The cationic dye crystal violet is used to stain the nucleic acids of the micro-organisms and background tissues. The crystal violet staining is then laked with iodine, forming a black complex. Certain micro-organisms resist differentiation due to the impermeability of their cell walls. However, using a suitable differentiator, (eg alcohol, aniline, or acetone), the tissue background and certain species of micro-organisms lose their staining, but take up a cationic dye of contrasting colour (usually red) subsequently applied.
The blue-black staining micro-organisms are termed "Gram positive", whereas the micro-organisms that take up the counterstain (red) are termed "Gram negative".
Gram positive micro-organisms Gram negative micro-organisms
Staphylococcus spp Coli-typhoid-dysentry group
Streptococcus spp Gonococcus spp
Pneumococcus spp Meningococcus spp
B Diphtheria Pasteurella pestis
Chlostridium spp Brucella spp
B Anthracis Salmonella spp
Lactobacillus spp Vibrio cholera
1. A known positive control section must be used to ensure correct differentiation has been achieved.
2. Step 6 - Differentiation with acetone is very rapid being only one or two seconds. The acetone should be poured liberally over the slide to ensure even decolourisation.
1. Bring sections to distilled water
2. On a rack, flood with filtered crystal violet 10 sec
3. Wash briefly in water to remove excess crystal violet
4. Flood with Gram’s iodine 10 sec
5. Wash briefly in water, do not let the section dry out.
6. Decolourise with acetone until the moving dye front has passed the lower edge of the section
7. Wash immediately in tap water
8. Note : If the section appears too blue repeat steps 6 and 7
9. Counterstain with safranin 15 sec
10. Dehydrate absolute alcohol, clear and mount.
Gram positive micro-organisms............................... blue/black
Filaments of nocardia and actinomyces mycelia............ blue
Gram negative micro-organisms, Actinomyces clubs....... red
1. Crystal violet stain
Obtain prepared commercially, or
crystal violet (CI 42555 ) 2.0 g
95% alcohol 20.0 ml
ammonium oxalate 0.8 g
distilled water 80.0 ml
The solution is prepared by dissolving the dye in the alcohol, the ammonium oxalate in the distilled water, and mixing the two solutions together. The mixture is stable for two to three years.
2. Gram's iodine
Obtain prepared stain commercially, or
potassium iodide 2.0 g
distilled water 300.0 ml
Dissolve the potassium iodide in 2 mls to 3 mls only of the distilled water - the crystals will dissolve and the solution will become very cold. Dissolve the iodine crystals in the concentrated potassium iodide solution. Dilute the mixture with the remainder of the distilled water.
CARE - Fire hazard
Obtain commercially – Medvet - Oxoid
Lillie RD,(1977), The Gram Stain. A quick method for staining gram positive organisms in tissue. Arch Path., V5,p828-834.