It is a method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative bacteria.

1. BACTERIAL GRAM
STAINING
HARINI.V
2ND MSc Zoology
2019-2021
Reg no: 1913392077003
MEDICAL LABORATORY TECHNOLOGY

2. INTRODUCTION
 GRAM STAINING is a method of
differentiating bacterial species into 2 large
groups ( GRAM-POSITIVE & GRAM-
NEGATIVE)
 It was developed by CHRISTIAN GRAM in
1884.

3. CONTENT
PRINCIPLE REAGENTS PROCEDURE
WORKS EXAMPLES SUMMARY

4. DIFFERENTIAL STAINING
 Differential staining is a
staining process which
uses more than one
chemical stain.
 It distinguishes organisms
based on their
interactions with multiple
stains.

5. PRINCIPLE
 GRAM POSITIVE- When the bacteria is stained with primary stain crystal
violet and fixed by the mordant, some of the bacteria are able to retain the
primary stain and some are decolorized by alcohol. The cell walls of gram
positive bacteria have a thick layer of protein-sugar complexes called
peptidoglycan and lipid content is low. Decolorizing the cell causes the thick
cell wall to dehydrate and shrink, which closes the pores in the cellwall and
prevents the stain from exiting the cell. So the ethanol cannot remove the
Crystal Violet-Iodine complex that is bound to the thick layer of peptidoglycan
of gram positive bacteria and appears blue or purple in colour.
 GRAM NEGATIVE- cell wall also takes up the CV-Iodine complex but due to
the thin layer of peptidoglycan and thick outer layer which is formed of lipids,
CV-Iodine complex gets washed off. When they are exposed to
alcohol, decolorizer dissolves the lipids in the cell walls, which allows the
crystal violet-iodine complex to leach out of the cells. Then when again
stained with safranin, they take the stain and appears red in color.

7. REAGENTS
•Crystal Violet, the
primary stain
•Iodine, the mordant
•A decolorizer made of
acetone and alcohol
(95%)
•Safranin, the
counterstain

8. PROCEDURE
 Take a clean, grease free
slide and prepare a bacterial
smear.

9. PROCEDURE
 Air dry and heat fix

10. . Crystal Violet was poured and kept for about 30 seconds to 1 minutes and rinse
with water.

11. Flood the gram’s iodine for 1 minute and wash with water.

12. Then ,wash with 95% alcohol or acetone for about 10-20 seconds and rinse with
water.

13. Add safranin for about 1 minute and wash with water.

14. Air dry, Blot dry and Observe under Microscope.

16. GRAM STAINING WORKS
1.The primary stain (crystal violet) binds to peptidoglycan, coloring cells purple. Both gram-
positive and gram-negative cells have peptidoglycan in their cell walls, so initially, all
bacteria stain violet.
2.Gram's iodine (iodine and potassium iodide) is applied as a mordant or fixative. Gram-
positive cells form a crystal violet-iodine complex.
3.Alcohol or acetone is used to decolorize the cells. Gram-negative bacteria have much less
peptidoglycan in their cell walls, so this step essentially renders them colorless, while only
some of the color is removed from gram-positive cells, which have more peptidoglycan (60-
90% of the cell wall). The thick cell wall of gram-positive cells is dehydrated by the
decolorizing step, causing them to shrink and trapping the stain-iodine complex inside.
4.After the decolorizing step, a counterstain is applied (usually safranin, but sometimes
fuchsine) to color the bacteria pink. Both gram-positive and gram-negative bacteria pick up
the pink stain, but it is not visible over the darker purple of the gram-positive bacteria. If the
staining procedure is performed correctly, gram-positive bacteria will be purple, while
gram-negative bacteria will be pink.

17. EXAMPLES
Gram Positive Bacteria: Actinomyces, Bacillus, Clostridium, Corynebacterium,
Enterococcus, Gardnerella, Lactobacillus, Listeria, Mycoplasma, Nocardia,
Staphylococcus, Streptococcus, Streptomyces ,etc.
Gram Negative Bacteria: Escherichia coli (E. coli), Salmonella, Shigella, and other
Enterobacteriaceae, Pseudomonas,Moraxella, Helicobacter, Stenotrophomonas, Bdello
vibrio, acetic acid bacteria, Legionella etc.

19. THANK YOU