Gram negative coccobacilli (Escherichia coli)
Gram-positive cocci (Staphylococcus lentus)
Gram Staining
Hans Christian Gram 1884
DESCRIPTION
The technique is used for the differentiation of Gram-positive and Gram-negative bacteria, usually as a first step in bacteria
identification. The cell walls of Gram-positive bacteria are different from those Gram-negative. Gram-positive walls have a thick layer
of peptidoglycan associated with teichoic acids and in Gram-negative walls lipoprotein and lipopolysaccharide are associated with a
thin peptidoglycan layer.
The structural difference between cell walls results in a different ability to retain
certain dyes and a different ability to resist decolorization.
There are four basic steps of the Gram stain, which include applying a primary stain
(crystal violet), followed by the addition of a mordant (Gram's iodine), rapid
decolorization with alcohol or acetone, and counterstaining with safranin, neutral red
or basic fuchsin.

REAGENTS
Aniline crystal violet solution: aniline 40 ml, water 1000 ml, crystal violet, saturated
alcoholic solution (about 10 g per 100 ml 95% alcohol) 114 ml.
Gram's iodine: Iodine 1 g, Potassium iodide 2 g, water 300 ml.
Alcohol-acetone solution:     3:1
Safranin: safranin saturated alcoholic solution  (about 2.5 grams per 100 ml 95%
alcohol) 10 ml, water 90 ml.
NOTES
Extended discoloring time will result in the crystal violet stain removal from both
Gram-positive and negative cells.
Gram-variable bacteria: some germs with Gram-positive type of wall may not express
this character in particular circumstances (growth conditions, staining procedure,
lesions of the cell wall in the oxygen presence) and may stain  Gram-negative.
Iodine solution is oxidized quite fast and loses it's efectiveness as a mordant. Some
commercial kits use a modified iodine complex formulation:
L-polyvinylpyrrolidone-iodine, which is more stable.
PROCEDURE
Dry and heat-fix the smear then cover it with crystal violet solution. Wait 1 minute then
rinse with distilled water.
Add Gram's iodine (Lugol solution). Wait 1 minute. Remove the mordant and add
alcohol-acetone solution.
Wait few seconds then rinse with distilled water. Cover the slide with safranin or basic
fuchsin.
Wait 1 minute then rinse with distilled water and dry the smear with filter paper.
RESULTS
The Gram-positive cells are not discolored and remain purple.
The Gram-negative cell loses its purple color. Applying safranin or basic fuchsin,
Gram-negative bacteria become pink or red.

QUALITY CONTROLS
Escherichia coli ATCC 25992 for Gram-negative rods,
Staphylococcus aureus ATCC 25923 for Gram-positive cocci.
REFERENCES
1. H. Raducanescu, V. Bica-Popii,1986. Bacteriologie veterinara, Ed. Ceres, Bucuresti.
2. Margaret Barnett, 1992. Microbiology Laboratory Exercises. Wm. C. Brown Publishers.
3. Augustus B. Wadsworth: Standard Methods of the Division of Laboratories and Research of the New York State Department of
Health. 1947, The Williams & Wilkins Company.
4. Dumitru Buiuc, Carmen Panzaru: Coloratii, coloranti si reactivi pentru microscopie, micrometrie. In Dumitru Buiuc, Marian Negut:
Tratat de microbiologie clinica ed. III, 2009, 1173-1215.
A demonstration of Gram staining procedure is available here:
Gram staining technique video.
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