Fundamentals of Microbiology (2+2)

Procedure

A.   Preparation of Smear

1.      Prepare a smear of the broth culture of Bacillus subtilis

2.      Air-dry and heat-fix the smear by passing the slide over the upper part of the flame of a Bunsen burner

B.      Endospore - Staining Procedure

1.        Place the slide on a holder and cover the smear with a 5 percent aqueous solution of malachite green and allow to react at room temperature for 30 to 60 seconds

2.            Place a strip of coarse filter paper over the smear flooded with the dye. Keep the slides over the flowing steam of a beaker containing boiling water.  Heat the slides until it steams three or four times (about 5 min). Do not allow the slides to go dry. Keep the slides moist by adding the dye.

3.            Allow the slides to cool

4.            Wash the slides in tap water for approximately 30 seconds

5.            Cover the smear with a 0.5 percent aqueous solution of safranin and allow to stain for 30 seconds

6.            Wash gently with tap water

7.            Blot dry

Observation of Endospore Stain

1.            Place a drop of oil on the smear and observe with the oil immersion objective

2.            Endospores appear green in color while the vegetative cells appear pink.

RESULT: