KOLAWOLE BISOLA MERCY

BACTERIOLOGY ASSIGNMENT

Activity 1

Reagents used for Capsule Staining

Crystal Violet (1%)

Crystal Violet (85% dye content) = 1 gm

Distilled Water = 100 ml

Nigrosin

Nigrosine, water soluble = 10 gm

Distilled Water = 100 ml

Procedure of Capsule Staining

• Place a small drop of a negative stain (India Ink, Congo Red, Nigrosin, or
Eosin) on the slide.
• Congo Red is easier to see, but it does not work well with some strains. India
Ink generally works, but it has tiny particles that display Brownian motion
that must be differentiated from your bacteria. Nigrosin may need to be kept
very thin or diluted.
• Using sterile technique, add a loopful of bacterial culture to slide, smearing it
in the dye.
• Use the other slide to drag the ink-cell mixture into a thin film along the first
slide and let stand for 5-7 minutes.
• Allow to air dry (do not heat fix).
• Flood the smear with crystal violet stain (this will stain the cells but not the
capsules) for about 1 minutes. Drain the crystal violet by tilting the slide at a
45 degree angle and let stain run off until it air dries .
• Examine the smear microscopically (100X) for the presence of encapsulated
cells as indicated by clear zones surrounding the cells.
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 • Capsule: Clear halos zone against dark background
• No Capsule: No Clear halos zone
https://m.youtube.com/watch?v=0_cEdk1gx2A

Activity 2

1. Cells are stained with crystal violet dye. Next, a Gram's iodine solution
(iodine and potassium iodide) is added to form a complex between the
crystal violet and iodine. This complex is a larger molecule than the original
crystal violet stain and iodine and is insoluble in water.
2. A decolorizer such as ethyl alcohol or acetone is added to the sample, which
dehydrates the peptidoglycan layer, shrinking and tightening it. The large
crystal violet-iodine complex is not able to penetrate this tightened
peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria.
Conversely, the outer membrane of Gram-negative bacteria is degraded and
the thinner peptidoglycan layer of Gram-negative cells is unable to retain
the crystal violet-iodine complex and the color is lost.
3. A counterstain, such as the weakly water-soluble safranin, is added to the
sample, staining it red. Since the safranin is lighter than crystal violet, it does
not disrupt the purple coloration in Gram positive cells. However, the
decolorized Gram-negative cells are stained red.

Assignment 1

Bacterial endospores are special tough, dormant and resistant spores

produced by some Gram-positive bacteria of Firmicute family during
unfavorable environmental conditions. Endospores are developed within the
vegetative cells (hence the name, endo = inside). They help the bacteria to
endure the unfavorable environmental conditions.

Another importance of endospores is that it can be easily dispersed by wind,

water and through the gut of animals. Bacillus and Clostridium are the most
studied endospore forming bacterial genera. Bacillus enters into endospore
formation cycle when the carbon or nitrogen source is getting limited in the
growing medium.

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Important Properties
• Endospores are exceptionally resistant to stressful environmental
conditions such as heat, ultraviolet radiation, gamma radiation,
chemical disinfectants and desiccation.
• Most of the endospores are viable for many years, even for 10, 000
years or more.
• Due to this long viability and their adaptations to stress conditions,
most of the endospores producing bacteria are notorious pathogens.
• Wiping with alcohol or hydrogen peroxide or boiling at 100oC will not
kill the bacterial endospores.
• However, endospores can be killed by autoclaving (at 121oC).
• Endospores can be visualized under light and electron microscope.
• Endospores will NOT take the usual bacterial stains such a safranin
used in Gram-staining.
• Specific stains and special staining techniques are required to stain the
endospores.
• The classically used stain to visualize endospore is Malachite Green
and the staining procedure is known as Schaeffer–Fulton Staining.
• The endospore-producing mother cell is called sporangium.
• Sporangium shows distinct differences from other vegetative cells.
• These characteristics are used for the identification purpose in
bacterial taxonomy.
• The position of the endospore within the spore mother cell also varies.
• Based on the position of spores, the sporangium/spores may be
Central spore, Sub-terminal spore, Terminal spore or Terminal spore
with swollen sporangium.
• Both aerobic and anaerobic bacteria (of Gram-positive type) can
produce endospores.
• No Archaebacteria are known to produce endospores.

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Ways to demonstrate its presence
1. By staining
2. Microscopically
Procedure of Endospore Staining
1. Take a clean grease free slide and make smear using sterile technique.
2. Air dry and heat fix the organism on a glass slide and cover with a square of
blotting paper or toweling cut to fit the slide.
3. Saturate the blotting paper with malachite green stain solution and steam for
5 minutes, keeping the paper moist and adding more dye as required.
Alternatively, the slide may be steamed over a container of boiling water.
4. Wash the slide in tap water.
5. Counterstain with 0.5% safranin for 30 seconds. Wash with tap water; blot
dry.
6. Examine the slide under microscope for the presence of endospores.
Endospores are bright green and vegetative cells are brownish red to pink.
Result of Endospore Staining

Endospores: Endospores are bright green.

Vegetative Cells: Vegetative cells are brownish red to pink.

The stained slide can be viewed under the microscope.

Assign 2.
Size - As mentioned, although they are both shorter and generally smaller compared
to flagella, pili are longer than fimbriae with a hair-like appearance. Fimbriae, on the
other hand, are shorter with a bristle-like appearance.

Bacteria - Although some Gram-positive bacteria have been shown to possess pili,
these structures are commonly found in Gram-negative bacteria. Fimbriae, on the
other hand, can be found in both Gram-positive and Gram-negative bacteria where
they are involved in adhesion and biofilm formation.

Distribution - Unlike pili, fimbriae are numerous in number and tend to be evenly
distributed on the surface of bacterial cells. Whereas a single bacterial cell may

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contain between 200 and 400 fimbriae on its surface, the number of pili may range
from less than 5 to about 10 in total.

Function - While fimbriae are primarily involved in attachment, which promotes

biofilm formation, pili are involved in attachment, motility as well as gene transfer
from one bacterial cell to another.

Fimbriae have a more solid structure (because they are primarily involved in
attachment) while pili, especially sex pili, have a hollow tubular structure that allows
for genetic material to be transferred from one cell to another.