Discussion

Exercise 7 discussed the bacterial capsule of three bacteria. The bacterial capsule is made
up of polysaccharides. The capsule is believed to prevent the cell from drying out, protect the
cell from injury, attach the cell to substrate, resist phagocytosis and contribute to biofilm
formation. Bacterial capsule is helpful in bacterial identification and virulence determinants.
Capsulated bacteria are more resistant to pathogens and phagocytosis compared to non-
capsulated bacteria. Generally, the bacterial capsule is non-ionic which makes it difficult to stain.
Staining procedures done to visualize it involves staining the background to create a contrast of
color to see the faint bacterial capsule. The synthesis of the bacterial capsule is affected by
environmental conditions such as temperature, oxygen tension, and concentration of certain ions.
In this exercise, three methods of staining were done to visualize the bacterial capsule.
The first staining technique used was negative staining. Negative staining uses Nigrosin – an
acidic dye which is expected to be repelled by the negatively-charged capsule caused by its
acidic sugar components. This dark stain produces a dark background to see capsules which will
appear as clear zones around cells. Using negative staining is advantageous because the smear
does not need to be heat fixed thus less distortion and damage of the capsule can be eliminated.
Heat fixing is not done in all the stains because the capsule contains highly hydrated
polysaccharides that can shrink when heat is applied to it. After negative staining, unknown
bacterium 2 cells did not show capsules. Rod-shaped cells in chains were clear zones but are no
capsules were seen around it. Smear from the NAG slant showed more chains of cells compared
to that of NAS. The control organisms used were Pseudomonas aeruginosa and Bacillus cereus.
Both control organisms are positive for bacterial capsule and both are made of rod-shaped cells.
Theoretically, cells of both are expected to be clear zones that are much thicker and prominent
compared to unstained cells on a dark background.
The second staining technique used was Anthony’s Method. This is a differential staining
technique which uses a combination of glucose, crystal violet, and copper sulfate solution. 6%
glucose was used to emulsify the cells then the primary stain- crystal violet was introduced. The
crystal violet is taken up by the cell and most of the smear. Copper sulfate solution is then
employed to act as a decolorizer and a negative stain. The copper sulfate solution will decolorize
the capsule by washing off the primary stain. The cell itself will keep the dark blue color. As the
copper sulfate decolorizes the capsule, it also stains it. The dark blue cells are expected to be
surrounded by a faint blue halo- the capsule. The smear from NAG slant of unknown bacterium
2 showed a significant difference from that of NAS. The NAG smear had more chains of cells
and it also had more spores in the background compared to the smear from NAS. The chains of
rod-shaped cells were dark purple and had no faint blue surrounding which could mean that this
unknown does not possess a bacterial capsule. Both the control organisms theoretically possess
light blue to clear zones around a dark blue rod-shaped cell.
The last capsule staining method used was Maneval staining. It is a negative staining
method which uses congo red – an acidic stain which stains the background instead of the
capsule. It also uses Maneval stain. This stain was introduced, and it has various components
needed in order to view the bacterial capsule. Ferric Chloride is a chemical fixative which helps
the stain be fixed on the cell. Phenol acts as an agent of the stain to help in penetrate the cell.
Fuchsin, a basic dye primarily stains the cell. The last component of Maneval stain is glacial
acetic acid which makes the smear acidic. The shift in the pH of the smear together with the
congo red theoretically turns the red background to blue. The cells are expected to be red or pink
in color surrounded by a colorless capsule. Unknown bacteria 2 smear had a red background and
its cells were light red in color. More chains of cells were observed in the smear coming from the
NAG slant compared to the one from NAS. Both the control organisms did not show capsules
during the experiment but according to literature of these organisms possess a bacterial capsule.
B. cereus should be pink with colorless capsule above a purple background.
Nutrient Agar with 2.0% sucrose is theoretically more favorable for capsule formation.
Sucrose can be broken down to glucose and fructose which makes its sugar concentration higher.
Because it can be broken down to simpler compounds, more material is utilized by the bacteria
thus capsule synthesis is favored.
There is not a clear answer whether prolonged incubation affects capsule formation;
however, one result of a study done said that the prolonged incubation made the culture more
viscous and slimy which can be attributed to prominent capsule formation. Also, depending on
physiological factors, prolonged incubation can lead to loss of capsule.

References:
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Cowan, K.M. (2012) Microbiology: A System’s Approach 3rd Edition. McGraw-Hill
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capsule-structure-function-examples-capsulated-bacteria/
Hoogerheide, J. (1939). The conditions under which klebsiella pneumoniae forms
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