TITLE:
Armstrong Atlantic State
University
Medical Technology
Program
Savannah, GA
3200
Microbiology
Clinical Microbiology
Anaerobic Gram-Positive
Spore Forming Bacilli
Clostridium
Clostridium is the only genus of spore-forming anaerobic bacilli. The three best known
species are C. perfringens (cause of clostridial myonecrosis, referred to as gas gangrene), C.
botulimum (cause of botulism), and C. tetani (cause of tetanus).
A Gram-positive anaerobic bacillus is placed in the genus Clostridium if it produces spores.
(The genus Bacillus is the spore-forming bacillus that grows aerobically). The gram stain is
usually sufficient for demonstrating spores, which will appear clear and unstained.
The tests in this lab are for the identification of C. perfringens, the most commonly isolated
Clostridium species in the clinical lab.
Organisms: Clostridium perfringens, Clostridium sporogenes.
Part 1- Perform all gram stains individually. In groups of three set up the aerotolerance test,
egg yolk agar test, and the reverse camp test using appropriate media and organisms. After
setting up your tests, bring them to the instructor to receive a set of incubated plates. As a
group interpret and record the results for all tests.
1. Using sterile technique make a gram stain of each organism from the stock cooked
meat cultures. Cooked Meat Medium supports the growth of spore-forming and nonsporeforming obligate anaerobes, but will encourage the formation of spores in sporeforming anaerobes.
2. Using one anaBAP(CDC) and one chocolate plate, setup the aerotolerance test for
both organisms.
Lecithinase and Lipase Production
Lecithinase and lipase are two enzymes that break down lipids. Their production is
determined on EYA and used for the identification of Clostridium,
Fusobacterium, and Bacteroides.
Interpretation:
a) Lecithinase: Lecithin, a normal component of egg yolk, is split by the enzyme
lecithinase. The result is an opaque, whitish precipitate within the agar beneath
the colonies and surrounding the colonies. C.perfringens is lecithinase-positive.
b) Lipase: Free fats present in egg yolk are broken down by the enzyme lipase to
produce glycerol and fatty acids. The fatty acids appear as an iridescent sheen
(like oil on water) or pearly layer on the surface of the agar. (The plate must be
held at an angle to detect the reaction.) C. sporogenes is lipase-positive.
C. perfringens is lipase-negative.
4. Inoculate organisms on Egg Yolk Agar (EYA) for lecithinase and lipase tests.
Split the EYA agar in half and label one side with C.perfringens and the other side
with C. sporogene. Streak a straight line of C.perfringens on the half corresponding
to the organism. Repeat the same procedure for the other organism.
Reverse CAMP TEST
The reverse CAMP test is used for the presumptive identification of C. perfringens. It is
called the reverse CAMP test because the center streak is a group B beta-hemolytic
Streptococcus rather than Staphylococcus aureus as in the conventional CAMP test.
Procedure for Reserve CAMP: Streak a group B beta-hemolytic streptococci down the center
of an SBA plate. Inoculate a single streak of the Clostridium at a 90-degree angle. Incubate
anaerobically. An arrowhead of hemolysis indicates a positive test.
C. perfringens is usually positive. No other Clostridium species is positive.
5. Follow the procedure for setting a reverse camp test for C. perfringens
Part 2
After setting up your tests, bring them to the instructor to receive a set of incubated
plates. As a group interpret and record the results for all tests.
1. Record results of colonies on anaerobe plates. Is there hemolysis?
C. perfringens usually demonstrates a characteristic double zone of hemolysis after overnight
incubation on blood agar. The hemolytic pattern consists of an interior narrow zone of
complete hemolysis with a much wider zone of incomplete hemolysis on the exterior. This
reaction is best observed within 24 hours and often fades by 48 hours.
2. Observe lecithinase and lipase on EYA.
Table 1. Clostridium
Test
Clostridium
Perfringens
Clostridium sporogenes
Lecithinase
Lecithinase
Lipase
Lipase
Gram Stain
Colony morphology
Aerotolerance
Test
Egg Yolk Agar
Reverse Camp Test