Gram’s Staining

This is the most useful and frequently employed staining technique in bacteriological laboratories. It is a differential staining.It gets its name from the Danish bacteriologist Hans Christian Gram who first introduced it in 1882.Bacteria generally categorized as either Gram positive or Gram negative.

Gram-positive bacteria have a thick peptidoglycan layer and less lipid.With the use of solvent(alcohol), the lipid layer is dissolved. When alcohol is used for destaining, only narrow pores are formed in the cell wall of gram positive bacteria.As a result they retain the primary stain (Crystal violet) after decolorization.Gram-negative bacteria on the other hand possess very thin peptidoglycan layer but thicker lipid layer.When alcohol is used for destaining, lipid dissolves in alcohol making wide\ pores in cell wall through which primary stain leaks out. As a result they become totally decolorized after alcohol washing and finally retain the counter stain (safranin/diluted carbol fuchsin).Some laboratories use safranin as a counterstain; however, basic fuchsin stains gram-negative organisms more intensely than safranin. Similarly, Hemophilus spp., Legionella app, and some anaerobic bacteria stain poorly with safranin.

The length of decolorization is a critical step in gram staining as prolonged exposure to a decolorizing agent can remove all the stains from both types of bacteria.

Reagents (Composition):
a. Hucker’s ammonium oxalate crystal violet
Solution 1
Crystal violet 2 g
Ethanol (95%) 20 ml
Solution 2
Ammonium oxalate 0.8 g
Distilled water 80 ml
Mix solution 1 and solution 2 and filter.
b. Gram’s iodine solution
Iodine crystal 1 g
Potassium iodide 2 g
Distilled water 200 ml

Dissolve the ingredients. Filter and store in amber colour bottle. It acts as ‘mordant’.
c. Decolourizer
It may be absolute alcohol or a mixture of acetone and alcohol (1:1 v/v)
d. Safranin (counter stain)
Safranin (2.5% solution in 95% ethanol) 10 ml
Distilled water 100 ml
Dissolve the stain and filter.
1. Bacterial culture.
2. Reagent a, b, c, d

1. Prepare a bacterial smear upon a grease free slide and fix over flame following standard technique.
2. Flood the smear with ammonium oxalate crystal violet solution and allow to act for 2-3 minutes.
3. Wash under gently running tap water.
4. Fixing of dye-Flood the smear with Gram’s iodine and keep it for 30 sec.Crystal violet- iodine complex is formed.
5. Wash with gently running tap water.
6. Decolourize with available decolourizer by gently agitating the slide till no colour comes out from the smear. Wash with gently running tap water.
7. Flood the smear with safranin/diluted carbol fuchsin and keep it for 30 sec.
8. Wash under gently running tap water, air dry and examine under oil immersion lens.
Gram positive bacteria will be deep violet and Gram negative bacteria will be red/pink in colour.