Demonstration of Capsule

y to define capsule, its chemical composition,

importance and how it is formed

y to observe capsule on bacterial cells using Negative

Staining (Duguid s Method and Maneval s Staining Method) and Structural Staining (Anthony s Method)
y to determine the medium which is more favorable for

observation of capsule NAG or NAS

y layer of gelatinous material or viscous substance

produced or secreted by many bacterial cells which accumulates around the cells and coat the cell wall
y most capsules are composed of polysaccharides or of

polypeptides
y Importance: protection from dessication, ability to

resist phagocytosis

Duguid s Method
y made use of nigrosin (acidic dye) y coloring portion of dye bears a negative charge, does

not stain cells due to repulsion of charges, stain is deposited outside the cell
y Result: refractile cells surrounded by a clear halo against

a dark background

Maneval s Staining Method

y made use of congo red and Maneval s solution to give y y y y y

color to the cells and background Congo red: pH indicator Maneval s solution: phenol, acetic acid, ferric chloride Acid fuchsin phenol + ferric chloride, responsible for the red color of bacterial cells Acetic acid causes a drop in pH Results: cells are stained red, capsules are colorless, background can either be blue or red

y Anthony s Method
y uses several reagents/solutions y crystal violet: basic dye, stains the cell but not the

capsule y glucose solution: used to emulsify the cells y copper sulfate: replaces water for washing so that capsule will not be dissolved (capsules: non-ionic); gives a bluish hue to the capsule due to the reaction of Cu2+ with saccharides y Result: cells are stained blue, capsules are light blue to colorless

y Why are smears for capsule staining not fixed by heat?

y Capsule is made up mostly by water, if fixed by heat, it

will be denatured.
y Heat fixing will cause shrinkage of cells. This will affect

the appearance of capsule on bacterial cell.

y Which of the two media was more favorable for

capsule formation of B. megaterium?

y NAG: less capsule formed due to lower sugar

concentration y NAS: greater capsule formation due to higher sucrose concentration

y What would be the effect of prolonged incubation on

capsule formation?
y Prolonged incubation will cause bigger and larger

capsule. It will be easier to observe and distinguish.