Gram's Stain

The most common and useful staining procedure used in bacteriologic work is that of Gram. It is most likely to yield valuable information and should be done in all cases when staining is indicated. It is also used for the examination of cultures to determine purity and for purposes of identification.

Hucker's Modification of Gram's Stain Solution

Crystal violet/ammonium oxalate solution (primary stain):
Solution A:
  • 2 gm Crystal violet (certified)
  • 20 ml Ethyl alcohol (95 percent)
 Solution B:
  • 0.8 gm Ammonium oxalate
  • 80 ml Distilled water
Mix solutions A and B, store for 24 hours, filter, and store at room temperature, in a dark bottle, in a dark place, away from direct sunlight.

Yeast contamination is common and the stain must be filtered before use. Use only certified crystal violet. Gentian violet and methyl violet are not recommended because they contain impurities.


 

Iodine solution (mordant):
  • Iodine crystals (USP) 1 gm
  • Potassium iodide 2 gm
  • Distilled water300 ml
Grind iodine and potassium iodide in mortar. Dissolve potassium iodide in a flask in as small amount of water as possible. Add iodine crystals to potassium iodide solution. When dissolution is completed, add remainder of distilled water. Mix and let stand at room temperature for 24 hours. Filter and store in a dark bottle, away from direct sunlight.

 

Decolorizer:
  • Acetone 1 volume
  • Ethyl alcohol (95 percent) 1 volume
Mix 1 volume of acetone with 1 volume of ethyl alcohol and store in a tightly sealed bottle.

 

Safranine 0 counterstain:
  • Safranine 0 0.25 g
  • Ethyl alcohol (95 percent) 10 ml
  • Distilled water 90 ml
Dissolve dye in ethyl alcohol, then add distilled water to dye solution and let stand at room temperature for 24 hours. Filter and store away from direct sunlight.

Procedure for Gram's Staining

After the smear has been dried, heat-fixed, and cooled off, proceed as follows:

  1. Place slide on staining rack and cover specimen with crystal violet. Let stand for 1 minute.
  2. Wash briefly in tap water and shake off excess.
  3. Cover specimen with iodine solution and let stand for 1 minute.
  4. Wash with water and shake off excess.
  5. Tilt slide at 45° angle and decolorize with the acetone-alcohol solution until the purple color stops running. Wash immediately with water and shake off excess.
  6. Cover specimen with safranine and let stand for 30 seconds to 1 minute.
  7. Wash with water, shake off excess, and gently blot dry. The smear is now ready to be read. (Use oil immersion lens.)

Principle of Gram's Stain

The crystal violet stain is the primary stain, which stains everything in the smear blue. The Gram's iodine acts as a mordant that causes the crystal violet to penetrate and adhere to the gram-positive organisms. The acetone-alcohol mixture acts as the decolorizer that washes the stain away from everything in the smear except the gram-positive organisms. The safranine is the counter-stain that stains everything in the smear that has been decolorized: pus cells, mucus, gram-negative organisms. The gram-negative organisms will stain a much deeper pink than the pus cells, and mucus will stain even lighter pink than the pus cells.

Reading and Reporting Smears

Place a drop of oil in the slide and, using the oil immersion objective of the microscope, read the smear. All body discharges contain extraneous materials, such as pus cells and mucus. Of interest, however, are the types of bacteria that may be present. The stained smear reveals only two things: the morphology and the staining characteristics of the bacteria present. Positive identification requires cultures and further studies.

The hospital corpsman reports only what he or she sees.

Example: "Smear shows numerous gram-negative bacilli." If two or more types of bacteria are seen in a smear, the rule is to report them in order of predominance, for example:

  1. Numerous gram-positive cocci in clusters
  2. Few gram-negative bacilli

Gram-positive organisms are easy to see because they stain a deep blue or blue-black. Gram-negative organisms stain a deep pink, but since the background material is also pink, minute and detailed inspection is necessary before reporting the results.

In the presence of gonorrhea the smear will reveal large numbers of pus cells with varying numbers of intracellular and extracellular gram- negative, bean-shaped cocci in pairs. Such a finding can be considered diagnostic. It is important to point out that only a few of the thousands of pus cells on the slide may contain bacteria, and sometimes it requires considerable search to find one.

Source: Naval Education and Training Command: Hospital Corpsman 1 & C: August 1986, Chapter 7: Clinical Laboratory