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25-03: Gram Stain

Camiron L. Pfennig; B. Ethan Brown

Uses

The 1st step in the identification of a predominant bacterial organism in a specimen. Classifies an organism by its cell wall’s ability to retain crystal violet dye during solvent treatment. Morphology of the organism is also identified.

Materials

Freshly collected specimen to be examined, glass microscope slide, crystal violet, Gram iodine, acetone-alcohol (acetone, 30 mL, and 95% alcohol, 70 mL), safranin, and Bunsen burner.

FIGURE 25.11

Gram Stain—Streptococcus pneumoniae. Gram-positive, kidney-shaped diplococci of S pneumoniae. (Photo contributor: Roche Laboratories, Division of Hoffman-LaRoche Inc. Nutley, NJ.)

A slide under the microscope shows irregular red, kidney shaped structures and clusters of tiny black dots..
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FIGURE 25.12

Gram Stain—Staphylococcus aureus. Small clusters of gram-positive cocci seen in S aureus infection. (Photo contributor: Roche Laboratories, Division of Hoffman-LaRoche Inc. Nutley, NJ.)

A slide under the microscope shows irregular red cells and multiple clusters of dark dot-like structures..
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Method

  1. Put specimen on dry, clean glass microscope slide and allow to air dry.

  2. Heat-fix specimen by gently passing over flame.

  3. Cover specimen with crystal violet for 1 minute.

  4. Rinse off completely with water; do not blot.

  5. Cover specimen with Gram iodine for 1 minute.

  6. Rinse off completely with water; do not blot.

  7. Decolorize for 30 seconds with gentle agitation in acetone-alcohol.

  8. Rinse off completely with water; do not blot.

  9. Cover with safranin for 10 to 20 seconds.

  10. Rinse off completely with water and let air dry.

FIGURE 25.13

Gram Stain—Gram-Negative Rods. Gram-negative rods of Pseudomonas aeruginosa. (Photo contributor: Roche Laboratories, Division of Hoffman-LaRoche Inc. Nutley, NJ.)

A slide under the microscope shows red, rod-shaped structures on a yellow background..
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FIGURE 25.14

Gram Stain—Neisseria gonorrhoeae. Multiple gram-negative, intracellular diplococci from a patient with N gonorrhoeae. (Reproduced with permission, from Morse SA, Holmes KK, Ballard RC, Moreland AA (eds). Atlas of Sexually Transmitted Diseases. London, United Kingdom: Mosby-Wolfe; 1990.)

A slide under the microscope shows red cells and clusters of paired red dot-like structures..
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