Hans Christian Gram developed a staining technique in 1884 that allows differentiation of bacteria into two types - Gram positive and Gram negative. The Gram staining technique involves staining a bacterial sample with crystal violet, adding iodine to fix the stain, washing with decolorizer such as alcohol or acetone, and counterstaining with safranin. Gram positive bacteria will appear purple due to retention of crystal violet stain, while Gram negative bacteria will appear pink from the counterstain. This simple staining method provides vital information for identifying and diagnosing bacterial infections.

2. Introduction:
Hans Christian Gram (1884)
A technique discriminate between two types of
bacteria
 Streptococcus pneumoniae (also known as the
pneumococcus) and Klebsiella pneumoniae bacteria

3. Applications of Gram staining:
1) Differentiation of bacteria into Gram positive and Gram negative
2) First step towards identification of bacteria in cultures
3) Provides a vital clue in the diagnosis of infectious diseases
4) Total count of bacteria

4. Materials recquired:
Tissue sample
Glass slides
Pipette
A source of flame
Distilled water
Crystal violet
Iodine
Decolorising agent (alcohol or acetone)
Safranin(counter stain)

7. Prepare for laboratory work.
Put on gloves
Tie back long hair to prevent
Check the bunsen burner and microscope are
functional before you begin.

8. Step 1:
Transferring the specimen
Spread the specimen
Step 2:
Fix the specimen by heating
Not to hold the slide over the flame too long

9. Step 3:
 Drop a crystal violet stain (enough to cover the
specimen)
Stand for 20 seconds
Pour off the crystal violet stain and rinse the excess
stain with distilled water

10. Objective:
 Crystal violet stain to bind to the peptidoglycan
molecules
 Crystal violet stain to bind to the lipopolysaccharide
molecules

13. Step 4:
Drop a iodine solution on the smear
Stand for 20 seconds
Pour off the iodine solution
Rinse slide with distilled water
Objective :
Fix crystal violet to the peptidoglycan molecules on the
Gram + bacteria

14. Step 5:
 Drop a decolorizer (acetone or ethanol)
 Trickle down off the slide
Objective :
Dissolve the lipopolysaccharide membrane
Expose the thin peptidoglycan layer

15. Immediately rinse the slide off
Pouring too much decolorizer will cause the
decolorization of the Gram + bacterial cells
If decoloriser ran too long,
see false negative results

16. Step 6:
Drop a basic counterstain (safranin)
Sit for 20 seconds
Wash off the solution
Objective :
Stain the peptidoglycan layer of the Gram - bacteria

17. Addition of iodine to the crystal violet in Step 4
binds the crystal violet stain in the Gram +
bacteria, so the counterstain is unable to bind to
the peptidoglycan wall in the Gram + bacteria in
the specimen

18. Prepare the light microscope
Place the slide under light microscope
Use magnification vary from 400x to 1000x

19. Identify gram positive and gram negative
bacteria
Gram positive bacteria appear purple
Gram negative bacteria appear pink or red

20. Gram positive cocci are either Staphylococci (meaning
cocci in clusters) or Streptococci (meaning cocci in
chains)
Gram positive rods include Bacillus, Clostridium,
Corynebacterium, and Listeria
Gram negative (pink-stained) bacteria are into three groups.
Cocci are spherical bacteria, rods are long, thin bacteria, and
coccoid rods
Gram negative rods include E. coli, Enterobacter, Klebsiella,
Citrobacter, Serratia, Proteus, Salmonella, Pseudomonas

21. THANK YOU…….