Name:___________________________________ Date:____________

Group No._______________
Rating:_________

Laboratory Exercise No .2
BACTERIAL STRUCTURES

Objectives: At the end of the exercise, the students should be able to:
1.perform out a standard procedure for the demonstration of bacterial spores.
2. identify other specific structural components of the bacterial cell and give their functions.
a.capsule
b. flagella
c.metachromatic granules
3.identify the special stains used to demonstrate these special structure.
4.perform a negative staining technique

A.Special Staining: Spore Staining Method

Certain bacteria form spores within the cell called endospores. Endospores are remarkably
resistant to conditions that would otherwise quickly kill the bacteria. Practically only members of
the Genera Bacillus and Clostridium form endospores.

Endospores resist the usual stains such as Gram stain. One such stains, they appear as
relatively transparent spots in the cell. A special staining procedure must be used to stain the
spores properly. Once stained, these organisms are difficult to decolorize.

In this activity, culture of Bacillus subtilis is flooded with malachite green and steamed three
to four times by passing over a flame. Then the stain is washed with water and the counterstain
safranin is applied.

Materials:
5% aqueous solution of malachite greens
Safranin red solution
Culture of Bacillus subtilis

Procedure: Spores are generally very difficult to stain but once stained ,they are likewise
difficult to decolorize. Hence ,special stains have to be used to demonstrate these structures
1, Make a smear on a clean glass slide
2. Flood the smear with malachite green.
3. Heat the flooded smear to gentle steaming for five minutes. Do not allow the smear with stain
to dry up.
4. Pour off excess stain and rinse thoroughly with tap water.
5. Flood the smear with safranin red for 1 minute.
6. Rinse thoroughly with tap water.

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7. Blot dry between sheets of filter paper.
8. Examine under OIO.
9. Observe for the presence of spores and their location.
10. Draw and label the spore which is colored green and the vegetative cell which is colored
red.

Or
2. SPORE STAINS
.
a.Make a thin smear.Air dry and fix.
b.Steam with carbol fuschin for 5 minutes.
c.Decolorize with 5% acetic acid until the film assumes a high pink color.
d. Stain with Loeffler’s alkaline methylene blue for 3 minutes.
e.wash off the excess stain with tap water.
f. Blot or air dry and examine under the oil immersion objective.

B.Capsule Stain (Hiss Capsule Method)

The glycocalyx is currently defined as any polysaccharide-containing structure outside of

the bacterial cell wall.It may be composed of fibrous polysaccharides or globular glycoproteins.
A few bacterial species produce such extracellular structures entirely composed of protein . The
term glycocalyx is generally used for a structural mass of extracellular polysaccharide that
attaches tightly to the cell wall. Less organized and loosely attached extracellular
polysaccharide is referred to as a slime..One type of glycocalyx is the capsule. Capsules vary in
thickness, may be rigid or flexible,and may or may not be closely associated with the bacterial
cell surface.

The glycocalyx has several important functions. With this structure, bacteria can adhere to
other bacteria and to the surface of inert materials (soil,sand,etc) and of animal and plant cells. In
this way, bacteria form microcolonies that are the major type of bacterial growth in nature and in
several diseases. The glycocalyx also provides bacteria with some protection from antibacterial
agents such as antibiotics , bacteriocins, ,immunoglobulins, and phagocytes. This exercise is
concerned with one type of glycocalyx :the bacterial capsule.

Bacterial capsules are indistinct in some organisms and well developed in others . The latter
include Streptococcus pneumonia, Clostridium perfringers, and Klebsiella pneumonia. Capsules
appear to increase the virulence of organisms by protecting them from the defense mechanisms
of their hosts. In addition, capsules impart specific immunologic properties to some
microorganisms. Pneumococci,for example, are differentiated on the basis of the antigenic
characteristics of their capsules, which are polysaccharide in nature. The presence of
pneumococcus capsules is demonstrated by means of a serologic test known as the Quellung
reaction , first described by Neufeld in 1902. The reaction is characterized by the occurrence of
capsular swelling when pneumococci of a specific type are mixed with homologous antisera. In
this exercise, the presence of capsules will be demonstrated by simpler, nonspecific method
called the negative stain.

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 The negative stain technique incorporates material such as India ink, which is composed of
particles too large to enter a cell. A small amount of culture is mixed with India ink, and the
resulting smear is stained with a dye, safranin, which penetrates the bacterial cell. On
examination of the preparation, the capsule will appear as a clear zone surrounding the cell wall.
One cannot state with certainty that all the clear zones observed are capsules, because
shrinkage of cells or withdrawal and cracking of the India ink may cause irregular results. In
general, however, when a treated smear contains many uniformly shaped clear zones, it is
probable that they are actual capsules and not artifacts.

Procedures:
1.CAPSULE STAIN (HISS CAPSULE METHOD)
a. place a drop of the bacterial culture in the center of a glass slide.
b.add a drop of animal serum to it and mix well to make a thin smear.
c. air dry and heat fix very gently by passing the slide over a Bunsen flame for only a few
seconds.
d.cover the smear with crystal or gentian violet. Hold it over a Bunsen burner flame for only a
few seconds.
Note: the crystal violet is used here as a contrast stain. The bacteria will be stained violet (like
the background) but the capsule will appear as clear zone or halo around the bacterial cell.
e.wash off the excess stain with a 20% aqueous solution of copper sulfate.
f.blot or air dry and examine under oil immersion lens.

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 Laboratory Exercise No .2
BACTERIAL STRUCTURES

Name:______________________________________________
Rating_______________________

Results: Draw and label the special structures you have seen. Use colored pencil.

Spore Spore

Stain used:___________________________ Stain used:______________

Functions:_________________________ Functions:_____________

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 Capsule

Stain used:___________________________
Functions:_________________________

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