Download Exercise 14: Bacterial Endospores

Bacterial Endospores
• Endospores are a dormant stage of some
bacterium that allows it to survive conditions that
would normally kill bacteria such as extreme
drought or heat
• Endospores provide resistance against:
• drying
– Low nutrient conditions
– Radiation
– High temperaturesand various chemical disinfectants
Endospores form within the Cell
The Vegetative Cell Gives Rise to
One Spore
Bacterial
CellCell
Bacterial
Spore
Bacterial Cell
The endospore is able to survive for long periods
of time until environmental conditions again become favorable for growth.
The endospore then germinates, producing a single vegetative bacterium.
Endospore Function
• Endospores are ultimately protection for
the bacterial genome
• Spores form within the cell and contain a
full copy of the bacterium’s genome
• Endospores are not a form of
reproduction, because only one new cell
germinates from each spore
• Spores can be variable in size and
location within the cell
Not all bacterial species can form
spores
• A few genera of bacteria produce
Endospore such as Clostridium
(gangrene) and Bacillus (anthrax), both of
them are gram + rods
• (Endospore production is associated with
Gram Positive bacteria)
• Since not all bacteria form endospores, we
can use this as an identification factor
The shape of the spore is an
identifying characteristic
• Swelled vs. Not swelled
spore
Bacterial cell
spore
Bacterial cell
The location of the spore is also an
identifying characteristic
• Central, Sub-Terminal, and Terminal
spores
Some spore forming bacteria are
capable of causing disease
•
•
•
•
Clostridium botulinum – botulism
Clostridium perfingens – gas gangrene
Clostridium tetani – tetanus
Bacillus anthracis – Woolsorter’s Disease
and wound infections
• The Schaeffer-Fulton Stain Procedure is used to
differentiate between endospores and vegetative
cells
Schaeffer-Fulton Stain Procedure
1. Make a smear. Air Dry. Heat fix
2. Flood the smear with Malachite Green
stain
3. Cover the flooded smear with a square of
filter paper
4. Steam slide for 10 minutes (every minute,
add a few more drops of Malachite Green
stain)
5. Allow slide to cool (after the 10 min.
steam process)
Schaeffer-Fulton Stain Procedure
(continued)
6. Drain slide and rinse for 30 seconds with
DI water (discard filter paper)
7. Put slide on steam rack
8. Flood smear with Safranin (counter stain).
This stains the vegetative cell.
(Leave for 1 minute)
9. Drain the slide and rinse with DI water
10. Blot Dry
11. Use oil immersion objective to view
Endospore Stain Example
Spores: Green
Cell: Red or Pink
Each Person will make a smear and Endospore
stain of: Bacillus pumilus or Bacillus subtilis
BACTERIAL MOTILITY
Examination for Motility by
Hanging Drop Technique
–Many bacteria show no motion and
are termed nonmotile. However, in
environment these bacteria appear to
be moving erratically. This erratic
movement is Brownian movement.
Brownian movement results from the
random motion of the water
molecules bombarding the bacteria
and causing them to move.
• True motility has been recognized
in other bacteria and involves
several different mechanisms. Most
motile bacteria propel themselves by
special organelles termed flagella.
• The above types of motility or nonmotility
can be observed in a hanging drop slide.
Hanging drop slides are also useful in
observing the general shape of living
bacteria and the arrangement of bacterial
cells.
• A ring of Vaseline around the edge of the
cover-slip keeps the slide from drying out
PROCEDURE
•
1. With a toothpick, spread a small ring of Vaseline around the concavity of
a depression slide.
•
2. use the inoculating loop to aseptically place a small drop of one of the
bacterial suspensions in the center of a coverslip .
•
3. Lower the depression slidedown onto the coverslip
•
4. Turn the hanging drop slide over and place on the stage of the
microscope so that the drop is over the light hole.
•
5. Examine the drop by first locating its edge under low power and focusing
on the drop. Switch to the high-dry objective and then, using immersion oil,
to the 100x objective. In order to see the bacteria clearly, close the
diaphragm as much as possible for increased contrast. Note bacterial
shape, size, arrangement, and motility. Be careful to distinguish between
motility and Brownian movement
The arrangement of the flagella about the
bacterium is of use in classification and
identification
• 1. monotrichous - a single flagellum at
one pole
• 2. amphitrichous - single flagella at both
poles
• 3. lophotrichous - two or more flagella at
one or both poles of the cell
• 4. peritrichous - completely surrounded
by flagella