2015

Diagnostic Microbiology
Laboratory Manual

Prepared by

Majdi F.Bilbisi Mcls

 CONTENTS

1. Staphylococci

2. Streptococci

3. Enterobacteriaceae

4. Nonfermentative gram Negative Bacilli.

5. Aerobic Gram positive Bacilli

6. Haemophilus

7. Campylobacter

8. Sexually Transmitted Diseases

9. Anaerobic organisms

10.Laboratory Procedure for Mycobacteria

11.Other Gram Negative Bacilli

12.Antimicrobial Susceptibility Testing.

13.Media Selection Guide

14.Appendix

15.Index.
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 Staphylococci

Staphylococci are gram-positive organisms; grow on any

nutrient medium containing peptone such as:
 Selective media include phenyl alcohol (PEA) agar
 Columbia nalidixic acid (CAN) agar
 Mannitol salt agar (MSA).
Staphylococci are facultative anaerobes, tolerate up to
10% sodium chloride.
Staphylococcus and Micrococcus species are the most
common isolates in the genera Micrococcaceae. These
two genera are differentiated by:

Micrococcus Staphylococcus
QF-dextrose O/Asaccharolytic Fermentative
Resistance to
Furazolidone (100 ug disk) + -
Bacitracin (0.04 ug disk) - +
Modified oxidase (6%) + -

Catalase Test

Most cytochrome aerobic & facultative anaerobic bacteria

possess the catalase enzyme. The catalase enzyme is capable
of degrading hydrogen peroxide to water and oxygen. The
presence of the enzyme is detected by adding H2O2to a
culture of the test organism and observing of bubbles of
oxygen.
Catalase
2H2O2 2H2O + O2

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 PROCEDURE:

A. 1. Transfer a colony of the test organism to a clean glass slide.

2. Add one drop of 3% H2O2, and observe for an immediate
development of bubbles.

B. Add 1 ml of 3% H2O2 to an ager slant culture of the test organism and again
C.

1. With a glass capillary tube (10cm×1.5mm) scrape a small amount of the colony
onto the end of the tube, taking care not to take of any of the underlying
medium.
2. Dip the other end of the onto 3% H2O2 and allow the tube to half-fill by
capillary action.
3. Invert the tube allowing the H2O2 to flow down into contact with the bacteria;
observe the production of gas.
4. Positive and negative controls should be run with the tested organism.
Observing for production of bubbles.

PRECATIONS:

1. Blood ager plates are not usually used for this test because red blood cells may
show catalase activity.
2. The order of the procedure (addition of H2O2to slide before organism) false
positive result. Nichrome wire does not cause bubbling.
3. Older colonies may lose their catalase activity, so they should be 18-24 hrs
cultures.
4. H2O2 solution must be fresh and kept refrigerated gentle shaking of the reagent is
done prior to its use.

All the Staphylococcus and Micrococcus species are catalase positive. These can be
differentiated from the streptococcus species which are catalase negative.

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 COAGULASE TEST

This test is used to distinguish coagulase producing Staphylococcus aureus from other
species of Staphylococcus. CNS (coagulase negative Staphylococci).

The enzyme coagulase exists in two forms. One form, bound coagulase, is bound to the
cell wall of S.aureus, is detected with a slide test, and is not present in broth culture
filtrates. Bound coagulase acts directly on fibrinogen to produce an insoluble fibrin clot.
The second form, extracellular coagulase, excreted by the cell, is detected with a tube test,
and is present in culture filtrates. The extracellular coagulase reacts with coagulase-
reacting factor (CRF) to produce coagulase CRF complex. This acts on fibrinogen to
produce an insoluble fibrin clot.

PROCEDURE:

Slide test for Bound Coagulase.

1. Emulsify a dense suspension of the test organism
in a drop 85% saline on a clean glass slide. If auto
agglutination occurs do not continue, use the tube
method.
2. Mix a loopful of undiluted ethylenediaminetetra
acetate (EDTA) treated rabbit plasma into
suspension and observe for formation of a white, flaky fibrin precipitate.
3. Development of the precipitate constitutes a positive test for production of bound
coagulase within 20 seconds.
Negative or delayed results (20-40 seconds) should be confirmed with the tube test.

Tube Coagulase Test:

1. Add 0.5 ml diluted rabbit plasma to a sterile tube.

2. Inoculate a loopful of the test organism growing on
an agar plate or (0.1 ml of a broth culture).
3. Incubate the culture at 35 c in a water bath and
observe every 30 minutes during the first 4 hours.
4. Evidence of clot formation, constitutes a positive
finding.
5. Negative tubes are incubated for overnight at room
temperature for delayed or weak coagulase.

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Quality Control

Tubes of plasma should be tested for proper reactivity by adding one drop of 5% CaCl2to
0.5 ml plasma and observe for clotting. Coagulase positive staphylococci should be run
with the tested organism.

PRECAUTION:

1. Young cultures should be used.

2. Fresh plasma id unstable and may itself coagulase.
3. Plasma should be not filtered.
4. Normal quality control organism should be tested. This problem can be alleviated by
diluting the plasma.
5. Slide test id only a presumptive procedure.
6. Do not use growth from an inhibitory medium containing salt; e.g., mannitol salt agar
due auto agglutination.
7. When performing the tube method do not shake or agitate the tube.
8. Citrated plasma are not recommended since certain species may use the citrate and
give false positive test.

DNase Test:

This is an extracellular enzyme produces by S. aureus, anuclease (end nuclease) which is

specific for hydrosis of nucleic acid.

PROCEDURE:

1. Inoculate (spot inoculation) a heavy inoculate into the center of the DNase agar
plate, or Band line streak inoculations is used.
2. Incubate at 35 c for 24-hrs.
3. Add 1 N HCl reagent directly to incubated plates.
4. Clearing zone surrounding the inoculums constitutes a positive test. While no
clearing around the colonies or hazy precipitate around the colony shows a
negative test.

Positive control: Nonpigmented Serratia marcescens and negative control other

Enterobacteriaceae should be run.

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FURAZOLIDONE DISK TEST

This test is performed as a disk susceptibility. Staphylococci are susceptible to this compound while
Micrococci are resistant.

PROCEDURE:

1. Prepare a 0.5 McFarland turbidity standard from the tested organism.

2. Spread the organism suspension onto one half of a blood agar plate.
3. Aseptically place an FX disk (100ug) in the center of the inoculated area.
4. Incubate at 35c for 24 hrs.
Micrococcus species are resistant while Staphylococcus are sensitive, more than 15 mm in
diameter.

NOVOBIOCIN TEST

The coagulase negative staphylococci (CNS) can be divided into novobiocin susceptible and resistant
species Staphylococcussaprophyticus is one the urinary tract pathogens which is usually resistant to
5 ug disks of novobiocin.

PROCEDURE:

1. Prepare a 0.5 Macfarland turbidity standard of the tested organism.

2. With a sterile swab spread the suspension of the organism on a half of a blood agar plate.
3. A septically place a novobiocin disk on the inoculated area & gently tamp the disk with sterile
forceps to ensure contact with the agar surface.
Zones of 16 mm or more are considered as susceptible and the organism is a CNS other
thenStaphylococcussaprophyticus.

STAPHAUREX TEST

Testing of protein A is an alternative to the coagulase slide test. This is good for testing sticky
colonies or autoagglutinators.

Polystyrene latex coated with human fibrinogen and immunoglobulin G will react with both clumping
factor and protein A to create an agglutination reaction.

This is a rapid test which gives results within 30 seconds.

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PROCEDURE:

1. Place a drop of milky regent in the circular black region of the reaction card.
2. Transfer a few suspect staphylococcal colonies to the card, and mix them with wooden
applicator stick.
3. Rotate the card gently for 20 seconds and observe for agglutination. S.aureus will produce
positive test and CNS will produce negative result.

Staphylase test:

This is another alternative to the coagulase test. Red blood cells are sensitized with the
immunoglobulin G instead of latex particles. Procedure and results are interpreted the same as
staphaurex test.

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 STREPTOCOCCI

Streptococci are gram positive cocci, facultative anaerobes, catalase negative, grow in pairs, short or
long chains. Hemolytic patters of the Streptococci

Alpha (α) Greenish or brownish zone surrounding colony indicates partial hemolysis of erythrocytes.

Beta (β) clear or yellowish zone surrounding colony indicates complete hemolysis of erythrocytes.

Gamma (γ) No zones of hemolysis.

Lance field has divided the B-hemolytic isolates into groups (A-G).

Species Lance field Group Main Habitat

S.pyogenes A Human
S. agalactiae B Human & Cattle
S. equisimilis C Human, many animals
S. equi C Horses
S. anginosus(melleri) F,G Human
Enterococcus faecalis D Human
Enterococcus durans D Dairy Product, milk

Once an isolate has been determined to be a member of the genus Streptococci either by Gram
staining, colony characteristics or catalase test. The following tests are recommended:

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Bacitracin and Trimethoprim Sulfamethoxazole Susceptibility Test

These two testes are used for the presumptive identification of groups A and B beta hemolytic
streptococci (BHS).

PROCEDURE:

1. Streak a pure culture of a β –hemolytic streptococcus onto each half of a sheep blood agar
plate.
2. Place a 0.4 unit bacitracin disk on one half of the inoculated area and an SXT
(sulfamethaxzole 23.75 ug\disk and trimethroprim 1.25ug\disk) on the other half.
3. Incubate the culture in air for 18-24 hrs at 35c.
Any zone of growth inhibition around either disk is interpreted as susceptible to the agents.

Bacitracin SXT Presumptive Identifications

S R Group A
R R Group B
R S Neither A or B
S S Rule out group A by
serologic tests.

CAMP TEST

This is used to determine the hemolytic phenomenon

which was described in 1944 by Christie, Atkins,
and Munch-Peterson, and it is their names that
provide the acronym CAMP for the test. The
hemolytic activity of B-hemolysin produced by most
Staphylococcus aureus in enhanced by an
extracellular protein produced by group B
streptococci. The interaction of the B-hemolysin
with this factor causes “Synergistic hemolysis”
which is easily seen on a blood agar plates.

PROCEDURE:
1. On the blood agar plate, make a single streak of B-hemolysin producing Staphylococcus aureus.
2. In a perpendicular position, streak at B-hemolytic streptococcus to be identified taking care not
to intersect the staphylococcal streak. N.B:different isolates can be streaked in parallel lines on
the same plate.
3. Incubate the plates at 35c for 18-24 hrs in an ambient air.
4. An increased hemolysis which is arrow shaped is produced at the intersect point. This test along
with Bacitracin and SXT resistant B-hemolytic streptococcus can be used to identify group
B(Streptococcus agalactiae).

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Bile esculin Test:

Certain bacteria are able to hydrolyseesculin(aglycoside)in the

presence of 4%bile salts.
The bile esculin positive bacteria are able to grow in the presence
of bile salts. Hydrolysis of esculin results in the formation of
glucose and esculetin which in turn, reacts with Ferric ions in the
medium and form a diffusable black complex.

PROCEDURE:

1. With an inoculated loop, streak a slant of bile esculin agar in

the form of a zigzag shape.
2. Incubate at 35c for 24 hrs in an ambient air incubator.
The formation of black diffusable color indicate the test is
positive.
Control= group D Streptococcus: Enterococcus faecalis are
positive, non group D streptococcus are negative.

SALT TOLERANCE TEST

Ability of the organism to grow in the presence of variable amounts of sodium chloride (NaCL), is
useful in the presumptive identification of the group D Enterococcus.

6.5% Nacl is incorporated either in a broth (TSB) or on an agar medium.

PROCEDURE

1. Inoculate the provided medium with the test organism.

2. Incubate for 24 hours at 35c.
Presence of obvious bacterial growth indicates a positive test (Enterococcus group) while
negative test indicate the organism is group D non enterococcus like Enterococcus bovis.

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PYR TEST PYR TEST
This is a rapid test used for the presumptive
identification of group A β-hemolytic Streptococci
and Enterococci. The substrate used for the PYR test
is L-pyrrolinodony-β -naphthylamide. This
compound is hydrolyzed by the bacterial specific
amino peptidase enzyme, which releases a free B-
naphthylamide byproduct.
This compound can be detected by the addition of
N,N-dime-thylaminocinnamaldehyde which couples
and form a red shift base.

PROCEDURE

1. PYR broth (Todd-Hewitt broth with (0.01%

PYR) dispensed into 2 ml volumes is
inoculated with the tested organism.
2. Incubate the tube at 35 c for 4 hours.
3. Add one drop of the PYR reagent and observe for color change.
4. The development of a deep cherry-red within a minute of reagent addition indicates
positive test. Yellow or orange color is considered negative.
HIPRATE HYDROLYSIS TEST

Group B beta hemolytic Streptococci can produce the enzyme hippuricase which hydrolyzes sodium
hippurate and leads for the formation of sodium benzoate and glycine.
This reaction can be detected by one of the following methods:
A. Benzoate Test
1. Inoculate a tube of sodium hippurate medium with the tested organism.
2. Incubate at 35 c for 4 hours.
3. Centrifuge the medium to pack the cell, pipette 0.8 ml of the suppurate into a clean test
tube.
4. Add 0.2ml of the FeCL3reagent and mix well.
5. A heavy precipitate will form upon the addition of the reagent, if this precipitate persists
beyond 10 minutes then the sodium benzoate has been formed and the test is positive.

B. Glycine Test

1. Inoculate the tested organisms into 0.4ml aliquot of 1% sodium hippurate reagent in a
small test tube.
2. Incubate the tube for 2 hours at 35 c.
3. Add 0.2ml of the ninhydrin reagent and mix well.
Appearance of a deep purple color within 10 minutes from the addition of the ninhydrin
reagent indicates presence of glycine in the mixture and thus the test is positive.
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Optochin is the compound ethylhydrocupreine hydrochlorides (aquinine derivative) which
selectively inhibit the growth of Streptococcus pneumonia (5ug/ml) and differentiate it from other
α-hemolytic streptococci (Resistant).
Filter paper disks impregnated with Optochin are used.

PROCEDURE:
1. Select 3-4 colonies of the tested organism and streak them onto one-half to a one third of
sheep blood agar plate.
2. With the help of a flamed forceps transfer an optochin disk (5ug) to the center of the
inoculated agar surface.
3. Incubate the culture for 18-24 hours at 35 in 5% CO2.
4. Examine the culture and look for the zone of inhibition.

susceptible 6mm disk 14mm zone

10mm disk 16 mm zone

resistant 6mm disk No zone

10mm disk No zone

N.B. Zones of 13mm with the 6mm disk and 15mm disks with the 10mm disk are questionable to
confirm the result bile solubility test should be done.

BILE SOLUBILITY TEST

Surface active agents such as bile salts, saponins, and other cationic detergents alter this surface of
S. pneumonia sodium salts (sodium taurocholate and sodium deoxycholate) activates the enzymes
autolysins (usually causing a central depression of old colonies) which results in the lysis of the cell.

PROCEDURE:

RAPID AGAR COLONY TEST

1. Add a loopful of 2% sodium deoxycholate solution to a growing colony on sheep blood agar
(bottom of the colony on the plate is marked).
2. Incubate the plate agar side up, at 35 c for 30 minutes.
Bile soluble (positive test) colonies disappear, leaving a partially hemolyzed area while bile
insoluble colonies remain intact& visible.

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BROTH TEST

1. Add 1 ml of dense physiologic saline suspension of the test organism to a test tube or 1ml of
an overnight. Todd Hewitt broth culture to the tube.
2. Add one drop of phenol red solution (0.04%) and adjust the pH to 7 with 0.1N NaOH.
3. Transfer 0.5ml aliquots of this mixture to each of two tubes.
4. Dispense 0.5ml of 10% sodium deoxycholate to one tube, labeled”test” and 0.5ml saline to
other tube labeled “control”. Shake each tube and incubate at 35c for 3 hours.
Clearance of the mixture in the test tube and turbidity in the control tube indicate the test is
positive and tested organism is bile soluble.

Positive and negative controls of Streptococcus pneumonia and viridian’s streptococci should be run
with the test.

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 ENTEROBACTERIACEAE
This is comprised of a large group of gram negative, non-spore forming bacilli that are oxidase
negative, catalase positive, ferment glucose with the production of acid and often gas. Reduce
nitrate to nitrite (with the exception of biogroups of Enterobacter agglomerans and members of the
genus Erwinia).

Many Genera of this family are inhabitants of the intestinal tract of humans and animals
(Escherichia, Enterobacter, and Klebsiella). Different selective as well as differential media are used
for the isolation of these organisms from clinical specimens. MacConkey agar and Eosin Methylene
blue agar considered as low selective agars.

Salmonella-Shigella (SS) agar, Deoxycholate Citrate agar, Hekten enteric (He) agar, and Xylose –
Lysine-deoxycholate (XLD) agar are said to be moderately selective while Brilliant green (BG) agar
and Bismuth sulfite (BS) agar are highly selective.

A similar growth is also obtained on blood agar by all the different species with large and dull gray to
watery or mucoid colonies. Hemolysis on blood agar is usually variable and not distinctive.

Proteus mirabilis, Proteus penneri, and Proteus vulgaris tend to swarm on blood and other moist
agar (thin film of growth on the surface).

Members of the Klebsiella, produce large, mucoid colonies which forms a string when touched with
a loop. Yersinia pestis grows from pinpoint colonies at 24 hrs to larger ones at 48 hrs.

BIOCHEMICAL IDENTIFICATION

From the first look at the colonies growing on the different types of media, it can be identified as
lactose or non-lactose fermenter, Escherichia, Enterobacter and Klebsiellawhich are examples of
lactose fermenters. So, their colonies appear colored due to the production of acids. While
Salmonella, Shigella, Proteus and Providencia are non-lactose fermenters and therefore their
colonies remain colorless. To rule out, these nonfermenter organisms whether they belong to the
Enterobacteriaceae or not. The oxidase or nitrate test should be performed. Gram staining id of no
value in the process of identification in this family.

OXIDASE TEST

Aerobic bacteria utilize the process of oxidative phosphorylation

for respiration and energy production. Cytochrome C oxidase is the
terminal cytochrome being produced.
Detection of the cytochrome oxidase system depends on the use of
reagents which are normally colorless but, become colored when
oxidized. Di-or tetramethyl-p-phenylendiamine dihydrochloride are
normally colorless but become purple or blue-black when oxidized.
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PROCEDURE:

To filter paper saturated with 1% solution of the above mentioned reagent, and with a
plastic needle or a wooden stick touch the tested colony and transfer it to the filter paper.
Development of a blue-black or purple color within 10 second is a positive test.

The test may be performed by dropping the reagent directly on colonies growing on blood or
chocolate agar.

The development of a purple or blue black color is positive. Reagent impregnated filter paper strips
or disks and disposable glass ampoules are also available.

All the Enterobacteriaceae are oxidase negative while many other bacteria like Pseudomonas,
Neisseria, Moraxella, Vibrio, Aeromonas, and Alkaligenes are positive.

PRECAUTIONS

1. Don’t perform the test on colonies growing on medium containing lactose because its
fermentation will inhibit oxidase activity and result in false negative BA, TSA, HIA and NA are
usually used.
2. Nichrome loop or needle may produce false positive results.
3. Don’t label the reagent as “Kovac’s” reagent only, but Kovac’s oxidase reagent the reagent
will auto-oxidize this can be reduced by the addition of 0.1% solution of ascorbic acid, if any
precipitate is formed the reagent is discarded.

INDOLE TEST:

Certain species of this family can oxidize tryptophan into indole, skatole and indole acetic acid.
Organisms are grown in tryptophan rich broth, when combined with certain aldehydes, indole yields
a red colored product.

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PROCEDURE

1. Inoculate a typotophan broth and incubate

for 24 hrs at 35c
2. Add 15 drops of Kovacs reagent down the
inner wall of the tube a red ring at the
interface of the reagent and the broth
indicate the test is positive.

METHYL RED

Methyl Red Test

The MR test is designed to detect organisms capable of
performing a mixed acid fermentation, which overcomesthe phosphate buffer in the medium and lowers the
pH . The acids produced by these organisms tend to be stable, whereas acids produced by other organisms
tend to be unstable and subsequently are converted to more neutral products.
Mixed acid fermentation is verified by the addition of methyl red indicator dye following incubation.

Methyl red is red at pH 4.4 and yellow at pH 6.2. Between

these two pH values, it is various shades of orange. Red
color is the only true indication of a positive result. Orange
is negative or inconclusive. Yellow is negative Glucose
supplied in the medium is metabolized by the bacteria
and produce a high yield of acids: lactic, succinic, acetic
and formic acid.The methyl red indicator is red at pH 4.4
and yellow at pH 6. So produce a positive test, the
organism should produce large quantities of acids from
the carbohydrate substrate.

PROCEDURER

1. Inoculate the MR/VP broth with a pure

culture of the tested organism.
2. incubate the broth at 35c for 48-72 hrs.
3. Add 5 drops of the methyl red reagent directly to the broth.
Development of a stable red color in the surface of the medium indicates sufficient acid
production to lower the pH to 4.4 and constitutes a positive test.
An orange color is considered as negative.

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VOGES-PROSKAUER TEST

Certain organisms can metabolize glucose to pyruvic acid. This is

further metabolized through a number of metabolic pathways,
depending on the enzyme system possessed by bacteria. One such
pathway results in the production of acetoin (acety-mehty1
carbinol). Organisms like Klebsiella-Enterobacter-Hafnia-Serratia
group produce acetoin as the chief and product of glucose
metabolism. In the presence of O2 and 4% KOH, acetoin is
converted to diacety1 and a –naphtho1 serves as a catalyst to bring
out a red color complex.

PROCEDURE

1. Inoculate a tube of MR/VP broth with a pure culture of the test organism.
2. Incubate at 35 c for 24 hrs.
3. Into 1 ml broth and 0.6ml of 5% a –naphthol followed by 0.2ml of 40% KOH.
Shake the tube to expose it into oxygen and allow it to stand for 10-15 minutes.
4. Development of red color indicate the presence of diacetyl and thus the test is positive.
Most entrobacteriaceae demonstrate either one or the other metabolic pathway, but rarely
both pathways.

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CITRATE UTILZATION:

This test is used to determine if an organism is capable of utilizing citratr as the sole source of
carbon for metabolism with resulting alkalinity.
Citrate medium is used. This citrate is cleaved to oxaloacetate and acetate oxaloacetates are
converted to pyruvates and CO2.
CO2 combines with sodium and water to from Na2CO3. An alkaline compound, the pH of thr
medium rises and the indicator (bromthymol blue) changes from green to blue.

Citrate permease
Sodium citrate Pyruvic acid + Oxalacetic acid + CO2
Citrase

Excess sodium from Sodium Citrate + CO2 + H2O Na2CO3 (pH ) (green blue)

PROCEDURE:
1. Inoculate the test organism to the surface of
citrate medium.
2. Incubate at 35 c 24-48 hrs.
The presence of blue color indicate positive test.

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KLIGLER IRON AGAR

Kligler iron agar is used to differentiate among-negative bacteria based on their ability to ferment
dextrose and to produce gas H2S. KIA and TSI are similar except the TSL contains 10g sucrose in
addition to glucose and lactose. The glucose is a 0.1% concentration, and lactose is 1.0%
concentration.

Some organisms are able to ferment glucose, glucose adnd lactose, others are unable to ferment both.
This type of fermentation may occur either with or without gas production (CO2 + H2S) fermentation
occurs both aerobically (on the slant) and anaerobically (in the butt).
This medium is dispensed in the tubes forming a butt and slanted positions.
Inoculations are usually performed by the stabbing of the butt and streaking of the slant with the same
needle. Reactions are usually read after 18-24 hrs),when the slant is red and the butt is yellow this is
one of the typical Results of the non-lactose fermenter Enterobacteriaceae/when the slant is Alkaline (
red ) this indicates the aerobic degradation of glucose has occurred. After 18-24 hrs of incubation the
low glucose concentration (0.1%) is completely used up and the organism stars to utilize the peptones
present in the medium to obtain its growth nutrients. .Catabolism of the peptone results in the release
of ammonia (NH3) yielding an alkaline pH with phenol red, the pH indicator incorporated in the
medium.
In the butt, a yellow color exists due to anaerobic degradation of glucose, acid end products are
formed yielding an acid pH and this acid reaction is maintained in the butt due to lower oxygen
tension. If the glucose-fermenting organism on KIA were read earlier, e.g., 12 hr or less, the slant
would be acid since in this short time period the glucose has not yet been completely depleted.The
something ‘if the tubes are not read until after incubation of 48 hrs or longer, the slant and butt may
both be alkaline (red). This indicates that the reaction is alkaline\alkaline and the result is interpreted
as false positive nonfermenter.

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Some organisms can ferment both glucose and lactose, and the tubes as yellow on both the butt and
the slant even after 18-24 hrs because of the 10 times the concentration of lactose to glucose and this
is sufficient to keep the pH in the acid rang but if results are read after 48 hrs, again they appear as
alk/alk.

Certain bacteria are unable to ferment both glucose and lactose because of their inability to derive
their nutrients from the carbohydrates present. They rely on peptone either aerobically or
anaerobically.

Gas (Co2+H2) is also detected as a result of carbohydrate metabolism, this is evident by splitting of
the medium, a single gas bubble, complete displacement of the medium from the bottom of the tube
leaving a clear area, or a slight indentation of the medium from the side of the tube.

In the medium, salts as ferric ammonium citrate and sodium thiosulfate are incorporated in order to
detect H2S production. This occurs as follows bacterium (acid environment) will act on sodium
thiosulfate which causes the production of H2S gas. This is a colorless gas which combines with
ferric ions forming ferrous sulfide which is an insoluble black precipitate. This precipitate may Mask
the acid condition produced in the KIA butt. Therefore, if H2S is produced an acid condition exists
even if not observable.

ONPG
For bacteria that ferment lactose but lake permease O-
nitrophenyl-B-D galactosidase test is useful for rapid
detection of the ability of an organism to ferment lactose.
The lactose fermenting organism possesses two distinct
enzymes:

1. Permease is responsible for the active transport of

the carbohydrate into the bacterial cell.

2. B-galactosidase degrades the lactose into glucose

and galactose. The galactose fermenting organisms
produce both enzymes especially when the substrate is available. Other bacteria possess B-
galactosidase and this appear as non-lactose fermenter in the standard lactose fermentation
tests. This test is used to detect this of organisms.

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The ONPG-P is a compound similar in structure to lactose. It enters the bacterial cell rapidly without
the aid of permease enzyme. Then, it is hydrolyzed by the B-galactosidase resulting in the formation
of galactose& orthonitrophenol. This compound is yellow at an alkaline pH. So, the development of
a yellow color indicates the presence of the enzyme B-galactosidase and indicates that the organism is
able to ferment lactose.

PROCEDURE:

1. Prepare a dense saline suspension of the tested organism.

2. Add one drop of toluene to the suspension. Mix thoroughly to enhance the release of B-
galactosidase.
3. Add 0.2ml of ONPG solution to the suspension & incubate at 35 c.
4. Examine the tubes for the development of a yellow color after 1 hour of incubation. If
negative, hold the tubes for 18-24 hrs and reexamine for color change.

N.B. ONOG-impregnated strips, disks and tables are commercially available.

CARBOHYDRATE FERMENTATION TESTS

One of the purposes of these tests is to determine the ability of an organism to ferment a specific
carbohydrate in a basal medium and produce acid or acid with visible gas.

A variety of carbohydrates may be utilized & generally 8-10 sugars are used: glucose, lactose,
sucrose, manitaol, dulcitol, salicin, adonitol, inositol, sorbitol, arabinose, raffinose, rahmnose, xylose,
trehalose, cellobiose, galactose, inulin, fructose and melibiose.

The sugar concentration is usually 1% (10g of sugar\liter of base).

The base is phenol red broth base with a pH of 7.4. this is yellow when the pH is 6 and red at 8.4 the
uninoculated media is reddish-orange.

The media is dispensed into tubes with 4-5ml and in the glucose tube insert a sterile Durham tube in
an inverted position to trap any gas produced.

PROCEDURE:

1. Roll a swab over the growth on KIA or other suitable culture after it has been moistened with
sterile saline.
2. Roll swab against the side of each carbohydrate just above the liquid level.
3. Slant each carbohydrate tube in order to pick up inoculum. Then shake each gently.

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 A single inoculum is usually sufficient a battery of 8-10 tubes of carbohydrates without any
flaming between inoculations. If needle or a loop is being used there is no problem of a tube
containing a mixture of carbohydrates.

4. Incubate at 35 c for 18-24hrs.

Development of yellow color in any tube indicates that the organism can ferment that particular
sugar.

UREASE TEST

This is an enzyme secreted by certain bacteria that hydrolyses

urea into ammonia.
Urea is provided as a substrate in liquid from like Stuart’s urea
broth with a phenol red as indicator or Christensen’s urea agar
slants.
3ml quantities of the broth are dispensed into serial tubes and 4-
5ml per tube to form a long slant and a short butt in case of the
solid medium.
1. Inoculate the urea broth or streak the slant of the urea agar do
not stab the butt.
2. Incubate at 35 c for 18-24hrs
Strong unease producer organisms can form positive results within 1-2 hrs.
Red color indicates that the test is positive.
Positive and negative control organisms should be run with each new batch of medium.

Positive Control- Proteus species

Positive (Weak)- Klebsiella species
Negative control- E.coil

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 Decarboxylases

Decarboxylases are a group of substrate specific enzymes which are capable of reacting with
the carboxyl portion of amino acids forming an alkaline amine.

The three amino acids routinely used are

lysine, ornithine and orginine, and their
specific amine products are:

Lysine Cadaverine
Ornithine Putrescine
Arginine Citruline

The conversion of arginine to citruline is a two-step method in which arginine is converted by

adihydrolase into citruline which is again converted to ornithine, which then undergoes
decarboxylation to form putrescine. Moeller decarboxylase medium is the base
Commonly used for the determination of decarboxylase activity.
The amino acid to be tested is incorporated into the media prior to inoculation.
Control tube should be run with the test tube, but it should be free of amino acids.
Both tubes (test control tube) should be incubated anaerobically by overlaying with mineral oil.
The medium contains 0.5g\L of glucose. During the initial stages of incubation, the organism starts
using the glucose, & then turns yellow. If the organism is able to decarboxylase the amino acid,
alkaline amine is formed & the medium revert to its original purple color.

PROCEDURE:

1. Inoculate tow tubes of Moeller decarboxylase medium one with the amino acid to be tested
and the other without to be used as a control.
2. Overlay both tubes with sterile oil to cover about 1cm of the surface
3. Incubate at 35 c for 18-24 hrs.
Formation of a yellow colorin the control tube indicates the organism is viable.
Reversion of test tubes to blue-purple color (original color) indicates the test is positive.
Any trace of purpul color denotes a positive test if it has been incubated for 24 hrs. if two layers are formed
one is yellow and the other is purple, mix the tube before attempting to read it. Although if it is difficult to
interpret, compare you’re your results to inoculated tubes.

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Tubes should be labeled properly prior to their use a C for Control, L,O and A. A positive control tube (purple)
invalidates the results.

PHENYLALANINE DEAMINATION TEST

This test is used to determine the ability of the bacteria to deaminate the
amino acid phenylalanine into the keto acid Phenylpyruvic acid. This
byproduct reacts with ferric chloride and forms a green product.

This test is used to differentiate Proteus, Morganella and Providencia

from other Enterobacteriaceae.

PROCEDURE:
1. Inoculate (streak) the agar slant with the tested organism.
2. Incubate a 35 c for 18-24 hrs.
3. Add 4-5 drops ferric chloride reagent directly to the surface of the slant – rotate the tube to dislodge
the surface colonies.
Immediate appearance of an intense green color indicates the presence of phenyl – pyretic acid and a
positive test.

GELATIN LIQUEFACTION TEST

Gelatin is a protein derivative of animal collagen. This substance is

added into several types of media to determine the ability of the
organism to produce photolytic – like enzymes which digest or
liquefies the gelatin present.

Denatured gelatin charcoal is used. Once this inoculated and

incubated for 24 hrs a positive test will cause the liquefaction, then
the charcoal particles will be released Nutrient gelatin stab medium
is also used, this is stabbed with a heavy inoculums & incubated at
35 c for 24 hrs.

Tubes are then placed in a refrigerator or ice bath for 2 hrs. then
observe for liquefaction which indicates a positive test. Uninoculated tubes are then compared with the
stabbed tubes to be used as a control.

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MOTILITY TEST

This test is performed to determine if the organism is motile on no

motile. A small amount of the agar is added to this medium to render
it semisolid. The medium is distributed into tube (5ml per tube), then
id is stabbed by the tested organism as a straight line. The initial
growth occurs along the line of inoculation, but the motile species of
bacteria are also able to move laterally away from the inoculation
line. This appear as a haze (Turbidity) around the inoculums.

1% of 2,3,5 Triphenyltetrazolium chloride (5m1/liter ) is added to the medium which causes reddening of
the medium in which bacteria are growing, so it facilitate the interpretation of the result. This colorless
soluble chemical is reduced to insoluble red pigment (formazan).
PROCEDURE:

1. With a sterile needle, touch a colony and stab the needle to the deep agar region
of the medium.
2. Incubate the media at 35 c for 24 hrs.
Diffuse growth around the line of inoculation indicates the test is positive.

LYSINE IRON AGAR (LIA)

This medium is useful in the study of gram

negative enteric bacilli. It permits the
differentiation of these organisms on the basis of
their ability to decarboxylase lysine,
deaminate lysine and produce hydrogen sulfide.
Acid/ alkaline: yellow butt and violet slant
indicate that the organism is glucose fermented
&cannot degrade the lysine.
Purple color throughout the medium = lysine
decarboxylation. This is due to production of
cadaverine.
A red slant shows that the organism can

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 delaminate lysine, yellow butt: organism is also glucose fermenter H2S causes blackening of the
butt same as the KIA.

PROCEDURE:

1. Stab the butt twice completely to the bottom in two different areas and streak the slant with a straight
wire.

Result: alk/Alk (k/K): Decarboxylation (anaerobic)

Alk / acid (K/A): Glucose fermenter only.
Red / Acid (A/A): Deamination (aerobic),

This test is useful for the identification of salmonella species which give H2S and lysine
decarboxylase (a black deep and a purple slant).

Also Proteus &Providencia species delaminate rather than Decarboxylate amino acids and
they form red slant.

Reactions in Lysine Iron Agar

Genus Slant Butt H2S

Arizona AIK AIK +
Salmonella AIK AIK +
Proteus Red A +or-
Providence Red A -
Citrobacter AIK A +
Escherichia AIK a/NC -
Shebelle AIK A -
Klebsiella AIK AIK -
Enterobacter AIK A -

A: acid (yellow)
Alk: Alkali (purple)
Nc: no change
H2S+ Blackening

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MOTILITY INDOLE ORNITHINE MEDIUM (MIO)

This medium permits the differentiation of organism on the basis of

their ability to produce insole from the amino acid tryptophan.
Decarboxylase the amino acid ornithine producing putrescence, and
demonstrate motility.

Motility and decarboxylase activity should be read before

determining insole production. Motility is indicated by generalized
turbidity or growth extending from the line of inoculation. Ornithine
decarboxylase activity is indicated by a purple color throughout the
tube. A yellow color in the bottom of the tube (even with a narrow
band of purple at the top) indicates a negative test.

Indole production is detected by adding three to four drops of Kava's reagent to the medium. The
development of red to pink color indicates insole production. When only small amounts are
produced, the color varies from flesh color to orange. A yellow color indicates a negative reaction.

PROCEDURE: stab the center of the medium completely to the bottom.

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 Biochemical identification of Enterobacteriaceae
I. Proteus
Biochemical test Proteus p. vulgar is p. penneri p. myxofaciens
Mirabilis Bio 2 Bios3
Indole - + + - -
Or. + - - - -
Decarboxylation
Esculin hydrolysis - + - - -
Maltose - + + + +
Xylose + + + + -
Chloramphenicol. S V S R S

II. Genus Serratia

Biochemical test S. marcescens S. liquefaciens s. fonticola S. ficaria S. polymathica
DNase (25c) + V - + +
Lipase + V - V V
Gelatinase at 22c + + - + V
Lysine + + + - -
Ornithine + + + - -
Odor of potatoes - - - + -
Arabians - + + + +

III. Genus Providencia

Biochemical p. alkalifaciens p. rustigianii p. stuartii p. rettgeri
Test
Urea hydrolysis - - V +
Citrate + - + +
Fermentation
Inositol - - + +
Adonitol + - - +
Trehalose - - + -
Galactose - + + +

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 IV. Genus Yersinia
Biochemical Y. pestis Y.pseudo- Y. Y. frederiksenii Y.
Test Tuberculosis enterocolitica intermidia
Indole - - V + +
Ornithine - - + + +
Motility 25c - + + + +
Sucrose - - + + +
Rhamonose - + - + +
Cellobiose - - + + +
Sorbital - - + + +

V. Enterobacter
Biochemical E. aerogenes E. cloacae E. sakazaki E. gergoviae
Test
Methyl red - - - -
VoguesProskauer + + + +

Lysine + - - +
Arginine - + + -
Ornithine + + + +
Urease - V - +
Motility = + + +
Fermentation + + + V
Of lactose
Sucrose + + + +
Adonitol + V - -
Orbital + + - -
Yellow- - - + -
Pigment

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 VI. Klebsiella

Biochemical Test k. pneumonia k.ozaenae k.oxytoca k.rhatinoscleromatis

Indole - - + -
Methyl red - + V +
Voges Proskauer + - + -
Urease + - + -
Lysine + V + -
Ornithine - - - -
ONPG + V + -
Malonate + - + +

VII. Citrobacter
Biochemical Test C.freundii C.diversus C.amalonaticus C.amaBiogroupl
Indole - + + +
H2S V - - -
Growth in + - + +
KCNmedium
Adonitol - + - -
Raffinose V - - +
Melibiose V - - +

VIII. Shigella

Biochemical Test S.dysentriae S.flexneri S.boydii S.sonnei

Orinithinedecar - - - +
Mannitol - + + +
Reffinose - D - -
Xylose - - D -

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