BAM Chapter 16: Clostridium

perfringens
Bacteriological Analytical Manual (BAM) Main Page
(/food/laboratory-methods-food/bacteriological-
analytical-manual-bam)

January 2001

Authors: E. Jeffery Rhodehamel (ret.) and Stanley M.

Harmon (ret.)

For additional information, contact Shashi Sharma

(mailto:[email protected])

Food poisoning caused by Clostridium perfringens may

occur when foods such as meat or poultry are cooked and
held without maintaining adequate heating or
refrigeration before serving. The presence of small
numbers of C. perfringens is not uncommon in raw meats,
poultry, dehydrated soups and sauces, raw vegetables,
and spices. Because the spores of some strains are
resistant to temperatures as high as 100°C for more than l
h, their presence in foods may be unavoidable.
Furthermore, the oxygen level may be sufficiently
reduced during cooking to permit growth of the
clostridia. Spores that survive cooking may germinate
and grow rapidly in foods that are inadequately
refrigerated after cooking. Thus, when clinical and
epidemiological evidence suggests that C. perfringens is
the cause of a food poisoning outbreak, the presence of
hundreds of thousands or more of these organisms per
gram of food substantiates the diagnosis.

Illness typically occurs 8-15 h after ingestion of the

contaminated food. The symptoms, which include intense
abdominal cramps, gas, and diarrhea (nausea and
vomiting are rare), have been attributed to a protein
enterotoxin produced during sporulation of the organism
in the intestine. The enterotoxin can be detected in
sporulating cultures, and a method for this purpose is
included. A high correlation has been established
between the ability of C. perfringens strains to produce
enterotoxin and their ability to cause food poisoning.
However, it is difficult to obtain consistent sporulation
with some strains.

C. perfringens cells lose their viability when foods are

frozen or held under prolonged refrigeration unless
special precautions are taken. Such losses may make it
difficult to establish C. perfringens as the specific cause of
a food poisoning outbreak. It is recommended that
samples which cannot be examined immediately be
treated with buffered glycerin-salt solution and stored or
shipped frozen to the laboratory as described below.

A. Sampling
Sample the entire portion of food (whole roast,
chicken, gravy, etc.) or take representative samples
of 25 g each from different parts of the suspect food
because contamination may be unevenly
distributed.

B. Transporting and storage of samples

Transport and examine samples promptly without
freezing, if possible, and store at about 10°C until
examined. If analysis cannot be started within 8 h or
if the sample must be shipped to the laboratory for
analysis, treat it with sterile buffered glycerin-salt
solution, store immediately at -70 to -90°F, and
transport it to the laboratory with dry ice, as
described below.
Use aseptic technique to prepare sample for storage
or shipment. Transfer 25 g portion of sample (sliced
beef, turkey, hash, etc.) to sterile 150 ml container,
such as plastic Whirl-Pak bag. Add 25 ml buffered
glycerin-salt solution, exclude air from bag, and mix
the sample well with glycerin solution. Liquid
 samples such as gravy or beef juice should be mixed
well with equal volume of double strength buffered
glycerin-salt solution.
Store glycerin-treated samples immediately at -70
to -90°F in low temperature freezer or with dry ice
so that freezing occurs as quickly as possible.
Maintain samples at this temperature until analysis.
Thaw samples at room temperature and transfer
sample and glycerin-salt solution to sterile blender
jar. Add 200 ml peptone dilution fluid to blender jar
and proceed with examination.

If sample must be shipped to the laboratory, follow

procedures above and pack frozen sample in
contact with dry ice to maintain temperature as low
as possible during shipment. Pack sample in a
container such as a paint can or Nalgene bottles
which are impervious to CO gas, because absorption
of CO2 by the sample could lower the pH and
diminish the viability of C. perfringens. Store sample
at -70 to -90°F on receipt and keep at this
temperature until examined, preferably within a
few days.

Cultural Methods for Enumeration and Identification

of Clostridium perfringens in Foods

A. Equipment and materials

1. Pipets, 1.0 ml with 0.1 ml graduations, and 10.0

ml with 1.0 ml graduations
2. Colony counter

3. High speed blender, Waring or equivalent, and

1 L glass or metal blender jars with covers; 1
jar required for each sample

4. Anaerobic jars, BBL GasPak, or Oxoid

anaerobic jars equipped with GasPak H2 + CO2
generator envelopes and catalyst

5. Incubator, 35°C
6. Petri dishes, sterile 15 × 100 mm
 7. Platinum loop, 3 mm id
8. Water bath, 46 ± 0.5°C

9. Reversed passive latex agglutination (RPLA)

test kit for C. perfringens enterotoxin (Oxoid
USA, Columbia, MD)

B. Media (/food/laboratory-methods/media-index-bam)
and reagents (/food/laboratory-methods/reagents-
index-bam)

1. Tryptose-sulfite-cycloserine (TSC) agar (M169

(/food/laboratory-methods/bam-media-m169-
tryptose-sulfite-cycloserine-tsc-agar))

2. Egg yolk emulsion, 50% (M51

(/food/laboratory-methods/bam-media-m51-
egg-yolk-emulsion-50))

3. Chopped liver broth (M38 (/food/laboratory-

methods/bam-media-m38-chopped-liver-
broth)) or cooked meat medium (modified)
(M43) (chopped liver is preferred)

4. Thioglycollate medium (fluid) (M146

(/food/laboratory-methods/bam-media-m146-
thioglycollate-medium-fluid-ftg))

5. Iron milk medium (modified) (M68

(/food/laboratory-methods/bam-media-m68-
iron-milk-medium-modified))

6. Lactose-gelatin medium (for C. perfringens)

(M75 (/food/laboratory-methods/bam-media-
m75-lactose-gelatin-medium-clostridium-
perfringens))
7. Sporulation broth (for C. perfringens) (M140
(/food/laboratory-methods/bam-media-m140-
sporulation-broth-clostridium-perfringens))

8. Motility-nitrate medium, buffered (for C.

perfringens) (M102 (/food/laboratory-
methods/bam-media-m102-motility-nitrate-
medium-buffered-c-perfringens))
 9. Spray's fermentation medium (for C.
perfringens) (M141 (/food/laboratory-
methods/bam-media-m141-sprays-
fermentation-medium-clostridium-
perfringens))

10. AE sporulation medium, modified (M5

(/food/laboratory-methods/bam-media-m5-ae-
sporulation-medium-modified-cperfringens))

11. Duncan-Strong sporulation medium, modified

(M45 (/food/laboratory-methods/bam-media-
m45-duncan-strong-ds-sporulation-medium-
modified-c-perfringens))

12. Peptone diluent (R56 (/food/laboratory-

methods/bam-r56-peptone-diluent-01))
13. Nitrite detection reagents (R48
(/food/laboratory-methods/bam-r48-nitrite-
detection-reagents))
14. Glycerin-salt solution (buffered) (R31
(/food/laboratory-methods/bam-r31-glycerin-
salt-solution-buffered))

15. Gram stain reagents (R32 (/food/laboratory-

methods/bam-r32-gram-stain))

16. Fermentation test papers. Saturate 15 cm

Whatman No. 31 filter paper disks with 0.2%
aqueous bromthymol blue solution adjusted to
pH 8-8.5 with ammonium hydroxide. Air-dry
the disks and store for later use.

17. Bromthymol blue, 0.04% aqueous solution (R10

(/food/laboratory-methods/bam-r10-004-
bromthymol-blue-indicator)).
C. Cultural and isolation procedures

Prepare Gram stain of sample and examine for large

Gram-positive rods.

Plate count of viable C. perfringens. Using aseptic

technique, place 25 g food sample in sterile blender
jar. Add 225 ml peptone dilution fluid (1:10 dilution).
Homogenize 1-2 min at low speed. Obtain uniform
homogenate with as little aeration as possible. Using
1:10 dilution prepared above, make serial dilutions
from 10-1 to 10-6 by transferring 10-90 ml peptone
dilution fluid blanks. Mix each dilution thoroughly
by gently shaking before each transfer. Pour 6-7 ml
TSC agar without egg yolk into each of ten 100 × 15
mm petri dishes and spread evenly on bottom by
rapidly rotating dish. When agar has solidified, label
plates, and aseptically transfer 1 ml of each dilution
of homogenate to the center of duplicate agar
plates. Pour additional 15 ml TSC agar without egg
yolk into dish and mix with inoculum by gently
rotating dish.

An alternative plating method preferred for foods

containing other types of sulfite-reducing organisms
is to spread 0.1 ml of each dilution with sterile glass
rod spreader over previously poured plates of TSC
agar containing egg yolk emulsion. After inoculum
has been absorbed (about 5 min), overlay plates
with 10 ml TSC agar without egg yolk emulsion.
When agar has solidified, place plates in upright
position in anaerobic jar. Establish anaerobic
conditions and place jar in 35°C incubator for 20-24
h. (TSC agar containing egg yolk is incubated 24 h.)
After incubation, remove plates from anaerobic jar
and select those containing 20-200 black colonies for
counting. C. perfringens colonies in egg yolk medium
are black with a 2-4 mm opaque white zone
surrounding the colony as a result of lecithinase
activity. Using Quebec colony counter with white
tissue paper over counting area, count black
colonies and calculate number of clostridia cells/g
food. Save plates for identification tests (see D,
below).
Prepare chopped liver broth (or cooked meat
medium) for inoculation by heating 10 min in boiling
water or flowing steam and cooling rapidly without
agitation. Inoculate 3 or 4 broth tubes with 2 ml of
1:10 homogenate as back-up for preceding plating
 procedure. Incubate these tubes 24-48 h at 35°C in
standard incubator. Disregard if plate counts for
viable C. perfringens are positive.
D. Presumptive confirmation test
Select 10 typical C. perfringens colonies from TSC or
TSC-egg yolk agar plates and inoculate each into a
tube of freshly deaerated and cooled fluid
thioglycollate broth. Incubate in standard incubator
18-24 h at 35°C. Examine each culture by Gram stain
and check for purity. C. perfringens is a short, thick,
Gram-positive bacillus. If there is evidence of
contamination, streak contaminated culture(s) on
TSC agar containing egg yolk and incubate in
anaerobic jar 24 h at 35°C. Surface colonies of C.
perfringens are yellowish gray with 2-4 mm opaque
zones caused by lecithinase activity. This procedure
is also used for isolating C. perfringens from chopped
liver broth whenever the organism is not detected
by direct plating on TSC agar.

Iron-milk presumptive test. Inoculate modified

iron-milk medium with 1 ml of actively growing
fluid thioglycollate culture and incubate medium at
46°C in a water bath. After 2 h, check hourly for
"stormy fermentation." This reaction is
characterized by rapid coagulation of milk followed
by fracturing of curd into spongy mass which
usually rises above medium surface. Remove
positive tubes to prevent spilling over into water
bath. For this reason, do not use short tubes for the
test. Cultures that fail to exhibit "stormy
fermentation" within 5 h are unlikely to be C.
perfringens. An occasional strain may require 6 h or
more, but this is a questionable result that should be
confirmed by further testing. Some strains of C.
baratii react in this manner, but this species can be
differentiated by its inability to liquefy gelatin in
lactose-gelatin medium. The rapidity with which the
"stormy fermentation" occurs depends on the strain
and the initial population. Therefore, only actively
 growing cultures are appropriate for this test. The
presumptive test in iron-milk medium may be
sufficient for some purposes. However, the
completed test must always be performed with
isolates associated with food poisoning outbreaks.
The following tests must be included for the
completed test.

E. Completed confirmation test

Stab-inoculate motility-nitrate (buffered) and
lactose-gelatin media with 2 mm loopfuls of pure
fluid thioglycollate medium culture or portion of
isolated colony from TSC agar plate. Stab lactose-
gelatin repeatedly to ensure adequate inoculation,
and then rinse loop in beaker of warm water before
flaming to avoid splattering. Incubate inoculated
media 24 h at 35°C. Examine lactose-gelatin medium
cultures for gas production and color change from
red to yellow, which indicates acid production. Chill
tubes 1 h at 5°C and examine for gelatin
liquefaction. If medium gels, incubate an additional
24 h at 35°C and examine for gelatin liquefaction.
Inoculate sporulation broth with 1 ml fluid
thioglycollate medium culture and incubate 24 h at
35°C. Prepare Gram stain of sporulation broth and
examine microscopically for spores. Store
sporulated cultures At 4° if further testing of isolates
is desired.

C. perfringens is nonmotile. Examine tubes of

motility-nitrate medium for type of growth along
stab line. Nonmotile organisms produce growth only
in and along stab. Motile organisms usually produce
diffuse growth out into the medium, away from the
stab.

C. perfringens reduces nitrates to nitrites. To test for

nitrate reduction, add 0.5 ml reagent A and 0.2 ml
reagent B (R48) to culture in buffered motility-
nitrate medium. Violet color which develops within
5 min indicates presence of nitrites. If no color
develops, add a few grains of powdered zinc metal
and let stand a few minutes. A negative test (no
violet color) after zinc dust is added indicates that
nitrates were completely reduced. A positive test
after addition of zinc dust indicates that the
organism is incapable of reducing nitrates.

Tabulate results. C. perfringens is provisionally

identified as a nonmotile, Gram-positive bacillus
which produces black colonies in TSC agar, reduces
nitrates to nitrites, produces acid and gas from
lactose, and liquefies gelatin within 48 h. Some
strains of C. perfringens exhibit poor sporulation in
sporulation medium or weak lecithinase reactions
on TSC agar containing egg yolk. Organisms
suspected to be C. perfringens which do not meet the
stated criteria require additional testing for
confirmation.
Subculture isolates which do not meet all criteria
for C. perfringens into fluid thioglycollate medium.
Incubate 24 h at 35°C, prepare Gram stain, and
examine for purity and typical cell morphology.
Inoculate 0.1 ml pure fluid thioglycollate culture
into 1 tube of freshly deaerated Spray's
fermentation medium containing 1% salicin, 1 tube
containing 1% raffinose, and 1 tube of medium
without carbohydrate. Incubate media 24 h at 35°C
and examine medium containing salicin for acid and
gas. Test for acid by transferring a 2 mm loopful of
culture to bromthymol blue test paper. Use only a
platinum loop. No color change or development of a
slight green color indicates that acid was produced.
Alternatively, transfer 1.0 ml of culture to test tube
or spot plate and add 1 or 2 drops of 0.04%
bromthymol blue. A light green or yellow color
indicates that acid was produced. Incubate media
for another 48 h and test for acid production. Salicin
is rapidly fermented with production of acid and gas
by culturally similar species but usually is not
fermented by C. perfringens. Acid is usually
produced from raffinose within 3 days by C.
 perfringens but is not produced by culturally similar
species. A slight change in pH can occur in the
medium without fermentation of carbohydrates.

Some species of Clostridium occasionally isolated

from foods have characteristics which differentiate
them from C. perfringens.

C. paraperfringens and C. baratii – slender cells

frequently in filamentous chains with large
spherical bodies in cooked meat or other media
containing carbohydrate; nitrite weak or absent
after 18 h; very weak lecithinase production; gelatin
never liquefied.

C. absonum or C. sardiniensis – young cultures may

exhibit weak motility; gelatin slowly liquefied;
strong lecithinase production; nitrite production
weak or absent after 18 h.

C. celatum – similar to C. paraperfringens, except that

cells form large mass in bottom of tube; usually
grows very slowly; all reported isolates of C. celatum
are from feces. C. celatum differs from C.
paraperfringens by the absence of lecithinase
activity and by the production of acid from starch.

Calculate number of C. perfringens cells in sample on

the basis of percent of colonies tested that are
confirmed as C. perfringens. Example: If average
plate count of 10-4 dilution was 85, and 8 of 10
colonies tested were confirmed as C. perfringens, the
number of C. perfringens cells/g food is 85 × (8/10) ×
10,000 = 680,000. NOTE: The dilution factor with
plates containing egg yolk is tenfold higher than that
of the sample dilution because only 0.1 ml was
plated.

F. Culturing procedures for sporulation and

enterotoxin production
If isolates are to be tested immediately for
sporulation and enterotoxin production, subculture
in fluid thioglycollate broth as described above.
Cultures to be stored or shipped to another
 laboratory for testing should be subcultured in Difco
cooked meat medium and incubated for 24 h at
35°C, followed by an additional 24 h at room
temperature. Store cooked meat culture at 4°. To
subculture for sporulation and enterotoxin
production, mix cooked meat culture with Vortex
mixer and transfer 0.5 ml of the mixture to each of
two tubes containing 10 ml of freshly steamed fluid
thioglycollate medium. Heat one tube in a beaker of
water or in a water bath at 75°C for 10 min, and
incubate at 35°C for 18 h. Incubate the second tube
at 35°C for 4 h, and use this culture to inoculate
modified AE sporulation medium. For best results
use 0.75 ml of 4 h thioglycollate culture to inoculate
15 ml of modified AE or modified Duncan-Strong
sporulation media. Incubate inoculated spore broth
at 35°C in anaerobic jar or incubator for 18-24 h.
Check resulting culture for spores by using a phase-
contrast microscope or by examining stained
smears. Fewer than 5 spores per microscopic field is
not considered good sporulation.

Centrifuge a portion of the sporulated culture for 15

min at 10,000 × g and test cell-free culture
supernatant for enterotoxin by using reversed
passive latex agglutination (RPLA) test kit.

Hypertext Source: Bacteriological Analytical Manual, 8th

Edition, Revision A, 1998. Chapter 16.