Demonstration of Capsule

to define capsule, its chemical composition, importance and how it is formed

to observe capsule on bacterial cells using Negative Staining (Duguid’s Method and Maneval’s Staining Method) and Structural Staining (Anthony’s Method)

to determine the medium which is more favorable for observation of capsule – NAG or NAS

layer of gelatinous material or viscous substance produced or secreted by many bacterial cells which accumulates around the cells and coat the cell wall

most capsules are composed of polysaccharides or of polypeptides

Importance: protection from dessication, ability to resist phagocytosis

Duguid’s Method

made use of nigrosin (acidic dye)

coloring portion of dye bears a negative charge, does not stain cells due to repulsion of charges, stain is deposited outside the cell

Result: refractile cells surrounded by a clear halo against a dark background

Maneval’s Staining Method

made use of congo red and Maneval’s solution to give color to the cells and background

Congo red: pH indicatorManeval’s solution: phenol, acetic acid, ferric

chlorideAcid fuchsin – phenol + ferric chloride,

responsible for the red color of bacterial cellsAcetic acid – causes a drop in pHResults: cells are stained red, capsules are

colorless, background can either be blue or red

Anthony’s Methoduses several reagents/solutionscrystal violet: basic dye, stains the cell but not

the capsuleglucose solution: used to emulsify the cellscopper 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

Result: cells are stained blue, capsules are light blue to colorless

Why are smears for capsule staining not fixed by heat?

Capsule is made up mostly by water, if fixed by heat, it will be denatured.

Heat fixing will cause shrinkage of cells. This will affect the appearance of capsule on bacterial cell.

Which of the two media was more favorable for capsule formation of B. megaterium?

NAG: less capsule formed due to lower sugar concentration

NAS: greater capsule formation due to higher sucrose concentration

What would be the effect of prolonged incubation on capsule formation?

Prolonged incubation will cause bigger and larger capsule. It will be easier to observe and distinguish.