Spore Stain and Capsule Stain
Copyright ©2009 by Gary Fromert, M.S.
PETRI Project, Northampton Community College
After completing this exercise, you should be able to:
1.
2.
3.
4.
5.
6.
Explain what is meant by a vegetative cell, endospore and spore.
Explain what is meant by a capsule.
Explain what is meant by a negative stain.
Explain why the cultures used in the capsule staining exercise are not heat-fixed.
Explain why the cultures used in the spore staining exercise are 48 hour and 5 day.
Demonstrate an understanding of the steps and usage of the spore staining and
capsule staining techniques.
We know that the conditions of our surroundings are always changing. For instance, the temperature
may be "warm" one day and be "cold" the next. And when we are exposed to this "cold" day it might
require us to put on a sweater or a jacket. When the temperature rises and gets "warm" again, we can
remove the sweater or jacket. We have the ability to adapt to these changing conditions in our
surroundings as long as the changes are not too extreme. If these conditions in our surroundings, more
aptly referred to as environmental factors, change to the point where they are out of the range of our
adaptability, we would most likely perish if sustained for extended periods of time.
Microorganisms are confronted by similar environmental factors and can adapt to changes within a
reasonable range and period of time. Otherwise, if the conditions do not return to a favorable range, the
microorganisms may also perish. But some microorganisms, and in this case certain bacteria, have a
mechanism to survive extreme conditions for extended periods of time until conditions return to
favorable. A cleanroom is one such environment in which bacteria can be exposed to extreme
conditions especially lacking a nutritional source and being exposed to harsh cleaning chemicals.
But even without proper nutrition and exposure to chemicals some bacteria will survive by forming
spores.
Bacterial spores are a dormant form of a bacterial cell produced by certain bacteria when starved or
exposed to extremes. A metabolically active bacterial cell is referred to as a vegetative cell. When
environmental conditions become unfavorable the cells undergo sporogenesis that gives rise to an
intercellular structure called an endospore. If conditions continue to worsen, the endospore is released
from the degenerating vegetative cell and becomes a new independent structure called a spore. The
spore is resistant to adverse conditions (including high temperatures and organic solvents). The spore
cytoplasm is dehydrated and is not metabolically active. Spores are commonly found in the genera
Bacillus and Clostridium. With the return of favorable environmental conditions, the free spore can
revert to a metabolically active vegetative cell through a process called germination.
In this laboratory exercise you will be using a standard spore staining procedure to differentiate
between vegetative cells, endospores, and spores.
Many bacterial cells are surrounded by a gelatinous or mucoid structure. These structures surround the
outside of the cell envelope. When more defined, they are referred to as a capsule when less defined as
a slime layer or glycocalyx. They usually consist of polysaccharide; however, in certain bacilli they are
composed of a polypeptide. They are not essential to cell viability and some strains within a species
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will produce a capsule, whilst others do not. Capsules of certain bacteria inhibit antibiotics from
entering the cell and can afford some protection to other unfavorable conditions.
Staining of the bacterial capsule cannot be accomplished by simple staining procedures. This is due in
part to the capsule materials being water-soluble and in part to the cells shrinking when heat-fixed
which leaves an artifactual white halo around the cell that might be interpreted as a capsule. But more
importantly, the capsule resists staining. To overcome this problem, the capsule "staining" procedure
utilizes a negative staining technique. In this lab exercise, the negative stain, Congo red or Nigrosin,
colorizes the background and the counterstain, Maneval's stain, colorizes the bacterial cell; leaving the
capsule unstained. Thus, the capsule is observed indirectly by staining everything else but the capsule
itself.
In this laboratory exercise you will also be using a standard capsule staining procedure to indirectly
observe capsules.
Materials (per student)
1 Pure Culture Slant of 48 hour and 5 day Bacillus subtilis or Bacillus cereus
1 Pure Culture Slant of Enterobacter aerogenes or Serratia marcescens
1 Inoculating/Transfer Loop
4 Glass Microscope Slides
3 Glass Cover Slips
Spore Stain Reagents (Schaeffer-Fulton Method)
Capsule Stain Reagents (Congo red or Nigrosin and Maneval's stain)
1 Compound Light Microscope with Oil Immersion Lens
Immersion Oil
Bibulous Paper of Paper Towel
Kimwipes
Lens Paper
China Marker or Sharpie
Bunsen Burner
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Procedure 1
Spore Stain
Acquire a pure culture slant of 48 hour and 5 day Bacillus subtilis or Bacillus cereus. You will prepare
individual heat-fixed slides for each 48 hour and 5 day cultures.
Note: Refer to the Gram staining section of Lab 3 for the heat-fixation procedure.
1. Setup and light a Bunsen burner.
2. Setup a steam bath using approx. 100mL in a 250mL beaker over the Bunsen burner.
3. Label the edge of the slide with the name of the organism and your initials.
4. Place the labeled slide on the bench.
Note: Refer to Figure 1 for steps 5, 6, 7, 8, 9, 10, and 11.
5. Place one drop of sterile water in the center of the slide.
6. Aseptically transfer a small inoculum to the drop of water and swirl with the loop to create the
smear and heat-fix the slide.
7. Cover the smear with a strip of paper towel or bibulous paper. Place the slide over the steam bath
across the opening of the beaker.
8. Apply Malachite green stain to the paper. Steam the stain for 5 minutes. Do not allow the paper to
dry out, keep moist with additional stain.
9. Remove the slide from the steam with a slide holder. Remove the paper and dispose of properly.
10. Gently rinse the excess stain with water from the tap or from a plastic wash bottle.
11. Cover the smear with the counterstain, safranin. Let stand for 60 seconds. Gently wash off the stain
with water. Blot the entire slide within the leaves of bibulous paper to remove the excess water.
Alternatively, the slide may be shaken to remove most of the water and air-dried.
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Figure 1. Spore Stain (http://www.slic2.wsu.edu:82)
Viewing a Spore Stained Slide
Note: Refer to the Gram staining section of Lab 3 for applying the cover slip, applying the
immersion oil to the slide, and viewing the specimen with the high magnification oil immersion
lens.
1. Observe under oil immersion. Refer to Figure 2. Make your observations and record your results.
Note: Discard your used oil immersion slides as instructed by your instructor!
Figure 2. Spore Staining. (http://ftp.ccccd.edu).
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Procedure 2
Capsule Stain
Acquire a pure culture slant of Enterobacter aerogenes or Serratia marcescens.
1. Setup and light a Bunsen burner.
2. Label the edge of the slide with the name of the organism and your initials.
3. Place the labeled slide on the bench.
Note: Refer to Figures 3 and 4 for steps 4, 5, 6, and 7.
4. Place one drop of Congo red or Nigrosin stain at one end of the slide.
5. Aseptically transfer a small inoculum to the drop of stain and swirl with the loop to create the smear.
6. Using a second clean slide, hold this spreader slide at one end from edge-to-edge, at a 45 degree
angle to the surface of the bottom slide, lower the edge of the spreader slide so that it touches the outer
perimeter of the stain droplet and makes contact with the surface of the bottom slide. Note that that
stain will disperse along the edge of the spreader slide.
7. While maintaining contact with the bottom slide, push the spreader slide toward the opposite end of
the bottom slide producing a smear. Dispose of the spreader slide as instructed by your instructor.
8. Air dry or very gently heat-fix the slide. Note: If the heat applied to the slide during heat-fixing is
too intense, the capsule/slime-layer will be destroyed.
9. Cover the smear with Maneval's stain. Let stand for 60 seconds. Pour off the stain and gently rinse
the excess stain with water from the tap or from a plastic wash bottle.
10. Blot the entire slide within the leaves of bibulous paper to remove the excess water. Alternatively,
the slide may be shaken to remove most of the water and air-dried.
Figure 3. Spreading a Smear. (http://www.ruf.rice.edu).
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Figure 4. Capsule Staining. (http://www.slic2.wsu.edu:82)
Viewing a Capsule Stained Slide
Note: Refer to Lab 3 for applying the cover slip, applying the immersion oil to the slide, and
viewing the specimen with the high magnification oil immersion lens.
1. Observe under oil immersion. Make your observations and record your results.
Note: Discard your used oil immersion slides as instructed by your instructor!
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Results and Observations
For the Spore stained slides record your observations below:
Slide
Spore (+) or (-)
Spore Stain Observations
48 hour
B. subtilis or B. cereus
5 day
B. subtilis or B. cereus
For the Capsule stained slide record your observations below:
Slide
Capsule (+) or (-)
Capsule Stain Observations
E. aerogenes or S. marcescens
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Laboratory Review
1. Explain why it is important to avoid heat-fixing bacteria to the microscope slide when performing
the capsule stain:
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
2. Explain the purpose of a capsule:
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
3. In your own words, explain what is meant by a negative staining technique:
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
4. Explain why the cultures used in the spore staining exercise are incubated for 48 hours and 5 days.
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
5. Describe and define each of the following:
Vegetative Cell __________________________________________________________________________________
__________________________________________________________________________________
Endospore __________________________________________________________________________________
__________________________________________________________________________________
Spore __________________________________________________________________________________
__________________________________________________________________________________
6. Briefly explain why a metabolically active bacterial cell would form a spore:
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
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