Procedure

Procedure

 Preparation of smear

  • Take a clean microscope slide 
  • Place a loopful of water at the centre 
  • Disperse a small amount of culture with inoculating needle 
  • Spread to form a thin smear
  • Air dry and heat fix

Staining method

1.  Cover the smear with crystal violet. Let stand for 30 to 60 seconds

2.  Briefly wash off the stain using a wash bottle and drain off excess water

3.  Cover the smear with Lugol's iodine (Gram's iodine) solution (mordant). Allow standing for one minute.

4. Pour off iodine solution and rinse.

5.  Flood the smear with 95% ethyl alcohol for 10 to 20 seconds. (This step is critical. Thick smears will require more time than thin ones)

6. Rinse the slide with water

     7. Cover the smear with safranin for 30 seconds.

8. Wash gently for a few seconds. Blot with paper and allow to dry at room temperature.

9. Examine the smear under oil immersion using 100x  objective lens.

10.Report the culture as Gram positive if it looks purple or as Gram negative if it appears pink or red.

 Note

i.  Do not make the smears too thick

ii.  Use only young cultures. Old cultures of gram positive bacteria tend to decolourize more rapidly and thus may appear to be gram negative.

 Observation:

 

 

 

 

RESULT: 

 
Last modified: Tuesday, 20 December 2011, 6:53 AM