Spore-Forming Bacteria: The Spore Staining Method

Introduction

There are two genuses of bacteria that are capable of forming spores. The
aerobic spore formers are the genus Bacillus; the anaerobic spore formers are the
genus Clostridium.

Spore formation is an activity performed by bacteria, not for reproductive

purposes, but for survival purposes. The loss or reduction of availability of as few as
one nutrient or alteration in one environmental event is capable of triggering
sporulation. Spores can survive conditions that range from the incredible (King Tut's
tomb) to the sublime (August in Washoe Valley) for whatever period of time
necessary for nutrients or the environment to recover to the growth requirements for
these bacteria.

As spores are formed in the bacteria in response to any of the above

challenges, they develop a thick protective wall. By the stage of wall development,
these "pre"-spores are called endospores and the rest of the bacterium is called a
sporangium. The wall around the endospore makes it particularly difficult to stain.
In order to force a stain to penetrate the endospore wall, it is necessary do heat the
slide and the stain (like in the acid-fast stain) to prod the wall to allow the stain to
enter. The sporangium is counter-stained for contrast and examined microscopically.

Representative bacteria in the anaerobic spore forming bacteria include C.

tetani (causative agent of tetanus), C. perfringens (involved in soft tissue destruction
and the formation of gas gangrene), C. difficile (causative agent of nosocomially
acquired diarrhea) and C. botulinum (causative agent of botulism). It is of interest to
note that the exotoxin produced by C. botulinum is so poisonous that a 6 ounce bottle
of this exotoxin is enough to decimate the world's population.

Representative bacteria in the aerobic spore forming bacteria include B.

anthracis (causative agent of anthrax; wool sorter's disease), B. subtilis and cereus
(cause eye infections, endocarditis, bacteremia, food poisoning) and B. megaterium
(a non-pathogenic bacteria that is used in teaching labs for the purposes of studying
spores).

The purpose of this experiment is to introduce you to spore staining and

reinforce your previous exposure to the Gram stain.
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 Materials and Methods

Materials

Gram stain supplies Basic fuchsin Agar plates with bacteria

Malachite green Microscope slides (2) Wax marking pencil
Staining rack Hot plate Beaker and water
Bibulous paper Lens paper Microscope
Bunsen burner Striker Wire loop and water
Immersion oil Drawing supplies

Method

Mark a target circle on your microscope slides. On one of the slides,

streak B. megaterium and spread with water. On the other slide, streak more
B. megaterium and spread with water. To the first slide, add malachite green
after the slide is placed over a beaker containing boiling water. Maintain wet
stain conditions for 10 minutes. Remove the slide from the water bath and
allow to cool to room temperature. Once the slide has reached room
temperature, rinse it with tap water.

Once the malachite green has been removed from the slide,
counterstain the slide with basic fuchsin. After 1 minute, rinse the slide and
dry with bibulous paper. To the remaining slide, perform the Gram stain.
Examine both slides under the microscope. Record your observations in the
space provided below:

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 B. megaterium – Spore Stain B. megaterium – Gram Stain

What differences do you observe between the staining methods?

REFERENCES

1.Beishir, L.: Microbiology in Practice: A Self-Instructional

Laboratory Course, Fifth Edition. (Harper Collins: New York)
©1991.

2.Gardner, P. and Provine, H.T.: Manual of Acute Bacterial

Infections: Early Diagnosis and Treatment. (Little, Brown and
Co.: Boston) ©1975.

3.Sherris, J.C., Editor: Medical Microbiology: An Introduction to

Infectious Diseases, Second Edition. (Elsevier: Amsterdam)
©1990.
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