Diagnostic Medical

Microbiology & Clinical

Correlation
22 Safar 1428H
18 Febuary 2009
 Principles of Diagnostic Medical
Microbiology
► Physicians who deal with infectious process must
know when and how to take specimens, what
laboratory examinations to request for.
► Laboratory procedures:
► Morphologic identification of agent
► Culture isolation and identification
► Detection of Ag from the agent
► DNA-DNA or DNA-RNA hybridization
► Ab or cell-mediated immune responses
 Bacterial & Fungal Infections
► Specimens : direct tissue, or fluid samples ( are collected
from normally sterile tissue eg. and body fluids
eg. and indirect samples (of
inflammatory exudates eg. that have
passed through sites known to be colonised with normal
flora.
► Specimen collection  isolation of organism
► Recovery of bacteria and fungi vs. normal microbial flora.
► A few general rules: i) adequate quantity; ii) sample
representative of the infectious process; iii) avoid
contamination; iv) prompt examination and v) meaningful
specimens must be secured before the administration of
antimicrobial drugs.
 Microscopy & Stains
►A relatively simple and No. Effective

inexpensive method, but much >105 Likely to be seen

less sensitive than culture. on smear

► Gram-staining : note on 105 Liquid medium

does not appear
Gram reaction, morphology. turbid
The appearance of bacteria on
<10 Growth in liquid
Gram-stained smears provide media
suggestion of organism, but
102 - Growth on solid
not definitive. Some non-viable 103 media
G+ can stain gram negatively.
 Morphology & Stains
► Bacterial morphology has been defined to bacteria grown
on agar, however in body fluids and tissue, morphology of
bacteria are highly variable.
► Mycobacteria: acid-fast organisms (Ziehl-Neelsen stain).
Other stains used is auramine-rhodamine but requires
fluorescence microscopy.
► Immunofluorescent (IF) antibody staining: more specific;
fluorescein-labelled Abs are made from antisera produced
by injecting animals with whole organisms or complex Ag
mixtures  results in polyclonal Abs. Control to minimise
nonspecific IF, or use monoclonal Abs.
► IF is more useful for specific organisms, isolated on culture
media, than for organisms directly isolated from patients.
 Morphology & Stains
► For fungi or other parasites,

Stain Binding affinity

Calcofluor white Cellulose and chitin eg. spherules

with endospores in Coccidiodes
immites
Methenamine Silver Pneumocystis jiroveci cysts
Periodic acid-Schiff Stain tissue sections when fungal
(PAS) infection is suspected

► Specimens to examine fungi is possible after

treatment with 10% _____, which breaks down
 Gram-staining
► 1. Fix smear by heat.
► 2. Cover with crystal violet.
► 3. Wash with water. Do not blot.
► 4. Cover with Gram’s iodine.
► 5. Wash with water. Do not blot.
► 6. Decolorise for 10-30s with gentle agitation in
acetone (30mL) and alcohol (70mL).
► 7. Wash with water. Do not blot.
► 8. Cover for 10-30s with safranin.
► 9. Wash with water and let dry.
 Mycobacteria staining
► Ziehl-Neelsen acid-fast stain Kinyoun
► 1. fix smear by heat. carbolfuchsin
► 2. Cover with carbolfuchsin, steam 1. Formula: 4g
gently for 5 min over direct flame (or basic fuchsin,
20min over a water bath). 8g phenol,
► 3. Wash with water. 20mL 95%
► 4. Decolourise in acid-alcohol until alcohol, 100mL
only a faint pink colour remains. distilled water.
► 5. Wash with water. 2. Stain fixed
► 6. Counterstain for 10-30s with
smear for 3min
Loeffler’s methlene blue. (no heat necs)
► 7. Wash with water and let dry.
cont with Z-N
stain.
 Ziehl-Neelsen staining
► Identification of acid-fast bacilli
 Culture Systems
► Standard medium is blood agar, 5% sheep blood for
most aerobic and facultatively anaerobic organisms.
► Chocolate agar, medium with heated blood.
► Selective medium (MacConkey or eosin-methylene blue
[EBM] agar) for enteric Gram- rods.
► Obligate anaerobes must be plated on at least 2 types of
media: i) highly supplemented agar such as brucella
agar with hemin, vit K; and ii) selective medium
containing substances that inhibit the growth of other
non-obligates.
► Specialised media - depend on clinical diagnosis and the
organism under consideration. Eg. freshly made Bordet-
Gengou or charcoal-containing medium to culture for B.
pertussis.
MacConkey’s Agar
 Culture Systems
► Broth cultures is highly enriched media, important
for back-up cultures of biopsy tissues and body
fluids.
► Many yeasts will grow on blood agar.
► Biphasic and mycelial phase fungi grow better on
media designed specially for fungi eg. Brain-heart
infusion agar (+/- antibiotics) and inhibitory mold
agar.
► Fungi also grow in media made with plants and
vegetable materials.
 Antigen Detection
► Immunologic systems to detect antigens of
microorganisms. IF test is one example.
► Enzyme immunoassays (EIA) inc enzyme-linked
immunosorbent assays (ELISA) and agglutination tests.
► In latex agglutination, an antigen-specific antibody
(either polyclonal or monoclonal) is fixed to latex beads.
Most useful to detect carbohydrate Ags of encapsulated
microorganisms.
► Immunoblotting (“Western blot”) whereby defined
antigens are placed on strips of nitrocellulose paper.
Following incubation, the strip is treated with an enzyme-
labeled Ab. Addition of the substrate for the enzyme allows
detection of Ag-specific bound Ab by colorimetric reaction.
Useful for Abs in HIV infection and Lyme disease.
Principle of Enzyme Immunoassays

First Ab Add antigen

Second antibody
for the Ag,
labelled with
enzyme
 Molecular Diagnostics
► Hybridisation of a characterised nucleic acid probe
(primer, oligonucleotides) to a specific nucleic acid
sequence in a test specimen followed by detection
of the paired hybrid.
► The nucleic acid probe typically is labeled with
enzymes, antigenic substrates, chemiluminescent
molecules or radioisotopes to facilitate detection of
the hybridisation product.
 Molecular Diagnosis
► A. Identifying bacteria using 16S rRNA
► The 16S rRNA has conserved portions of the sequence.
► Labeled probe specific for the 16S rRNA of a species are
added and then measured. This allows the identification of
Mycobacterium sp., Coccidioides immitis, Histoplasma
capsulatum.
► Portions of the 16S rRNA are conserved across many
species and its amplification using primers allows isolation
and sequencing of the variable regions of the molecules.
These genus- or species-specific allows the identification of
pathogens that are impossible or difficult to culture. Eg.
Tropheryma whipplei the cause of Whipple’s disease.
 Molecular Diagnosis
► B. Target amplification systems
► The polymerase chain reaction (PCR) is used to amplify
extremely small amounts of specific DNA.
► This technique uses DNA polymerases through alternate
changes in temperature to initiate replication in either the
3’ or 5’ direction. The specificity is provided by primers that
recognise a pair of unique sites on the chromosome so that
the DNA between them can be replicated.
► PCR can also be performed on RNA targets, which is called
reverse transcriptase PCR. The rev transcriptase is use to
transcribe RNA into complimentary DNA for amplification.
► PCR assays available commercially for Chlamydia
trachomatis, Neisseria gonorrhoeae, Mycobacterium
tuberculosis, cytomegalovirus and enteroviruses.