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GRAM Micro-Organisms Staining Protocol
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GRAM Micro-Organisms Staining Protocol

Prepared by


IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011


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

Technical Points

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


Reagent Formulae

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

iodine crystals 1.0 g
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.

3.      Acetone
     CARE - Fire hazard

4.      Safranin

Obtain commercially – Medvet - Oxoid


Gram C,(1884),Fortschr.Med.,2,185

Lillie RD,(1928),Archs.Path.,

Lillie RD,(1977), The Gram Stain. A quick method for staining gram positive organisms in tissue. Arch Path., V5,p828-834.

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