Gram-stain

This is the most important stain in bacteriology and is so central to identification that it should be practised until the operator is fully competent. A number of different variations are found, and the laboratory should standardise on one method. 

It is essential that young cultures (24 hrs) are used as old cultures may give false negative results. 

Cover the slide with an ammonium oxalate, crystal violet preparation and leave for one minute. This is prepared by dissolving 20 g of crystal violet in 200 ml of methanol, and adding 800 ml of 1 % ammonium oxalate. 

Decolourise with 95% ethanol for approximately 15 seconds, rinse with water and blot. 

One of the most commonly used procedures, the Gram stain, differentiates bacteria based upon cell wall characteristics. The procedure calls for initial 

Staining of cells with crystal violet and fixation with iodine. This reaction is neutralized, decolorized, counterstained with safranin, rinsed, and allowed to dry. The preparation is then examined directly (without a coverslip) using oil immersion and brightfield illumination. One should not use phase-contrast optics when its necessary to observe color. 

Interpretation. Gram-positive cells (lactic acid bacteria) retain the crystal violet primary stain upon decolorization and appear violet-blue when examined under oil immersion (brightfield). 

Supplemental Notes. Most suppliers of scientific equipment and supplies market prepackaged Gram stain kits (sufficient to do hundreds of Gram stains) at nominal prices (approx 30). It is recommended that interested parties purchase these and follow the instructions provided with the kit. Aside from procedural matters relative to staining, several additional issues merit consideration. 

Staining should always be done on young cultures of approximately the same age. 

Prepare a smear of the bacterium, making sure to heat-fix the slide prior to staining (Section 12.4). Be sure to use bacterial cultures of the same age (incubation time) and avoid staining very dense preparations. 

Flood the smear with crystal violet and set aside for 1 min. 

Lightly wash off excess crystal violet using cold tap water. 

Flood smear with iodine and allow the iodine to remain on slide for 1 min. 

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Gram negative. Bacteria that fail to retain Gram stain. This group includes the genera Salmonella, Pseudmonas, Pasteurella, Escherichia, and Brucella. 

Gram-Fkrbung, /. (Bact.) Gram staining. Gramm-aquivalent, n. gram equivalent. 

Histochemistry of the Gram-staining Reaction for Micro-organisms, H. Henry and M. Stacey, Nature, 151 (1943)671. 

Recent Advances in Microbiological Methods. Chemistry of Gram Staining and of the Feulgen and Dische Reactions for Nuclear Material, M. Stacey, Nature, 171 (1953) 507. 

Same pathogenic bacteria seen on Gram-stain, add 1 additional point... 

Sputum Gram stain less than 10 epithelial cells, greater than 25 WBCs, predominance of gram-negative bacilli... 

Microbiologic studies that allow for direct examination of a specimen (e.g., sputum, blood, or urine) also may aid in a presumptive diagnosis and give an indication of the characteristics of the infecting organism. Generally, microbial cultures are obtained with a Gram stain of the cultured material. 

FIGURE 66-2. Important bacterial pathogens classified according to Gram stain and morphologic characteristics. (Reprinted from Rybak MJ, Aeschlimann JR. Laboratory tests to direct antimicrobial pharmacotherapy. In In DiPiro JT, Talbert RL, Yee GC, et al, (eds.) Pharmacotherapy A Pathophysiologic Approach. 6th ed. New York McGraw-Hill 2005 1894.)... 

Empirical therapy should be directed at the most likely pathogen (s) for a specific patient, taking into account age, risk factors for infection (including underlying disease and immune dysfunction, vaccine history, and recent exposures), CSF Gram stain results, CSF antibiotic penetration, and local antimicrobial resistance patterns. 

CSF stain Negative Positive Gram stain (60%-90%) Negative Positive India ink stain (Cryptococcus) Positive acid-fast bacilli stain... 

Gram stain (adequate for diagnosis in 60% to 90% of patients with bacterial meningitis)... 

If CSF Gram stain and/or culture is negative, rapid diagnostic tests (such as latex agglutination) may be useful these tests are positive even if bacteria are dead. 

Sputum Gram stain many gram-negative bacilli, many WBCs... 

Gram stain, culture, and sensitivities of ear fluid if draining spontaneously or obtained via tympanocentesis (not performed routinely in practice)... 

Folliculitis presents as small, pruritic, erythematous papules. Location of the lesions and a good patient history are often all that are required in the diagnosis of folliculitis. While the papules may be cultured and Gram stains or potassium hydroxide stains done to help determine causative agent, it is not generally required because folliculitis often resolves spontaneously within a few days. 

Deep tissue specimens obtained during surgical irrigation and debridement should be sent for Gram stain, culture, and sensitivity.3 Imaging Studies... 

The patient was admitted and placed on respiratory isolation. Three separate sputum Gram stain specimens were reported to contain 3+ AFB. A PPD tuberculin skin test was placed. Sputum samples were sent for AFB, fungi, and bacterial cultures and sensitivities. After 48 hours, the PPD skin test was read as a 12-mm area of induration. 

Ascitic fluid usually contains greater than 300 leukocytes/ mm3, and bacteria may be evident on Gram stain of a centrifuged specimen. 

Based on culture and Gram stain (if available), is your empiric selection reasonable ... 

Several laboratory tests are available to aid in the diagnosis of gonorrhea and include gram-stained smears, culture, or the DNA hybridization probe. 

Alternatively, a Gram stain vaginal smear may be used to diagnose BV using the Nugent criteria. This relies on estimating the proportions of bacteria morphotypes to provide a score between 0 and 10. A score of less than 4 is normal, 4 to 6 is intermediate, and greater than 6 is consistent with BV. 

Sepsis The systemic inflammatory response syndrome and documented infection (culture or Gram stain of blood, sputum, urine, or normally sterile body fluid positive for pathogenic microorganisms Severe sepsis Sepsis associated with organ dysfunction, hypoperfusion, or hypotension (systolic blood pressure less than 90 mm Hg). Hypoperfusion and perfusion abnormalities may include, but are not limited to, lactic acidosis, oliguria, or acute alteration in mental status. 

Tlie morphology of some bacteria, especially those that form spores, is distinctive enough under the light microscope to have value for identification. This means that differential staining techniques, such as the Gram stain or acid-fast stain, and fluorescence microscopy may help to determine the iden-... 

Holm, C. Jespersen, L. A flow-cytometric gram-staining technique for milk-associated bacteria. Appl. Environ. Microbiol. 2003, 69, 2857-2863. 

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