short Demonstration of gram staining for MBBS stude

1. GRAM STAINING
BY : DR GAURAV MATHUR

2. • Most commonly used DIFFERENTIAL Stain
• Introduced by HANS CHRISTIAN GRAM in
1884

3. Principle
• CRYSTAL VIOLET serves as the primary stain ,
binding to bacterial cell wall after treatment with
a weak solution of IODINE (mordant of dye)
• Some bacterial species because of the chemical
nature of their cell wall have ability to retain the
dye iodine complex even after treatment with an
organic decolorising agent like 95% ETHYL
ALCHOL or ACETONE

4. • Dye Retaining bacteria stains dark purple ( k/a –
GRAM POSITIVE )
• Bacteria which are decolorised takes the counter
stain – Safranin , and appears pink (k/a –
GRAM NEGATIVE )

5. MECHANISM OF GRAM STAIN
LIPID CONTENT THEORY
Gram positive organism contains more protein
while cellwall of gram negative contains more
lipid .During decolorizing the lipid get dissolved
, thus leaking the dye iodine complex out of the
cell,
thus Gram negative organism take the counter
stain. In Gram positive the protein in cell wall
gets dehydrated , shrinking the pore size. Thus
donot get decolorized and take the primary stain

8. PROCEDURE
“COME IN AND STAIN”
COME – CRYSTAL VIOLET (Primary stain)
IN – IODINE (Mordant)
AND- ACETONE ( Decolorizer)
STAIN – SAFRANIN (Counter stain )

9. 1. Fix the slide
2. Cover the smear completey with crystal violet ,
keep it for 1 min
3. Wash the smear under running tap water and
flood the slide with Iodine solution and wait
for1 min
4. Gently drain of iodine and rinse with running
tap water
5. Decolorize rapidy for few seconds with
ACETONE

10. 6. Wash the slide under running tap water and
drain
7.Pour SAFRANIN solution on the slide and keep
it for 30 seconds
8 Wash the slide with tap water – air dry it
9.Examine the smear microscopically with oil
immersion objective lense

12. RESULTS
• Gram positive organism – DARK PURPLE
• Yeast cell – DARK purple
• Gram negative – Pale to dark RED
• Epithelial cell – Pale red

13. Staphylococcus – Grape like clusters –
Gram positive cocci

14. Streptococcus – Chain like – GRAM
positive cocci

15. Gram Negative coccus - NISSERIA

16. Gram positive bacilli – Cornybacterium
Nocardia Listeria Actinomyces

17. • GRAM negative bacilli – PSEUDOMONAS
VIBRIO Legionella Enterobacteriace

18. THANK YOU