TO STUDY CAPSULE STAINING | PHARMACEUTICAL MICROBIOLOGY PRACTICAL

 

S.Y B.PHARM SEMESTER III

PHARMACEUTICAL MICROBIOLOGY PRACTICAL.

                                                               EXPERIMENT NO. 05

AIM: To study capsule staining technique.

REFERENCE: -

1.       Experimental Microbiology book (As Per PCI Syllabus) by Savita Mandan, Umesh Laddha, Sanjay Surana, Published by Career Publication, First edition, Page No 51 - 52.

2.      Pharmaceutical Microbiology (As Per PCI Regulation) by Prof Chandrakant Kokare, Published by Nirali Prakashan, First Edition, Page No. 6.3 – 6.6.

REQUIREMENTS:

CULTURES: Bacterial Culture: 24 hours truth Bacterial culture.

STAIN: Crystal violet, Nigrosin.

APPARATUS: Staining tray, glass slides, Bunsen burner, inoculating loop, glass slide.

EQUIPMENT’S: Microscope.

INTRODUCTION: Capsule is gelatinous outer layer secreted by microorganism which surrounds to the cell wall. It is made up from polysaccharide and some are made up from peptidoglycan. The main purpose of capsule stain is to distinguish capsular material from the bacterial cell. Bacteria are classified into two types depending on the presence or absence of capsule:

i)       Capsulated bacteria which possess capsule.

ii)    Non capsulated bacteria which do not possess capsule. 

PRINCIPLE: Capsules are fragile and can be diminished or destroyed by heating. So drop of serum can be used during smearing to enhance the size of capsule and easily observed by dyes like crystal violet or methylene blue. In this staining like a background staining, capsule remains unstained while the background is stained by either Nigrosin or India ink. 

PROCEDURE:

1.       The slide is cleaned properly under tap water.

2.      Wipe out adhering water from slide surface and air-dry the slide.

3.      A smear of bacteria is prepared at the center of marking area on glass slide.

4.      The smear is air dried, heat fixation is not necessary. Heat treatment may result into cell shrinkage and hence capsular damage. .

5.      The smear is then flooded with primary stain crystal violet for 5-7 minutes.

6.      Wash the smear with 20% copper sulphate solution.

7.      The slide is blotted dry with filter paper.

8.     Observe the smear under low power and then high power objective. 

OBSERVATION AND RESULT: Capsule appears colorless which surrounds the red cells against dark background while non-capsulated bacteria which appear dark purple or blue.