| 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 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.
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.
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.
| Gram positive cocci Staphylococcus lentus |
| Gram negative rods Escherichia coli |
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.
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.
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.
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.
Gram staining technique video.
(c) Costin Stoica
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