Diagnostic Principles in

Microbiology

Michael M. Navera, MD
Associate Dean
BUCM

Role of the Clinical Microbiology Laboratory

To

determine whether potential

pathogens are present in tissues,
body fluids or secretions of patients
and if present, to identify them.
To predict the course of an infection
Guide in the selection of appropriate
therapy.

Diagnostic Methods
(1)
(2)
(3)
(4)

Microscopic examination of patient

samples
Cultivation and identification of
microorganisms from patient samples.
Measurement of a pathogen-specific
immune response in the patient.
Detection of pathogen-specific
macromolecules in patient samples.

Laboratory Test Performance Assessment

No laboratory test is perfect.
To interpret the results of tests, clinicians must have a
sense of how reliable they are.

Test Results
True positive : A positive test in the presence of a
pathogen.
True negative : A negative test obtained in the absence of a
pathogen.

Inaccuracies:
False positive : A positive test in the absence of a
pathogen.
False negative : A negative test in the presence of a
pathogen

Sensitivity vs Specificity

Sensitivity (of the test) is the likelihood that the test is

positive when the pathogen is present.

Specificity (of the test) is the likelihood that it will be

negative if the pathogen is not present.

In clinical practice, diagnostic tests with 100% sensitivity

and 100% specificity do not exist.

Sensitivity and specificity values does not take into account

the prevalence of the infection in the population.

Sensitivity vs Specificity

true positives
Sensitivity = _____________________
true-positives + false-negatives

true-negatives
Specificity = _____________________
true-negatives + false-positives

X 100%

X 100%

Screening vs Confirmatory Tests

Screening tests used to rule out a suspected

diagnosis. E.g., EIA

Confirmatory tests used to rule in a suspected

diagnosis. E.g., Western blot

A test that is very sensitive but not particularly specific can

be useful in screening for the presence of an infection --E.g., VDRL, RPR

Predictive Value (PV)

PV - an expression of the likelihood that a given test result

correlates with the presence or absence of disease.

The PV of a diagnostic test is influenced by the frequency of

the infection in the population being tested.

The interpretation of a lab test result depends not only on the

technical accuracy of the method but also the prevalence of
the infection in the population to which the patient belongs.

Predictive Value
true-positives
Positive PV = _____________________
true-positives + false-positives

x 100%

true-negatives
Negative PV = _____________________
true-negatives + false-negatives

x 100%

Diagnosing Infections by Microscopy

Some

pathogens can be accurately

identified by direct microscopic
examination of clinical material.

For

certain infections, microscopic

diagnosis is highly sensitive and
specific.

rapid technique that sometimes

permits the physician to initiate
treatment without waiting for the
results of a culture.

Most

helminth and protozoal infections

are routinely diagnosed by microscopy.

Diagnosing Infections by Microscopy

Many

fungal pathogens also have

characteristic morphologic features.

Cryptococcus

neoformans meningitis is
diagnosed rapidly by finding encapsulated
yeast in CSF.

Diagnosing Infections by Microscopy

Syphilis can easily be diagnosed

by observing the helical form and
bending motions of spirochetes
from fresh scrapings of primary or
secondary lesions.

Diagnosing Infections by Microscopy

Viruses

cannot be seen in the light

microscope.

Virus-induced

changes in host cell

morphology may be diagnostic.

E.g.,

multinucleated giant cells in

scrapings from herpes simplex or
varicella-zoster virus lesions (the Tzanck
Smear) and the specific intracellular
inclusion bodies in tissues that are
actively infected by cytomegalovirus
(CMV)

What is a stain?
Stain

- is a chemical substance that adheres to a cell, giving

the cell color.

The

presence of color gives the cells significant contrast so are

much more visible.

Different

stains have different affinities for different

organisms, or different parts of organisms.

They

are used to differentiate types of organisms or to view

specific parts of organisms.

Bacterial

pathogens may be visualized and assigned to

morphologic and functional groups using special stains.

Principle of Staining

Bacteria have nearly the

same refractive index as
water, therefore, when
they are observed under a
microscope they are
opaque or nearly invisible
to the naked eye.
Bacteria are slightly
negatively charged at pH
7.0
Basic dye-stains bactria
Acidic dye stains
background

Different stains react or

concentrate in different
parts of a cell or tissue,
and these properties are
used to advantage to
reveal specific parts or
areas

Classes of Stains
Simple

stain provide the color

contrast but impart the same color
to all organisms in the smear.
Ex. Methylene blue, Basic fuchsin
Differential

Stain uses two or

more stains and allows the cells to
be categorized into various groups
or types.

Gram Stain
Named after Hans Christian Gram
Gram staining is a differential staining
technique that differentiates bacteria into
two groups: gram-positives and gramnegatives.

The Gram stain is a very important

preliminary step in the initial characterization
and classification of bacteria.

Is much less useful when a sample is

obtained from a nonsterile body site due to
microbiota contamination.

Gram Staining Steps

Crystal

violet acts as the primary stain.

Grams

iodine acts as a mordant (helps to

fix the primary dye to the cell wall.

Decolorizer

is used to remove the primary

stain (crystal violet) from Gram-negative
bacteria (those with LPS imbedded in their
cell walls). Decolorizer is composed of an
organic solvent, such as acetone or 95%
ethanol or combination of both.

Finally,

a counter stain, Safranin, is

applied to stain those cells (Gramnegative) that have lost the primary stain
as a result of decolorization.

Gram Staining Procedure

Step 1. Preparation. Smear and heat fix the bacteria as

described above (steps 1 through 3) for the simple stain.

Step 2. Primary stain. Cover the smear with crystal violet and
incubate for 30 seconds. Rinse the dye off with distilled water
from the squeeze bottle.

Step 3. Mordant. Cover the smear with grams iodine. After 20

seconds, rinse the slide with distilled.

Step 4. Decolorization. Rinse the stain with 95% ethanol. This

step must be done very carefully.

Step 5. Counterstain. Cover the bacteria with safranin for 30

seconds. Rinse with distilled water and dry.

Gram

positive bacteria:

Stain

dark purple due to

retaining the primary dye
called Crystal Violet in the
cell wall.

Gram

negative bacteria:

Stain

red or pink due to

retaining the counter staining
dye called Safranin.

Example:

Example:

aureus

Staphylococcus

Escherichia coli

Importance of Gram Staining

Gram stain can yield the following information:

1. The presence of bacteria in a normally sterile

body fluid (e.g., CSF, pleural fluid, urine).

2. Staining properties and morphology of the

organisms in a sample or culture that can direct
further efforts at species identification or the
empirical selection of antibiotics for the patient.

3. For certain clinical specimens, a diagnosis.

E.g., Gram-negative diplococci inside the
leukocytes of urethral pus.

Antibody-Based Identification
Enhances

the accuracy of microscopic

identification of bacterial and viral
pathogens.

Specific

antibodies are used in

conjunction with direct microscopy
involve the attachment (or conjugation)
of a detectable substance to antibody
molecules so that the microscopist can
see where the antibodies bind.

The

specificity of Ab-based identification

of pathogens depends on the specificity
of the antibodies used.

Direct Fluoresent Antibody (DFA) Test

For the detection of Cryptosporodium
protozoa in stool.
Uses a monoclonal antibody.
Examined using a fluorescent
microscope.
Cysts appear as bright yellow-green
glowing spheres when illuminated with
light of appropriate wavelength.

Diagnosing Infections by Culture

Culturing

the most specific way to

establish the presence of a particular
pathogen in a patient sample.

Culture

is routine for most bacterial and

fungal but is rarely performed for
helminthic and protozoal pathogens.

Some

m.o. cannot be cultured at all in

clinical microbiology lab (E.g., hepatitis
virus, T. pallidum, M. leprae and P.
jiroveci).

Chlamydia

and viruses are obligate

intracellular pathogens, they can be
propagated and identified only in
appropriate cell cultures.

Treponema pallidum

General Principles in Culture Method

The

choices of culture method and

medium are tailored to the nature and
source of the specimen and the question
that the culture is meant to address.

To

look for etiologic agents, the sample

can be:
a. cultured on a variety of agar based
media and in broth medium under
both aerobic and anaerobic conditions.
b. Cultured on a selective media when
the specimen is obtained from
nonsterile body sites (E.g., ThayerMartin)

General Principles in Culture Method

A negative culture does

not rule out the agent as
the cause. E.g., bacterial
pneumonia --- the
sensitivity of sputum for
this fastidious pathogen
may be 50% or lower in
some clinical settings.
Positive cultures always
raise the question of
whether the isolate is the
actual cause of the patients
illness.

In

cultures from
nonsterile body sites,
the problem is whether the
isolate represents normal
microbiota (colonization)
or an etiologic agent
(infection).

In

cultures from normally

sterile body sites,
contamination can occur
when patient samples are
obtained using faulty
sterile technique.

General Principles in Culture Method

Specimens obtained by needle

puncture are contaminated with
normal skin microbiota (e.g., S.
epidermidis, diphtheroids,
streptococci opportunists)

Almost all voided urine

collections have some degree of
contamination; true infection is
associated with a relatively high
concentration of bacteria in the
specimen.

Blood Culture

The simplest blood culture involves the

direct inoculation of blood sample into
nutrient broth, followed by incubation
at 37oC and periodic checks for an
indication of microbial growth.

Once growth is detected, m.o. from

cultures are transferred to agar plates,
or subcultured, to permit species
identification.

Lysis-centrifugation technique works

well for fungi, mycobacteria and certain
fastidious pathogens.

Culture Identification

Classically, bacteria cultured from

clinical specimens are identified by
determining phenotypic properties,
such as motility, utilization of
various nutrient substances,
production of enzymes, toxins, or
byproducts of metabolism.

m.o. in culture are sometimes

identified more rapidly using Abbased techniques.

Types of Culture Media

Media are of different types on
consistency and chemical composition.
A. On Consistency:
1. Solid Media.
Any liquid medium can be rendered
by the addition of certain solidifying
agents (e.g., agar, egg yolk, and
serum)
Advantages of solid media:
a. Bacteria may be identified by
studying the colony character
b. Mixed bacteria can be separated.

c. Solid media is used for the

isolation of bacteria as pure culture.

Types of Culture Media

2. Liquid Media.

These are available for use in testtubes, bottles or flasks.

Liquid media are sometimes referred

as broths (e.g., nutrient broth).

In liquid medium, bacteria grow

uniformly producing general turbidity.

Mixed organisms cannot be separated.

Types of Culture Media

3. Semisolid Media
Made

by reducing the amount of agar to 0.2-0.5%

renders a medium semi-solid.

Such

media are fairly soft and are useful in

demonstrating bacterial motility and separating
motile from non-motile strains (U-tube and
Cragies tube).

E.g.,

transport media: Stuarts and Amies media

Hugh

& Leifsons oxidation fermentation test

medium as well as mannitol motility medium
are also semi-solid.

Types of Culture Media

4. Biphasic media
Consists of both liquid and solid medium
in the same bottle.
E.g., Castaneda system for blood culture
B. On Chemical Composition:
(1) Routine Laboratory Media
(2) Synthetic Media.
These are chemically defined media
prepared from pure chemical substances.
It is used in research work.

Media Classification Based on Nutritional

Component
1. Simple media
can support most non-fastidious bacteria
E.g., peptone water, nutrient agar
2. Complex media
have ingredients whose exact
components are difficult to estimate.
E.g., blood agar
3. Synthetic or defined media
are specially prepared media for research
purposes where the composition of every
component is well known
such as Davis & Mingioli medium

Media Classification Based on Functional Use or

Application
1) Basal media
are basically simple media that supports
most non-fastidious bacteria.
E.g., peptone water, nutrient broth and
nutrient agar considered basal medium.
2) Enriched media
addition of extra nutrients in the form of
blood, serum, egg yolk etc, to basal medium
makes them enriched media.
enriched media are used to grow nutritionally
exacting (fastidious) bacteria.
E.g., blood agar, chocolate agar, Loefflers
serum slope

Classification Based on Functional Use or

Application
3) Selective Media

Agar-based media which

favor the growth of a
particular bacterium by
inhibiting the growth of
undesired bacteria and
allowing growth of
desirable bacteria.
Examples: MacConkey
agar, Lowenstein-Jensen
media, tellurite media

4) Indicator (differential)
Media
An indicator is included in the
medium.
different bacteria can be
recognized on the basis of
their colony colour.
E.g., : MacConkeys agar,
CLED agar, TCBS agar, XLD
agar

Classification Based on Functional Use or

Application
5. Transport Media

6. Storage Media

Media used for storing the

media are used when
specimen cannot be cultured soon
bacteria for a long period of
after collection.
time.
E.g., Egg saline medium, chalk
E.g., Cary-Blair medium, Amies
cooked meat broth.
medium, Stuart medium
These

Venkatraman

Ramakrishnan
medium (transport feces from
suspected cholera patients)

Sachs

buffered glycerol saline

(transport feces from patients
suspected to be suffering from
bacillary dysentery)

7. Anaerobic media
low

oxygen content, reduced

oxidation reduction potential
and extra nutrients.
supplemented with nutrients
like hemin and vitamin K.

Blood Agar
Most

commonly used medium. 5-10% defibrinated sheep or

horse blood is added to melted agar at 45-50C.
Blood acts as an enrichment material and also as an indicator.
Certain bacteria may or may not cause hemolysis when grown
in blood agar
Types of Hemolysis:
1) Beta haemolysis. The colony is surrounded by a clear zone
of complete haemolysis, e.g. Streptococcus pyogenes is a beta
haemolytic streptococci
2) Alpha (a) haemolysis. The colony is surrounded by a zone
of greenish discolouration due to formation of biliverdin, e.g.
Viridans streptococci,
3) Gamma haemolysis. No haemolysis. There is no change
in the medium surrounding the colony.

MacConkey Agar

Most

commonly used for enterobacteriaceae.

It contains agar, peptone, sodium chloride, bile salt, lactose
and neutral red.
It is a selective and an indicator medium
(1) Selective as bile salt does not inhibit the growth of
enterobactericeae but inhibits growth of many other bacteria.
(2) Indicator medium:
a. lactose fermenter - colonies of bacteria that
ferment
lactose take a pink color due to production of acid. Acid turns
the indicator neutral red to pink. e.g. Escherichia coli.
b. non-lactose fermenter - colorless colony indicates that
lactose is not fermented. e.g.
Salmonella, Shigella, Vibrio.

MacConkey

Blood Agar

Chocolate Agar or Heated Blood agar

Prepared by heating blood agar.
It is used for culture of pneumococcus,
gonococcus, meningococcus and
Haemophilus.
Heating the blood inactivates inhibitor
of growths.

Mueller Hinton Agar

Disc diffusion sensitivity tests for
antimicrobial drugs should be carried
out on this media as per WHO
recommendation to promote
reproducibility and comparability of
results.

Tellurite Blood Agar

It

is used as a selective medium

for isolation of Corynebacterium
diphtheriae.

Tellurite

inhibits the growth of

most secondary bacteria without
an inhibitory effect on diphtheria
bacilli.

It

is also an indicator medium as

the diphtheria bacilli produce
black colonies.

EMB (Eosin-methylene blue)

Agar
A

selective and differential

medium for enteric Gramnegative rods.

Lactose-fermenting

colonies are
coloured and nonlactosefermenting colonies are
nonpigmented.

Selects

against gram positive

bacteria.

SS (Salmonella-Shigella) Agar
It is a selective medium used to isolate
Salmonella and Shigella species.
SS Agar with additional bile salt is used
if Yersinia enterocolitica is suspected.

Motility Indole Urea (MIU) Medium

This is used to differentiate

enterobacteria species by their motility,
urease, and indole reactions.

Antimicrobial Sensitivity Testing

The most important advantage of culturing is that isolate can

be tested for susceptibility to antimicrobial agents.

Measuring the Antibody Response to Infection

Serologic tests
measure the patients
humoral immune response
to an infection.
are designed to measure
only those directed at
specific microbial antigens.

Formats for serology of

infectious diseases (i.e.,
agglutination, complement
fixation, neutralization, and
indirect fluorescent
antibody tests).

Positive titer the

highest dilution of the
patients serum that still
can exert the measured
function, or endpoint.

Immunoblots
Blotting
is a powerful and sensitive technique for
identifying the presence of specific
biomolecules within a sample.
Subtypes of blotting are differentiated by
the target molecule that is being sought.
The

basic principles underlying these

techniques are virtually identical.
(1) First, the target molecules (DNA, RNA, or
protein) are separated by a combination of their
size and charge using the appropriate method of
gel electrophoresis.
(2) Separated molecules are transferred to a
membrane; and third, the membrane is queried
with a probe directed against the specific
molecule of interest.

Immunoblots
Southern blot (Edward M. Southern)
detection specific DNA sequences
Northern blot
detection of RNA
Western blot
detection of protein
Eastern blot
detection of posttranslationally modified proteins
Southwestern blot
detection of DNA binding proteins