The Gram stain test is the most extensively performed procedure in diagnostic microbiology. It is used to classify bacteria as either gram-positive or gram-negative based upon their ability to retain the crystal violet stain following decolorization. In addition, the Gram stain provides vital diagnostic information, aids in the selection of culture media, and dictates initial selection of antibiotics for treatment and antimicrobial susceptibility testing.
The Gram stain is used to detect the presence of bacteria, yeast, and other cells in direct smears prepared from swabs, aspirates, secretions, etc. from any part of the body where infection is suspected. Direct smears are often made of throat swabs, sputum, genital swabs, wounds, abscesses, cerebrospinal fluid (CSF), serous fluids, joint fluid, urine, and stool. Gram stain is also performed to help identify colonies isolated from cultures. In addition to gram-negative or gram-positive, organisms are evaluated for size, shape, arrangement, number, and any special characteristics such as bipolar staining and the presence of spores. These characteristics often point the way to the most efficient selection of biochemical tests needed to identify the organism. The finding of organisms on direct examination of some specimens is sufficient to establish a preliminary diagnosis and justify immediate antibiotic treatment pending confirmation by culture or other means. The Gram stain is very useful in identifying anaerobic bacteria by comparing the microscopic morphology and number of organisms to culture results. Significant numbers of characteristic bacteria on Gram stain not appearing on aerobic culture often signals the presence of an anaerobic infection.
The Gram stain will identify male patients with Neisseria gonorrhoeae genital infections with a specficity approaching 100% and a sensitivity above 90%. In female patients, the sensitivity and specificity are lower owing to the presence of other genital flora, but the test is still sufficiently specific to justify immediate antibiotic therapy when symptoms of pelvic inflammatory disease are present. The presence of bacteria on Gram stain of concentrated CSF is presumptive evidence of bacterial meningitis and reason to begin antibiotic therapy. The Gram stain is positive in the majority of bacterial meningitis cases. Recovery of bacteria from other normally sterile fluids including exudative plueral, pericardial, and abdominal fluid and inflammatory joint fluid is also presumptive
Crystal violet—A rosanaline dye staining all bacterial cells but retained only by gram-positive bacteria.
Decolorizer—A solution of alcohol or acetone-alcohol which removes crystal violet from the cell wall of gram-negative bacteria.
Gram stain—A test for classifying and characterizing bacteria by microscopic analysis following staining by the procedure developed by Hans Christian Gram.
Gram's iodine—A solution of iodine and potassium iodide which causes crystal violet to adhere more tightly to the bacterial cell wall.
Safranin O—A water soluble counterstain that is used to visualize gram-negative bacteria.
evidence of an infectious process. Gram stain detects approximately 70% of pleural fluids and 50% of pericardial fluids that are infected.
The Gram stain was discovered by Hans Christian Gram in the late 1800s. There are several modifications but all Gram stain procedures involve four steps: staining with crystal violet, reaction with Gram's iodine, decolorization, and counterstaining. Material from a direct specimen or culture is transferred to the slide using a dropper, needle, or swab. The material is spread in the center of a glass slide until visible, and allowed to air dry. The slide is heat-fixed and allowed to cool before staining. The slide is flooded with an alcoholic solution of basic crystal violet for 30 seconds. This dye stains all cells a bright blue or purple color. Excess dye is removed with a stream of tap water and the slide is flooded with Gram's iodine (mordant) solution which should remain on the slide for one minute. The iodide displaces chloride in the dye causing it to adhere more strongly to the cell wall. The slide is rinsed in tap water to remove the mordant. Next, a stream of decolorizer is used to rinse the slide for 10 seconds followed immediately by tap water. The most commonly used decolorizer is a 1:1 solution of acetone and 95% ethanol. Gram-positive cells have a thick cell well rich in teichoic acid. The gram-negative bacteria have a thin cell wall that is rich in lipids. The decolorizer is able to penetrate through the thinner lipopolysaccharide-rich cell wall of the gram-negative bacteria and wash away the crystal violet. The slide is flooded with Safranin O counterstain and then blotted or air-dried.
The slide is examined using a light microscope first under low power and then using an oil immersion lens at 1000X. The nature and number of leukocytes and epithelial cells are recorded along with the number, size, shape, and gram-stain reaction of any bacteria. Gram-positive bacteria and yeast stain blue or purple while gram-negative bacteria, white cells, epithelial cells, and trichomonads stain red. Some gram-positive bacteria, especially old colonies and those from treated patients, may not resist decolorization and appear gram-negative. Some species, for example, Mycobacterium tuberculosis may stain gram-negative or gram-positive and are referred to as gram-variable. In addition to staining, bacteria are classified according to shape as round (coccus) or rod-shaped (bacillus). This gives four general categories; gram-positive cocci (eg. Staphylococcus spp.), gram-positve bacilli (e.g. Clostridium spp.), gram-negative cocci (e.g. Neisseria spp.), and gram-negative bacilli (Salmonella spp.). Other organisms may fall between these shapes (coccobacilli) or may be variable (pleomorphic) or cigar-shaped (fusiform). Such characteristics are important leads to identification.
For collection of samples, patients may need to be properly positioned and given specific instructions (e.g. how to produce sputum). Urine specimens should be collected from a catheter or using the midstream void technique after cleansing of the outer genitalia. Blood, CSF, synovial and serous fluids, and aspiration needle biopsy specimens require disinfection of the puncture site with alcohol followed by an iodine solution. Specimens should be transported in a syringe or transferred to an airtight sterile container. Swabs should be placed in transport medium to prevent drying. When collecting specimens (with the exception of urine), health care workers must follow standard precautions for the prevention of occupational exposure to bloodborne pathogens.
All positive Gram stain results should be confirmed by culture, DNA analysis, or serological evidence. However, in the appropriate clinical setting (e.g. the finding of bacteria in cerebrospinal fluid) antibiotics should be administered immediately. In these situations the Gram stain result provides critical information for the selection of proper antibiotic therapy. For example, gram-positive bacteria are most likely susceptible to penicillins, cephalosporins, vancomycin, and erythromycin.
Health care team roles
Specimens may be collected or collection assisted by a physician, nurse, or physician assistant. Gram stain is performed by a clinical laboratory scientist, NCA(CLS) or medical technologist, MT(ASCP) or by a clinical laboratory technician, NCA(CLT) or medical laboratory technician, MLT(ASCP).
Koneman, E.W., et. al. Color Atlas and Textbook of Diagnostic Microbiology, 5th Ed. Philadelphia: Lippincott, 1997.
Murray, P.R., et. al. Manual of Clinical Microbiology. Washington, DC: American Society for Microbiology, 1995.
American Society for Microbiology. 1752 N Street NW, Washington, DC 20036. (202) 737-3600. <http://www.asmusa.org>.