Diagnostic microbiology

Contents
• An overview
• Specimen, collection, and
processing

• Methods in diagnostic
microbiology
An overview
Diagnostic or clinical microbiology:
• Is the field of medical microbiology that deal with laboratory diagnosis of
infectious diseases caused by pathogenic microorganisms

• Diagnostic medical microbiology is concerned with identification of the

etiologic that cause infection and their treatment (susceptibility profile of
pathogens)

• Diagnostic microbiology is the back bone of medicine.

Identification of the organism causing an infectious process is usually
essential for effective antimicrobial and supportive therapy.

The patient's history and symptoms (sign and symptoms) provide the first
clues to the diagnosis of an infection.
• Diagnostic laboratory procedures are performed
basically to:
1. Confirm the diagnosis by identifying the agent
2. Determine appropriate antimicrobial and
supportive therapy.
SPECIMEN. SELECTION, COLLECTION, TRANSPORTATION
AND PROCESSING
Common samples used for isolation and identification of
pathogens
• Blood
• Urine
• Stool
• Rectal swab
• Sputum
• CSF
• Mycology samples
• Wound swabs
Specimen: is a sample or part of a thing or several things, which is
taken as a representative to show or determine the characteristics of
the whole.

• The proper collection, handling/ transportation and reliable

processing (procedure followed) of clinical specimen is a vital part of
medical laboratory diagnostic service .

On Specimen collection give attention on:-

• Type of specimen

• Time of collection

• Quality of specimen collected

• Specimen labeling
General information
1. Collect the specimen from the actual site of infection, avoiding
contamination from adjacent tissues or secretions.
3. Collect the specimen at optimal times (i.e. early morning sputum
for AFB culture).
4. Collect a sufficient quantity of material.
5. Use appropriate collection devices: sterile, leak proof specimen
containers.
6. Use appropriate transport media.
6. Whenever possible, collect specimens prior to administration of

antibiotics.

7. Properly label the specimen including the date, time etc.,

8. Minimize transport time.

9. If appropriate, decontaminate the skin surface.

• Use 70-95% alcohol and 1-2% tincture of iodine to avoid

contaminants (blood)
.
 Transport medium

• Allows organisms to survive

•Non-nutritive - does not allow organisms to
proliferate
•For bacteria – i.e., Cary Blair
•For viruses - virus transport media (VTM)
• -Amie 's transport Medium: Gonococcal infection
 Specimens collection (Summary)

General rules
It should be the right specimen;
Sufficient material;
Avoid the contamination of specimens;
Be sent to the lab immediately in an appropriate method
and examined ASAP.
Be collected before using antimicrobial agents, e.g.
antibiotics.

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Label High risk Specimens
• Sputum with
suspected Tuberculosis
• Fecal samples
suspected with
Cholera, Typhoid,
• Serum when
suspected with
HIV/ HBV/HCV,
infections
Specimen should be sent with request form
Methods in Diagnostic Microbiology

1. Direct morphologic identification (microscopy and macroscopic) of

the agent in stains of specimens or sections of tissues.
2. Culture isolation and identification of the agent.
3. Detection of antigen from the agent by immunologic assay.

4. Detection and amplification of organism nucleic acid in patients'

specimens.
5. Demonstration of meaningful antibody immune responses to an
infectious agent.
Diagnostic Bacteriology
Laboratory investigation of Bacterial disease

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Note that:
• All laboratory studies must be directed by:
oPatient's history and
oPhysical examination
Microscopic examination
• provide a rapid presumptive diagnosis of
an infection,
e.g. pulmonary tuberculosis using the
Ziehl-Neelsen staining technique
•Helps to study the morphology, structure and
differentiate wither the bacteria is
- Gram positive or gram negative
- Acid fast or non acid fast bacteria
- Motile or not
Microscopic examination

Stained Preparations for Microscopic

examination

 Gram staining
 Acid-fast staining
 simple staining
 India ink preparation
Others…..
Staining
•Bacteria are colorless and cannot be seen under microscope.
•Staining is giving color to bacteria so that can be seen
Uses of staining
1. To observe the morphology (shape, size and arrangement) of bacteria
2. To differentiate one group of bacteria from the other group
Steps in staining
a. Collect specimen
Preparation of smear
a. drying
b. Fixation
c. Staining
Type of staining methods

1. Simple staining method

2. Differential staining method

3. Special staining method

Differential staining method

• Commonly or routine used methods

• A method in which multiple stains (dye) are used to

distinguish different group of bacteria. e.g.

1. Gram’s stain,

2. Ziehl-Neelson stain.
Gram staining
1. Helps to classify bacteria in to two groups
Gram positive
Gram negative
2. To study morphology
shape :- rods, cocci, spiral
Arrangement:- diplococci, streptococci, staphylococci
The process includes the use of:
a primary stain (crystal violet)
a mordant (helper) iodine solution,
a decolorizer (95% ethanol),
a counterstain (safranin).
Acid-fast stain

used to classify bacteria in to acid fast (AFB) and non acid fast
bacilli
• Used to detect Mycobacterium species (M. tuberculosis)
• Mycobacterium species has special cell wall
• Contain high lipid (60%, Mycolic acid)
• Can not stain with gram stain
Acid fast bacteria are red color ( take color of CF)
NAFB become Blue( take color of MB)
Results
• AFB............. Red, straight or slightly curved rods,
occurring single or in a small groups
• Cells......................................... Blue
• Background Material ……….. Blue

Fig. AFB under the microscope

Simple stain
a. used to study the morphology (shape, size and arrangement of bacteria)
-b. used only one type of chemical or stain
 Simple staining method
• There are two kinds of simple staining methods
A. Positive staining (direct)
B. Negative staining (indirect)

A. Positive staining: The bacteria or its parts are stained by the

dye. e.g. Methylene blue stain, Crystal violent stain
B. Negative staining-
• The dye stains the background and the bacteria
component remain unstained. e.g. Indian ink stain
Special staining :- is used to see
Or to stain bacterial capsule, flagella and spore
Auramine-phenol fluorochrome technique
used to detect M. tuberculosis in sputum, CSF etc.,
Methylene blue staining
Used to stain capsules of B. anthracis
Wayson’s bipolar staining
Used to stain clearly bipolar staining of bacteria such as Yersinia pestis
Albert stianing
Used to stain volutins/metachromatic granules (energy storage) of C.
diphtheriae
H. Giemsa stain
Used to stain Chlamydia trachomatis inclusion bodies, Borrelia species,

Wet mount preparation (saline)

- Looking for motile bacteria
 Culturing of bacteria
Culturing:- is growing of bacteria in the laboratory on different culture
media(containing all substances needed for growth of bacteria)
Purpose of Culturing is :
- identification of pathogenic organisms
- Antimicrobial sensitivity tests
 Culture…
• For a culture medium to be successful in growing the pathogen of interest is
, it must
o Provide all essential nutrients, ions, and moisture,
o Maintain the correct pH and osmotic pressure,
o Neutralize any toxic materials produced.

• It is also essential to incubate the medium:

o In the correct atmosphere (aerobic, anaerobic, carboxyphilic)
o At the optimum temperature (psychrophilic, mesophilic, thermophilic)
o For an adequate period.
Bacterial growth patterns (detection of growth)
• In liquid medium:
Superficial growth;
Turbidity/diffuse;
Precipitate growing;
(sediment)

 In solid medium:
• Formation of colony
(Size, shape, color),
• change of color of Media etc.,
(hemolysis, pigment..)

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Considerations during culturing microbes

• Collection of specimens
• Growth requirements
• Environmental requirements (Oxygen, Temperature
etc.,)
• Media used

Success of culturing depends on:

oAppropriate collection and transport of specimen
oSelection of appropriate culture media
 Types of solid media
1. simple media:
Nutrient agar (grow most microorganisms)

2- Enriched media: media of high nutritive value

. Blood agar
. Chocolate agar
. Loffler’s serum

3- Selective media: allow needed bacteria to grow

Lowenstein–Jensen medium used to grow M. tuberculosis
.MacConkeys agar to grow gram negative bacteria
.Mannitol Salt Agar used to grow S. aureus

4- differential media: allow to differentiate one group of bacteria from other

. MacConkeys medium (lactose ferementer vs nonfermenter)
5. Transport media
6. Identification media (biochemical testing media)

5-
Biochemical Testing

Based on enzyme they produce, metabolic waste

product, sugar utilization

1. Carbohydrate fermentation test

•Different bacteria ferments different carbohydrates

•When carbohydrates are fermented the following end products

may be produced:
1. acid end products, or
2. acid and gas end products.
The fermentation solution/ media contains
CarbohydrateEnergy source
pH indicator (phenol red)  acid production
Durham tubes  detection of gas production
-Inoculate bacteria and incubate at 37 for 24 hours and see for
acid, gas, or both production
•If the particular carbohydrate is fermented by the bacterium, acid
end products will be produced which lowers the pH, causing the pH
indicator to change color (phenol red turns yellow).
•If gas is produced along with the acid, it collects in the Durham
tube as a gas bubble. If the carbohydrate is not fermented, no acid
or gas will be produced and the phenol red will remain red.
2) Indole test
o Principle:
- Demonstrates the ability of certain bacteria to produce
tryptophanase and decompose the amino acid tryptophan present
in peptone water to indole.
- Indole production is then tested for by adding few drops of Kovac’s
reagent which gives a pink ring in the presence of indole.

o Procedure:
- The organism is inoculated in peptone water and after incubation at
37 degree for 24 hours, Kovac’s reagent is added.

o Interpretation:
- If a pink ring is produced, the organism is indole +Ve (E. coli).
- If a yellow ring is produced, the organsim is indole –Ve (Klebsiella).
3. Urease test:
o Principle:
- Some organisms produce urease enzyme.
- This enzyme splits urea with the release of ammonia.
- Ammonia causes alkalinity and increases pH of the medium.
- The phenol red indicator turns deep pink.
o Procedure:
- The bacteria is grown on a medium containing urea and
phenol red indicator for 24 hours.
o Interpretation:
- Urease positive organisms such as proteus will turn the
medium deep pink.
+Ve -Ve
4. Oxidase test:
o Is enzyme found in ETS
Principle:
- Some bacteria produce oxidase enzyme which reduces the
oxidase reagent (tetramethyl-p-phenylene-diamine
hydrochloride) to a deep purple color.
o Procedure:
- Done by picking up a portion of the colony tested and
smearing it on a filter paper impregnated with oxidase
reagent.
o Interpretation:
- The immediate development of a deep purple color indicates
positive test.
- Examples: neisseria & pseudomonas & vibrios.
5. Catalase test:

- Tests the ability of some bacteria to produce catalase enzyme.

o Procedure:
- Pick up the test colony on a platinum loop and immerse it in
few drops of 3 % H2O2 (hydrogen peroxide).
o Interpretation:
- Rapid effervescence indicates oxygen production and a
positive test.
- This test is used to differentiate between staphylococci (+Ve)
and streptococci (-Ve).
 1. Catalase test …Cont’d

Figure: Catalase tube test.

Right: Shows a positive test.
Left: Shows a negative test.

Results
• Active bubbling ----- Positive test- Catalase produced
• No release of bubbles ----- Negative test - No catalase produced
 Serological identification
A- Direct serological tests:
- Identification of unknown organism (antigen)
- Detection of microbial antigens by using specific
known antibodies
B- Indirect serological tests:
- Detection of antibodies produced against bacteria and which is present
in patients blood

Common Methods

a. Agglutination/ precipitation

b. Immunofluresence tests

c. Enzyme linked immunosorbent assay

d. Complement fixation tests

Figure 17.7 Basic principles of serological testing using antibodies and antigens
Direct immunofluorescence
First antibody is labeled
known Antibody + unknown antigen
Indirect immunofluorescence
• Detection of unknown organism
• Known antibody
• Second antibody is labeled
Known antigen
Unknown Ab
ELISA Procedures

Figure 5-19a Figure 5-19b

Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third
Edition . ASM Press, 2000.
Complement Fixation
• Lysin mediated hemolysis

• Test uses four components

• Antigen, antibody, complement, and sensitized sheep RBCs

• Steps of test

1. Test antigen reacts with test antibody

2. Contents of tube from (1.) are mixed with sheep RBCs
• Complement used up in first stage, no hemolysis
• Unfixed complement, hemolysis
 Medical Virology

Diagnostic Methods in Medical Virology

 Types of Virus Detection
Wide range of techniques available for diagnosis of viral
infections
VIRUS ISOLATION
•Animal Culture
•Egg Culture
•Cell culture

SEROLOGY VIRUS DETECTION

•Acute/Convalescent •Electron Microscopy
•Antibody Detection •Immunofluorescence
•IgM / IgG •Viral Antigen Detection
•RNA /DNA Detection
Immunofluorescence (IF)

 Use fluorescein labeled Abs

 Infected cells placed on a microscope slide flooded with Abs

 Binding of Ab to viral proteins is signaled by fluorescence

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Cytopathic effect (CPE)
 swelling, shrinking, complete destruction of the infected cell
which are cytological changes

or

 cell lysis / necrosis / giant cell formation / cytoplasmic

vacuolization) inside cell

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Molecular Methods
Polymerase chain reaction (PCR)
 it is possible to detect viral nucleic acid in infected cells rapidly
 Amplifies a DNA target by repetition of cycle
 Use a known sequence of primer complementary to the DNA of the
pathogen to be detected.

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LABORATORY DIAGNOSIS OF MYCOSES (fungal
infection)
Laboratory diagnosis of fungal disease depends on proper
1.Specimen Selection
2. Specimen Collection
3. Specimen Transport
4. Specimen Processing
5. Specimen Examination
Mycological   specimens

• Specimens used for diagnosis of fungal infection include:

• Respiratory tract secretions

• Cerebrospinal fluid (CSF)

• Blood and bone marrow aspirates

• Tissue biopsies from visceral organs

• Urine and urogenital specimen

• Skin, nail, hair scrapings and swabs

LABORATORY DIAGNOSIS OF MYCOSES
• Direct microscopic examination
Gram, potassium hydroxide (KOH), calcofluor
white, India ink
• Culture
Sabouraud dextrose agar
• Biochemical test (enzyme, CHO utilization)
• Serology (antibody, antigen detection)
• Molecular techniques

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• The cultures are examined macroscopically and

microscopically.

• The cultures are not considered negative for

growth until after 4 weeks of incubation (takes longer time to grow)

Germ Tube Test
• Used for presumptive identification of Candida species 
• culture of Candida species is placed in test tube contain in serum
• incubated at 37°C for 2-4 hours.
• A drop of suspension is examined on the slide under a microscope.
• The germ tubes are seen as long as tube-like projections extending from the
yeast cell

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Serology
serological tests for fungal antibodies and are based on latex agglutination,
immunodiffusion, complement fixation and enzyme immunoassays.