Microbiological Diagnostic Principles

MICROBIOLOGICAL
DIAGNOSTIC
PRINCIPLES
INTRODUCTION
• Medical microbiology is essentially concerned with diagnosis of
microbial infections.
• What are the duties of a clinical microbiologist?

• To test specimens for microorganisms
• To provide information about in vitro activity of antimicrobial drugs
against the microorganisms when appropriate.
• To confirm clinical diagnosis of infectious disease with bacterial
aetiology.
• To advise the physician.
• Participate in decisions regarding diagnostic studies to be performed.
• Give advise on type and timing of specimens to be collected.
• Mode of transportation and storage.
• Provide interpretation of lab results.
LABORATORY PROCEDURES
• Morphological identification of the agent in specimen using light or
electron microscopy.
• Culture isolation and identification of the agent.
• Detection of antigen by immunologic assays.
• PCR method
• Antibody demonstration.
SPECIMEN SELECTION, COLLECTION
& PROCESSING
• Proper specimen collection is the most important step in diagnosis of
disease.
• And is additionally dependent on selection, timing and method of
collection.
• The site most likely to yield the agent is most preferred.
• The specimen handling should favour an organisms survival and
growth.
GENERAL RULES TO ALL SPECIMEN
• Adequate quantity
• Representative of infectious process.
• Use sterile containers and observe aseptic precautions.
• Appropriate transportation of specimen to the laboratory and TAT.
• Samples ought to be take or collected before drug administration.
• Specimens required through operation should be enough and
requires special attention.
MICROBIOLOGICAL EXAMINATION &
STAINS
• Direct examination of specimen frequently provides the most rapid
indication of microbial infection. E.g. microscopic, immunologic
techniques have been developed for rapid diagnosis.
MACROSCOPIC EXAMINATION
• Colour, consistency, presence of blood.
MICROSCOPIC EXAMINATION
• WBCs, RBCs, parasites and bacteria.
STAINING METHODS
• Staining is of primary importance for recognition of microorganisms.
Fluorescent microscopy using
auramine-O
• Used for microscopic examination of Mtb
• TB MICROCOPY STAINING PROCEDURE
• QUALITY CONTROL
• With each new batch of stains, known positive smears of low
positivity and known negative smears should be stained to check the
stain, the technique and microscopic examination.
• Distilled water should be used for rinsing because tap water reduces
the fluorescence.
PROCEDURE
• Gently fix the smears with the flame or on the heating block
• Flood the smear with Auramine O with solution and allow staining for 15 minutes
• Rinse smear with distilled water and drain. Tap water contains chlorine, which
may interfere with fluorescence
• Flood (decolorize) with 0.5% acid alcohol for 2 minutes
• Rinse with distilled water and drain
• Flood smear with potassium permanganate * and counter for minute' Time is
critical with potassium permanganate because counterstaining for longer time
may quench fluorescence of the acid-fast bacilli
• Rinse with distilled water and drain
• Allow smears to air dry and, do not blot dry or dry using heat. Read as soon as
possible (Microscopy) or keep the smears in the dark so that fluorochrome
doesn't fade
• *Acridine Orange may be used in place of potassium permanganate as a
counterstain
Giemsa stain
• Named after Dr Gustav Giemsa from Germany.
• Used for the diagnosis of malaria.
PROCEDURE
• Pipette 3 mls of buffered water into a coupling jar
• Add 6 drops of giemsa stock solution
• Flood the slide (thick and thin) with giemsa working solution
• Stain for 10 minutes
• Wash off the slide with buffered water
• Air dry/dry at 370c
• Examine the slide using x100
CULTURE
• Involves the isolation and culturing of microorganisms in artificial
(broth or agar)media, tissue cultures or hosts.
• Solid media provides recognition of morphology of colonies od
species.
• One can take advantage of fermentation capabilities for differential
media.
• Culture can also be selective by incorporation of antimicrobial agents
that inhibit indigenous flora and permitting the growth of specific
ones resistant to the inhibitors (Thayer-Martin for N. gonorrhoeae).
Containing vancomycin to inhibit G+ bacteria, colistin for G- bacilli,
Bactrim for proteus species and anisomycin for fungi.
• The number of bacteria in specimens is useful in determination of
infection.
• 3+, 2+ or 1+……
• Chlamydiae and viruses are cultured in cell cultures but may
occasionally require inoculation into animals.
• Ricketsial infection is usually diagnosed serologically due to its
isolation difficulty and being particularly hazardous to the individual.
• Some viruses cannot be isolated in cell cultures (hepatitis) and as
such is dependent on detection of viral antigens or antibodies.
Incubation
• Generally incubated at 35 to 37 degrees Celsius.
• Supplementation of CO₂, reduced oxygen, no oxygen depend on
requirements of the microorganisms.
• The duration ranges from minimum of 18 to 24 hours to a maximum
of 6 to 8 weeks.
Interpretation
• Some are always considered clinically significant e.g S. dysentriae,
Mtb but others are ordinarily harmless depending on site of specimen
collection.
Antimicrobial susceptibility
• Many bacteria have unpredictable susceptibility to antimicrobial
agents.
SERODIAGNOSIS
• Infection may be diagnosed by antibody response to infecting
microorganism.
• Especially useful for those that cannot be isolated in culture e.g. EBV
• The technique for isolation of HIV in cell culture is demanding
therefore diagnosis is by detection of antibodies to the virus.
Disadvantage
• The lag between onset of disease and development of antibodies.
• Presence of antibodies may signify a past infection.
• Immunocompromised unable to mount antibody response.
MOLECULAR TECHNIQUES
• Involves the use of molecular technology in diagnoses of infectious
diseases through gene amplification techniques e.g. PCR.
• This approach has had a major applications in the detection of
infectious diseases that are difficult to culture (HIV, Influenza) or
those that are yet to be successfully cultured.
IMMUNOLOGIC ASSAY
• The most common are latex particle agglutination, coagglutination,
and ELISA (Enzyme-linked immunosorbent assay).
THE END

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MICROBIOLOGICAL

• What are the duties of a clinical microbiologist?

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