Other Herpes group: Varicella zoster, CMV,
EBV Myxoviruses: Influenza A and B,
2–20 years • Neisseria meningitidis: Most common parainfluenza virus, and mumps virus
agent Arboviruses, and adenoviruses, Rubella
• Haemophilus influenzae viruses and HIV
• Streptococcus pneumoniae

> 20 years Streptococcus pneumoniae: Most Bacteria: Treponema pallidum, and Leptospira
(adults) common agent
Haemophilus influenzae Parasites-Naegleria species,
Neisseria meningitides Acanthamoeba species and Toxoplasma
gondii
Overall Most common agent is Fungi: Cryptococcus neoformans
Streptococcus pneumoniae

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Collection, transport and examination of effusions (synovial,pleural,
pericardial and ascitic fluids)
An effusion is fluid which collects in body cavities.
Fluid which collects due to an inflammatory process is referred to as an exudates and that
which forms due to a non-inflammatory condition is referred to as a transudates. If the
effusion is all exudates, it is important to investigate whether the inflammatory process is all
infective one. Effusions sent to the laboratory for investigation include:
1. Cell count
2. Protein estimate
3. Microscopy
4. Culture

Table (2-17): Shows origin of body fluids

Fluid Origin

Synovial From joint

Pleural From the pleural (space between the lungs and the inner chest
wall)
Pericardial From the pericardial sac (membranous sac surrounding the
health)
Ascitic From the peritoneal (abdominal) cavity

Hydrocele Usually from the sacs surrounding the tests.

1.In a hospital with a microbiology laboratory:

After aspiration, aseptically dispense the fluid as follows:
A. 2–3 ml into a dry, sterile, screw-cap tube or bottle to observe for clotting.
B. 9 ml into a screw-cap tube or bottle which contains 1 ml of sterile tri-sodium citrate.
Mix the fluid with the anticoagulant.
(Tri-sodium citrate prevents clotting, especially of exudates. The sterile citrated sample can
be used to estimate cell numbers, protein concentration, and for microscopy and culture fluid
is carried out by a medical officer).
C. Label, and as soon as possible deliver the samples with a completed request form to the
laboratory.

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LABORATORY EXAMINATION OF EFFUSIONS
1 -Describe the appearance of the specimen Report:
A. Color of the effusion
B. –Whether it contains blood
C. –Whether it is clotted (sample without anti-coagulant)
Purulent effusion: When the specimen is pus or markedly cloudy, examine and report a
Gram stained smear as soon as possible .Whether it is clear, cloudy, or purulent (like pus)

2 -Examine the fluid for cells

Estimate the number of white cells in the fluid.

3- Estimate the protein

Dilute the fluid 1 in 99 ml in physiological saline (0.1 ml effusion mixed with 9.9 ml saline).

Possible pathogens
(Synovitis and Infective arthritis)
Gram positive
Staphylococcus aureus, Streptococcus pyogenes , Streptococcus pneumoniae, Anaerobic
streptococci, Actinomycetes.
Gram negative
Neisseria gonorrhoeae , Neisseria meningitides(rare), Haemophilus influenzae, Brucella
species, Salmonella serovars, Escherichia coli, Pseudomonas aeruginosa, Proteus,
Bacteroides, Mycobacterium tuberculosis.

4 -Culture the specimen

Centrifuge the citrated sample in a sterile tube at high speed for about 20 minutes to
sediment the bacteria.
Remove the supernatant fluid (do not discard) and resuspend the sediment.

Culture the sediment as follows:

Chocolate agar, blood agar and MacConkey agar
 Inoculate the sediment on chocolate (heated blood) agar, blood agar, and MacConkey
agar.
 Incubate the chocolate agar plate in a carbon dioxide enriched atmosphere at 35–37 C for
up to 48 hours , checking for growth after overnight incubation.
 Incubate the blood agar plate and MacConkey agar plate aerobically at 35–37 C for up to
72 hours, examining for growth after overnight incubation.

159
ADDITIONAL
Culture of specimen when tuberculosis is suspected Isolation, identification, and sensitivity
testing of M. tuberculosis and other mycobacteria require the facilities of a Tuberculosis
reference laboratory

5- Examine the specimen microscopically

Gram smear
 Make a thin evenly spread smear of a purulent effusion or sediment from a centrifuged
non-purulent sample (use the citrated specimen).
 When fix the smear with methanol for 2 minutes, and stain it by the Gram technique).
 Examine the smear for pus cells and bacteria using the 40 and 100 objectives.

Look especially for :

 Gram positive cocci that could be S. aureus.
 Gram positive streptococci that could be S. pyogenes, or possibly enterococci.
 Gram positive diplococci or short chains that could be pneumococci.
 Gram negative rods that could be enterobacteria, Pseudomonas, H. influenzae
especially if the rods are pleomorphic.
 Gram negative intracellular diplococci that could be gonococci when the fluid is from
a joint.
 Gram positive branching threads that could be actinomycetes.

Ziehl-Neelsen smear
Make a smear on a slide using several drops of sediment from the centrifuged fluid. Fix the
dried smear and stain by the Ziehl-Neelsen AFB are usually few.

6 -Examine and report the cultures

Chocolate agar, blood agar, and MacConkey agar cultures

Look especially for colonies that could be:

 Staphylococcus aureus
 Streptococcus pyogenes
 Streptococcus pneumoniae
 Haemophilus influenzae
 Enterobacteria
 Pseudomonas aeruginosa
 Neisseria species

160
Collection, Transport and Examination of urogenital specimens

Sexually Transmitted Infections

URETHRITIS & CERVICITIS

Genital Ulcer Disease
1. Chancres caused by Syphilis
2. Chancroid caused by Haemophilus ducreyi
3. Lymphogranuloma caused by Chlamydia trachomatis

Non-Sexually Transmitted Infections

Vaginitis is an inflammation of the vagina that can result in discharge, itching and pain. The
cause is usually a change in the normal balance of vaginal bacteria or an infection.
The most common types of vaginitis are:
1. Bacterial vaginosis, which results from overgrowth of one of several organisms normally
present in Vagina.
2. Yeast infections
3. Trichomoniasis

Vaginitis & Vaginosis

Vaginitis : Is caused by a limited number of infectious agents . The disease often involve the
vulva (vulvovaginitis)
Bacterial Vaginosis
 Gardnerella vaginalis
 Peptocococcus and Mobiluncus produce the characteristic malodorous discharge.
Possible pathogens
S. pyogenes ,other B – haemolytic Streptococci ,enterococci, S. aureus , Listeria
monocytogenes , Bacteriodes species , Proteus species, E. coli & other coliform.
Neisseria gonorrhoeae, T. palladium,C. trachomatis,Calymmatobacterium granulomatis
(Klebsiella granulomatis) H. ducreyi.

Specimen Collection
Cervical or high vaginal swabs are preferred to lower vaginal swabs. Specimens should be
collected using sterile swabs and placed into Amies transport medium (+/- charcoal).
Vaginal flora: Vaginal flora (e.g. lactobacilli, Prevotella spp). Coagulase negative
Staphylococci and diphtheroids aerobic, non-sporulating, pleomorphic Gram-positive bacilli
which are more uniformly stained than Corynebacterium diphtheriae, lack the metachromatic
granules are the commonest aerobes.

161
Infections may be characterised by pain, irritation, and discharge due to:
1. Organisms normally present in low numbers which can cause symptoms when found as
the predominant isolate (e.g. Staphylococcus aureus).
2. Organisms not normally isolates, whose presence is usually associated with disease (e.g.
Neisseria gonorrhoeae.
T. vaginalis,candida species,Gardnerella vaginalis).
Fluid and pus from genital ulcers
T. palladium,C. trachomatis,Calymmatobacterium granulomatis (Klebsiella granulomatis)
H. ducreyi.

Collection and transport of urogenital specimen

Amies medium is the most efficient medium for transporting urethral, cervical and vaginal
swabs.

Figure (2-92) : Amies medium

Specimen required for diagnosis of gonorrhea

Male patients: Smears (GM stain) of urethral discharge ,Rectal swab from homosexual
patient.
Female patients: Smears (GM stain) of mucous from the cervix and urethra.

Collection of Urethral specimens

1. Cleans the urethral opening using a swab moistened with sterile physiological saline.
2. Gently massage the urethra from above downwards, and collect a sample of pus on a
sterile cotton wool swab.
The patient should not have passed urine preferably for 2hours before the specimen is
collected.
3. Make a smear of the discharge on a slide for staining by the Gram technique and label the
specimen.
Collection of Cervical specimen
1. Moisten a vaginal specimen with sterile warm water and insert into the vagina.
2. Pass a sterile cotton wool swab into the endocervical canal and gently rotate the swab to
obtain a specimen.
Amies transport: Make a smear of the cervical mucopus for Gram staining and label the
specimen.

162
Collection of vaginal specimens
Using sterile swab, collect a sample of vaginal discharge.Amies transport medium.Smear
and label the sample.

Laboratory examination of urogenital specimen

Routine culture
MNYC medium (Modified New York City medium)
Incubate in moist CO2 atmosphere ⇒Neisseria gonorrhoeae.

Figure (2-93) :The growth of Neisseria gonorrhoeae colonies on New York City medium agar

Blood agar (aerobic and anaerobic), macCokey agar, Sabouraud medium, if vaginal
candidiasis is suspected and yeast cell not detected microscopically ,Serum culture, if
chancroid is suspected ⇒H. ducreyi.
Haemophilus ducreyi is a fastidious gram-negative coccobacillus bacteria. It causes
the sexually transmitted disease chancroid, a major cause of genital ulceration in developing
countries characterized by painful sores on the genitalia. Chancroid starts as an erythematous
papular lesion that breaks down into a painful bleeding ulcer with a necrotic base and ragged
edge.

Figure (2-94) : Haemophilus ducreyi bacteria , the causative agent of chancroid - stained
with Gentian Violet.

163
Microscopy
Gram smear:
Look for pus cells and bacteria ,
Suspected gonorrhoeae :Look for intracellular gram negative diplococci.
vaginitis: Look for yeast cells, and epithelial cells in the gram variable coccobacilli.
Suspected puerperal sepsis or septic abortion
Gram stain: Look for gram positive rods Closteridium perfringens , streptococci and gram
negative rods.

Suspected chanchroid Look for Gram negative coccobacilli showing bipolar staining.
Wet preparation, if trichomoniasis is suspected, mix the swab with a drop of sterile saline
on a clean microscopic slide. Place a coverslip over the wet inoculum and examine with the
low power objective lens.
Giemsa stained smear: If donovanosis is suspected
Dark field preparation, if syphilis is suspected.

Culture
1. Blood agar (35 - 37 5 - 10% CO2 18 - 24h) for beta-haemolytic Streptococci Group
Bbeta-haemolytic In pregnant women Streptococcus a galactiae.
2.Chocolate agar (35 - 37 5 - 10% CO2 24 - 48h) Haemophilus ducreyi.
3. Sabouraud agar (35 - 37 Air 24 - 48h) Yeasts .
4. Modified New York City (MNYC) or Thayer Martin medium For isolation of
N.gonorrhoeae and incubate in moist carbon dioxide enriched atmosphere at (35 – 37 C for
up to 48 hr agar 35 - 37 5 - 10% CO2 24 - 48h).

Significant Isolates:
 Group beta-haemolytic streptococci in pregnant women
 Haemophilus ducreyi which cause the sexually transmitted infections Chancroid (It is
characterized by painful necrotizing genital ulcers that may be accompanied by inguinal
lymphadenopathy. It is a highly contagious but curable disease.)
 Neisseria gonorrhoeae
 Yeasts: report to the “Candida ssp in culture.
 Enterobacteraceae identify, do antimicrobial susceptibilities and report only if heavy
pure growth.
 Staphylococcus aureus: do antimicrobial susceptibilities and report only if heavy or
pure growth.

Reporting Wet preparation results: presence or absence of Trichomonas vaginalis and

fungal elements.
Gram stain results: WBC and organisms detected. The presence of intra-cellular Gram
negative diplococci should be communicated to the clinician urgently.

164
Culture results: presence of significant isolates (e.g. Neisseria gonorrhoeae);
N.gonorrhoeae produces small raised . Grey shiny colonies on MNYC medium and Thayer
Martin medium after overnight incubation .
 If growth pathogenic organism: Growth of (Microorganism) was isolated.
 If not growth pathogenic organism: Growth of normal vaginal flora.
Clue cells are vaginal squamous epithelial cells coated with the anaerobic gramvariable
coccobacilli Gardnerella vaginalis and other anaerobic bacteria causing bacterial vaginosis.

Whiff test: This test, called the whiff test, is performed by adding a small amount of
potassium hydroxide 10% to a microscopic slide containing the vaginal discharge. A
characteristic fishy odor is considered a positive whiff test and is suggestive of bacterial
vaginosis.

Biochemical Tests of N.gonorhoea

I. Oxidase Positive
II. Ferments glucose but not maltose, sucrose, or lactose.
III. DNase negative
IV. Beta-galactosidase (ONPG) negative.

Table (2-18): Preparing and Scoring Vaginal Stains for BacteriaVaginosis

Organism morphology Number Score
More than 30 0
Lactobacillus 5-30 1
1-4 2
Less than 1 3
0 4

More than 5 2
Mobiluncus 1–4 1
(G-ve curved rode) 0 0

More than 30 4
Gardnerella 5 – 30 3
(G-ve rode) 1–4 2
Less than 1 1
0 0

165
Table (2-19) : Comparison of STDs producing genital ulcer
Feature Syphilis Herpes Chancroid LGV Donovanosis

Incubation period 9–90 days 2–7 days 1–14 days 3 days–6 1–4 weeks
weeks (up to 6 months)

Genital ulcer Painless, Multiple, painful Painful, soft Painful, Painless

indurated, Single or soft
single multiple

Lymphadenopathy Painless, hard, Absence or Painful, soft, marked Painless Absent (pseudo bubo
moderate swelling moderate swelling swelling leads to bubo may be present due to
(no bubo) (no bubo) formation subcutaneous
swelling)

Collection, Transport and Examination of Blood Culture

Blood is a sterile body fluid so Blood is cultured to detect and identify bacteria or other
cultivable microorganisms (yeasts, filamentous fungi). The presence of such organisms in
the,blood is called bacteraemia or fungaemia, and is usually pathological. In healthy
subjects, the blood is sterile. However, there are a few exceptions: transient,bacteraemia
often occurs shortly after a tooth extraction or other dental,or surgical manipulation of
contaminated mucous membranes, bronchoscopy,or urethral catheterization. This type of
transient bacteraemia is generally due,to commensal bacteria and usually resolves
spontaneously through phagocytosis,of the bacteria in the liver and spleen.,Septicemia is a
clinical term used to describe bacteraemia with clinical,manifestations of a severe infection,
including chills, fever, malaise, toxicity,,and hypotension, the extreme form being shock.
Shock can be caused by,toxins produced by Gram-negative rods or Gram-positive cocci.
It is important that blood specimens for culture are collected before initiating empirical
antimicrobial therapy. If necessary, the choice of antimicrobial can be adjusted when the
results of susceptibility tests become available.

Quantity of blood
Because the number of bacteria per millilitre of blood is usually low, it is important to take a
reasonable quantity of blood: 10 ml per vein puncture for adults; 2–5 ml may sufficient for
children, who usually have higher levels of bacteraemia;
For infants and neonates, (1–2) ml is often the most that can be obtained. Two tubes should
be used for each vein puncture: the first a vented tube for optimal recovery of strictly aerobic
microorganisms, the second a non-vented tube for anaerobic culture.

Common pathogens
 Streptococcus spp
 Staphylococcus aureus
 Listeria monocytogenes
 Coagulase negative staphylococci
 Haemophilus influenza
 Non fermenter gram negative bacilli
166
  Salmonella typhi
 Pseudomonas aeruginosa
 Enteric gram negative bacill
 Neisseria meningitides
 Bacteroides fragilis and other anaerobic bacteria

Fungi
Candida albicans ,Cryptococcus neoformans ,Other candida spp ,

Specimen collection
Patient preparing
The major difficulty in interpretation of blood cultures is potential contamination by skin
flora. This difficulty can be markedly reduced by careful attention to the details of skin
preparation and antisepsis prior to collection of the specimen.

Skin disinfection
The skin at the vein puncture site must be meticulously prepared using a bactericidal
disinfectant: 2% tincture of iodine, 10% polyvidone iodine, 70% alcohol, or 0.5%
chlorhexidine in 70% alcohol. The disinfectant should be allowed to evaporate on the skin
surface before blood is withdrawn.
If tincture of iodine is used, it should be wiped off with 70% alcohol to avoid possible skin
irritation.Even after careful skin preparation, some bacteria persist in the deeper skin layers
and may gain access to the blood, e.g. S. epidermidis, Propionibacterium acnes, and even
spores of Clostridium. Pseudobacteraemia (false-positive blood culture) may result from the
use of contaminated antiseptic solutions,syringes, or needles. The repeated isolation of an
unusual organism (e.g. Burkholderia cepacia, Pantoea (Enterobacter) agglomerans, or
Serratia spp.) in the same hospital must raise suspicion of a nosocomial infection and
promote an investigation.
Another source of contamination is contact of the needle with non-sterile vials (or solutions),
if the same syringe is first used to provide blood for chemical analysis or measurement of the
erythrocyte sedimentation rate.

Figure (2-95): Shows blood collection

167
Timing of Blood Collection
Whenever possible, blood should be taken before antibiotics are administered. The best time
is when the patient is expected to have chills or a temperature spike. It is recommended that
two or preferably three blood cultures be obtained, separated by intervals of approximately 1
hour (or less if treatment cannot be delayed). More than three blood cultures are rarely
indicated.
The advantages of repeated cultures are as follows :
 The chance of missing a transient bacteraemia is reduced.
 The pathogenic role of “saprophytic” isolates (e.g. Staphylococcus epidermidis) is
confirmed if they are recovered from multiple venepunctures.
Choice of broth medium The blood-culture broth and tryptic soy broth (TSB) should be able
to support growth of all clinically significant bacteria. The bottles containing 0.05% sodium
polyanethol sulphonate (SPS), (The use of sodium polyanethol sulfonate (SPS) as an
anticoagulant is recommended because it also inhibits the antibacterial effect of serum and
phagocytes.) .
Ideally, the blood should be mixed with 10 times its volume of broth (5 ml of blood in 50 ml
of broth) to dilute any antibiotic present and to reduce the bactericidal effect of human
serum. Specimen transport and storage After inoculation, bottles should be transported to the
microbiology laboratory without delay in a sealed plastic bag or rigid specimen container. If
a delay in processing is unavoidable, then inoculated bottles should be maintained at ambient
temperature (and must not be refrigerated).

Laboratory diagnosis
Processing of blood cultures
Incubation time
Blood-culture bottles should be incubated at 35–37 C0 and routinely inspected twice a day (at
least for the first 3 days) for signs of microbial growth. A sterile culture usually shows a
layer of sedimented red blood covered by a pale yellow transparent broth. Growth is
evidenced by:

1. A floccular deposit on top of the blood layer

2. Uniform or subsurface turbidity
3. Haemolysis
4. Coagulation of the broth
5. A surface pellicle
6. Production of gas
7. White grains on the surface or deep in the blood layer.
Whenever visible growth appears, the bottle should be opened aseptically, a small amount of
broth removed with a sterile loop or Pasteur pipette, and a Gram-stained smear examined for
the presence of microorganisms.

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Subcultures are performed by streaking a loopful on appropriate media:
1. For Gram-negative rods: macconkey agar, Kligler iron agar, motility,indole–urease
(MIU) medium, Simmons citrate agar.
2. For staphylococci: blood agar, mannitol salt agar.
3. For streptococci: blood agar with optochin, bacitracin, and tellurite discs, sheep blood
agar for the CAMP test, and bile esculin agar.
4. For Haemophilus influenza Chocolate Agar in (CO2).

For routine examinations, it is not necessary to incubate blood cultures beyond 7 days. In
some cases, incubation may be prolonged for an additional 7 days, e.g. if Brucella or other
fastidious organisms are suspected, in cases of endocarditis, or if the patient has received
antimicrobials.

Blind subcultures and final processing

Some microorganisms may grow without producing turbidity or visible,alteration of the
broth. Other organisms, e.g. pneumococci, tend to undergo,autolysis and die very rapidly.
For this reason some laboratories perform,routine subcultures on chocolate agar after 18–24
hours of incubation. Ablind,subculture may be made at the end of 7 days of incubation, by
transferring,several drops of the well-mixed blood culture (using a sterile Pasteur pipette)
into a tube of thioglycollate broth, which in turn is incubated and observed,for 3 days.
All culture results should be reported to the clinician, including the presumed contaminants.
The identification of two or more agents may indicate polymicrobial bacteraemia, which can
occur in debilitated patients, but may also be due to contamination.“Anaerobic” bacteraemia
is often caused by multiple pathogens;for example, one or more anaerobes may be associated
with one or more aerobes,in severe fulminating bacteraemia associated with severe trauma or
surgery involving the large intestine.

Culture media
1. Aerobic Blood culture bottle,Anaerobic Blood culture bottle.
2. MacConkey Agar,Blood Agar,Chocolate Agar in (CO2).
3. Blood is injected to both aerobic and anaerobic bottles and incubated for up to
7 days at 37 Co.
In some cases, incubation may be prolonged for an additional 7 days, e.g. if Brucella or
other fastidious organisms are suspected, in cases of endocarditis, or if the patient has
received antimicrobials. Interpretation of Positive blood Cultures.

Interpretation of Positive blood Cultures

1. Virtually any organism, including normal flora, can cause bacteremia
2. A negative culture result does not necessarily rule out bacteremia;
3. false-negative results occur when pathogens fail to grow
4. A positive culture result does not necessarily indicate bacteremia;false-positive results
occur when contaminants grow.
5. Gram-negative bacilli, anaerobes, and fungi should be considered pathogens until
proven otherwise.
6. The most difficult interpretation problem is to determine whether an organism that is
usually considered normal skin flora is a true pathogen.
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BacT/Alert 3D Blood Culture System
BacT/Alert is an automated microbial detection system based on the colorimetric detection
of C02 produced by growing microorganisms. Results of an evaluation of the media, sensor,
detection system, and detection algorithm indicate that the system reliably grows and detects
a wide variety of bacteria and fungi.
Results of a limited pilot clinical trial with a prototype research instrument indicate that the
system is comparable to the radiometric BACTEC 460 system in its ability to grow and
detect microorganisms in blood. On the basis of these initial findings, large-scale clinical
trials comparing BacT/Alert with other commercial microbial detection systems appear
warranted.
BacT/ALERT Microbial Detection Systems and culture bottles provide both a microbial
detection system and a culture media with suitable nutritional and environmental conditions
for organisms which might be present in the test sample. Inoculated bottles are placed into
the instrument where they are incubated and continuously monitored for the presence of
microorganisms that will grow in the BacT/ALERT bottles.

Description of the prototype BacT/Alert system

)i( Media.Two different broth media were evaluated: one for growing common aerobic,
microaerophilic, and fastidious bacteria and common yeasts and the other for growing
anaerobic bacteria. The proprietary media are based on a tryptic soy broth supplemented with
complex amino acids and carbohydrates and are designed both to support growth and to
ensure optimal C02 production.
(ii) Colorimetric detector and instrument. The prototype BacT/Alert system is a self-
contained incubator (the temperature can be adjusted between 35 and 37°C ± 0.5°C), shaker,
and detector. end, rock continuously at a rate of 60 rpm. Each well contains a colorimetric
detector .
The instrument scans each well once every 10 min. After amplification and filtering, voltage
signals are digitized and transmitted to a microcomputer for analysis. The system uses a
colorimetric sensor and reflected light to determine the amount of carbon dioxide (CO2) that
is dissolved in the culture medium. If micro-organisms are present in a blood sample, CO2 is
produced as the organisms metabolize the substrates in the culture medium.
When growth of the micro-organisms produces CO2, the colour of the sensor in the bottom
of each culture bottle changes from grey to yellow. The light reflected is measured by a
photodetector. As more CO2 is generated, more light is reflected. This information is
transmitted to a computer where it is compared to the initial CO2 level in the bottle. If there
has been a sustained acceleration in the rate of CO2 production, high initial CO2 content,
and/or an unusually high rate of CO2 production, the sample is determined to be positive.

170
 Figure (2-96) : BACT Alert 3D 120 Microbial Detection System

Figure (2-97): A laboratory worker unloads blood culture bottles from a BACT/Alert machine

171
 Figure (2-98) : BacT/ALERT Culture Media

(iii) Instrument operation. Bottles are logged into the system by entering the sample
accession number and patient identifier into the computer. The computer display then
prompts the user to place the bottles into the assigned wells. These wells are also identified
by the illumination of a small green light adjacent to each well. After the bottles are placed in
the wells, the lights are shut off and the computer records when the bottles were placed in the
instrument.
When an increasing concentration of C02 is detected in a bottle, the light adjacent to the well
is illuminated and the computer prints out the accession number, patient identifier, well
number, time growth was detected (i.e., when the bottle became positive), and time to
positivity. After the bottle is removed from the instrument, the light is shut off and that
wellcan then be used for new cultures.
False-positive bottles (bottles flagged as positive but with negative Gram-stained smears)
can be returned to the system for additional incubation and testing. Bottles that do not
become positive remain in the system for 5 days. After 5 days, the computer prints out a list
of negative bottles and illuminates the light adjacent to each well containing a negative
bottle. These bottles are removed from the system and discarded.

Interpretation of BacT/Alert Blood Culture Bottles

The BacT/Alert System will continuously rock the blood culture bottles at 60 cycles per
minute and scan all bottles (every 10 minutes) for evidence of growth.
The machine will automatically flag any positive blood cultures. Blood culture discarded all
negative bottle after 5 days incubation and issue a negative report.
SBE (subacute bacterial endocarditis) and PUO (pyrexiaofunknown origin) / Brucella after
21 days incubation, discard all negative bottle and issue a negative report. If only one bottle
has been flagged positive leave the companion bottle in the BacT/Alert until completion of
its incubation time, then discarded if negative.

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Process all suspected positive bottle as follows:
Gram stain
Sub-culture onto the following (whole) plates :
 Blood Agar (BA) CO2 35°C x 24, 48 hours.
 Chocolate Agar (CHOC) CO2 35°C x 48 hours.
 MacConkey Agar (MAC) O2 35°C x 24 hours

Incubated all plate overnight , and 48hrs, for plate anaerobically in carbon dioxide
atmosphere. Examination and report cultures: Examen all culture plate , Performing the
antimicrobial sensitivity test .

Colony appearance
Staphylococcus, S.typhi, Brucella and most coliform can be usually be seen easily ,where as
colony of S. pneumoniae , Neisseria species. Pyogenes and Y. pestis are less easily seen.
Pseudomonasand Proteus spp., produce a film of growth on the agar.
Report all positive microbiology results. Perform antibiotic susceptibility testing by standard
guidelines;
Record the negative result, dispose of the bottle according to your lab‟s standard procedures
(e.g. autoclave and discard in biohazard waste).

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Collection, Transport and Examination of Seminal Fluid Analysis

Introduction
During ejaculation, semen is produced from a concentrated suspension of spermatozoa,
stored in the paired epididymides, mixed with, and diluted by, fluid secretions from the
accessory sex organs. It is emitted in several boluses. Comparison of pre- and post-
vasectomy semen volumes reveals that about 90% of semen volume is made up of secretions
from the accessory organs, mainly the prostate and seminal vesicles, with minor
contributions from the bulbourethral (Cowper‟s) glands and epididymides.

Figure (2-99): Shows morphology of spermatozoa

Semen has two major quantifiable attributes:

1. The total number of spermatozoa: this reflects sperm production by the testes and the
patency of the post-testicular duct system.
2. The total fluid volume contributed by the various accessory glands: this reflects the
secretory activity of the glands.
The nature of the spermatozoa (their vitality, motility and morphology) and the composition
of seminal fluid are also important for sperm function.
During sexual intercourse, the initial, sperm-rich prostatic fraction of the ejaculated semen
may come into contact with cervical mucus extending into the vagina, with the rest of the
fluid remaining as a pool in the vagina.
In contrast, in the laboratory setting, the entire ejaculate is collected in one container, where
spermatozoa are trapped in a coagulum developed from proteins of seminal vesicular origin.
This coagulum is subsequently liquefied by the action of prostatic proteases, during which
time its osmolality rises.

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The results of laboratory measurements of semen quality will depend on:
1. Whether a complete sample is collected. During ejaculation the first semen fractions
voided are mainly sperm-rich prostatic fluids, whereas later fractions are dominated by
seminal vesicular fluid. Therefore, losing the first (sperm-rich) portion of the ejaculate has
more influence on the results of semen analysis than does losing the last portion.
2. The activity of the accessory sex glands, the fluids of which dilute the concentrated
epididymal spermatozoa at ejaculation. Sperm concentration is not a direct measure of
testicular sperm output, as it is influenced by the functioning of other reproductive organs;
however, the total number of sperm ejaculated (sperm concentration multiplied by semen
volume) is.
For example, sperm concentrations in semen from young and old men may be the same, but
total sperm numbers may differ, as both the volume of seminal fluid and total sperm output
decrease with age, at least in some populations.
3. The time since the last sexual activity. In the absence of ejaculation, spermatozoa
accumulate in the epididymides, then overflow into the urethra and are flushed out in urine.
Sperm vitality and chromatin are unaffected by increased length of abstinence unless
epididymal function is disturbed.

Semen analysis involves the following steps:

In the first 5 minutes:
Placing the specimen container on the bench or in an incubator (37 °C) for liquefaction.

Between 30 and 60 minutes:

 Assessing liquefaction and appearance of the semen.
 Measuring semen volume.
 Measuring semen pH (if required).
 Preparing a wet preparation for assessing microscopic appearance, sperm motility and the
dilution required for assessing sperm number.
 Assessing sperm vitality (if the percentage of motile cells is low).
 Making semen smears for assessing sperm morphology.
 Making semen dilutions for assessing sperm concentration.
 Assessing sperm number.
 Performing the mixed antiglobulin reaction (MAR) test (if required).
 Assessing peroxidase-positive cells (if round cells are present).
 Preparing spermatozoa for the immunobead test (if required).
 Centrifuging semen (if biochemical markers are to be assayed).
Within 3 hours:
Sending samples to the microbiology laboratory (if required).
After 4 hours:
Fixing, staining and assessing smears for sperm morphology.

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Later on the same day (or on a subsequent day if samples are frozen):
 Assaying accessory gland markers (if required).
 Performing the indirect immunobead test (if required).

Sample collection
Preparation
1. The sample should be collected in a private room near the laboratory, in order to limit the
exposure of the semen to fluctuations in temperature and to control the time between
collection and analysis.
2. The sample should be collected after a minimum of 2 days and a maximum of 7 days of
sexual abstinence. If additional samples are required, the number of days of sexual
abstinence should be as constant as possible at each visit.
3. The man should be given clear written and spoken instructions concerning the collection
of the semen sample. These should emphasize that the semen sample needs to be complete
and that the man should report any loss of any fraction of the sample.
4. The following information should be recorded on the report form: the man‟s name, birth
date and personal code number, the period of abstinence, the date and time of collection, the
completeness of the sample, any difficulties in producing the sample, and the interval
between collection and the start of the semen analysis.

Collection of semen for diagnostic or research purposes

1. The sample should be obtained by masturbation and ejaculated into a clean, wide-mouthed
container made of glass or plastic, from a batch that has been confirmed to be non-toxic for
spermatozoa.
2. The specimen container should be kept at ambient temperature, between 20 °C and 37 °C,
to avoid large changes in temperature that may affect the spermatozoa after they are
ejaculated into it. It must be labelled with the man‟s name and identification number, and the
date and time of collection.
3. The specimen container is placed on the bench or in an incubator (37 °C) while the semen
liquefies.
4. Note in the report if the sample is incomplete, especially if the first, sperm-rich fraction
may be missing. If the sample is incomplete, a second sample should be collected, again after
an abstinence period of 2–7 days.

Sterile collection of semen for microbiological analysis

The man should:
1. Pass urine.
2. Wash hands and penis with soap, to reduce the risk of contamination of the specimen with
commensal organisms from the skin.
3. Rinse away the soap.
4 .Dry hands and penis with a fresh disposable towel.
5. Ejaculate into a sterile container.
Note: The time between collection of the semen sample and the start of the investigation
by the microbiological laboratory should not exceed 3 hours.
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Collection of semen at home
1. A sample may be collected at home in exceptional circumstances, such as a demonstrated
inability to produce a sample by masturbation in the clinic or the lack of adequate facilities
near the laboratory.
2. The man should be given clear written and spoken instructions concerning the collection
and transport of the semen sample. These should emphasize that the semen sample needs to
be complete,
i.e. all the ejaculate is collected, including the first, sperm-rich portion, and that the man
should report any loss of any fraction of the sample. It should be noted in the report if the
sample is incomplete.
3. The man should be given a pre-weighed container, labelled with his name and
identification number.
4. The man should record the time of semen production and deliver the sample to the lab.
5. During transport to the laboratory, the sample should be kept between 20 °C and 37 °C.
6. The report should note that the sample was collected at home or another location outside
the laboratory.

Initial macroscopic examination

Semen analysis should begin with a simple inspection soon after liquefaction, preferably at
30 minutes, but no longer than 1 hour after ejaculation, to prevent dehydration or changes in
temperature from affecting semen quality.

Liquefaction
Immediately after ejaculation into the collection vessel, semen is typically a semisolid
coagulated mass (coagulum).Due to that seminal vesicles produce coagulation proteins in
order to improve fertilization by avoiding the loss of seminal fluid.
Within a few minutes at room temperature, the semen usually begins to liquefy (become
thinner), at which time a heterogeneous mixture of lumps will be seen in the fluid. As
liquefaction continues, the semen becomes more homogeneous and quite watery, and in the
final stages only small areas of coagulation remain. The complete sample usually liquefies
within 15 minutes at room temperature.
Liquefaction occurs due to the action of prostate gland ( decoagulation ) by the production
of :
1. Prostate specific antigen (PSA).
2. Proteolytic enzymes (fibrinolysin, fibrinogenase, aminopeptidase), which leads to
complete liquefaction within 30 minute.
Although rarely it may take up to 60 minutes or more. If complete liquefaction does not
occur within 60 minutes, this should be recorded. Semen samples collected at home or by
condom will normally have liquefied by the time they arrive in the laboratory. Normal
liquefied semen samples may contain jelly-like granules (gelatinous bodies) which do not

177
liquefy; these do not appear to have any clinical significance. The presence of mucus strands,
however, may interfere with semen analysis.
Note : If the semen does not liquefy within 30 minutes, do not proceed with semen analysis
but wait for another 30 minutes. If liquefaction has not occurred within 60 minutes, proceed
as delayed liquefaction section below.

Delayed liquefaction
Occasionally samples may not liquefy, due to prostate glands abnormalities, making semen
evaluation difficult. In these cases, additional treatment, mechanical mixing or enzymatic
digestion may be necessary.
1. Some samples can be induced to liquefy by the addition of an equal volume of
physiological medium (e.g. Dulbecco‟s phosphate-buffered saline), followed by repeated
pipetting.
2. Inhomogeneity can be reduced by repeated (6–10 times) gentle passage through a blunt
gauge 18 (internal diameter 0.84 mm) or gauge 19 (internal diameter 0.69 mm) needle
attached to a syringe.
3. Digestion by bromelain, a broad-specificity proteolytic enzyme , may help to promote
liquefaction

Semen viscosity
After liquefaction, the viscosity of the sample can be estimated by gently aspirating it into a
wide-bore (approximately 1.5 mm diameter) plastic disposable pipette, allowing the semen
to drop by gravity and observing the length of any thread.
A normal sample leaves the pipette in small discrete drops. If viscosity is abnormal, the drop
will form a thread more than 2 cm long. Alternatively, the viscosity can be evaluated by
introducing a glass rod into the sample and observing the length of the thread that forms
upon withdrawal of the rod. The viscosity should be recorded as abnormal when the thread
exceeds 2 cm.
In contrast to a partially unliquefied sample, a viscous semen specimen exhibits
homogeneous stickiness and its consistency will not change with time. High viscosity can be
recognized by the elastic properties of the sample, which adheres strongly to itself when
attempts are made to pipette it.
High viscosity can interfere with determination of sperm motility, sperm concentration,
detection of antibody coated spermatozoa and measurement of biochemical markers.

Appearance of the ejaculate

A normal liquefied semen sample has a homogeneous, grey-opalescent appearance. It may
appear less opaque if the sperm concentration is very low; the color may also be different,
i.e. red-brown when red blood cells are present (haemospermia), or yellow in a man with
jaundice or taking certain vitamins or drugs.

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Semen volume
The volume of the ejaculate is contributed mainly by the seminal vesicles and prostate gland,
with a small amount from the bulbourethral glands and epididymides. Precise measurement
of volume is essential in any evaluation of semen, because it allows the total number of
spermatozoa and non-sperm cells in the ejaculate to be calculated.

The volume can be measured directly.

 Collect the sample directly into a modifed graduated glass measuring cylinder with a
wide mouth. These can be obtained commercially.
 Read the volume directly from the graduations (0.1 ml accuracy).
Note: Measuring volume by aspirating the sample from the specimen container into a pipette
or syringe, or decanting it into a measuring cylinder, is not recommended, because not all the
sample will be retrieved and the volume will therefore be underestimated. The volume lost
can be between 0.3 and 0.9 ml .
Low semen volume is characteristic of obstruction of the ejaculatory duct or congenital
bilateral absence of the vas deferens a condition in which the seminal vesicles are also poorly
developed.
Low semen volume can also be the result of collection problems (loss of a fraction of the
ejaculate), partial retrograde ejaculation or androgen deficiency.
High semen volume may reflect active exudation in cases of active inflammation of the
accessory organs. (The lower reference limit for semen volume is 1.5 ml).

Semen pH
The pH of semen reflects the balance between the pH values of the different accessory gland
secretions, mainly the alkaline seminal vesicular secretion and the acidic prostatic secretion.
The pH should be measured after liquefaction at a uniform time, preferably after 30 minutes,
but in any case within 1 hour of ejaculation since it is influenced by the loss of CO2 that
occurs after production.

For normal samples, pH paper in the range 6.0 to 10.0 should be used.
 Mix the semen sample well.
 Spread a drop of semen evenly onto the pH paper.
 Wait for the color of the impregnated zone to become uniform (<30 seconds).
 Compare the color with the calibration strip to read the pH.

Note: The accuracy of the pH paper should be checked against known standards.
Reference values
There are currently few reference values for the pH of semen from fertile men. Pending more
data, this manual retains the consensus value of 7.2 as a lower threshold value.
Comment 1: If the pH is less than 7.0 in a semen sample with low volume and low sperm
numbers, there may be ejaculatory duct obstruction or congenital bilateral absence of the vas
deferens, a condition in which seminal vesicles are also poorly developed.

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Comment 2: Semen pH increases with time, as natural buffering decreases, so high pH
values may provide little clinically useful information.

Initial microscopic investigation

A phase-contrast microscope is recommended for all examinations of unstained preparations
of fresh semen, An initial microscopic examination of the sample involves scanning the
preparation at a total magnification of ×100

This provides an overview of the sample, to reveal:

 mucus strand formation;
 sperm aggregation or agglutination;
 the presence of cells other than spermatozoa, e.g. epithelial cells, “round cells”
(leukocytes and immature germ cells) and isolated sperm heads or tails.
The preparation should then be observed at ×200 or ×400 total magnification . This permits:
 assessment of sperm motility .
 determination of the dilution required for accurate assessment of sperm number.

Making a wet preparation

 Mix the semen sample well in the original container, but not so vigorously that air
bubbles are created. This can be achieved by aspirating the sample 10 times into a wide-
bore (approximately 1.5 mm diameter) disposable plastic pipette (sterile when
necessary). Do not mix with a vortex mixer at high speed as this will damage
spermatozoa.
 Remove an aliquot of semen immediately after mixing, allowing no time for the
spermatozoa to settle out of suspension.
 Remix the semen sample before removing replicate aliquots.
 The volume of semen and the dimensions of the coverslip must be standardized, so that
the analyses are carried out on a preparation of fixed depth of about 20 µm , which
allows the spermatozoa to swim freely.
 Place a standard volume of semen, e.g. 10 µl, onto a clean glass slide.
 Cover it with a coverslip, e.g. 22 mm × 22 mm for 10 µl, to provide a chamber
approximately 20 µm deep . The weight of the coverslip spreads the sample.
 Take care to avoid the formation and trapping of air bubbles between the coverslip and
the slide.
 Assess the freshly made wet preparation as soon as the contents are no longer drifting.

Note 1: A chamber depth of less than 20 _m constrains the rotational movement of

spermatozoa.
Note 2: If the chamber is too deep, it will be difficult to assess spermatozoa as they move in
and out of focus.
Note 3: If the number of spermatozoa per visual field varies considerably, the sample is not
homogeneous. In such cases, the semen sample should be mixed again thoroughly and a new
slide prepared as above.

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Note 4: Lack of homogeneity may also result from abnormal consistency, abnormal
liquefaction, aggregation of spermatozoa or sperm agglutination

Aggregation of spermatozoa
The adherence either of immotile spermatozoa to each other or of motile spermatozoa to
mucus strands, non-sperm cells or debris is considered to be nonspecific aggregation and
should be recorded as such.

Figure (2-100) : Non-specific aggregation of spermatozoa in semen, Views of spermatozoa

aggregated with an epithelial cell (a), debris (b) or spermatozoa (c, d).

Agglutination of spermatozoa
Agglutination specifi cally refers to motile spermatozoa sticking to each other, head-to-head,
tail-to-tail or in a mixed way. The motility is often vigorous with a frantic shaking motion,
but sometimes the spermatozoa are so agglutinated that their motion is limited. Any motile
spermatozoa that stick to each other by their heads, tails or midpieces should be noted.
The major type of agglutination (refl ecting the degree (grades 1–4) and the site of
attachment (grades A–E) should be recorded :
 Grade 1: isolated <10 spermatozoa per agglutinate, many free spermatozoa.
 Grade 2: moderate 10–50 spermatozoa per agglutinate, free spermatozoa.
 Grade 3: large agglutinates of >50 spermatozoa, some spermatozoa still free.
 Grade 4: gross all spermatozoa.
Note: Motile spermatozoa stuck to cells or debris or immotile spermatozoa stuck to each
other (aggregation) should not be scored as agglutination.

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A. Head-to-head

B. Tail-to-tail (heads
are seen to be free and
move clear of agglutinates)

C. Tail-tip-to-tail-tip

D. Mixed (clear headto-

head and tail-to-tail
agglutinations)

E. Tangle (heads and

tails enmeshed. Heads
are not clear of agglutinates
as they are in tailto-
tail agglutination)

Comment 1: The presence of agglutination is not sufficient evidence to assume an

immunological cause of infertility, but is suggestive of the presence of anti-sperm antibodies;
further testing is required.
Comment 2: Severe agglutination can affect the assessment of sperm motility and
concentration.

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Sperm motility
The extent of progressive sperm motility is related to pregnancy. Sperm motility within
semen should be assessed as soon as possible after liquefaction of the sample, preferably at
30 minutes, but in any case within 1 hour, following ejaculation, to limit the deleterious
effects of dehydration, pH or changes in temperature on motility.

Categories of sperm movement

A simple system for grading motility is recommended that distinguishes spermatozoa with
progressive or non-progressive motility from those that are immotile. The motility of each
spermatozoon is graded as follows :
 Progressive motility (PR): spermatozoa moving actively, either linearly or in a large
circle, regardless of speed.
 Non-progressive motility (NP): all other patterns of motility with an absence of
progression, e.g. swimming in small circles, the flagellar force hardly displacing the
head, or when only a flagellar beat can be observed.
 Immotility (IM): no movement.

Preparing and assessing a sample for motility

 If motility is to be assessed at 37 °C, turn the stage warmer on 10 minutes in advance, to
allow the temperature to stabilize.
 Prepare a wet preparation 20 µm deep.
 Examine the slide with phase-contrast optics at ×200 or ×400 magnification.
 Wait for the sample to stop drifting.
 Look for spermatozoa in an area at least 5 mm from the edge of the coverslip, to prevent
observation of effects of drying on motility.
 Systematically scan the slide to avoid repeatedly viewing the same area. Change fields
often. Avoid choosing fields on the basis of the number of motile sperm seen (field
choice should be random).

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  Start scoring a given field at a random instant. Do not wait for spermatozoa to swim into
the field or grid to begin scoring.
 Assess the motility of all spermatozoa within a defined area of the field. This is most
easily achieved by using an eyepiece reticle. Select the portion of the field or grid to be
scored from the sperm concentration, i.e. score only the top row of the grid if the sperm
concentration is high; score the entire grid if the sperm concentration is low.
 Scan and count quickly to avoid overestimating the number of motile spermatozoa. The
goal is to count all motile spermatozoa in the grid section instantly; avoid counting both
those present initially plus those that swim into the grid section during scoring, which
would bias the result in favour of motile spermatozoa.
 Initially scan the grid section being scored for PR cells. Next count NP spermatozoa and
finally IM spermatozoa in the same grid section. With experience, it may be possible to
score all three categories of sperm movement at one time, and to score larger areas of the
grid.
 Count the number of spermatozoa in each motility category with the aid of a laboratory
counter.
 Evaluate at least 200 spermatozoa in a total of at least five fields in each replicate, in
order to achieve an acceptably low sampling error .
 Calculate the average percentage and difference between the two percentages for the
most frequent motility grade (PR, NP or IM) in the replicate wet preparations.
 If the difference between the percentages is acceptable, report the average percentage for
each motility grade (PR, NP and IM). If the difference is too high, take two new aliquots
from the semen sample, make two new preparations and repeat the assessment .
 Report the average percentage for each motility grade to the nearest whole number.

Note 1: Assess only intact spermatozoa (defined as having a head and a tail), since only
intact spermatozoa are counted for sperm concentration. Do not count motile pinheads.

Note 2: If spermatozoa are being scored in two stages (i.e. PR first, followed by NP and IM
from the same area) and a count of 200 spermatozoa is achieved before all motility
categories from that area have been scored, counting must continue beyond 200 spermatozoa
until all categories have been counted, in order to avoid bias towards the motility category
scored first.

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Table (2-20) : Acceptable differences between two percentages for a given average, determined
from replicate counts of 200 spermatozoa (total 400 counted)

Average (%) Acceptable Average (%) Acceptable

Difference Difference
0 1
1 2 66–76 9
2 3 77–83 8
3–4 4 84–88 7
5–7 5
89–92 6
8–11 6
93–95 5
12–16 7
96–97 4
17–23 8 98 3
24–34 9 99 2
35–65 10 100 1

Note 3 : If the difference between the replicate percentages is less than or equal to that
indicated in Table 2.1 for the given average, the estimates are accepted and the average is
taken as the result.
Larger than acceptable differences suggest that there has been miscounting or errors of
pipetting, or that the cells were not mixed well, with non-random distribution in the
chamber or on the slide.
When the difference between percentages is greater than acceptable, discard the first two
values and reassess. (Do not count a third sample and take the mean of the three values, or
take the mean of the two closest values.)

The lower reference limit for total motility (PR + NP) is 40%.

The lower reference limit for progressive motility (PR) is 32%.

Sperm vitality
Sperm vitality, as estimated by assessing the membrane integrity of the cells, may be
determined routinely on all samples, but is especially important for samples with less than
about 40% progressively motile spermatozoa. This test can provide a check on the motility
evaluation, since the percentage of dead cells should not exceed (within sampling error) the
percentage of immotile spermatozoa. The percentage of viable cells normally exceeds that of
motile cells.
The percentage of live spermatozoa is assessed by identifying those with an intact cell
membrane, from dye exclusion or by hypotonic swelling. The dye exclusion method is based
on the principle that damaged plasma membranes, such as those found in non-vital (dead)
cells, allow entry of membrane-impermeant stains.

185
The hypo-osmotic swelling test presumes that only cells with intact membranes (live cells)
will swell in hypotonic solutions.
Sperm vitality should be assessed as soon as possible after liquefaction of the semen sample,
preferably at 30 minutes, but in any case within 1 hour of ejaculation, to prevent observation
of deleterious effects of dehydration or of changes in temperature on vitality.

Vitality test using eosin–nigrosin

This one-step staining technique uses nigrosin to increase the contrast between the
background and the sperm heads, which makes them easier to discern. It also permits slides
to be stored for re-evaluation and quality-control purposes

Procedure
1. Mix the semen sample well .
2. Remove a 50-µl aliquot of semen and mix with an equal volume of eosin– nigrosin
suspension.
3. Remix the semen sample before removing a replicate aliquot and mixing with eosin–
nigrosin and treating as in step 2 above.
4. For each suspension make a smear on a glass slide and allow it to dry in air.
5. Examine immediately after drying, or later after mounting with a permanent non-aqueous
mounting medium.
6. Examine each slide with brightfield optics at ×1000 magnification and oil immersion.
7. Count the number of stained (dead) or unstained (vital) cells with the aid of a laboratory
counter.
8. Evaluate 200 spermatozoa in each replicate, in order to achieve an acceptably low
sampling error.
9. Calculate the average and difference of the two percentages of vital cells from the
replicate slides.

Figgure (2-101) : Eosin–nigrosin smear observed in brightfield optics ,Spermatozoa with red (D1) or dark
pink (D2) heads are considered dead (membrane-damaged), whereas spermatozoa with white heads (L) or
light pink heads are considered alive (membrane- intact).

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Scoring
1. The nigrosin provides a dark background that makes it easier to discern faintly stained
spermatozoa.
2. With brightfield optics, live spermatozoa have white heads and dead spermatozoa have
heads that are stained red or dark pink . Spermatozoa with a faint pink head are assessed as
alive.
3. I f the stain is limited to only a part of the neck region, and the rest of the head area is
unstained, this is considered a “leaky neck membrane”, not a sign of cell death and total
membrane disintegration. These cells should be assessed as alive.

Vitality test using eosin alone

This method is simple and rapid, but the wet preparations cannot be stored for quality control
purposes.

Preparing the reagents

1. NaCl, 0.9% (w/v): dissolve 0.9 g of NaCl in 100 ml purifi ed water.
2. Eosin Y, 0.5% (w/v): dissolve 0.5 g of eosin Y in 100 ml of 0.9% NaCl.

Note: Some commercially available eosin solutions are hypotonic aqueous solutions that will
stress the spermatozoa and give false-positive results. If using such a solution, add 0.9 g of
NaCl to 100 ml of solution to raise the osmolality.

Procedure
1. Mix the semen sample well .
2. Remove an aliquot of 5 µl of semen and combine with 5 µl of eosin solution on
a microscope slide. Mix with a pipette tip, swirling the sample on the slide.
3. Cover with a 22 mm × 22 mm coverslip and leave for 30 seconds.
4. Remix the semen sample, remove a replicate aliquot, mix with eosin and treat as in steps 2
and 3 above.
5. Examine each slide, preferably with negative-phase-contrast optics (positive phase-
contrast makes faint pink heads difficult to discern) at ×200 or ×400 magnification.
6. Count the number of stained (dead) and unstained (vital) cells with the aid of a laboratory
counter.
7. Evaluate 200 spermatozoa in each replicate, in order to achieve an acceptably low
sampling error .
8. Calculate the average and difference of the two percentages of vital cells from the
replicate preparations.
9. Report the average percentage of vital spermatozoa to the nearest whole number.

187
Scoring
1. Live spermatozoa have white or light pink heads and dead spermatozoa have heads that
are stained red or dark pink.
2. If the stain is limited to only a part of the neck region, and the rest of the head area is
unstained, this is considered a “leaky neck membrane”, not a sign of cell death and total
membrane disintegration. These cells should be assessed as alive.
3. If it is difficult to discern the pale pink stained head, use nigrosin to increase the contrast
of the background

Figure (2-102): Evaluation of human sperm viability using eosin- nigrosin staining; (a)
unstained (white) alive spermatozoa, (b) stained (red) dead spermatozoa.

The lower reference limit for vitality is 58% .

Sperm numbers
The total number of spermatozoa per ejaculate and the sperm concentration are related to
both time to pregnancy and pregnancy rates, The number of spermatozoa in the ejaculate
is calculated from the concentration of spermatozoa, which is measured during semen
evaluation.
For normal ejaculates, when the male tract is unobstructed and the abstinence time short, the
total number of spermatozoa in the ejaculate is correlated with testicular volume and thus is a
measure of the capability of the testes to produce spermatozoa and the patency of the male
tract. The concentration of spermatozoa in the semen, while related to fertilization and
pregnancy rates, is influenced by the volume of the secretions from the seminal vesicles and
prostate and is not a specific measure of testicular function.
Comment 1: The terms “total sperm number” and “sperm concentration” are not
synonymous. Sperm concentration refers to the number of spermatozoa per unit volume of
semen and is a function of the number of spermatozoa emitted and the volume of fluid
diluting them.

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Total sperm number refers to the total number of spermatozoa in the entire ejaculate and is
obtained by multiplying the sperm con- centration by the semen volume.

Determination of sperm number comprises the following steps :

 Examining a well-mixed, undiluted preparation of liquefied semen on a glass
slide under a coverslip, to determine the appropriate dilution and appropriate
chambers to use. This is usually the wet preparation used for evaluation of
motility.
 Mixing semen and preparing dilutions with fixative.
 Loading the haemocytometer chamber and allowing spermatozoa to settle in a
humid chamber.
 Assessing the samples within 10–15 minutes (after which disappearance has
noticeable effects on sperm position within the chamber).
 Counting at least 200 spermatozoa per replicate.
 Comparing replicate counts to see if they are acceptably close. If so, proceed- ing
with calculations; if not, preparing new dilutions.
 Calculating the concentration in spermatozoa per ml.
 Calculating the total number of spermatozoa per ejaculate.

Types of counting chamber

The use of 100-µm-deep haemocytometer chambers is recommended.

Figure (2-103) : Improved Neubauer chamber

Figure (2-104) : The improved Neubauer haemocytometer

Sketches of the inscribed area showing: all nine grids in one chamber of the haemocytometer
( left panel); the central grid (number 5) of 25 large squares (middle panel); and a
micrograph of part of a filled chamber (right panel), showing one of the 25 squares of the
central grid (the circled square in the middle panel) bounded by triple lines and containing 16
smaller squares.

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Using the haemocytometer grid
 Count only whole spermatozoa (with heads and tails).
 Whether or not a spermatozoon is counted is determined by the location of its head;
the orientation of its tail is unimportant. The boundary of a square is indicated by the
middle line of the three; thus, a spermatozoon is counted if most of its head lies
between the two inner lines, but not if most of its head lies between the two outer
lines.
 To avoid counting the same spermatozoon in adjacent squares, a spermato- zoon with
its head on the line dividing two adjacent squares should be counted only if that line is
one of two perpendicular boundary lines. For example, cells may be counted if most of
the sperm head lies on the lower or left center boundaries, which form an “L” shape,
but not if it lies on the upper or right center boundary line.

Figure (2-105) : Which spermatozoa to count in the grid squares.

The middle of the three lines defines the square‟s boundary (black line, left panel). All
spermatozoa within the central square are counted, as well as those with their heads between the
two inner lines (white circles), but not those whose heads lie between the outer two lines (black
circles). A spermatozoon with most of its head lying on the central line is counted only if that line
is the lower or left-hand line of the square (white circles, middle panel) but not if it is the upper or
right hand line of the square (black circles, right panel).

190
 Fixative for diluting semen
 97 ml distilled water
 1 ml formalin 35 %
 2 ml crystal violet stain
 Dissolve 5 g of sodium bicarbonate (NaHCO3 )
 Store at 4 °C.

Determining the required dilution

The dilution of semen required to allow sperm number to be measured accurately is assessed
from an undiluted semen preparation. This is usually the wet preparation used for evaluation
of motility.
 Examine one of the wet preparations, to estimate the number of spermatozoa per HPF
(×200 or ×400).
 One HPF is equivalent to approximately 16 nl (at ×200) or 4 nl (at ×400) .
 If spermatozoa are observed, count them, determine the necessary dilution from Table
2.3.
 If no spermatozoa are observed, examine the replicate wet preparation.

Table (2-21) : Semen dilutions required, how to make them, which chambers to use and
potential areas to assess
Spermatozoaper Dilution required Semen (l) Fixative (l) Area to beassessed
Chamber
×400 field

>100 1:20 (1 + 19) 50 950 Improved Grids 5

Neubauer
15 –100 1:5 (1 + 4) 50 200 Improved Grids 5
Neubauer
< 15 1:2 (1 + 1) 50 50 Improved Grids 5
Neubauer

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Low sperm numbers: Cryptozoospermia and suspected Azoospermia
If no spermatozoa are observed in the replicate wet preparations, azoospermia can be
suspected . Azoospermia remains a description of the ejaculate rather than a statement of its
origin or a basis for diagnosis and therapy. It is generally accepted that the term azoospermia
can only be used if no spermatozoa are found in the sediment of a centrifuged sample.
However, it should be borne in mind that :
 Whether or not spermatozoa are found in the pellet depends on the centrifugation time
and speed and on how much of the pellet is examined;
 Centrifugation at 3000g for 15 minutes does not pellet all spermatozoa from a sample ;
and
 After centrifugation, motility can be lost and concentration will be underestimated.

When an accurate assessment of low sperm numbers is not required

If the number of spermatozoa per HPF in the initial wet preparation is low (0 to 4 per ×400
HPF), several options are available:

1. Taking no further action

If the number of spermatozoa per ×400 HPF is <4 (i.e. < approximately 1 × 10 6 /ml), it is
sufficient for most clinical purposes to report the sperm concentration as <2 × 10 6 /ml
(Cryptozoospermia) (to take into account the high sampling error associated with low sperm
numbers), with a note as to whether or not motile spermatozoa were seen.

2. Examination of centrifuged samples to detect spermatozoa

When no spermatozoa are observed in either wet preparation, the sample can be centrifuged
to determine if any spermatozoa are present in a larger sample.
 Mix the semen sample well.
 Remove a 1-ml aliquot of semen and centrifuge at 3000 g for 15 minutes.
 Decant most of the supernatant and re-suspend the sperm pellet in the remaining
approximately 50 µl of seminal plasma.
 Place one 10 µl aliquot of the pellet on each of two slides under 22 mm × 22 mm
coverslips. This will create two wet preparations approximately 20 µm deep.
 Scan the entire coverslip systematically field by field. Start in one corner and scan
along the x-axis to the opposite side; then move one field along the y-axis and scan
back along the entire width. Continue in this zigzag fashion to make a complete and
systematic search of the entire aliquot . Keep observing the slide while changing
fields.
 With a ×20 objective and a ×10 ocular of 20 mm aperture, the microscope field has a
diameter of approximately 1000 µm. There will thus be approximately 484 fields (22
× 22) per 22 mm × 22 mm coverslip to be examined.
 The presence of spermatozoa in either replicate indicates Cryptozoospermia ( Less
than 1 million/ml).

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  The absence of spermatozoa from both replicates suggests Azoospermia ( No sperms
could be detected in the whole sediment material after centrifugation of this sample,
Examination of three successive Semen samples are recommended).

Preparing the dilutions and loading the haemocytometer chambers

 Make the haemocytometer surface slightly damp by breathing on it.
 Secure the coverslip on the counting chambers by pressing it firmly onto the chamber
pillars. Iridescence (multiple Newton‟s rings) between the two glass surfaces confirms
the correct positioning of the coverslip. The more lines there are, the better the fit;
only one or two lines may indicate problems with variation in chamber depth.
 Mix the semen sample well.
 Aspirate the appropriate volume of semen immediately after mixing, allowing no time
for the spermatozoa to settle out of suspension.
 Wipe the semen off the outside of the pipette tip, taking care not to touch the opening
of the tip.
 Dispense the semen into the fixative and rinse the pipette tip by aspirating and
expressing the fixative.
 Mix the semen sample well again, and prepare the replicate dilution following the
steps above.
 Mix then add 10 µl of fixed suspension, to avoid settling of the spermatozoa.
 Touch the pipette tip carefully against the lower edge of one of the chambers at the V-
shaped groove.
 Depress the plunger of the pipette slowly, allowing the chamber to fill by capillary
action. The coverslip should not be moved during filling, and the chamber should not
be overfilled (when the coverslip may be seen to move) or under- filled (when air
occupies some of the chamber area).
 Mix the second dilution, as above, and immediately remove a second 10 µl aliquot.
Load the second chamber of the haemocytometer following the steps above.
 Store the haemocytometer horizontally for at least 4 minutes at room temperature in a
humid chamber (e.g. on water-saturated filter paper in a covered Petri dish) to prevent
drying out. The immobilized cells will sediment onto the grid during this time.

Worked examples
Example 1: If the dilution (1: 20) used,
1:20 ( 1 semen +19 diluent) (50 semen + 950 diluent) On grid 5 ( R.B.C Squares on Hemocytometer)

Sperm Concentration (ml) = Total count X 20

5 X 0.004 (the size of each square of grid 5) X 1000

In other word , Sperm Concentration (ml) = Total count X 1000,000

193
Example 2: If the dilution (1: 5) used,
1:5 ( 1 semen +4 diluent) (50 semen + 200 diluent) On grid 5 ( R.B.C Squares on Hemocytometer )

Sperm Concentration (ml) = Total count X 5

5 X 0.004 (the size of each square of grid 5) X 1000

In other word , Sperm Concentration (ml) = Total count X 250,000

Example 3: If the dilution (1: 2) used,

1:2 ( 1 semen +1 diluent) (50 semen + 50 diluent) On grid 5 ( R.B.C Squares on Hemocytometer)

Sperm Concentration (ml) = Total count X 2

5 X 0.004 (the size of each square of grid 5) X 1000

In other word , Sperm Concentration (ml) = Total count X 100,000

Lower reference limit for sperm concentration

The lower reference limit for sperm concentration is (15 × 106 ) spermatozoa per ml.

Causes of Abnormal Sperm Count

As mentioned previously ,The lower reference limit for sperm concentration is (15 × 10 6 )
spermatozoa per ml.
The abnormal sperm count due to :

A. Oligozoospermia , low sperm count ( Sperm count = 1-15 × 106 / ml)

Oligozoospermia Classification :
1. Mild ( 10 -15 × 106 / ml )
2. Moderate (5- 10 × 106 / ml)
3. Sever ( 1 – 5 × 106 / ml)

Oligozoospermia Causes :
1. Pretesticular , caused by :
 Hypogonadism , decreased functional activity of the gonads (testes) to produce
hormones (testosterone).
 Drugs, Alcohol and smoking.
 Strenuous riding, such as bicycle riding and horseback riding.
 Medication (Androgen)

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 2. Testicular , caused by :
 Age.
 Genetic defect on Y chromosome.
 Klinefelter syndrome (Abnormal genetic condition where a male has an additional
copy of the X chromosome.The primary features are infertility and small, poorly
functioning testicles).
 Neoplasms (Seminoma, which is a malignant germ cell tumor that involves most
commonly the testicle).
 Cryptorchidism (also known as undescended testis, is the failure of one or both
testes to descend into the scrotum. It is the most common birth defect of the male
genital tract).
 Varicocele (is an enlargement of the veins within the loose bag of skin that holds
the testicles (scrotum). These veins transport oxygen-depleted blood from the
testicles. A varicocele occurs when blood pools in the veins rather than circulating
efficiently out of the scrotum).
 Trauma.
 Hydrocele ( produced by fluid in the sac which normally surrounds the testicle. It
often presents as painless swelling in the scrotum).
 Mumps.
 Malaria.

3. Post- Testicular, caused by :

 Vas deferens obstruction (the epididymis becomes blocked, preventing sperm from
entering the vas deferens and getting into the ejaculate. A blockage can occur on
one side or on both sides).
 Infection ( Prostatitis ).
 Ejaculation duct obstruction (EDO) is the blockage of one or both ejaculatory
ducts, which are the tubules that sperm travels through.
 Idiopathic , 30%.

B. Cryptozoospermia, extremely low concentration of sperm (< 1 × 106 / ml)

Cryptozoospermia Causes :

1. Pretesticular :
 Hormonal defects.

2. Testicular , caused by :

 Genetic abnormalities.
 Injuries.
 Infections.

195
  Cryptorchidism (also known as undescended testis, is the failure of one or both
testes to descend into the scrotum. It is the most common birth defect of the
male genital tract).
 Varicocele.

3. Post- Testicular, caused by :

 Ejaculation duct obstruction (EDO) is the blockage of one or both ejaculatory
ducts, which are the tubules that sperm travels through.

C. Azoospermia
Is the medical term used when there are no sperm in the ejaculate( No detected sperms).
It can be “obstructive,” where there is a blockage preventing sperm from entering the
ejaculate, or it can be “Non-obstructive” when it is due to decreased sperm production by
the testis.

Non-obstructive Azoospermia Causes :

1. Pretesticular, caused by :
 Inadequate stimulation of otherwise normal testicles and genital tract.
 Follicle-stimulating hormone (FSH) levels are low (hypogonadotropic)
matching with inadequate stimulation of the testes to produce sperm. Examples
include hypopituitarism , hyperprolactinemia, and exogenous FSH suppression
by testosterone.
 Chemotherapy may suppress spermatogenesis.
 Pretesticular azoospermia is seen in about 2% of azoospermia.

2. Testicular , caused by :
 Testicular azoospermia means the testes are abnormal, atrophic, or
absent, and sperm production severely disturbed to absent. FSH levels
tend to be elevated (hypergonadotropic) as the feedback loop is
interrupted (lack of feedback inhibition on FSH).
 The condition is seen in 49–93% of men with azoospermia.
 Genetic conditions (e.g. Klinefelter syndrome).
 cryptorchidism or Sertoli cell-only syndrome as well as acquired
conditions by infection (orchitis), surgery (trauma, cancer).
 Radiation.

3. Post- Testicular, caused by :

 In post-testicular azoospermia, sperm are produced but not ejaculated, a
condition that affects 7–51% of azoospermic men.
 The main cause is a physical obstruction (obstructive azoospermia) of the
post-testicular genital tracts. The most common reason is a vasectomy
done to induce contraceptive sterility.

196
  Other obstructions can be congenital (for example, agenesis of the vas
deferens as seen in certain cases of cystic fibrosis) or acquired, such as
ejaculatory duct obstruction for instance by infection.
 Ejaculatory disorders include retrograde ejaculation and anejaculation; in
these conditions sperm are produced but not expelled.
 Idopathic.

D. Polyzoospermia : Is a condition of abnormally high sperm count in the ejaculate .

According to the World Health Organization (WHO) criteria, men with sperm counts
more than 250 million / ml are referred to as polyzoospermics.

Causes :
 Idiopathic
 Long period sexual abstinence

Calculation of the total number of spermatozoa in the ejaculate

It is recommended to calculate and report the total number of spermatozoa per ejaculate, as
this parameter provides a measure of the capability of the testes to produce spermatozoa and
the patency of the male tract. This is obtained by multiplying the sperm concentration by the
volume of the whole ejaculate.

Total Sperm Count = Sperm Concentration (ml) X semen volume (ml of semen sample /
ejaculate)

Lower reference limit for total sperm number

The lower reference limit for total sperm number is (39 × 106 ) spermatozoa per ejaculate.

Classification of abnormal sperm morphology

Human semen samples contain spermatozoa with different kinds of malformations.
Defective spermatogenesis and some epididymal pathologies are commonly associated with
an increased percentage of spermatozoa with abnormal shapes. The morphological defects
are usually mixed. Abnormal spermatozoa generally have a lower fertilizing potential,
depending on the types of anomalies, and may also have abnormal DNA.
Morphological defects have been associated with increased DNA fragmentation , an
increased incidence of structural chromosomal aberrations , immature chromatin and
aneuploidy . Emphasis is therefore given to the form of the head, although the sperm tail is
also considered.

197
The following categories of defects should be noted (see Figure 2-106).
 Head defects: large or small, tapered, pyriform, round, amorphous, vacuolated (more
than two vacuoles or >20% of the head area occupied by unstained vacuolar areas),
vacuoles in the post-acrosomal region, small or large acrosomal areas (<40% or >70%
of the head area), double heads, or any combination of these.

 Neck and midpiece defects: asymmetrical insertion of the midpiece into the head,
thick or irregular, sharply bent, abnormally thin, or any combination of these.

 Principal piece defects: short, multiple, broken, smooth hairpin bends, sharply
angulated bends, of irregular width, coiled, or any combination of these.

 Excess residual cytoplasm (ERC): this is associated with abnormal spermatozoa

produced from a defective spermatogenic process. Spermatozoa characterized by large
amounts of irregular stained cytoplasm, one third or more of the sperm head size,
often associated with defective midpieces are abnormal. This abnormal excess
cytoplasm should not be called a cytoplasmic droplet .

A. Head defects

(a) (b) (c) (d) (e) (f)

Tapered Pyriform Round Amorphous Vacuolated Small
acrosomal
No area
Small
acrosome

B. Neck and midpiece defects C. Tail defects D. Excess residual

cytoplasm

neck

Figure (2-106): Schematic drawings of some abnormal forms of human spermatozoa

198
Pathogens isolated from semen:
1. Nesseria.gonorrheae
2. Streptocoocus. faecalis
3. Proteus
4. E.coli

Fungi (on sabouraud dextrose agar) Candida albicans.

1. Chlymydia trachomatis
2. Treponema pallidum
3. Trichomonas vaginalis
4. Mycoplasma
5. Ureaplasma urealyticum
6. Corynebacterium
7. Herpes virus
8. Cytomegalovirus

Table (2-22): Microorganisms capable of causing infection in seminal tract

Classical germs Aerobic, Aerobic, Anaerobic
Gram -ve Gram +ve
Chlymydia E. Coli Gardnerella Bacteroides
Trachomatis vaginalis
Neisseria Enterobacter Streptococcus Bifidobacterium
gonorrhoeae faecalis
Treponema Klebsiella Staphyloccocus Fusobacterium
pallidum aureus

Trichomonas Proteus Streptococcus Lactobacillus

vaginalis epidermidis

Mycoplasma Pseudomonas Streptococcus Peptococcus

agalactiae
Ureaplasma Streptococcus Propionibacterium
Urealyticum Saprophyte
Corynebacterium Peptostreptoccus

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 CHAPTER
3

ISOLATION AND IDENTIFICATION OF BACTERIA

GRAM POSITIVE COCCI

1. Genus Staphylococci
2. Genus Streptococci

Catalase test differentiates

• Staphylococcus (positive)

• Streptococcus (negative)

GENUS: STAPHYLOCOCCi
Characteristics:
1. Gram positive non spore-forming non-motile, spherical cells, usually arranged in grape-
like clusters.
2. Single cocci , pairs, tetrads and chains are seen in liquid cultures.
3. Young cocci stain strongly gram-positive, on aging many cells become gram-negative.
4. The three main species of clinical importance
(Staphylococcus aureus , Staphylococcus epidermidis & Staphylococcus saprophyticus).

Laboratory Diagnosis
Specimen: Surface swabs, pus, blood, sputum, cerebrospinal fluid.
Gram stain: Gram positive cocci in clusters, singly or in pairs.
Culture: Grow well aerobically and in a CO2 enriched ordinary media at an optimal
temperature of 35C0-37C0.

Cultural characteristics of Staphylococcus aureus

1. Staphylococci grow readily on most bacteriologic media under aerobic or
microaerophilic conditions.
2. Colonies on solid media are round, smooth, raised, and glistening.
3. S. aureus usually forms gray to deep golden yellow colonies.
4. Mannitol Salt Agar: circular, 2–3 mm in diameter, with a smooth, shiny surface;
colonies appear opaque and are often pigmented golden yellow.
5. Tryptic Soy Agar: circular, convex, and entire margin.
6. Blood Agar: beta-hemolysis.
7. Brain heart infusion agar: Yellow pigmented colonies.

200
Mannitol salt agar or MSA is a commonly used selective and differential growth medium
in microbiology. It encourages the growth of a group of certain bacteria while inhibiting the
growth of others. It contains a high concentration (about 7.5–10%) of salt (NaCl) which is
inhibitory to most bacteria - making MSA selective against most Gram-negative and
selective for some Gram-positive bacteria (Staphylococcus, Enterococcus and
Micrococcaceae) that tolerate high salt concentrations.
It is also a differential medium for mannitol-fermenting staphylococci, containing the sugar
alcohol mannitol and the indicator phenol red, a pH indicator for detecting acid produced by
mannitol-fermenting staphylococci.
Staphylococcus aureus produces yellow colonies with yellow zones, whereas other
coagulase-negative staphylococci produce small pink or red colonies with no colour change
to the medium.

Figure (3-1): An MSA plate with Micrococcus sp. (1), Staphylococcus epidermidis (2)
and S. aureus colonies (3).

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 Figure (3-2): Staphylococcus aureus in Mannitol Salt Agar.

Figure (3-3) : Staphylococcus aureus colonies on blood agar, beta-hemolysis.

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 Figure (3-4) : Staphylococcus aureus on Tryptic Soy Agar

S.saprophyticus: may be white or yellow, non-hemolytic.

Biochemical characteristics of Staphylococcus aureus

 Catalase positive
 Oxidase negative
 OF test – fermentative
 Coagulase positive: the presence of free and /or bound coagulase
 Indole negative
 Gas negative
 Hydrogen sulfide negative
 Methyl red positive
 VP positive
 Nitrate reduction positive
 Gelatin hydrolysis positive
 Beta hemolysis on Blood agar
 Citrate positive
 Motility negative
 PYR negative
 Urease positive

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Biochemical reaction
1. Catalase test
Active bubbling: Catalase producing Bacteria, catalase +ve (Staphlococci),
No active bubbling: Non-catalase producing bacteria,catalase -ve (streptococci).

Gram positive cocci

Catalase test

Catalase +ve (staph/micrococcus) Catalase –ve (strepto/enterococcus/pneumococcus)

Hemolysis

Staph. Micrococcus

OF test Fermentative Oxidative

Furazolidone Sensitive Resistant β-Strepto Nonhemolytic α-Pneumococcus

enterococcus
Bacitracin Resistant Sensitive group A or B str. viridans

2. Coagulase test
A. Slide test: To detect bound coagulase, Clumping with in 10 seconds ( S.aureus), No
clumping within 10 seconds ... CONS (Coagulase negative staphylococci).

B. Tube test: To detect free coagulase

Fibrin clot… (S.aureus)
No fibrin clot.. Coagulase Negative Staphylococcus (CONS)

Sensitivity testing
Novobiocin sensitive : S.aureus and S.epidermidis.
Novobiocin resistant : S.saprophyticus.

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 Figure (3-5) : Staphylococcus spp in clusters (Gram stain)

Table (3-1): Differentiation of staphylococcus species

Organism Colony Catalase Coagulase Novobiocin Hemolysis on

appearance Production production sensitivity Blood agar

S. aureus Golden Positive Positive Sensitive Positive

S. epidermidis White-Gray Positive Negative Sensitive Negative

S. saprophyticus White-Gray Positive Negative Resistant Negative

GENUS: STREPTOCOCCI
Family Streptococcaceae are catalase negative gram-positive cocci, arranged in pairs or
chains (due to single plane of division).
• Streptococcus, Enterococcus and Pneumococcus are the important members of this
family.
• However, according to the molecular structure, Enterococcus is now reclassified under
separate family Enterococcaceae.

CLASSIFICATION
On the basis of hemolysis, streptococci can be divided into 3 groups
1. α Hemolytic: (Partial or green hemolysis), e.g. Streptococcus Viridans, Streptococcus
pneumoniae
2. β Hemolytic: (Complete or yellowish hemolysis), e.g. β haemolytic Streptococcus
3. γ Hemolytic: (no hemolysis is seen), e.g. Enterococci

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 Lancefield’s grouping (for β haemolytic streptococci)
Based on carbohydrate antigen in cell wall, the β haemolytic streptococci are further
divided into 20 serogroups: Group A to V except I and J.
Carbohydrate antigen extracted by HCl (Lancefield‟s method), Formamide (Fuller‟s
method), Enzymatic (Maxted‟s) or autoclaving.
Streptococcus group A (S. pyogenes) is further subdivided based on
1. Griffith typing: Based on M protein (> 100 M serotypes) or
2. . emm typing: Based on gene coding for M protein, > 124 emm genotypes identified.

Characteristics
1. They are non-motile, non-sporulating, gram- positive facultative anaerobes.
2. Spherical or oval cells characteristically forming pairs or chains during growth.
3. Grow well on ordinary solid media enriched with blood, serum or glucose.
4. Most streptococci grow in solid media as discoid colonies.
5. Capsular streptococcal strains give rise to mucoid colonies.
6. They are aerobic bacteria in which growth is enhanced with 10% carbondioxide.
7. They are catalase-negative.
8. They are widely distributed in nature and are found in upper respiratory tract,
gastrointestinal tract and genitourinary tract as normal microbial flora.
9. They are heterogeneous group of bacteria, and no one system suffices to classify them.
10. The currently used classification is based on colony growth characteristics, pattern on
blood agar.

Antigenic composition of group specific cell wall substance and biochemical reaction.
Lancefield grouping of streptococci:
Streptococci produce group specific carbohydrates identified using group specific
antiserum.It is designated A-H and K-V.The clinically important streptococci are grouped
under A,B,C,D,F and G.

The main species and groups of medical importance :

1. S. pyogenes ( Lancefield group A)
2. S. agalactiae ( Lancefield group B)
3. Enterococci (Lancefield group D)
Viridans streptococci and are not grouped under Lancefield Classification.

Viridans streptococci
1. Streptococcus mitis
2. Streptococcus mutans
3. Streptococcus salivarius
4. Streptococcus sanguis

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Nonsuppurative Complications
Streptococcal antigens show molecular mimicry with human antigens. Due to antigenic
cross reactivity, antibodies produced against previous streptococcal infections cross react
with human tissues to produce lesions. This accounts for a number of nonsuppurative
complications such as:
1. Acute rheumatic fever
2. Poststreptococcal glomerulonephritis (PSGN)
3. Guttate psoriasis
4. Reactive arthritis
5. Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus
pyogenes (PANDAS)

Table (3-2): Antigenic cross reactivity between streptococcal antigens and the
corresponding human antigens
Streptococcal Ag Mammalian Ag
Cell wall M protein (of serotypes M1, M5, M6, Myocardium (tropomyosin and myosin)
and M19)
Cell wall C carbohydrate Cardiac valves
Cytoplasmic membrane Glomerular vascular intima
Peptidoglycan Skin antigens
Hyaluronic acid Synovial fluid

Table (3-3): Differences between acute rheumatic fever and poststreptococcal

glomerulonephritis
Acute rheumatic Poststreptococcal glomerulonephritis (PSGN)
Feature fever (ARF)
Prior history of infection Pharyngitis strains Mainly pyodermal strains, or rarely
With pharyngitis strains
Serotype Most of the strains Pyodermal strains: 49, 53–55, 59–61 and
of S. pyogenes Pharyngitis strains: 1, 12
Immune response Marked Moderate
Complement level Unaltered Low (due to deposition in glomeruli)
Genetic susceptibility Present Absent
Repeated attacks Common Uncommon
Penicillin prophylaxis Indicated Not indicated
Course Progressive Spontaneous resolution
Prognosis Variable Good
Hypersensitivity reaction Type II Type III

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Laboratory Diagnosis
Specimen
S. pyogenes, Throat swab, pus, blood.
S. agalactiae , High vaginal swab , blood and cerebrospinal fluid of new born.
Enterococci , Blood, pus.
Viridans streptococci , Blood.

Gram stain : Non-motile gram-positive cocci in chains

Culture,biochemical tests and sensitivity testing :
Grow in aerobic and anaerobic environment at a temperature of 35-37 0c.
Grow in ordinary media with shiny or dry colonies with grey-white or colorless appearance.
S. pyogenes shows clear zone of hemolysis in blood agar plate. Does not grow in
MacConkey agar plate, Bacitracin sensitive.

Laboratory Diagnosis of Streptococcus pyogenes

1. Transport medium: Pike‟s medium
2. Direct smear microscopy: Pus cells with gram-positive cocci in short chains
3. Culture:
 Like most Streptococcus species, Streptococcus pyogenes also don‟t grow well on
a simple medium with basic nutrients. The growth, however, occurs well on agar
media supplemented with blood.
 The use of blood agar facilitates the observation of β-hemolysis, which
differentiates S. pyogenes from other Streptococcus species.
 S. pyogenes isolated from oral swabs grow well on sucrose-containing agar
medium like Trypticase Yeast Extract Cystine with 5% sucrose.
 Other selective media for S. pyogenes include the agar medium like Columbia
agar with colistin commonly used for the culture of Gram-positive bacteria.
 In the case of clinical samples, ample growth of typical S. pyogenes typical
colonies can be seen after 24 hours at 35-37°C.
 The incubation of blood agar plates in anaerobic or CO2-rich environment often
leads to the isolation of non-S. pyogenes β-hemolytic streptococci.
 Strep Selective agar is best for the suppression of commensal respiratory
microbiota, including the species of the same genus.
 The optimum temperature for the growth of S. pyogenes is at 37°C, but growth
can be seen between 15°C to 40°C.
 The inability of the bacteria to grow at 10°c and 45°C as well as at 6.5% NaCl and
40% bile helps differentiate S. pyogenes from other Streptococcus species.
 The bacteria is an aerobic or facultatively anaerobic bacteria that can tolerate
comparatively higher levels of oxygen and can also grow at a low level of oxygen.
About 5-10% CO2 during incubation promotes hemolysis on blood agar.
 S. pyogenes also grows well in liquid culture media like Nutrient agar and
Glucose broth. The growth is observed in the form of granular turbidity with a
powdery deposit as a result of heavy bacterial chains that settle down and form
powdery deposits.

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The following are some cultural characteristics of S. pyogenes on different culture media:
A. Nutrient Agar
 The colonies of S. pyogenes on NA appear circular pinpoint with an average diameter
of 0.5-1 mm.
 The colonies are light yellow to yellow colored semi-transparent to opaque with low
convex or convex elevation with matt surface (in the case of virulent strains) or glossy
(in the case of non-virulent strains) and mucoid (in the case of capsule producing
strains).
B. Blood Agar
 On blood agar, S. pyogenes form circular pinpoint colonies that are similar in
morphology to the colonies formed on other solid agar media.
 Light golden yellow colonies are formed that are surrounded by a clear zone
exhibiting β-hemolysis.
 The surface of the colonies differs in different species based on their virulence and
production of the capsule.

Figure (3-6): S.pyogenes cultivated on CAP agar (sheep blood agar with colistin +
aztreonam). Cultivation 24 hours in an aerobic atmosphere, 5% CO2. Colonies are
surroundend by a zone of beta-hemolysis.

C. PNF medium
 Circular pinpoint colonies of S. pyogenes are observed on PNF medium that are
yellow-colored.
 Like on blood agar, S. pyogenes also produces β-hemolysis around the colonies on the
PNF medium.

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4. Biochemical identification : Following biochemical tests are useful for the identification
of Streptococcus pyogenes.

Name of the test S. pyogenes Identification

Useful to differentiate staphylococci from

Catalase test Negative
streptococci

Presumptive identification of group A streptococci

Bacitracin sensitivity test Sensitive
(GAS)

Pyrrolidonyl-β-naphthylamide (PYR) test Positive Presumptive identification of GAS and enterococci

CAMP test Negative GBS (S. agalactiae) is CAMP test positive.

Hippurate hydrolysis test Negative Streptococcus agalactiae is positive

5. Typing:
 Lancefield grouping: Shows group A Streptococcus
 Typing of group A Streptococcus: Griffith typing and emm typing.

6. Serology: ASO antibodies and Anti-DNase B antibodies.

Anti-Streptolysin O (ASO) Test: ASO titer is not done for the diagnosis of Streptococcal
sore throat but for sequelae (complications) that result due to previous infections with
Streptococcus pyogenes.
 Rheumatic fever
 Post streptococcal glomerulonephritis (PSGN)
 Scarlet fever
 Erysipelas
 ASO antibodies titer is elevated > 200 Todd unit/ml in most streptococcal infec- tions
except in pyoderma and Post streptococcal glomerulonephritis (PSGN) .
 Anti-DNase-B Ab – Titer > 300–350 units/ml is diagnostic of PSGN and pyoderma.
 Other antibodies elevated are Antihyaluronidase and antistreptokinase antibodies.

Figure (3-7): Streptococcus spp (Gram stain)

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 Figure (3-8) : Streptococcus pyogenes (Bacitracin susceptibility test)

Figure (3-9) : Streptococcus agalactiae on blood agar (β-hemolysis)

Viridans streptococci show greenish discoloration of blood agar plate.

 Optochin resistant.
 Not soluble in bile salts.
 Do not ferment inulin.

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 Figure (3-10) : Colonies of viridans streptococci on blood agar surroundend by a wide
zone of alpha-hemolysis.

Enterococci , Non-hemolytic or α hemolytic changes in blood agar Plate.

Grow in the presence of 6.5% NaCl.
Grows in Macconkey agar.
Identified by litmus milk reduction test.
Bile esculin-positive.

Figure (3-11) : (A) Bile Esculin test of Enteroccci

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 Figure (3-12) : Enterococcus faecalis on macconkey

Streptococcus pneumoniae
1. Fastidious, lancet-shaped gram positive diplococci.
2. Possess a capsule of polysaccharide that permits typing with specific antisera.
3. Found as a normal flora in the upper respiratory tract.

Virulence Factors and Pathogenesis

S. pneumoniae possesses a number of virulence factors such as:
• Capsular polysaccharide: It protects the cocci from phagocytosis:
○ It is type specific (> 90 capsular serotypes are recognized), serotypes are detected
by Quellung reaction.
○ Quellung reaction: Capsular swelling occurs when colonies are added with type-
specific antiserum and methylene blue dye.
○ It diffuses (soluble) into culture media, tissue and exudates, hence also called solu-
ble specific substance.
• C-carbohydrate antigen (C-polysaccharide or C-substance): It is species specific. CRP
(C-reactive protein) present in sera of patients with acute inflammation. It is so named
because; it precipitates with pneumococcal C-antigen.
• Pneumolysin: It is a membrane damaging toxin, inhibits neutrophil chemotaxis.
• Autolysin: It is an amidase enzyme that cleaves peptidoglycan leading to autolysis of
cells. This property is responsible for bile solubility and draughtsman appearance of
pneumococcal colonies.

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Laboratory Diagnosis
Specimen: Sputum, blood, cerebrospinal fluid, ear discharge and sinus drainage.
Gram stain: Lancet-shaped gram positive diplococci
Culture: Grow best in chocolate agar media in CO2 enriched atmosphere. Shows α-
hemolytic, draughts man colony appearance: Sunken centre colony due to spontaneous
autolysis of older bacteria.
Young colonies resemble dew-drops due to capsule.
Bile soluble, ferment inulin.
Optochin sensitive.
Serology: Quellung reaction Good for rapid identification of S.pneumoniae in fresh
specimen.

Table (3-4) : S. pneumoniae can be differentiated from Viridans streptococci by various features

Properties Pneumococcus Viridans streptococci

Morphology Lanceolate or flame shaped Round/oval

Arrangement Gram-positive cocci in pairs Gram-positive cocci in long

chains
Capsule Present Absent

On blood agar Draughtsman or carom coin Convex shaped colony

colony
Liquid medium Uniform turbidity Granular turbidity

Bile solubility Soluble in bile Insoluble in bile

Inulin fermentation Fermenter Nonfermenter

Optochin Sensitive Resistant

Mice Pathogenicity Pathogenic Nonpathogenic

Procedure of Quellung test

1. Mix specific serotype of S.pneumoniae with specific anti-polysaccharide serum of the
same serotype or with polyvalent anti-serum on a slide.
2. Look for the appearance of capsule swelling under the 100X objective microscope.

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 Figure (3-13) : Streptococcus pneumonia (Quellung reaction)

Figure (3-14): Streptococcus pneumoniae alpha hemolysis on blood agar plate. Note the
partial hemolysis accompanied by a greenish discolorization of the agar around the growth
and the mucoid, transluscent colonies of the pneumococcus.

215
Figure (3-15) : Streptococcus pneumonia (optochin test)

Figure (3-16) : Streptococcus pneumonia (Bile solubility test)

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 Table (3-5): Differentiation of streptococcus species
Species Catalase Bacitracin Optochin test Litmus milk Camp test
test test Bile solubility test reduction test
S. pyogenes -ve +ve -ve -ve -ve
S. agalaciae -ve -ve -ve -ve +ve
Enterococci -ve -ve -ve +ve -ve
Viridans -ve -ve -ve -ve -ve
streptococci
S.pneumoniae -ve -ve +ve -ve -ve

GRAM POSITIVE SPORE FORMING RODS

1. Genus: Bacillus 2. Genus: Clostridium

Genus: Bacillus
Characteristics
1. Aerobic, non-motile,spore-forming, gram-positive chain forming rods.
2. Bacillus species are ubiquitous saprophytes
3. Important human pathogen ( B. anthracis & B. cereus )

Laboratory diagnosis
Specimen: Fluid or pus from skin lesion, Blood, sputum.
Gram stain: Non-capsulated gram-positive rods with centrally located spores from culture
Large capsulated gram-positive rods without spores from primary specimen.
Culture: Grows aerobically in ordinary media, After overnight incubation at 35–37 °C on
horse or sheep blood agar (BA) non-hemolytic,large, dense, grey-white irregular colonies
with colony margin of “Medussa Head” or “curled-hair lock” appearance due to composition
of parallel chaining of cells.

Biochemical reaction
Gelatin-stab culture: Gelatin liquefaction. Growth along the track of the wire with lateral
spikes longest near the surface Providing “inverted fir tree” appearance (Figure 49-14).
Serology: ELISA has been developed to measure antibodies to edema toxin and lethal toxin.

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Figure (3-17): B. anthracis Gelatin-stab , characteristic inverted fir tree appearance

Figure (3-18) : B. anthracis on blood agar

Medusa head appearance colony

Figure (3-19) : B. anthracis (Gram stain)

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Bacillus cereus
General characteristics
Exhibit motility by swarming in semisolid media.
Produce β lactamase, so not sensitive to penicillin.

Lab. Diagnosis
>105 org/gm of food.
SPECIMENS: Faeces, vomitus, remaining food (if any), eye specimen (corneal swab).
Gram stain: the organisms appear as large gram-positive rods in singles, pairs, or serpentine
with square ends after Gram staining.
Endospores formation is seen as an unstained oval or round region within the center of the
cell. Spores are oval (ellipsoidal) and not swelling of the mother cell.

CULTURE
1. Growth on 5% sheep blood agar, chocolate agar, routine blood culture media, and
nutrient broths.
2. Detectable growth within 24 hours following incubation on media incubated at 35° C,
in ambient air, or in 5% carbon dioxide (CO2).
3. Colony character on blood agar: Large, feathery, spreading, dull, gray, granular,
spreading colonies and opaque with a rough matted surface and irregular perimeters,
beta-hemolytic.
4. Bacillus cereus can be isolated from feces by using selective media such as MYPA
(mannitol, egg yolk, polymyxin, phenol red, and agar), PEMBA (polymyxin, egg yolk,
mannitol, bromothymol, blue agar).
These media take advantage of the phospholipase C positive reaction on egg yolk agar, no
production of acid from mannitol, and incorporation of pyruvate or polymyxin as the
selective agents.

Figure (3-20) : Bacillus cereus on blood agar

219
 Figure (3-21) : Bacillus cereus (Gram stain)

Genus: Clostridium
Characteristics
1. Clostridia are anaerobic, spore-forming motile, gram-positive rods.
2. Most species are soil saprophytes but a few are pathogens to human.
3. They inhabit human and animal intestine, soil, water,decaying animal and plant matter
4. Spores of clostridia are wider than the diameter of organism and located centrally,
subterminally and terminally.

Species of medical importance

1. C. perfringens
2. C. tetani
3. C. botulinum
4. C. .difficile

Clostridium perfringens
Characteristics
1. Capsulated, non-motile, short gram-positive rods in which spores are hardly seen.
there are five toxigenic groups : A-E
2. Human disease is caused by C. perfringens type A and C.

Laboratory diagnosis
Specimen: Infected tissue, pus, vomitus, leftover food, serum.
Gram stain: Non-motile, capsulated, thick brick-shaped gram positive rods in smears from
tissue; spores are rarely seen.

Identification of C. perfringens
1. Thick, stubby, boxcar-shaped gram-positive bacilli without spore
2. Target hemolysis (double zone hemolysis)
3. Nagler‟s reaction Positive
4. Reverse CAMP test: Positive

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Culture
1. Blood agar medium anaerobic incubation.
2. β-hemolytic colonies are seen in blood agar in anaerobic atmosphere.Some strains
produce double zone of hemolysis.
3. Cooked meat medium (Chopped meat-glucose medium).
4. Thioglycolate medium .

Biochemical reaction
Nagler reaction
Principle:
C. perfringens produces opalescence in human serum or egg yolk media due to the
production of Lecithinase C (phospholipase). It's used in identification of alpha toxin of C.
perfringens , the addition of antitoxin to one half of egg yolk agar prevents visible opacity ,
Due to Lecithinase action which is normally observed around colonies.

Procedure
1. Streak colonies of C. perfringens on egg-yolk agar.
2. Cover half of the medium with C. perfringens antitoxin.
3. Look for dense opacity production by the growth of C. perfringens; but no opacity on the
area with antitoxin (Figure 49-18).

Figure (3-22) : Nagler reaction(Lecithinase test)

 Lactose fermentation: Reddening of the medium; red colonies when exposed to air.
 Litmus milk medium: “stormy- clot” formation due to acid and gas formation.
Identification of C.perfringens rests on colony form, hemolysis pattern, biochemical
reaction, and toxin production and neutralization by specific antisera (Figure 49-19).

221
 Figure (3-23) : Milk medium showing the classical stormy clot reaction of C. perfringens,
the tube on the left extrems is uninoculated.

GRAM POSITIVE NON-SPORE FORMING RODS

GENUS CORYNEBACTERIA , GENUS: LISTERIA

Medical important species: Corynebacteria diphteriae & C. diphteriae

Characteristics
1. Non-spore forming, non-capsulated, non-motile aerobe or facultative anaerobe gram-
positive rods.
2. Possess irregular swelling at one end that give them the “clubshaped” appearance.It tends
to lie in parallel (pallisades) or at acute angles to one another in stained smears, forming
V,L , W shapes, so called Chinese-character arrangement.

Laboratory diagnosis
Specimen: Swabs from the nose, throat, or suspected lesion.
Smears: Beaded rods in typical arrangement when stained with alkaline methylene blue or
gram‟s stain.

Culture
Small, granular,and gray, with irregular edges with small zone of hemolysis on blood agar
Selective media are necessary for isolation from cilincal specimens.

Selective media
1. Loeffler’s serum media : Grows rapidly with in 8 hrs. after inoculation and show typical
appearance.
2. Tellurite Blood Agar : Is a selective medium used for isolation and cultivation of
Corynebacterium species, Potassium tellurite acts as a selective agent and has inhibitory
activity against most gram-positive and gram-negative bacteria except Corynebacterium

222
species, C.diphtheriae reduces potassium tellurite to tellurium and thereby produce gray-
black coloured colonies.

Biochemical reaction
Acid production from a range of carbohydrate fermentation.

Typing
Serotyping by agglutination tests, phage typing and bacteriocin typing have been used to
subdivide strains of C.diphteriae for epidemiologic studies.

Toxin production
Responsible for virulence; can be demonstrated by guinea pig inoculation or by gel
precipitation test
1. Guinea pig inoculation: Inject suspension of the isolated strain of C.diphteriae into two
guinea pig, one protected with diphtheria antitoxin.Death in 2-3 days of the unprotected
animal.
2. Gel-precipitation (Elek) test: a filter paper strip previously immersed in diphteria
antitoxin is incorporated into serum agar; the strain of C.diphteriae under investigation is
then streaked onto the agar at right angles to the filter paper strip. Incubate at 37 c0 for 1-2
days, and observe for lines of precipitation in the agar indicating
toxin-antitoxin interaction.

Figure (3-24) : Corynebacterium diphteriae on blood tellurite agar

223
 Figure (3-25) : Corynebacterium diphteriae (Albert Stain)

GENUS: LISTERIA
Most important species : Listeria monocytogenes

General characteristics
Non-sporulating, facultative anaerobe, intracellular. Gram positive rods

Lab. Diagnosis
Specimen : Blood/ CSF
Culture: Grow in blood agar and demonstrate narrow zone of β-hemolysis. Produce
“umbrella” growth pattern below the motility media surface at room temprature
demonstrating motility at room temperature

Biochemical reaction
1. Catalase positive
2. Oxidase negative
3. Tumbling motility

224
 Figure (3-26) : Tumbling motility of Listeria monocytogenes

GRAM NEGATIVE DIPLOCOCCI

GENUS: NEISSERIA
Characteristics
1. They are non-motile, gram-negative intracellular diplococci.
2. Rapidly killed by drying, sunlight, heat, and disinfectants.
3. Ferment carbohydrate produucing acid but not gas.
4. Each cocci is Coffee bean or kidney-shaped with adjacent concave sides.
5. Grow best on complex media under aerobic conditions containing 5% Co2.
6. Oxidase positive.
7. Aerobic , Carpnophilic bacteria ( 5- 10 % Co2)
8. Ferment glucose
9. Fastidious Microorganisms (Require specific nutrients for growth , Enriched media )
10.Gonococci are not able to grow on common blood agar because :
a. Cannot cause blood lysis.
b. Cannot survive in blood inhibitors.
So, Heating blood agar 70-80°C for 10 min will release X and V factors ,which they
are an essential elements for Neisseria gonorrhoeae growth.
Selective medium is Thayer Martin medium (Chocolate blood agar+ Antibiotics) for
primary Neisseriae isolation.

Thayer Martin agar contain Antibiotics:

1. Colistin : To inhibit most gram negative bacteria.
2. Vancomycin : To inhibit most gram positive bacteria.
3. Nystatin : To inhibit yeast.
4. Trimethoprim : To inhibit Proteus.

225
Figure ( 3-27): N.gonorrhoea intracellular gram-negative diplococci within the cytoplasm
of the polymorphonuclear (PMN) cell marked with the small arrow.

The main species of medical importance are:

N. meningitidis
N.gonorrhoea. An obligate parasite of the human urogenital tract.

Route of infection: Sexual contact

Pathogenicity in males
• Urethritis, Leads to Dysuria (Painful urination) , Purulent discharge.
Multiplication in males
• Cystitis (inflammation of the bladder)
• Epididymitis (an inflammation of the small, coiled tube at the back of the testicle, It
carries and stores sperm cells that are created in the testes )
• Prostatitis (inflammation (swelling) of the prostate gland, The prostate's primary
function is to produce the fluid that nourishes and transports sperm )
• Epididymitis & Prostatitis could lead to infertility.

Pathogenicity in females
• Urethritis, Leads to Dysuria (Painful urination) , Purulent discharge.
• But , 50% Asymptomatic due to women produce some discharge normally to protect
and clean the uterus by removing bacteria and dead tissues.
• Abdominal discomfort

226
Multiplication in Females
• Fallopian tube obstruction (could lead to infertility).
• Ectopic pregnancy.
• Peritonitis.
• Fitz-Hugh-Curtis syndrome.

Pathogenicity in infant
(When delivered through the infected birth canal) Gonococcal ophthalmia neonatorum If
untreated and complicated leads to blindness. So, immediate treatmentwith 0.5%
Erythromyin ophthalmic Ointment during the first hour after birth is used to prevent
gonorrhea infection.

Figure (3-28): Neonatal conjunctivitis (ophthalmia neonatorum) caused by Neisseria

gonorrhoeae. Note purulent exudate, especially on lower right eyelid.

Laboratory diagnosis
Specimen : Urethral swab, cervical swab, eye swab
Smear : Gram-negative intracellular diplococcic.More than five polymorphs per high power
field.

Culture: Requires an enriched media like chocolate agar or thayer-martin agar. Grows best
in CO2 enriched aerobic atmosphere with optimal temperature of 35-37Oc.
Fastidious- Dies with exposure to sunlight, room temperature and drying. Small glistening
colonies.

227
Figure (3-29) : Typical colonies of Neisseria gonorrhea on modified Thayer martin media (MTM).

Biochemical reaction
1. Oxidase positive.
2. Ferment only glucose in carbohydrate utilization test.
Serology: Antibodies to gonococcal pili and Omps can be detected by immunoblotting,
RIA or ELISA tests.

Neisseria meningitidis
Characteristics
1. Gram-negative intra cellular diplococci.
2. Present in the nasopharynx in 5-10% of healthy people.
3. Ferment glucose and maltose
4. Capsular polysaccharide: It prevents the bacteria from phagocytosis, can be typed
into 13 serogroups, Only 5 serogroups account for the majority of cases: A, B, C, Y,
and W135;

Transmission
1. Droplet from case or carrier ,they colonize the membranes of the nasopharynx and
become part of the transient flora of the upper respiratory tract.
2. Carriers are usually asymptomatic.
3. From the nasopharynx, the organism can enter the bloodstream and spread to specific
sites, such as the meninges or joints, or be disseminated throughout the body
(meningococcemia).
4. About 5% of people become chronic carriers and serve as a source of infection for
others.

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Risk factors that promote colonization include:
• Overcrowding and semi-closed communities such as schools, military and refugee
• Travellers (Hajj pilgrims) and smoking

Symptoms
Fever , sore throat , sever headache , neck rigidity and coma.

Laboratory diagnosis
Specimen : Cerebrospinal fluid, blood.
Smear : Gram-negative intracellular diplococcic.
Culture : Transparent or grey, shiny, mucoid colonies in chocolate agar after incubation at
35-37c0 in a CO2 enriched atmosphere.

Diagnosis
1.Case : Blood and Lumbar puncture and examination the C.S.F.
C.S.F collection : CSF turbid and under tension (due to pus).
CSF examination: Biochemical analysis: ↑CSF pressure, ↑protein and ↓glucose in CSF.
2. For carriers: Nasopharyngeal swab.

Biochemical reaction
Oxidase positive.
Ferment glucose and maltose in carbohydrate utilization test.
Serology : Latex agglutination test/ Hemmagglutination test.

Figure (3-30) : Nasopharyngeal swab (Left), C.S.F collection (Right)

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GRAM NEGATIVE COCCOBACILLI
GENUS: HAEMOPHILUS
Characteristics
1. This is a group of small gram-negative, non-spore forming, non-motile, pleomorphic
bacteria that require enriched media for growth.
2. Growth is enhanced in CO2 enriched atmosphere.
3. Present in upper respiratory tract as a normal microbial flora in healthy people.
4. The group is fastidious requiring growth factors for isolation.The growth factors are
X-factor(Hematin) and V-factor (Diphosphopyridine nucleotide).

Haemophilus influenzae
Characteristics
1. Gram-negative cocobacilli.
2. Fastidious bacteria requiring growth factors for isolation.
3. Found in upper respiratory tract as normal flora in healthy people.

Laboratory diagnosis
1.Specimen collection and transport:
 CSF, blood, sputum, pus, aspirates from joints, middle ears or sinuses.
 As it is highly sensitive to low temperature, the specimens should never be
refrigerated.
2.Gram staining of CSF and other specimen shows pleomorphic gram-negative coccobacilli
3.Capsule detection: By Quellung reaction or Latex aggl. test
4.Culture: H.influenzae is largely aerobic, growth is enhanced by 5–10% CO2.
 Blood agar with S.aureus streak line: Colonies of H.influenzae grow adjacent to
S.aureus streak line (this property is called as satellitism). This is due to release of V
factor by lysis of RBCs mediated by S.aureus.
 Chocolate agar: It grows well on chocolate agar but sparsely on blood agar.
 Fildes agar and Levinthal‟s agar.

5.Disk test for X and V requirement:

 Require only X factor: H.ducreyi and H.aphrophilus
 Require only V factor: H.parainfluenzae, V.parahaemolyticus and H.paraphrophilus
 Require both X and V factor: H.influenzae, H.aegyptius and H.haemolyticus.

6.Biotyping: It is done by IOU tests (indole, urease test and ornithine decarboxylase test).

7.Slide agglutination test: Serotyping is carried out using type-specific antisera.

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Methods
1. Mix a loopful of haemophilus growth in 2ml of sterile saline.
2. Inoculate the bacteria suspension on a plate of blood agar using a sterile swab.
3. Streak a pure culture of S. aureus across the inoculated plate which provides V-factor for
H. influenzae.
4. Incubate the plate over night in a CO2 -enriched environment at 35-37 Oc.
5. Look for growth and satellite colonies in next morning, Colonies are largest nearest to
the S. aureus column of growth.

Serology : Quellung reaction (using specific antisera) , Immunofluorescence stain.

Figure (3-31) : Haemophilus influenza (satellitism test)

Table (3-6) : Hemohpilus species differentiation

Growth factor required Haemophilus species

X and V factor H. influenzae, H. aegyptius, H.

hemolyticus

X factor H. ducreyii

V factor H. parainfluenzae, H. parahemolyticus

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GENUS BRUCELLA
General characteristics
Gram-negative, non-motile, non-sporulating, zoonotic,obligate intracellular aerobic
coccobacilli , 3 major human pathogenic species.

Table (3-7) : Hosts of Brucalla species

Species Primary animal host

B.abortus Cattle

B. melitensis Goat / Sheep

B. suis Swine

B.canis Dogs

Lab. Diagnosis
Specimen : Blood, Biopsy material (Bone marrow, Lymphnodes),serum.
Culture: Grow in blood agar, chocolate agar, or brucella agar incubated in 10% CO2 at 35-
37 C0 for 3 weeks.
Gram stain: Gram negative, non-motile, non-sporulating, zoonotic, obligate intracellular
aerobic coccobacilli

Biochemical reaction
1. Non-hemolytic
2. Catalase positive
3. Oxidase positive
4. Urease test positive
5. Dye inhibition test positive

Serology: Agglutination test , IgG agglutination titer >1:80 indicate active infection.

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 Figure (3-32) : Brucella abortus in blood agar

Figure (3-33) : Brucella abortus (Gram stain)

GRAM NEGATIVE RODS

It comprises the following bacterial groups
1. Oxidase negative
Enterobacteriaceae
A . Lactose-fermenters
1. Escherichia spp.
2. Klebsiella spp.
3. Enterobacter spp.
4. Citrobacter spp.

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B. Non-lactose fermenters
1. Salmonella spp.
2. Shigella spp.
3. Proteus spp.

2. Oxidase Positive
1. Pseudomoas
2. Vibrio
3. Campylobacter
4. Helicobacter

ENTEROBACTERIACEAE
Characteristics
1. Named, as well coliforms or enterobacilli.
2. Found as normal flora in intestinal tract of humans and animals.
3. Gram-negative, non-spore forming, aerobic and facultative anaerobic bacteria.
4. Most are motile.
5. Grow over a wide range of temperature in ordinary media.
6. All ferment glucose with acid production.
7. Oxidase negative.

Escherichia coli
Characteristics
E.coli is the most important species encountered as human pathogen.
 It is also the commonest aerobe to be harbored in the gut of humans and animals.
 After excreted in feces, it remains viable only for some days in the environment. Hence
detection of fecal E.coli (thermotolerant E.coli that survives at 44°C) is taken as an indicator
of recent contamination of drinking water with human or animal feces.
• Normal flora in human and animal gastrointestinal tract.
• Found in soil, water and vegetation.
• Most are motile; some are capsulated.

Laboratory diagnosis
Specimen: Urine, pus, blood, stool, body fluid.
Smear : Gram-negative rods.
Culture : Grow on blood and macconkey media.Lactose-fermenting pink colour mucoid
colonies on macconkey agar and some strains are hemolytic on blood agar .

Biochemical reaction: Produce indole from tryptophan containing peptone water.Motility

test positive,Ferment glucose and lactose producing acid and gas by in Triple sugar iron agar
medium.

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Clinical Manifestations of E.coli Infection
1. Urinary tract infection (UTI): Caused by uropathogenic E.coli (UPEC).
2. Diarrhoea: Caused by six types diarrheagenic E.coli .
3. Other syndromes:
• Abdominal infections: E.coli is the most common cause of both primary bacterial
peritonitis (occurs spontaneously) and secondary bacterial peritonitis (occurs secondary
to intestinal perforation. It also causes visceral abscesses, such as hepatic abscess.
• Pneumonia (especially in hospitalized patients: ventilator associated pneumonia)
• Meningitis (especially neonatal meningitis)
• Wound and soft tissue infection.
• Osteomyelitis
• Endovascular infection and bacteremia.

Urinary Tract Infection (UTI)

E.coli (uropathogenic E.coli or UPEC) is the single most common pathogen, accounting for
85–95% of all cases of UTI. UPEC serotypes O1, O2, O4, O6, O7 and O75 are responsible
for most UTIs.
Route of Spread
 Ascending route: After colonizing the periurethral area, E.coli ascends the urinary tract
to reach bladder and later to kidney.
 Descending route: Hematogenous seeding of E.coli into kidneys result in
pyelonephritis.

Types of UTI
Table (3-8) : Depending on the site involved, there are two types of UTI: Lower and upper UTI
Lower UTI Upper UTI

Syndromes Cystitis and urethritis Pyelonephritis

Symptoms Local manifestations: Systemic manifestations:
dysuria, urgency, Fever, vomiting, abdominal pain
frequency,
Route of spread Ascending route Both ascending (common) and
descending route
Occurrence More common Less common
Virulence factors Fimbriae (e.g. P fimbriae) Capsular K antigen
Common virulence factors responsible are:
• Cytotoxins (CNF 1-cytotoxic necrotizing factor 1 and SAT-Secreted auto transporter toxin)
• Hemolysins

Predisposing Factors that Promote UTI

 Females: Due to short urethra and close proximity to anus.
 Pregnancy: Physiological obstruction in urinary tract due to growing fetus may lead to
prolonged stasis of urine and asymtpomatic bacteriuria.
 Others: Presence of urinary catheters, urinary stones or prostate enlargement.
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Laboratory Diagnosis of UPEC
 Specimen collection:
1. Clean voided midstream urine: It is the most common specimen for UTI.
2. Suprapubic aspiration is the most ideal specimen (for coma or infants).
3. In catheterized patients: Collected from the catheter tube and not from the bag.
• Transport: Stored in refrigerator or stored by adding boric acid
• Direct examination: Screening tests done are as follows:
1. Wet mount examination is done to demonstrate the pus cells in urine. Pyuria of > 8
pus cells/mm3 or 4 lakh pus cells excreted in urine/hour is taken as significant.
2. Others: Leukocyte esterase test, Nitrate reduction test (Griess test), Catalase test
3. Gram staining of urine is not a reliable indicator as (i) low bacterial count in urine,
(ii) Pus cells rapidly deteriorate and may not be seen well. It may be limited to
pyelonephritis and invasive UTI cases and a count of ≥ 1 bacteria/oil immersion filed is
taken as significant.

• Culture:
1. Culture Media: Urine is inoculated onto:
 MacConkey agar and blood agar combination or
 CLED (cysteine lactose electrolyte deficient) agar.
2. Kass concept of significant bacteriuria:
 A count of ≥ 105 colony forming units (CFU)/ml of urine is considered as significant
– indicates infection (referred as „significant bacteriuria‟ developed by Kass)
 Low count of ≤ 104 CFU/ml is due to commensal bacteria (due to contamination
during voiding) and is of no significance. However, low counts may be significant in
following conditions:
a. Patient on antibiotic treatment or on diuretic drugs
b. Infection with some gram-positive bacteria like S.aureus, and Candida.
c. Pyelonephritis and acute urethral syndrome
d. Sample taken by suprapubic aspiration
3. Quantitative culture is done to count the number of colonies. This is done by:
 Semi quantitative method, such as standardized loop technique
 Quantitative method, such as pour plate method.
 Antibody coated bacteria test is used to differentiate upper and lower UTI.

Diarrhea (Diarrheagenic E. coli)

Enteropathogenic E. coli (EPEC)
EPEC frequently cause infantile diarrhea (outbreaks) and rarely sporadic diarrhea in adults.
• It is nontoxigenic and noninvasive
• Mechanism of diarrhea:
1. Adhesion to intestinal mucosa, mediated by plasmid coded bundle-forming pili
2. A/E lesions (attaching and effacing lesions) on the intestinal epithelium.

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Enterotoxigenic E. coli (ETEC)
ETEC is the most common cause of traveler’s diarrhea causing 25–75% of cases:
1. It causes acute watery diarrhea in infants and adults.
2. Common serotypes associated are: O6, O8, O15, O25, O27, O153, O159, etc.
3. It is toxigenic but not invasive

Pathogenesis:
1. Attachment to intestinal mucosa is mediated by CFA (Colonization Factor Ag)
2. Toxins: (i) heat labile toxin or LT (↑cAMP), (ii) heat stable toxin or ST (↑cGMP)
3. Diagnosis is done by detection of toxins by in vitro and in vivo methods.

Enteroinvasive E. coli (EIEC)

Common serotypes associated with EIEC are O28, O112, O114, O124, O136, O152, etc.
• Pathogenesis: EIEC is not toxigenic but invasive. The epithelial cell invasion is
mediated by a plasmid coded antigen called virulence marker antigen (VMA).
• EIEC is biochemically, genetically and pathogenically closely related to Shigella.
 Manifestations include ulceration of bowel, dysentery (resembling shigellosis).

Diagnosis:
1. Detection of VMA by ELISA
2. Human cervical cancer cell (HeLa), cell invasion assay used in scientific study.
3. Sereny test (inoculation into guinea pig eyes produces conjunctivitis)
4. EIEC are biochemically atypical being nonmotile and lactose nonfermenters.

Enterohemorrhagic E. coli (EHEC)

 Serotypes associated with EHEC are:
1. O157: H7 (most common serotype)
2. Other serotypes are rare (O26: H11, O6, O55, O91, O103, O111 and O113).
 EHEC is usually transmitted by contaminated food, i.e. lettuce, spinach, sprouts and
undercooked ground beef.
 Prevalent in industrialized countries (other Diarrheagenic E.coli are common in
developing regions)
 Low infective dose (< 102 CFU) of EHEC can also initiate the infection.
 Pathogenesis: EHEC secretes a toxin called verocytotoxin or Shiga-like toxin ( refer
table for its mechanism)
 Manifestations: VT has predilection for endothelial cells causing capillary
microangiopathy which leads to:
1. HC (hemorrhagic colitis): Manifests as gross bloody diarrhea, abdominal pain and fecal
leukocytosis but no fever.
2. Hemorrhagic uremic syndrome (HUS): Injury to small vessels of the kidney and brain
can lead to bloody diarrhea, thrombocytopenia, renal failure and encephalopathy but
without fever. It is more common in children.

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  Diagnosis:
1. Sorbitol MacConkey agar: EHEC in contrast to other E.coli, does not ferment sorbi- tol
2. Toxin detection:
▪ Cytotoxicity in Vero cell lines (gold standard method)
▪ Fecal toxin antigen detection by ELISA or rapid tests
3. PCR can be used to detect gene coding for VT.

Enteroaggregative E. coli (EAEC)

 It is so named because it adheres to HEp-2 cells in a stacked-brick fashion
 Most strains are „O‟ untypeable but „H‟ typeable

Pathogenesis:
1. Colonization mediated by aggregative adhesion fimbriae I (regulated by aggR
gene)
2. Produce EAST 1 toxin (enteroaggregative heat stable enterotoxin 1)

Manifestations: Persistent and acute diarrhea are common; in developing countries

 E. coli O104: H4 is an enteroaggregative strain that has caused major outbreaks in
Germany in 2011. Peculiarity is, it produces Shiga toxin and can cause HUS.

Diffusely Adherent E. coli (DAEC)

 It is characterized by Ability to adhere to HEp-2 cells in a diffuse pattern
 Expresses diffuse adherence fimbriae which contribute to the pathogenesis.

Figure (3-34) : E. coli in macconkey agar

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Figure (3-35) : Escherichia coli colonies on sheep blood agar after 24 h at 37°C. Fig. A, B:
Most strains of E.coli produce smooth, circular, low-convex colonies with entire edge that
are about 3-4 mm in diameter. They are greyish, butyrous and readily emulsified. Fig. B, G:
Partial digestion of erythrocytes may cause more or less profound discoloration of agar under
colonies and in their vicinity (Fig. F). Isolates from urinary tract are quite often beta-
hemolytic. Fig. C, H: Less common are higly mucoid strains, typically isolated from urine.
Fig. D, I: or small-colony variants, previously known as dwarf colonies, typically isolated
from urine. Fig. J: urinary isolate - Rough colonies are quite uncommon in clinical samples.

Figure (3-36) : E. coli (Gram stain)

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Genus: Klebsiella
Characteristics
 Nonmotile, lactose fermenter.
 Gram-negative rods.
 Capsulated, produce mucoid colonies.
 K. pneumoniae: Urease positive. It causes pneumonia, UTI, abdominal, wound and
surgical site infection.
 K.ozaenae: It causes ozaenae (foul smelling nasal discharge)/atrophic rhinitis.
 K.rhinoscleromatis: causes rhinoscleroma.

Laboratory diagnosis
Specimen : Sputum, urine, pus, CSF, body fluid
Smear : Gram-negative rods.
Culture: Grow on blood and macconkey media.Large, mucoid, pink colour lactose-
fermenting colonies on mac conkey agar.

Biochemical reaction
Not produce indole from tryptophan containing peptone water. Non motile, Ferment glucose
and lactose producing acid and gas in Triple sugar iron agar medium.

Serology: Capsular polysaccharide serotyping.

Figure (3-37) : Klebsiella pneumonia in macconkey agar

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 Figure (3-38) : Klebsiella pneumonia (Gram stain)

GENUS:ENTEROBACTER
Characteristics
It is gram-negative lactose fermenting motile rods.
Laboratory diagnosis
Smear : Gram-negative rods.
Culture: Grow on blood and Macconkey media.Large, mucoid, pink colour lactose-
fermenting colonies on Macconkey agar. Enterobacter can be differentiated with a few
specific tests from Klebsiella species, Enterobacter organisms are motile, and urease-
negative.
Biochemical reaction : Not produce indole from tryptophan containing peptone
water,motile and ferment glucose and lactose producing acid and gas in Triple sugar iron
agar medium.

Figure (3-39) : Enterobacter spp. on MacConkey agar

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 Figure (3-40) : Enterobacter spp. (Gram stain)

GENUS CITROBACTER
It is gram-negative lactose fermenting motile rods. Medical important species is
Citrobacter freundii.

Laboratory diagnosis
Smear: Gram-negative rods.
Culture: Grow on blood and macconkey media shows pink colour lactose-fermenting
colonies on mac conkey agar.
Biochemical reaction: Not produce indole from tryptophan containing peptone water,motile
and ferment glucose and lactose producing acid , gas from glucose and H2S in Triple sugar
iron agar medium.

Figure (3-41) : Citrobacter freundii in Macconkey agar

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 Figure (3-42) : Citrobacter freundii (Gram stain)

GENUS: SALMONELLA
Laboratory diagnosis
Specimen: Blood, Bone marrow, stool, urine and serum
for enteric fever.
1. Blood – 80% positive in the first week.
2. Stool- 80% positive in the second and third week.
3. Urine- 25% positive in the third and fourth week.
4. Serum for widal test- positive after the second week of illness.
5. Stool for gastroenteritis.

Gram reaction : Gram-negative rods.

Culture: Bacteriologic methods for salmonella isolation.
1. Differential medium
For rapid isolation of lactose non-fermenters Ex. EMB agar,MacConkey
agar,Deoxycholate agar.
2. Selective medium
favor growth of salmonella and shigella over other enterobacteriaceae
Ex. SS agar, Hekton Enteric agar,XLD agar, Deoxycholate-Citrate agar
3. Enrichment cultures
Inhibit replication of normal intestinal flora and permit replication of salmonella
Ex. Selenite F broth,Tetrathionate broth. , Non-lactose fermenting in MacConkey agar.

Biochemical reaction: Generally produce gas and acid from carbohydrate; except S.typhi
which does not produce gas. H2S producing colonies in Triple sugar iron agar.
Serology: (wiedal test): Tube dilution agglutination test Used to determine antibody titers in
patients with unknown illness.
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Figure (3-43) : Salmonella species on XLD agar with H2S

Figure (3-44) : Salmonella species on S-S agar with H2S

244
Figure (3-45) : Salmonella species on Hekton agar

Figure (3-46) : Salmonella species (Gram stain)

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GENUS: SHIGELLA
Shigella species are the agent of bacillary dysentery.
• Four species has been recognized: S. dysenteriae, S. flexneri, S. boydii, S. sonnei.
• Most hardier: S. sonnei.
• Most common species: In world: S. sonnei, In India: S. flexneri.

Species of medical importance are:

Subgroups
1. S. dysenteriae A
2. S. flexneri B
3. S. boydii C
4. S. sonnei D

Pathogenicity
• Transmission: (i) ingestion through contaminated fingers (MC), food, and
water or flies and (ii) rarely by homosexuals.
• Infective dose:
a. Shigella has a low Infective dose (10 to 100 bacilli). Others with low infective dose
include EHEC, Entamoeba histolytica and Giardia.
b. Salmonella Typhi: 103–106 bacilli.
c. Vibrio cholerae: 106–108 bacilli
• Bacilli enter the mucosa via M cells.
• Invasion: Mediated by a large virulence plasmid.
• Direct cell to cell spread: This occurs by inducing actin polymerization of host
cells.
• Exotoxins:
a. Shigella enterotoxin (ShET 1 and 2)- found essentially in S. flexneri
b. Shiga toxin is a cytotoxin, produced by S.dysenteriae type1.
• Endotoxin induces intestinal inflammation and ulcerations.

Laboratory Diagnosis
• Specimen: mucus flakes of stool, serum.
• Gram-negative non-motile rods.
• Biochemical reaction : It produces acid but not gas from glucose.
• Culture : Non-lactose fermenting colonies on Mac conkey agar and SS agar.
• Culture Media: (Common media for both Shigella and Salmonella):
1. Transport media: Sach‟s buffered glycerol saline.
2. Selective media: DCA (deoxycholate citrate agar), XLD, SS Agar.
3. Enrichment Broth: Gram-Negative broth, selenite F broth, tetrathionate broth.

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Important biochemical properties
1. Nonmotile, Nonlactose fermenter except: S. sonnei (Late lactose fermenter).
2. Catalase +ve except: S. dysenteriae type 1.
3. Mannitol fermenting except: S. dysenteriae.

Typing of Shigella
1. Serotyping S. dysenteriae: 15, S. flexneri 6 , S. boydii 19 serotypes, S. sonnei 1
2. Colicin typing (Bacteriocin typing) done for S. sonnei (has 26 colicin types).

Figure (3-47) : Salmonella & Shigella on (SS) Agar

GENUS: PROTEUS
Proteus species are found in the intestinal tract of humans and animals, soil, sewage and
water.
They are gram-negative, motile, non-capsulated , pleomorphic rods.
Species of medical importance:
1. P. mirabilis
2. P. vulgaris

Laboratory diagnosis
Specimen: Urine, pus, blood, ear discharge
Smear: Gram-negative rods
Culture: Produce characteristic swarming growth over the surface of blood agar.
Non-lactose fermenting colonies in mac conkey agar.
Proteus species have a characteristic smell.

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Biochemical reaction
 Proteus spp (Urease positive)
 P. vulgaris (Indole positive)
 P. mirabilis (Indole negative)

Figure (3-48): Proteus vulgaris in blood agar shows swarming

GENUS: PSEUDOMONAS
General characteristics
1. Gram-negative motile aerobic rods having very simple growth requirement.
2. Can be found in water, soil, sewage, vegetation, human and animal intestine.

Species of medical importance:

1. P. aeruginosa
2. P. pseudomallei

Pseudomonas aeruginosa
Laboratory diagnosis
Specimen: pus, urine, sputum, blood, eye swabs, surface swabs
Smear: Gram-negative rods.
Culture: Obligate aerobe, grows readily on all routine media over wide range of
temperature(5-42 C0). Bluish-green pigmented large colonies with characteristic “fruity”
odor on culture media.

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Biochemical reaction
1. Oxidase positive
2. Catalase positive
3. Citrate positive
4. Indole negative
5. Non-lactose fermenter.

NB: identification of the bacteria is based on colony morphology, oxidase-positivity,

characteristic pigment production and growth at 42 c0

Figure (3-49) : Pseudomonas aeruginosa in nutrient agar shows green pigment .

(pyocyanin&pyoverdine)

Figure (3-50) : Pseudomonas aeruginosa (Gram stain)

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GENUS: VIBRIOS
Actively motile, gram-negative curved rods.
Species of medical importance: Vibrio cholerae-O1,O139

CLASSIFICATION
Based on Salt Requirement
• Nonhalophilic vibrios: They cannot grow at higher salt concentrations. Examples,
V.cholerae and V. mimicus
• Halophilic vibrios: Salt is their absolute requirement. They cannot grow in the absence
of salt. They can tolerate and grow at higher salt concentration of up to 7–10, e.g.
V. parahaemolyticus, V. alginolyticus and V. vulnificus.

V. cholerae is Further Classified

A. Serogrouping: Based on somatic O antigen side chain of LPS (lipopolysaccharide),
V. cholerae can be grouped into more than 200 serogroups or serovars:
 O1 serogroup was responsible for all pandemics and most of the epidemics of
cholera.
 Nonagglutinable (NAG vibrios) or non-cholera vibrios (NCV)- refers to non-O1
serogroups.
 O139 serogroup was identified in 1992 and causes cholera outbreaks India and
Bangladesh.
 Non O1/O139 serogroups have occasionally caused sporadic outbreaks of diarrhea and
extraintestinal manifestations, but have never caused epidemic cholera so far.

B. Serotyping: O1 serogroup can be further divided into three serotypes: Inaba, Ogawa,
and Hikojima; based on minor antigenic differences of O antigen:
 Ogawa is the most common serotype isolated from clinical samples followed by
Inaba.
 However, during epidemics, shifting between Ogawa to Inaba shift can takes place.
 Hikojima represents an unstable transitional state where both Inaba and Ogawa
antigens are expressed.

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C. Biotyping: Serogroup O1 has two biotypes classical and El Tor; differentiated by various
biochemical reactions:
 Classical biotype was responsible for the first six pandemics of cholera.
 Currently, most of cholera cases are due to El Tor, although occasional classical
isolates are still seen.
 However, some isolates do not fit in to both the biotypes and are called as El Tor
variants.
 El Tor variants: Few variants of El Tor biotype have been described recently in
Bangladesh and in few other places of Asia and Africa which show properties
overlapping with that of the classical biotype, e.g. include:
i. Matlab variants (El Tor hybrid): These strains could not be biotyped because
they have a mixture of both classical and El Tor properties, were described first
in Bangladesh in 2002.
ii. Mozambique variant (2004–2005): It has a typical phenotypic properties and
genome of El Tor, except that the cholera toxin and its gene (CTX) are of classical
type.

Biotypes of V. cholerae O1 Classical biotype El Tor biotype

β hemolysis on sheep blood agar Negative Positive

Chick erythrocyte agglutination Negative Positive

Polymyxin B (50 IU) Sensitive Resistant

Group IV phage susceptibility Susceptible Resistant

El Tor Phage V susceptibility Resistant Susceptible

VP (Voges Proskauer) test Negative Positive

CAMP test Negative Positive

Cholera toxin gene CTX-1 CTX-2

Laboratory diagnosis
Specimens
• Freshly collected watery stool before starting the antibiotics.
• Rectal swab is preferred specimen for convalescent patients or carriers.

Transport/Holding Media
• Venkatraman-Ramakrishnan (VR) medium
• Alkaline salt transport medium
• Cary-Blair medium
• Autoclaved sea water.

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Direct Microscopy
• Gram staining of fecal smear reveals short curved comma-shaped gram-negative
rods, arranged in parallel rows, (fish in stream appearance)
• Darting motility or shooting star motility (actively motile frequently changing
their direction, also seen in Campylobacter and Aeromonas).

Figure (3-51) : Gram stain with comma-shaped Gram-negative bacilli suggestive of Vibrio
cholerae

Culture
Cultural conditions: V. cholerae is:
• Nonfastidious and strongly aerobic
• Hemodigestion on blood agar
• Growth is better in alkaline medium. The optimum pH is 8.2
• NaCl (0.5–1%) stimulates the growth, however, high concentrations of NaCl (> 6%) are
inhibitory.

Culture medium
• Enrichment broth:
○ Alkaline peptone water (APW) An enrichment broth, should also be inoculated and sub -
cultured in 6 to 8 hours.
○ Monsur‟s taurocholate tellurite peptone water (pH 9.0).

• Selective media:
○ Alkaline bile salt agar (BSA)
○ Monsur‟s gelatin taurocholate trypticase tellurite agar (GTTTA) medium.
○ TCBS-agar , the isolation of vibrios is favored by an alkaline (pH 8.6) liquid medium and
TCBS-agar (thiosulfate, citrate, bile salts, sucrose and pH of 8.6), V. cholerae produces
yellow Yellow, shiny colonies , 2-3 mm should be selected for further study with
biochemical and serologic tests. ,due to sucrose fermentation. Sucrose nonfermenters (V.
252
 mimicus and V. parahemolyticus) produce green colonies.
○ Vibrios grow well on blood agar, Vibrio cultures usually grow on MacConkey and will
appear as colorless (lactose negative) colonies.

Biochemical Reactions
Important biochemical properties of V. cholerae include:
• Catalase and Oxidase positive
• Indole and nitrate test positive (together called Cholera red reaction)
• String test positive
• Susceptible to O/129 (vibriostatic agent)
• Arginine negative
• Kligler iron agar :Acid / Alk . no gas no H2S

Serological test (Slide agglutination)

Fresh growth of suspect V. cholerae on a non selective agar medium may be tested in V.
cholerae O1 polyvalent antiserum. Usually after 5 to 6 hours of incubation, growth on the
surface of the slant is sufficient to perform slide serology with V. cholera O1 polyvalent
antisera. Isolates that agglutinate in polyvalent antiserum to the O1 serogroup are
presumptively identified as V. cholerae O1. Presumptive V. cholerae O1 may be confirmed
with agglutination in either monovalent Ogawa or Inaba antisera.

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Figure (3-52) : Vibrio cholera identification

Figure (3-53) : V. cholera (Positive String test )

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 Figure (3-54) : V. cholera in TCBS agar shows golden colonies

GENUS: CAMPYLOBACTER
Characteristics
1. Small, delicate, spirally curved gram-negative bacteria.
2. Motile bacteria with single polar flagellum.
3. Stricly microaerophilic bactria requiring 5-10% o2 and 10% co2 enriched environment.
4. Oxidase and catalase positive.

Species of medical importance

1. Campylobacter jejuni
2. Campylobacter coli

Characteristics
1. Gram-negative non-spore forming motile rods with comma, S or „gull-wing‟ shapes.
2. Requires selective media like skirrow‟s and Butzler‟s media for isolation of the
bacteria from faecal specimen.

Laboratory diagnosis
Specimen: Stool
Microscopy: Typical „gull-wing‟ shaped gram-negative rods.
Typical darting motility of the bacteria under dark field microscopy or phase contrast
microscopy.

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Culture: Colonies of C. jejuni are small, nonhemolytic, mucoid, usually grayish, and flat
with irregular edges. Campylobacter CSM Agar (Charcoal-Based Selective Medium) is a
blood free selective medium for the primary isolation of Campylobacter species from human
fecal specimens Grow best at 42c0 on selective media but can be cultured at 37c0.Watery and
spreading or round and convex colonies on solid media at low oxygen tension.

Biochemical reaction
C. jejuni … hydrolyzes hippurate.
C. coli … does not hydrolyze hippurate.
Both are oxidase positive.

Figure (3-55) : Compylobacter species (Gram stain)

Helicobacter pylori
General characteristics
Spiral-shaped gram negative, microaerophilic, motile rods with polar flagella.

Lab. Diadnosis
Specimen: Gatric biopsy, serum.
Smear: Giemsa or silver stain
Culture: Skirrow‟s media.Translucent colonies after 7 days of incubation.

Biochemical reaction:
1. Catalase positive
2. Oxidase positive
3. Urease positive

Serology: Detection of antibodies in the serum specific for H. Pylori, Detection of H. pylori
antigen in stool specimen.

256
Special tests
Urea breath test (UBT)
Patients swallow urea labelled with an uncommon isotope, either radioactive carbon-14 or
non-radioactive carbon-13. In the subsequent 10–30 minutes, the detection of isotope-
labelled carbon dioxide in exhaled breath indicates that the urea was split; this indicates that
urease (the enzyme that H. pylori uses to metabolize urea) is present in the stomach, and
hence that H. pylori bacteria are present.

Figure (3-56) : Principle of a urea breath test for H. pylori

GENUS: MYCOBACTERIA
Characteristics
1. Non-spore forming, non-motile, aerobic, Acid-fast bacilli.
2. Acid-fastness depends on the waxy envelope-mycolic acid of cell wall.
3. More resistant to chemical agents than other bacteria.
4. Once stained with primary stain, they resist decolorization by acid-alcohol.
5. All bacteria are decolorized by acid-alcohol except Mycobacteria.

Mycobacteria of medical importance

1. M. tuberculosis
2. M. leprae

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Mycobacterium tuberculosis
Characteristics
1. Strictly aerobic acid-fast bacilli.
2. The main reservoir is an infected human.

Laboratory diagnosis
Identification of M. tuberculosis
Specimen: Sputum; pleural, peritoneal and cerebrospinal fluid
Smear: Acid fast bacilli from primary specimen.
Cord forming acid-fast bacilli from culture.

Culture
Lowenstein-Jensen medium
It is the ordinary selective media for tubercle bacilli
Raised, dry, cream colored colonies of tubercle bacilli after 3-6 weeks of incubation.

Biochemical reaction
Niacin Test
The niacin test has been widely used since the 1960s to identify mycobacteria at the species
level in the clinical laboratory. The niacin test detects niacin (nicotinic acid)
in aqueous extracts of a culture. M. tuberculosis strains that test negative for the niacin test
are very rare. Redox reactions happening in Mycobacterium species produce niacin as a part
of energy metabolism. Even though all mycobacteria produce niacin, M.
tuberculosis accumulates an excess of niacin because of its inability to process niacin,
excreting the excess niacin into the culture media, thus allowing it to be detected using the
niacin test. The niacin test is typically only conducted on slow-growing, granular, tan
colored colonies, as these are the morphology characteristics of M. tuberculosis on an agar
plate.

Figure (3-57) : Mycobacterium tuberculosis (Niacin test), A negative niacin test on

the left, a positive niacin test on the right.

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 New techniques
1. Molecular probes (DNA probes)- It detects Mycobacterial RNA sequence.
2. High-performance liquid chromatography.
3. Polymerase chain reaction.
4. Enzyme immunoassay.

Figure (3-58) : Mycobacterium tuberculosis (Ziehl-Neelsen stain)

Mycobacterium leprae
Characteristics
1. Typical acid-fast bacilli, arranged in singly, parallel bundles or in globular masses.
2. Not grown in non-living bacteriologic media.
3. Characteristic lesions are grown in laboratory animals. Ex. Foot pads of mice.

Laboratory diagnosis
Specimen: Skin scrapings from the ear lobe.
Smear: Acid fast bacilli from the primary specimen.

Bacterial index (BI) indicates number of organisms present in a smear

Number of M. leprae bacilli found in smears are related to type of leprosy and effect of drug
therapy.This is an expression of the extent of bacterial loads. It is calculated by counting six
to eight stained smears under the 100 x oil immersion lens. in a smear made by nicking the
skin with a sharp scalpel and scraping it; the fluid and tissue obtained are spread fairly
thickly on a slide and stained by the Ziehl-Neelsen method and decolorized (but not
completely) which 1% acid alcohol. The results are expressed on a logarithmic scale.

 1+ At least 1 bacillus in every 100 fields.

 2+ At least 1 bacillus in every 10 fields.
 3+ At least 1 bacillus in every field.
 4+ At least 10 bacilli in every field.
 5+ At least 100 bacilli in every field.
 6+ At least 1000 bacilli in every field.

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SPIROCHETES
GENUS: TREPONEMA
Species of medical importance:
1. T. pallidum causes syphilis
2. T. pertenue causes yaws
3. T. carateum causes pinta
4. T. endemicus causes bejel

Treponema pallidum
Characteristics
1. Slender spiral, microaerophilic gram-negative rods.
2. Not cultured in artificial media, in fertilized eggs and tissue culture, but the
saprophytic Reiter strain grows in anaerobic culture.
3. Actively motile, rotating steadily around their endoflagella.
4. Remain viable in the blood or plasma store at 4 c0 at least for 24 hrs (transmitted via
blood transfusion).

Laboratory diagnosis
Specimen : Tissue from skin lesion
1. Dark field microscopy
Motile spirochetes in dark field illumination are observed.

2. Immunofluorescence stain Procedure:

• Put tissue fluid on a glass slide.
• Fix and stain with fluorescein-labeled anti treponeme serum.
• Observe fluorescent spirochetes in Immuno-fluorescence microscopy.

3. Serological tests for syphilis (STS).

Specimen: Serum
a. Non-treponemal antigen tests Antigen- Cardiolipin from beef heart.
1. Flocculation test :VDRL , RPR
2. Positive after 2-3 wks of untreated syphilitic infection.
3. Complement fixation test: Wasserman test; Kolmer test.

b. Treponemal antibody tests

1. Fluorescent treponemal antibody-absorption test (FTA-abs).
2. Treponema pallidum- particle agglutination test (TP-PA).
3. Treponema pallidum immmobilization test (TPI).

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Medical mycology and laboratory diagnosis
Mycology is the study of fungi, which are eukaryotic organisms. Fungal infections are
mycoses.

Collection of specimens:
Superficial mycoses:
1. The dermatophytic lesions usually collected by scraping outward from edges of lesion
with scalpel blade held at an angel of 90 deg to skin surface.
2. Hair should be plucked from scalp with the help of forceps.
3. Nail should be taken from discoloured part of the nail. We need to collect full thickness of
the nail and as far back as possible from the edge of the nail.

Subcutaneous mycoses:
The scrapings or crusts from superficial parts of subcutaneous lesions is enough for culture.
Systemic mycoses:
Here generally samples like pus, faeces, sputum, spinal fluid, blood and scrapings or swabs
from the edge of the lesions are considered. Most of the specimens can be examined in wet
mount after partial digestion with 10-20% KOH.

Laboratory Diagnosis
Laboratory diagnosis of fungal infections depends on:
1. Direct microscopy
2. Culture
3. Serological tests
4. Nonculture methods
5. Molecular methods

Microscopical Examination KOH Potassium hydroxide

KOH is the preliminary step for any samples that come for fungal examination. 10% KOH is
used for hair, tissues, skin scrapings and 20% KOH is used for nail clippings or any sample
which is difficult to dissolve.
Principle
KOH may be used to examine hair, nails, skin scrapings, fluids, exudates, or biopsies. KOH
helps in dissolving human cellular debris and helps in easier visualization of fungal
elements. Specimens placed in a drop of 10% KOH will dissolve at a greater rate than fungi
because fungi have chitinous cell walls. The clearing effect throughout the clinical specimen
can be accelerated by gently heating the KOH preparation.
Procedure
Emulsify the specimen in a drop of 10% KOH on a microscopic slide with the help of a
loop. Cover the smear with the cover slip. Leave it for 5-10 minutes. Examine the slide
under low(10X) and high power (40X) magnifications. Examine the slide for 15-20 minutes
for demonstration of shining fungal elements. .

261
Results and interpretations
Different fungi will have different morphological forms (yeast cells with pseudo hyphae,
budding septate and aseptate hyphae, granules, etc.) which can be clearly seen in a KOH wet
mount.

Gram stain for fungi

Gram stain is done mainly for the staining of Candida spp. The procedure is same as Gram
stain explained in bacteriology .The Candida spp looks like gram positive budding yeast
cells.

India ink test - demonstration of capsule

Principle: India ink is used as a negative stain preparation. When used in wet mount
preparation of the CSF, the background appears black, and the unstained capsule of
Cryptococcus neoformans appears as a white halo around the yeast cells in microscopy.

Lacto Phenol Cotton Blue Preparation (LPCB)

Lacto phenol cotton blue is the most commonly used method adopted in mycology
laboratory to identify filamentous fungi.
Principle
Identification of filamentous fungi is made by their characteristic microscopic morphology
such as shape, size, arrangement of spore and hyphae. LPCB can be used for demonstration
of fungal elements in
a. Scotch tape preparation
b. Slide culture preparation
 Phenol: It acts as a disinfectant by killing any living organisms
 Lactic acid: To preserve the fungal structures
 Cotton blue: To stain or give color to the chitin on the fungal cell wall and other
fungal structures The stain will give the fungi a blue-colored appearance of the fungal
spores and structures, such as hyphae.
 Calcofluor dye is a fluorescent dye that combines with fungal cell wall and is useful in
identification of fungi in tissue specimens.
 Methenamine silver stain is useful for demonstration of fungi in tissues.

Fungal culture
Fungal culture is a frequently used method for confirming the diagnosis of fungal infection.
SDA is the most commonly used medium for fungal culture. Other media include CHROM
agar, blood agar, etc. The low pH of the medium and addition of chloramphenicol and
cycloheximide to the medium inhibit the growth of bacteria in the specimen and thereby
facilitate the appearance of slow-growing fungi.
Fungal colony is identified by rapidity of growth, color, and morphology of the colony at the
obverse .
Microscopy of the fungal colony is carried out in lactophenol cotton blue (LPCB) mount to
study the morphology of hyphae, spores, and other structures. The appearance of the
mycelium and the nature of the asexual spores are very much helpful to identify the fungul
Infection.

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 Figure (3-59) : CHROM agar , Candida albicans → green Candida tropicalis → metallic blue

Germ tube test

Principle: Germ tube is an initial hypha from a sprouting conidia, spore or yeast. Formation
of germ tube can be demonstrated by inoculating serum with a small quantity of growth of
Candida spp.The suspension is incubated at 37 C0 for a minimum of 1 and half hours, after
which a drop is examined under the microscope for demonstration of germ tube.

Cutaneous Mycoses:
Diseases of the skin, hair, and nail. These infections are caused by a homogeneous group of
closely related fungi known as dermatophytes infect only superficial keratinized structures
such as skin, hair, and nail, but not deeper tissues .

Types of hair infections:

1. Ectothrix layer of arthrospores on the surface of hair shaft
2. Endothrix: The clusters of arthrospores are found entirely within the hair shaft
3. (Favus) is a chronic fungal infection of the scalp most commonly cause in children by
Trichophyton schoenleinii.
Wood‟s lamp examination should be performed routinely when dermatophytosis is
suspected. Wood‟s lamp examination is a useful technique. About 50% of M. canis strains
produce metabolites following hair invasion which fluoresce an apple-green colour when
illuminated by the lamp.

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 Figure (3-60) : The three patterns of hair invasion and the causative dermatophytes

Figure (3-61) : Dermatophytic folliculitis. Ectothrix type: mycelia and arthroconidia are seen on
the surface of the hair follicle (extrapilary). Endothrix type: hyphae and arthroconidia occur within
the hair shaft (intrapilary).

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Opportunistic Fungal infections
 Candida albicans
 Aspergillus spp.
 Penicillium marneffei
 various Zygomycetes

Morphology and cultural characteristics of Candida albicans:

 Microscopically:Candida albicans is a dimorphic fungi,reproduce mainly by
budding,but can produce pseudohyphae and true hyphae.
 Culture: after 34 hr. at 37 C0 or room temperature, white,creamy,rounded colonies with
feet projection or regular margin appears. Asexual germination of candida occurs by
production of blastospores or chlamydiospores
 Germ tube test: To differentiate c. albicans from other spp.

C.albicans produce germ tube(true hyphae) when incubated with serum at 37 C0 for 90
minutes.

Figure (3-62) : Candida albicans on Sabouraud-Dextrose Agar at 48 hours at 30C

Figure (3-63) : Candida albicans retaining crystal violet stain from routine gram stain taken from
SAB Agar

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 Figure (3-64) : Api Candida

Method for antifungal disk diffusion

Susceptibility testing of Candida albicans Mueller-Hinton Agar + 2% Glucose and 0.5
μg/mL Methylene Blue Dye (GMB) Medium - Of the many agar media available,
supplemented Mueller-Hinton agar to be a good choice for routine susceptibility testing of
yeasts. The agar medium should have a pH between 7.2 and 7.4 at room temperature after
gelling. *Moisture on agar surface: If excess surface moisture is present, the agar plates
should be dried in an incubator.

Turbidity standard for inoculum preparation

To standardize the inoculum density for a susceptibility test,turbidity equivalent to a
McFarland standard or its optical equivalent should be used.

Inoculum preparation: Direct colony suspension method

1. All organisms need to be subcultured onto blood agar or Sabouraud dextrose agar to
ensure purity and viability. The incubation temperature throughout must be 35°C (±2°C).
2. Inoculum is prepared by picking five distinct colonies of approximately 1 mm in diameter
from a 24-hour-old culture of Candida species. Colonies are suspended in 5 mL of sterile(
0.85% saline).
3. The resulting suspension is vortexed for 15 seconds and its turbidity is adjusted either
visually or with a spectrophotometer by adding sufficient sterile saline or more colonies to
adjust the transmittance to that produced by a 0.5 McFarland standard .

Inoculation of test plates

 Optimally, within 15 minutes after adjusting the turbidity of the inoculum suspension, a
sterile cotton swab is dipped into the suspension. The swab should be rotated several
times and pressed firmLy against the inside wall of the tube above the fluid level. This
will remove excess fluid from the swab.
 The dried surface of a sterile Mueller-Hinton + GMB agar plate is inoculated by evenly
streaking the swab over the entire agar surface. This procedure is repeated by streaking
two more times,rotating the plate approximately 60° each time to ensure an even
266
 distribution of inoculum. As a final step, the rim of the agar is swabbed.
 The lid may be left ajar for three to five minutes, but no more than 15 minutes, to allow
for any excess surface moisture to be absorbed before applying the drugimpregnated
disks.

Application of disks to inoculated agar plates

Antimicrobial disks are dispensed onto the surface of the inoculated agar plate. Each disk
must be pressed down to ensure its complete contact with the agar surface.
Whether the disks are placed individually or with a dispensing apparatus, they must be
distributed evenly so that they are no closer than 24 mm from center to center. Ordinarily, no
more than 12 disks should be placed on a 150-mmplate, or more than five disks on a 100-
mm plate. Because the drug diffuses almost instant aneously,a disk should not be moved
once it has come into contact with the agar surface. Instead, place a new disk in another
location on the agar.
Disk should be placed no less than 10 mm from the edge of the petri dish.The plates should
inverted and placed in an incubator set to 35°C (± 2°C) within 15 minutes after the disks are
applied. Reading plates Examine each plate after 20 to 24 hours of incubation.

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 CHAPTER
4

LABORATORY DIAGNOSTIC METHODS

VITEK 2 Compact
Fully automated microbial identification system .The efficiency of the VITEK 2 Compact
systems relies on the advanced colorimetry technology, the system reads the latest generation
VITEK test cards – containing 64 wells to ensure accuracy every 15 minutes using three
different wavelengths. With this technique, more data is analyzed, which increases results‟
accuracy .

Principle
The Vitek 2 Compact (30 card capacity) system uses a fluorogenic methodology for
organism identification and a turbidimetric method for susceptibility testing using a 64 well
card that is barcoded with information on card type, expiration date, lot number and unique
card identification number.
Test kits available include ID-GN (gram negative bacilli identification), ID-GP (gram
positive cocci identification), AST-GN (gram negative susceptibility) and AST-GP (gram
positive susceptibility).
The Vitek 2 ID-GN card identifies 154 species of Enterobacteriaceae and a select group of
glucose non-fermenting gram negative organisms within 10 hours.
The Vitek 2 ID-GP card identifies 124 species of staphylococci, streptococci, enterococci
and a select group of gram positive organisms within 8 hours or less. The Vitek 2
Antimicrobial Susceptibility Tests (AST) is for most clinically significant aerobic gram
negative bacilli, Staphylococcus spp., Enterococcus spp., and Streptococcus agalactiae.
Susceptibility results are available for bacteria in less than 18 hours.

There are currently four reagent cards available for the identification of different organism
classes as follows:
1. GN - Gram-negative fermenting and non-fermenting bacilli
2. GP - Gram-positive cocci and non-spore-forming bacilli
3. YST - yeasts and yeast-like organisms
4. BCL - Gram-positive spore-forming bacilli.

Specimen
Pure isolates of organisms to be tested may be taken from Trypticase Soy Agar with 5%
sheep blood (BAP), chocolate agar, Maconkey, , and Columbia Sheep Blood Agar (CBA).

268
Figure (3-65): The Vitek 2 Compact system

Figure (3-66) : VITEK 2 Identification Cards

269
ANTIGEN ANTIBODY REACTIONS
ANTIGEN ANTIBODY REACTIONS
Antigen (Ag): Antibody(Ab) reactions are characterized by the following general properties:
• Specific: Involves specific interaction of epitope of an antigen with the corresponding
paratope of its homologous antibody.
• Non-covalent interactions exist between antigen and its antibody such as:
○ Hydrogen bonds
○ Electrostatic interactions
○ Hydrophobic interactions
○ van der Waal forces
• Strength: The strength or the firmness is influenced by the affinity and avidity
○ Affinity: It refers to sum total of non-covalent interactions between a single epitope of
an antigen with its corresponding paratope present on antibody. It can be meas- ured
by: (i) by equilibrium dialysis and (ii) by surface plasmon resonance method
○ Avidity: It is a term used to describe the affinities of all the binding sites when mul-
tivalent antibody reacts with a complex antigen carrying multiple epitopes.

Marrack’s Lattice Hypothesis

When the sera containing antibody is serially diluted (in normal saline), gradually the
antibody level decreases. To such a set of test tubes containing serially diluted sera, when a
fixed quantity of antigen is added, then:
• In the middle tubes, Ag-Ab reaction occurs at its best, because the amount of antigen and
antibody are equivalent to each other (zone of equivalence).
• In the earlier test tubes, antibodies are excess, hence the Ag-Ab reaction does not occur:
This is called as prozone phenomenon.
• In the later test tubes, antigen is excess, hence the Ag-Ab reaction fails to occur: This is
called as postzone phenomenon.
This lattice hypothesis holds true for any Ag-Ab reactions.

TYPES OF ANTIGEN-ANTIBODY REACTIONS

• Conventional techniques: Precipitation, Agglutination reaction, Complement fixation test

and Neutralization test
• Newer techniques: ELIS, IFA, RIA, CLIA, Immunohistochemistry, Rapid tests (Lateral
flow assay or ICT and Flow through assay), Western blot and Immunoassays using
electron microscope.

Precipitation Reaction
When a soluble antigen reacts with its antibody in the presence of optimal temperature, pH
and electrolytes (NaCl), it leads to formation of the antigen-antibody complex in the form of
:
• Insoluble precipitate band when gel containing medium is used or
• Insoluble floccules when liquid medium is used.

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 A. Precipitation in Liquid Medium
• Ring test: Streptococcal grouping by Lancefield technique, and Ascoli‟s thermoprecipita-
tion test done for anthrax.
• Slide flocculation test: VDRL and RPR tests used for diagnosis of syphilis.
• Tube flocculation test: Kahn test used previously for syphilis.

B. Precipitation in Gel (Immunodiffusion)

It has many advantages over liquid medium: (i) Clear visible bands are formed, which can
be preserved for longer time, (ii) Individual antigens from a mixture can be
differentiated, e.g.
• Single diffusion in one dimension (Oudin procedure)
• Double diffusions in one dimension (Oakley–Fulthorpe procedure)
• Single diffusion in two dimensions (Radial immunodiffusion)
• Double diffusions in two dimensions (Ouchterlony procedure), e.g. Elek‟s test
(diphtheria toxin) and Eiken test (E.coli toxin).

C. Precipitation in Gel in Presence of Electric Current

The movement of Ag and Ab can be made faster if immunodiffusion in gel is carried out in
presence of electric current. Examples include:
• Electroimmunodiffusion (EID)
• CIEP (Countercurrent immunoelectrophoresis)
• Rocket electrophoresis.

Agglutination Reaction
When a particulate or insoluble antigen is mixed with its antibody in the presence of
electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated.
Agglutination is more sensitive than precipitation test and the clumps are better visualized
and interpreted.

Diagnostic Applications of Agglutination Tests

Slide agglutination: To confirm the identification and serotyping of bacterial colonies
grown in culture.

Tube agglutination: is routinely used for:

• Typhoid fever (Widal test): Detects Ab against both H (flagellar) and O (somatic) Ag
• Acute brucellosis (Standard agglutination test)
• Blood grouping (ABO and Rh grouping)
• Coombs test or Antiglobulin test: Detects incomplete Rh antibodies:
○ Direct Coombs test: Detects bound Rh antibodies in fetus/baby‟s serum
○ Indirect Coombs test: Detects free Rh antibodies present in maternal serum.
• Heterophile agglutination tests:
○ Typhus fever (Weil–Felix reaction)
○ Infectious mononucleosis (Paul Bunnell test)
○ Mycoplasma pneumonia (Cold agglutination test).

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Microscopic agglutination test (MAT) for leptospirosis
Indirect or passive agglutination test: Antigen is coated on carriers such as Latex or RBCs
to detect Ab in serum. Examples:
• Indirect hemagglutination test (IHA)
• Latex agglutination test (LAT) for antibody detection‟ e.g. ASO.

Reverse passive agglutination test: Antibody is coated on carriers such as Latex or RBCs
to detect Ag in serum. Examples:
• RPHA (Reverse passive hemagglutination assay), e.g. HBsAg detection
• Latex agglutination test for antigen detection, e.g. CRP, RA factor, capsular antigen in
CSF and streptococcal grouping.
• Coagglutination test (here, S. aureus protein A is used as carrier).

Complement Fixation Test

CFT detects complement fixing antibodies in patient‟s serum. It is now almost obsolete:
• Wasserman test was the most popular CFT, used for the diagnosis of syphilis.
• CFT was also widely used for detection of antibodies in Rickettsia, Chlamydia,
Brucella, Mycoplasma infections and some viral infections such as arboviruses, rabies,
etc.
• Indirect complement fixation test: Detects certain avian (e.g. duck, parrot) and
mammalian (e.g. horse, cat) serum antibodies which cannot fix guinea pig complement.
• Conglutination test: To perform CFT using nonhemolytic complements, e.g. horse
complements.

Complements are also used for various serological tests, other than CFT
such as:
• Treponema pallidum immobilization test (for detecting antibodies to T. pallidum)
• Sabin-Feldman dye test for detecting Toxoplasma antibodies.
• Vibriocidal antibody test for V. cholerae.

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Figure (3-67) : Complement fixation. Left: Positive reaction (i.e., the patient‟s serum
contains antibody). If a known antigen is mixed with the patient‟s serum containing antibody
against that antigen, then complement (solid circles) will be fixed. Because no complement
is left over, the sensitized red cells are not lysed. Right: Negative reaction. If a known
antigen is mixed with the patient‟s serum that does not contain antibody against that antigen,
complement (solid circles) is not fixed. Complement is left over and the sensitized red cells
are lysed. Ab, antibody; Ag, antigen.

Figure (3-68) : Complement Fixation Test in Microtiter Plate

• Non-hemolysis------- Positive result----- Ab against virus in patient serum
• Hemolysis-------------Negative result----No Ab against virus in patient serum.

Neutralization Test
Neutralization tests are also less commonly used in modern days. Examples include:
• Viral neutralization test: Detects viral neutralizing antibodies
• Plaque inhibition test: Done for bacteriophages.
• Toxin-antitoxin neutralization test:
○ Schick test for Corynebacterium diphtheriae.
○ Nagler‟s reaction: Due to α-toxin of Clostridium perfringens
○ ASLO detection in past (now it is done by latex agglutination)
• Hemagglutination inhibition (HAI) test.

NEWER TECHNIQUES OF ANTIGEN ANTIBODY REACTION

The newer techniques use a detector molecule to label antibody or antigen which in turn
detects the corresponding antigen or the antibody in the sample by producing a visible effect.
Most of the newer techniques use the same principle, but they differ from each other by the
type of labeled molecule used and the type of visible effect produced.

Abbreviation Immunoassay Molecules used for Type of visible effect

method labeling
ELISA Enzyme linked Enzyme Color change is detected by
immunosorbent spectrophotometer
assay
IFA Immunofluorescen Fluorescent dye Emits light, detected by
ce Assay fluorescence microscope

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Abbreviation Immunoassay method Molecules used for Type of visible effect
labeling
RIA Radioimmunoassay Radioactive isotope Emits β and γ radiations,
detected by β and γ
Counters
CLIA Chemiluminescence- Chemiluminescent Emits light, detected by
linked immunoassay compounds luminometer
IHC Immunohistochemistry Enzyme or Fluorescent Color change (naked
dye eye) or Fluorescence
microscope
WB Western blot Enzyme Color band (naked eye)

Rapid test Immunochromatographic Colloidal gold or silver Color band, (naked eye)
test
Flow through assay Protein A conjugate Color band, (naked eye)

IEM Immunoferritin Electron dense Appears as black dot

electron molecules (e.g under electron
microscopy ferritin) microscope

ELISA (Enzyme Linked Immunosorbent Assay)

This method can be used for the quantitation of either antigens or antibodies in patient
specimens. It is based on covalently linking an enzyme to a known antigen or antibody,
reacting the enzyme-linked material with the patient‟s specimen, and then assaying for
enzyme activity by adding the substrate of the enzyme. The method is nearly as sensitive as
RIA yet requires no special equipment or radioactive labels.

Figure (3-69) : Enzyme-linked immunosorbent assay (ELISA). The term enzyme-linked

refers to the covalent binding (linking) of an enzyme to antibody to human IgG. If the patient
has antibodies to the microbial or viral antigen, those antibodies will bind to the microbial or
viral antigens. The antibody to human IgG linked to the enzyme will then bind to the
patient‟s antibodies. Then when the substrate of the enzyme is added, the substrate changes
color, indicating that the patient‟s serum contained antibodies.

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For measurement of antibody, known antigens are fixed to a surface (e.g., the bottom of
small wells on a plastic plate), incubated with dilutions of the patient‟s serum, washed, and
then reincubated with antibody to human IgG labeled with an enzyme (e.g., horseradish
peroxidase). Enzyme activity is measured by adding the substrate for the enzyme and
estimating the color reaction in a spectrophotometer.
The amount of antibody bound is proportional to the enzyme activity. The titer of antibody
in the patient‟s serum is the highest dilution of serum that gives a positive color reaction.

ELISA is so named because of two of its components:

• Immunosorbent: It is an absorbing material used (e.g. polystyrene, polyvinyl), that
specifically absorbs the antigen or antibody present in serum.
• Enzyme is used to label one of the components of immunoassay (i.e. antigen or
antibody). ELISA is the method of choice in big laboratories as large number of samples
can be tested together using the 96 well microtiter plate. But it is not preferred in small
laboratories:
• It is economical, takes 2-3 hours (rapid tests take 10–20 min)
• ELISA is the most sensitive immunoassay, thus is the preferred screening test at blood
banks and tertiary care sites.
• Its specificity used to be low. But now, with use of more purified recombinant and
synthetic antigens, and monoclonal antibodies, ELISA has become more specific.
• It needs expensive equipments such as ELISA washer and reader.

Applications of ELISA test (mainly):

1- Measure of nanogram amounts.
2- Enzymeimmunoassays for antimicrobial Antibodies.
3- Antigens detection.
4- Quantity measure for hormones.
5- Quantity measure for drugs.
6- Pregnancy test (hCG Antigen).
7- Diagnosis of different infectious diseases.

ELISA type Used for detection of Enzyme is labeled with

Direct ELISA Antigen Primary antibody

Indirect ELISA Antibody or antigen Secondary antibody

Sandwich ELISA Antigen Primary antibody in sandwich direct ELISA

Secondary antibody in sandwich indirect ELISA

Competitive Antigen or antibody Secondary antibody

ELISA

ELISPOT Cells producing Primary antibody

antibody or cytokine

Note: Primary antibody is directed against the antigen, Secondary antibody is an anti-human
(or other species) Ig directed against Fc region of any human/other species Ig.

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 Figure (3-70) : Types of ELISA

Figure (3-71) : Direct ELISA

Figure (3-72) : Indirect ELISA(Antigen coated plate)

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Figure (3-73) : Competitive ELISA (Antigen coated plate)

Figure (3-74) : Sandwich ELISA (Antibody coated plate)

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Figur (3-75) : Microplate ELISA for HIV antibody colored wells indicate reactivity

Figure (3-76) : Elisa reader (spectrophotometry)

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MD MPH. Manual for the Laboratory Identification and Antimicrobial Susceptibility
testing of Bacterial Pathogens of Public Health Importance in the Developing World
(2003).

3. Abilo Tadesse, Meseret Alem.Medical bacteriology, University of Gondar (2006).

4. Review of Medical Microbiologx and Immunology ,Thirteenth Edition ,Warren

Levinson, MD, PhD Professor of Microbiology Department of Microbiology and
Immunology University of California, San Francisco San Francisco, California (2014)
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5. Review of Medical Microbiologx and Immunology , Sixth Edition, Apurba Sankar
Sastry MD (JIPMER) DNB MNAMS PDCR (2018) .
6. Streak Plate Method- Principle, Types, Methods, Uses, April 28, 2023 by Prashant
Dahal.
7. CLED Agar: Composition, Uses, Colony Characteristics ,Written by Acharya
Tankeshwarin Culture Media Last Updated November 5, 2022.
8. Streak Plate Method Principal and Types, by RBRFebruary 9, 2022Microbiology,
Microbiology Techniques.
9. Triple Sugar Iron (TSI) Agar: Principle, Results, and Interpretation,Written by
Acharya Tankeshwarin Biochemical Tests Last Updated November 5, 2022
10.WHO laboratory manual for the Examination and processing of human semen FIFTH
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12.A.Elmanama. Diagnostic Medical Microbiology.Medical Technology Department

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