Transcript Principle Diagnistic Microbiology

Principle Diagnostic
Microbiology
Concerned with the etiologic
diagnosis of infection.
• Laboratory procedures used in the diagnosis of
infectious disease in humans include the
following:
– (1) Morphologic identification of the agent in stains of
specimens or sections of tissues (light and electron
microscopy).
– (2) Culture isolation and identification of the agent.
– (3) Detection of antigen from the agent by
immunologic assay (latex agglutination, EIA, etc) or
by fluorescein-labeled (or peroxidase-labeled)
antibody stains.
– (4) DNA-DNA or DNA-RNA hybridization to detect
pathogen-specific genes in patients' specimens.
– (5) Detection and amplification of organism nucleic
acid in patients' specimens.
– (6) Demonstration of meaningful antibody or cellmediated immune responses to an infectious agent.
In the field of infectious diseases,
laboratory test results depend largely on:
• The quality of the specimen,
• The timing and the care with which it is
collected, and
• The technical proficiency and
experience of laboratory personnel.
• Diagnostic microbiology encompasses the
characterization of thousands of agents that
cause or are associated with infectious
diseases.
• The techniques used to characterize infectious
agents vary greatly depending upon:
– the clinical syndrome and
– the type of agent being considered, be it virus,
bacterium, fungus, or other parasite.
Communication between
Physician & Laboratory
• no single test will permit isolation or
characterization of all potential pathogens
• clinical information is much more important
for diagnostic microbiology than it is for
clinical chemistry or hematology.
• the physician should inform the laboratory
staff of the tentative diagnosis
– e.g. type of infection or infectious agent
suspected.
– Infected part to be sampled
Treatment of samples:--Proper labeling of specimens including:
– clinical data
– the patient's identifying data (at least two
methods of definitive identification)
– the requesting physician's name and pertinent
contact information.
• Many pathogenic microorganisms grow slowly,
and days or even weeks may elapse before they
are isolated and identified.
• the physician should begin treatment with drugs
aimed at the organism thought to be responsible
for the patient's illness.
• As the laboratory staff begins to obtain results,
they inform the physician, who can then
reevaluate the diagnosis and clinical course of
the patient and perhaps make changes in the
therapeutic program.
• The "feedback" information from the laboratory
consists of preliminary reports of the results of
individual steps in the isolation and identification
of the causative agent.
Diagnosis of Bacterial & Fungal
Infections
• Specimens
A few general rules apply to all
specimens:
• (1) The quantity of material must be adequate.
• (2) The sample should be representative of the infectious
process (eg, sputum, not saliva; pus from the underlying
lesion, not from its sinus tract; a swab from the depth of
the wound, not from its surface).
• (3) Contamination of the specimen must be avoided by
using only sterile equipment and aseptic precautions.
• (4) The specimen must be taken to the laboratory and
examined promptly. Special transport media may be
helpful.
• (5) Meaningful specimens to diagnose bacterial and
fungal infections must be secured before antimicrobial
drugs are administered. If antimicrobial drugs are given
before specimens are taken for microbiologic study, drug
therapy may have to be stopped and repeat specimens
obtained several days later.
Microscopy & Stains
• relatively simple and inexpensive
• much less sensitive method than culture for
detection of small numbers of bacteria.
• A specimen must contain at least 105 organisms
per milliliter.
• Specimens containing 102–103 organisms per
milliliter produce growth on solid media, and
those containing ten or fewer bacteria per
milliliter may produce growth in liquid media.
• Gram staining is a very useful procedure in
diagnostic microbiology.
• All specimens submitted when bacterial infection
is suspected should be smeared on glass slides,
Gram-stained, and examined microscopically.
Gram staining
• Gram reaction (purple-blue indicates grampositive organisms; red, gram-negative) and
morphology (shape: cocci, rods, fusiform, or
other) of bacteria should be noted.
• The appearance of bacteria on Gram-stained
smears does not permit identification of species.
• Reports of gram-positive cocci in chains are
suggestive of, but not definitive for, streptococcal
species; gram-positive cocci in clusters suggest
a staphylococcal species.
• Gram-negative rods can be large, small, or even
coccobacillary.
• Some nonviable gram-positive bacteria can stain
gram-negatively. Typically, bacterial morphology
has been defined using organisms grown on
agar. However, bacteria in body fluids or tissue
can have highly variable morphology.
• Specimens submitted for examination for
mycobacteria should be stained for acid-fast
organisms, using either Ziehl-Neelsen stain or
Kinyoun stain.
• An alternative fluorescent stain for mycobacteria,
auramine-rhodamine stain, is more sensitive
than other stains for acid-fast organisms but
requires fluorescence microscopy and, if results
are positive, confirmation of morphology with an
acid-fast stain
Table 47–1. Gram and Acid-Fast Staining Methods.
Gram stain
(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) Decolorize for 10–30 seconds with gentle agitation in acetone (30 mL) and alcohol (70 mL).
(7) Wash with water. Do not blot.
(8) Cover for 10–30 seconds with safranin (2.5% solution in 95% alcohol).
(9) Wash with water and let dry.
Ziehl-Neelsen acid-fast stain
(1) Fix smear by heat.
(2) Cover with carbolfuchsin, steam gently for 5 minutes over direct flame (or for 20 minutes over a water
bath).
(3) Wash with water.
(4) Decolorize in acid-alcohol until only a faint pink color remains.
(5) Wash with water.
(6) Counterstain for 10–30 seconds with Loeffler's methylene blue.
(7) Wash with water and let dry.
Kinyoun carbolfuchsin acid-fast stain
(1) Formula: 4 g basic fuchsin, 8 g phenol, 20 mL 95% alcohol, 100 mL distilled water.
(2) Stain fixed smear for 3 minutes (no heat necessary) and continue as with Ziehl-Neelsen stain.