Question

1. how important are capsules for bacteria to be able to cause disease?

2. why is it nessary to use a negative stain technique to view capsules? in you answer, be sure to include a definition of negative stain.

3. In preforming the spore stain, why is it necessay to heat malchite green?

4. In a natural habitat such as soil, what advantages does endospores production afford to members of the genera Bacillus and Clostridum over the nonsporing bacteria?

Answer 1

1. The capsule of the bacterium is the polysaccharide outermost covering in bacteria and is known as K antigen. It is the virulence factor of the bacterium. It is present just above the peptidoglycan layer. Most pathogenic bacteria such as Klebsiella pneumoniae, Neisseria meningitidis etc have capsule. Capsules are anti-phagocytic, as they limit the ability of phagocytes to engulf the bacteria. The phagocytes cannot adhere to the bacteria due to the smooth nature and negative charge on the bacteria. Loss of capsule causes loss of pathogenicity. Bacterial capsule prevents the direct access of lysosome contents with the bacterial cells. Hence, the bacteria cannot be killed. Capsules prevent complement mediated cell lysis as well as protect anaerobic bacteria from toxicity due to oxygen present in the host. Capsule help in colonization of surfaces due to formation of biofilms.

2. Negative stain stains the background dark but does not stain the cell. This staining is used to stain bacteria with heat fixation and harsh dyes that distorts the shape of the cells. Bacterial cells are non-ionic. Hence. they are not stained by acidic or basic dyes as these dyes do not adhere to the surface. The acidic dyes in the stain will only stain the background while the basic dye will stain the other components of the cells. Hence, the capsule will be visualized as a bright unstained part against a dark background, due to India ink and a purple color of bacterium (due to crystal violet).

3. Endospore staining by the Schaeffer-Fulton method involves use of malachite green. Endospores have a keratin covering and resist staining. Hence, heating of the cells, allows the malachite green to enter the endospore. Malachite green is water soluble with low affinity for cellular material. Malachite green stains both the endospore and vegetative cells. Decolorization of the cells will remove the stain from the vegetative cells but not the endospore. Hence, endospore can now be visualized easily.

4. Endospores are formed by bacteria when nutrients such as carbon and nitrogen are deprived in soil. Endospores are resistant to heat, dehydration, radiation and chemicals. Dipicholinic acid, a calcium ion chelator, is the molecule that gives resistance to spores. Hence, the bacterium can survive harsh environmental conditions in the soil for a long time. When the soil becomes rich in nutrients, the spore will reproduce again to form the bacteria.