The document discusses Gram staining, a technique used to differentiate between Gram-positive and Gram-negative bacteria based on differences in their cell walls. Hans Christian Gram, a Danish bacteriologist, developed the staining method in 1884 to classify bacteria. The Gram staining process involves staining a bacterial smear with crystal violet dye followed by iodine and decolorizing agent treatments, resulting in Gram-positive bacteria appearing dark purple and Gram-negative bacteria appearing pale to dark red under a microscope. Common Gram-positive and Gram-negative bacterial genera are also listed.

1. ALL ABOUT MICROBIOLOGY

2. GRAM STAINING
By Saba

3. INTRODUCTION
 Technique used to differentiate between Gram negative and
Gram positive bacteria.
 Difference on the basis of cell wall constitutes.
 Gram stain colour these cells red or violet

4. HISTORY
 Danish Bacteriologist
Hans Christian
Gram(1853-1938)
developed the
technique of gram
staining.
 Method published in
1884.

5. GRAM REACTION
 Difference in gram reaction is due to cell wall permeability.
Staining with basic dye crystal violet and treatment with iodine,
the complex of stain-iodine is easily removed from more
permeable cell wall as compared to less permeable cell wall of
gram-positive bacteria.

6. METHOD OF STAINING
 Heat fix the dried smear.
 Cover the fixed smear with crystal violet for 30-60 sec.
 Rapidly wash the stain with clean water.
 Tip off water, and cover the smear with Lugol’s iodine for 30 – 60
seconds.
 Wash off the iodine.

7. METHOD OF STAINING
 Decolorize rapidly with acetone alcohol.
 Wash immediately with clean water.
 Cover the smear with safranin/ neutral red stain for 2 minutes.
 Wash the stain.
 Wipe the back side and place in rank for smear to be air dry.
 Examine it microscopically.

9. RESULT
 Gram positive bacteria  Dark purple
 Gram negative bacteria  Pale to dark red
 Epithelial cell  pale red
 Pus cells  red
 Yeast cells  dark purple

10. REPORTING
Number of bacteria present, few, many and moderate.
Gram reaction whether gram negative or positive.
Morphology of bacteria
Presence of pus cells.
Presence of yeast and epithelial cell.

11. GRAM POSITIVE BACTERIA
 Staphylococcus
 Streptococcus
 Clostridium
 Corynebacterium

12. GRAM NEGATIVE BACTERIA
 Klebsiella
 Neisseria
 Salmonella
 Shigella
 Vibrio
 Brucella

13. THANK YOU