Name: KNEDRICK KEVIN C.

MACALMA Date: FEBRUARY 10, 2017

Group No. 5
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 Loefflers 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: KNEDRICK KEVIN C. MACALMA
Rating
Results: Draw and label the special structures you have seen. Use colored pencil.
B. subtilis spore
(x1000 magnification)
Stain used: malachite green
Functions:
Spores of Bacillus subtilis
- possess a thick protein coat that consists of an
electron-dense outer coat layer and a lamella-like
inner coat layer. The spore coat has been shown to
confer resistance to lysozyme and other sporicidal
substances.
Malachite green
- Stain used in Endospore staining that is water-
soluble and has a low affinity for cellular material,
so vegetative cells and spore mother cells can be
decolorized with water and counterstained with
Safranin. The spore is colored green and the
vegetative cell is colored red.
Prepared by:Ma.Tereza A. Blanco Med Micro Lab
B. subtilis spore
(x1000 magnification)
Stain used: carbolfuchsin
Functions:
Spores of Bacillus subtilis
- possess a thick protein coat that consists of an
electron-dense outer coat layer and a lamella-like
inner coat layer. The spore coat has been shown to
confer resistance to lysozyme and other sporicidal
substances.
carbolfuschin
- the dyes can be made to penetrate the spore by
heating the preparation. The same permeability then
serves to prevent decolorization of the spore by a
period of alcohol treatment sufficient to decolorize
vegetative cells. The latter can be counterstained.
The spore retain the carbolfuchsin stain.
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 S. pyogenes capsule
(x1000 magnification)
Stain used: crystal violet
Functions:
The capsule of S. aureus
- enhances staphylococcal virulence by
impeding phagocytosis, resulting in bacterial
persistence in the bloodstream of infected
hosts. S. aureus capsules also promote abscess
formation in rats. Although the capsule has
been shown to modulate S. aureus adherence
to endothelial surfaces in vitro, animal studies
suggest that it also promotes bacterial
colonization and persistence on mucosal
surfaces.
crystal violet
- used 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.
Prepared by:Ma.Tereza A. Blanco Med Micro Lab
S. aureus capsule
(x1000 magnification)
Stain used: Nigrosin
Functions:
The Streptococcus pyogenes capsule
- is required for adhesion of bacteria to virus-
infected alveolar epithelial cells and lethal
bacterial-viral infection.
Nigrosin
- used in negative staining; it is used to
determine the morphology and cellular
arrangement in bacteria that are too delicate to
withstand heat fixing. Also, where determining
the accurate size is crucial, a negative stain can
be used because it produces minimal cell
shrinkage.
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QUESTIONS:

1. Why is heat not used in capsule heating procedure using Nigrosin?

Heating causes the cells to shrink, leaving an artificial white halo around them that might be
interpreted as a capsule. In the place of heating, cells may be emulsified in a drop of serum to
promote their adhering to the glass slide.

2. How important are capsules in establishing a disease process?

The capsule is considered a virulence factor because it enhances the ability of bacteria to
cause disease (e.g. prevents phagocytosis). The capsule can protect cells from engulfment by
eukaryotic cells, such as macrophages. A capsule-specific antibody may be required
for phagocytosis to occur. Capsules also contain water which protects the bacteria
against desiccation. They also exclude bacterial viruses and most hydrophobic toxic
materials such as detergents. Immunity to one capsule type does not result in immunity to the
other types. Capsules also help cells adhere to surfaces.

The bacterial surface contains various structures that are capable of activating the host
defenses and consequently inducing host immune responses. Bacterial capsules are one of
the most external structures on the bacterial surface, which may completely surround all the
antigenic molecules or may be co-expressed with other bacterial antigens. They are involved
in a wide range of biological processes, such as prevention of desiccation, adherence and
resistance to nonspecific and specific host immunity.

Gramnegative and Grampositive capsular polysaccharides contribute to the bacterial

resistance of host immune responses by different mechanisms. Usually, capsular
polysaccharides that mask the underlying cell surface structures activate weakly or not at all
the immune system, whereas bacterial capsules co-expressed with other bacterial antigens
activate the immune system but mask opsonins and prevent complement attack complex
formation, as well as phagocytosis.

3. Are capsules found both in gram (+) and gram (-) bacteria?

The capsule can be found in both Gram-negative bacteria and Gram-positive bacteria.

4. Give 3 specific functions of capsules

a. The capsule is considered a virulence factor because it enhances the ability of bacteria to
cause disease (e.g. prevents phagocytosis).
b. The capsule can protect cells from engulfment by eukaryotic cells, such as macrophages.
A capsule-specific antibody may be required for phagocytosis to occur.

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 c. Capsules also contain water which protects the bacteria against desiccation. They also
exclude bacterial viruses and most hydrophobic toxic materials such as detergents.
Immunity to one capsule type does not result in immunity to the other types. Capsules
also help cells adhere to surfaces.

5. Can the presence of a capsule always be correlated with virulence? If not Give an example

Capsule (also known as K antigen) is a major virulence factor of bacteria, e.g. all of the
principal pathogens which cause pneumonia and meningitis, including Streptococcus
pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Klebsiella pneumoniae,
Escherichia coli, and group B streptococci have polysaccharide capsules on their surface.
Nonencapsulated mutants of these organisms are avirulent.

However, other factors can be attributed to virulence. These virulence factors include:
Adherence components (for attachment) and invasion enzymes (for the entry into host,
colonization).

Invasion enzymes include hyaluronidase that breaks down the polysaccharide that glues the
host cells together, collagenase that breaks down collagen network in connective tissues,
fibrinolytic enzymes (e.g., streptokinase) that destroys fibrin of blood clots (escape),
coagulase that promotes blood clotting (protection) and various other proteases, nucleases,
lipases.

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REFERENCES:

Merino, S., & Toms, J. M. (2015, July). Bacterial Capsules and Evasion of Immune Responses.
Retrieved February 05, 2017, from http://www.els.net/WileyCDA/ElsArticle/refId-a0000957.html

"Gram Stain, Capsule Stain and Endospore Stain." N.p., n.d. Web. 05 Feb. 2017.

Todar, Kenneth, PhD. "Structure and Function of Bacterial Cells." Structure and Function of
Bacterial Cells. Todar's Textbook of Bacteriology, n.d. Web. 05 Feb. 2017.

Acharya, Tankeshwar, and Tankeshwar Acharya I Am a Medical Microbiologist Cum Lecturer. I Am

Teaching Microbiology to Medical Students in Patan Academy of Health Sciences, School of
Medicine, Lalitpur, Nepal. "Bacterial Capsule: Structure, Importance and examples of capsulated
bacteria." Microbeonline. N.p., 29 June 2016. Web. 05 Feb. 2017.

Okamoto, S., Kawabata, S., Terao, Y., Fujitaka, H., Okuno, Y., & Hamada, S. (2004, October).
TheStreptococcus pyogenesCapsule Is Required for Adhesion of Bacteria to Virus-Infected Alveolar
Epithelial Cells and Lethal Bacterial-Viral Superinfection. Retrieved February 06, 2017, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC517596/

Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. (n.d.).
Retrieved February 06, 2017, from http://onlinelibrary.wiley.com/doi/10.1111/j.1365-
2672.2005.02736.x/full

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