Gram staining is the most widely used staining in microbiology. Different steps and reagents used in this method are described here. Basic principle behind the procedure is explained in detail.

1. GRAM STAINING TECHNIQUE
THINGS YOU MUST KNOW
Dr C R Meera
Assistant Professor & HOD
Department of Microbiology
St. Mary’s College, Thrissur-20,
Kerala, India

2. Gram Staining Technique, Dr C R Meera, Assistant Professor & HOD, Dept. of Microbiology, St Mary’s College, Thrissur-20, Kerala.
• Developed in 1880 by the Danish bacteriologist
Christian Gram
"I have therefore published the method, although I am aware that as yet it is very
defective and imperfect; but it is hoped that also in the hands of other investigators it
will turn out to be useful."
Gram Staining Technique

3. • Most important and widely used differential staining in
Microbiology
• Bacteria can be differentiated into two major groups called Gram
positive and Gram negative bacteria.
• 24 hr. old cultures are usually used for gram staining
Gram Staining
Gram Staining Technique, Dr C R Meera, Assistant Professor & HOD, Dept. of Microbiology, St Mary’s College, Thrissur-20, Kerala.

4. • Why bacteria stain differently in Gram Staining?
• The difference in the
chemical and physical nature
of the bacterial cell wall
• G –ive cell wall is thin,
complex, multi-layered,
relatively high lipid contents
and low peptidoglycan
content.
• G +ive cells have less lipid
and thick peptidoglycan
layer.
Gram Staining Technique, Dr C R Meera, Assistant Professor & HOD, Dept. of Microbiology, St Mary’s College, Thrissur-20, Kerala.

5. • What is Gram Staining?
• A type of differential staining
• Four reagents & Four steps
1. Primary stain - Crystal Violet
2. Mordant-Gram’s Iodine
3. Decolorizing agent- 95% ethanol or ethanol-acetone
4. Counter stain or secondary stain –Safranin
• G +ive cells- appear Violet in colour
• G –ive cells- appear Red in colour
Image courtesy: laboratoryinfo.com
Gram Staining Technique, Dr C R Meera, Assistant Professor & HOD, Dept. of Microbiology, St Mary’s College, Thrissur-20, Kerala.

6. • The heat fixed smear treated with
the primary stain called Crystal
violet 30 sec
• Crystal violet - A basic dye and
function is to impart its colour to
all cells
• At this stage all the organisms
appear violet in colour.
• First step in Gram Staining
Gram Staining Technique, Dr C R Meera, Assistant Professor & HOD, Dept. of Microbiology, St Mary’s College, Thrissur-20, Kerala.

7. • Second step in Gram Staining
• Smears are treated with Gram’s Iodine
• Gram’s Iodine - acts as the killing agent
as well as the mordant
• Mordant- a substance that increases the
cells’ affinity for a particular stain
• It binds with the primary stain and
forms an insoluble crystal violet- iodine
(CV-I) complex
• All cells appear violet or purple at this
stage.
Gram Staining Technique, Dr C R Meera, Assistant Professor & HOD, Dept. of Microbiology, St Mary’s College, Thrissur-20, Kerala.

8. • Third step in Gram Staining
• The smear is treated with the
decolorizing agent, like 95% ethanol or
ethanol-acetone solution
• Add ethyl alcohol drop by drop, until
no more colour flows from the smear
• Excess decolourization will make G +ive
organisms lose stain and give false
results
• G -ive bacteria lose the CV-I complex,
whereas G +ive cells retain the same
Gram Staining Technique, Dr C R Meera, Assistant Professor & HOD, Dept. of Microbiology, St Mary’s College, Thrissur-20, Kerala.

9. • Third step in Gram Staining (Conti..)
• Decolorizing agents act as both lipid solvent and
dehydrating agent
• G-ive bacteria, the decolorizing agent dissolves the higher
amount of lipids leading to the formation of large number
pores in the cell wall
• Dehydration and flattening of the cell wall proteins is
taking place, but do not close the pores on the cell wall
appreciably as numerous pores
• Through these pores the CV-I complex escape and the cells
become colourless
• G +ive cell walls are thick and chemically simple, composed
mainly of protein and cross-linked polypeptides
• Lipid is dissolved and few pores are produced
• Protein dehydration causes closure of cell wall pores,
preventing the loss of CV-I complex
Gram Staining Technique, Dr C R Meera, Assistant Professor & HOD, Dept. of Microbiology, St Mary’s College, Thrissur-20, Kerala.

10. • Also peptidoglycan content plays an important
role in this step
• G +ive bacteria peptidoglycan content is high
which is cross linked well
• Porosity is less to allow the escape of the CV-I
Complex
• G -ive bacteria, peptidoglycan content is less and
are poorly cross-linked
• Hence more porosity and CV- I complex can
escape easily
• Third step in Gram Staining (Conti..)
Gram Staining Technique, Dr C R Meera, Assistant Professor & HOD, Dept. of Microbiology, St Mary’s College, Thrissur-20, Kerala.

11. • Smear is treated with the counter stain or secondary
stain called Safranin for 30 sec
• Counterstain - a basic dye having a different colour
from that of the primary stain Crystal Violet
• The G -ive organisms take up this red dye through the
pores created by decolorizing agents and appear red in
colour
• The G +ive organisms which did not lose CV-I
complex will not take up the secondary stain and
remain violet in colour
• Fourth step in Gram Staining
Gram Staining Technique, Dr C R Meera, Assistant Professor & HOD, Dept. of Microbiology, St Mary’s College, Thrissur-20, Kerala.

12. 1. Gram Positive cells- walls retain Crystal Violet and appear deep violet in colour
2. Gram Negative cells- do not retain Crystal Violet and hence take up safranin
3. Gram non reactive organisms- do not stain or which stain poorly
Atypical bacteria remain colourless to Gram staining procedure.
Egs:Organisms under Chlamydiaceae and the Mycoplasmataceae (including mycoplasma)
Rickettsiaceae which are actually G-ive, but too small to stain well by the procedure
4. Gram variable organisms - which stain unevenly during Gram staining
Gram variable reaction - Very old cultures of Gram Positive bacteria
Changes in the environment of the organism
Slight changes in the staining technique
Gram staining procedure divide the bacteria into 4 groups
Gram Staining Technique, Dr C R Meera, Assistant Professor & HOD, Dept. of Microbiology, St Mary’s College, Thrissur-20, Kerala.