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.
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
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.
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
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 1030 seconds with gentle agitation in acetone (30 mL) and alcohol (70 mL). (7) Wash with water. Do not blot. (8) Cover for 1030 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 1030 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.