Gram staining is a differential staining technique developed by Christian Gram in 1880, crucial for classifying bacteria into gram-positive and gram-negative groups based on cell wall composition. The process involves four main steps: applying crystal violet, using gram's iodine as a mordant, decolorizing with ethanol, and counterstaining with safranin, with positive cells retaining the violet color and negative cells taking on a red hue. Understanding the chemical differences in bacterial cell walls is essential for interpreting gram staining results.

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.