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 cell- mediated 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 10 5 organisms per milliliter. Specimens containing 10 2 –10 3 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 gram- positive 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.