Gram staining

It is a differential stain used frequently to differentiate between gram positive (purple) and gram
negative (reddish pink) organisms, based on the physical and chemical properties of cell wall. The
staining method was devised by the histologist Hans Christian Gram in 1884.

 Reagents:
1. Primary stain:
To stain primarily the target organism
Ex: Crystal violet/ Methyl violet/ Gentian violet
2. Mordant:
To fix the primary stain with the cell wall or tissue
Ex: Gram’s iodine
3. Decolouriser:
To decolorize the background except the primary target organism
Ex: Alcohol/ acetone/Acetone alcohol
4. Counterstain:
To stain the background or other tissue/material to enhance contrast of primary stain in
target organism
Ex: Safranin/ Basic fuchsin/ Neutral red

 Smear preparation:

 One glass slide is taken

 It is made dust free and grease free
 Now one inoculating loop is made sterile
 One loopful of specimen is placed on the centre of the slide
 Then with concentric movement with the loop it was spread to make a smear of
approximately 1-2 cm in diameter.

(If the specimen is liquid, then one loopful of liquid specimen is to be placed again to make
layering or heaped up smear to enhance the chance of isolation)
 Then the smear is air dried.

An ideal smear should be 1-2 cm in diameter, in the centre of the slide. It should not be too thick
or too thin and printed letters can be seen through it.
  Fixation:
 The smear is heat fixed by passing the air-dried film above the flame three times with smear
side up.

Fixation of smear

Heat fixation Chemical fixation

1. Alcohol (Ethanol /
Methanol)
2. Formaldehyde
3. Chilled Acetone
By passing the air- By passing the air-dried
(Immunofluorescence)
dried film through the film above the flame
flame three times with 4. Formol sucrose
three times with smear
smear side down 5. Schaudin’s fixative
side up
6. Polyvinyl alcohol
7. 4% Potassium
permanganate (For
Anthrax bacilli)

Mechanism:
Denaturation and coagulation of bacterial protein over cell wall or cell membrane to fix the
organism with the slide.
Advantages:
i) It fixes the smear to get washed away while staining
ii) It kills the bacterium
iii) It permeabilises the cell to stain
iv) It inhibits autolytic enzymes

 Marking of slide: Marking of the smear should be done after preparation of smear, with
grease pencil or diamond marker on the film bearing side or on ground, matt surface at
one end.
  Procedure of Gram staining:

1.crystal violet is poured over the smear and kept for 1 min. The dye is decanted and the smear is
washed in water.

2.Gram’s iodine is poured over the smear and kept for 2 mins and then thrown off and smear is
washed in water.

3.One drop of decolorizing agent is put on the centre of the smear and tilted to spread the
Decolouriser over the smear and washed in tap water. Timing of decolourisation depends upon the
type of Decolouriser used-

a) Acetone  2-3 Seconds

b) Alcohol  10-30 Seconds

c) Acetone-alcohol 3-10 Seconds

4.Safranin is poured over the smear and kept for 1 min. Then the dye is thrown off and the smear
is washed in water.

5.Now the stained smear is air dried, a drop of cedar wood oil is put on the centre of the smear and
examined under oil immersion objective.
 Observations:
Gram positive bacteria are seen as purple coloured whereas gram negative bacteria are seen as
reddish pink coloured.

 Examples:
Bacteria

Gram positive Gram negative

Cocci Bacilli Cocci Bacilli

Staphylococci, Corynaebacterium Gonococci E. coli,
Streptococci, Clostridium, Meningococci Salmonella,
Pneumococci Bacillus Shigella,
Vibrio
 Gram positive cocci and Gram Negative bacilli arranged in cluster under oil-immersion field
(1000X)

 Principle of gram staining:

Although exact mechanism of gram staining is still unknown, various theories have been proposed
to explain why some organism retain the primary stain and some do not.
Primary stain used in Gram stain (Crystal violet or gentian violet) is a basic dye, which dissociates
in the aqueous solution (CVCl2  CV+ + Cl-) and crosses the cell wall layer to reach cytoplasm.
After using mordant (Gram’s iodine), it attaches with the mordant in the cytoplasm to form a larger
dye-iodine complex (CV+ + I-  CV-I Complex).
i) Acid protoplasm theory:
According to this theory, Gram positive bacteria have more acidic cytoplasm than gram negative.
Thus have more affinity towards the basic dye. Iodine also makes the cytoplasm more acidic. This
pH gradient makes the dye easier to cross the cell wall to reach to the cytoplasm. As the cytoplasm
in gram negative bacteria is less acidic, thus forming low pH gradient.
That is why, Gram positive bacteria retain the stain more strongly than Gram negative bacteria.
ii) Isoelectric pH theory:
Isoelectric point is the pH where ionization of amphoteric compound is maximum. It ranges in
Gram positive bacteria is 2-3 whereas in Gram negative bacteria is 4-5. So, there are more
unsaturated acids in Gram positive bacteria, which are oxidized after treatment with iodine to
produce more acidity in the cytoplasm, thus producing higher pH gradient.
 iii) Cell wall permeability theory:
According to this theory, the cell walls of Gram positive bacteria contain more peptidoglycan layer
(50-100 sheets) because of which it is thicker and stronger and is impermeable as compared to
Gram negative bacteria (2-3 sheets of peptidoglycan only).
After using decolorizer during staining procedure, dye-iodine complex diffuses freely out of the
cell in Gram negative cell wall whereas it Gram positive cell wall can retain it due to higher
thickness of cell wall.
iv) Lipid content theory (stern theory):
This is a more recent theory. According to this theory, Gram negative cell wall contain more lipid
content (20%) than Gram positive (4%) cell wall. After adding decolorizer while staining, lipids
are also washed out in alcohol or acetone. As a result, numerous larger pores are formed after
decolourisation, through which dye-iodine complex can be washed out easily. But due to less
amount of lipid in Gram positive cell wall, pore size and number are less and dye-iodine complex
is retained within the cell.