Uploaded by Dr. Sara Khalid !

2. by
Dr. Khalid Yousuf Memon
Lecturer Dept: of Pathology

3. Gram’s Staining
 OBJECT:
To study the identification &
morphological features of micro-
organisms by Grams’ staining

4. Gram’s Staining
 REQUIREMENTS:
Glass slide, specimen, wire loop, tripod
stand, crystal violet dye, gram’s iodine,
spirit, 1/10 % safranin, water, cedar wood
oil & Microscope

5.  THEORY:
The Gram stain procedure was originally
developed by the Danish Physician Hans
Christian Gram to differentiate
Pneumococci from Klebsiella Pneumonia. It
is also known as “ Differential staining”, as
it divides micro-organisms into two main
groups i.e. Gram’s +ve and Gram’s –ve
micro-organisms.

6. Gram’s Staining
 Crystal violet is a basic dye, it forms a
complex with iodine & get fixed
permanently in Gram’s +ve organisms. The
dye iodine complex is not washed out from
these organisms ever. They are not
decolorized by spirit use.

7. Gram’s Staining
 This is because these organisms contain
thick and multilayered peptidoglycan,
while Gram –ve organisms have single
layered peptidoglycan, therefore the dye is
not fixed permanently in them & is
removed by spirit.

9. Gram’s Staining
 Gram –ve organisms loose the dye and
become colorless while the Gram + ve
organisms retain the colour of crystal violet
so when stained with counter stain i.e. 1/10
% Safranin , Gram –ve organisms appear
pink in colour while Gram +ve organisms
appear violet in colour by retaining the
violet colour.

10. Gram’s Staining
 On the basis of Gram’s staining micro-
organisms can be divided into four groups;
according to their color & shape
 Gram’s +ve cocci: Streptococci, Staphylococci.
 Gram’s +ve bacilli: Corny bacterium Diphtheria,
Clostridia, Bacillus Anthrax.
 Gram’s –ve Cocci: Neisseria.
 Gram’s –ve Baccilli: E.Coli, Salmonella, Shigella,
Proteus, Pseudomonas

11. Procedure
1. Take specimen on the wire loop & spread it
on the slide.
2. Fix the smear by passing it over the flame 2-3
times.
3. Cover the smear with crystal violet for 1-2
minutes.
4. Wash it & than cover it with Gram’s staining
for 01 minute.
5. Wash the smear with spirit till the blue colour
of the slide stops to appear.

12. Procedure
6. Than wash the slide with water & cover it
with 1/10 % safranin red for about ½
minute. Than wash it with water.
7. Allow the slide to dry in air. Put a drop of
cedar wood oil.
8. Than see the slide under oil immersion
lens.

14. RESULT
 Gram +ve organisms appear violet in
colour, Gram –ve organisms appear pink in
colour. The epithelial cells and pus cells are
also pink in appearance.

15. Gram +ve Bacilli

16. Gram +ve & -ve organisms

17. Gram +ve in chains
(S. Pneumoniae)

18. Gram +ve Bacilli
Clostridium Species

19. Gram +ve cocci
Staph: aureus

20. Gram +ve Diplococci
( Pneumococci)

21. Gram +ve rods
( Diphtheroids)

22. G+ve & G-ve Bacilli