12 Clostridium perfringens
12.1 Introduction reduction (positive) (Labbe, 2001, Rhodehamel and
Clostridium perfringens is a pathogenic bacteria, which
causes foodborne diseases classified by the International
Commission on Microbiological Specifications for
Foods (ICMSF, 2002) into two risk groups. The very
common disease caused by the strains of type A is clas-
sified in risk group III: “diseases of moderate hazard
usually not life threatening, normally of short dura-
tion without substantial sequelae, causing symptoms
that are self-limiting but can cause severe discomfort”.
The much more rare disease caused by the strains of
type C (necrotic enteritis) is classified in risk group IB:
“diseases of severe hazard for restricted population; life
threatening or resulting in substantial chronic sequelae
or presenting effects of long duration”.
12.1.1 Main characteristics
of C. perfringens
According to Rainey et al. (2009) the cells of C. per-
fringens are Gram-positive rods, nonmotile, strictly
anaerobic, catalase-negative. Growth is stimulated by
the presence of a fermentable carbohydrate and is not
inhibited by 20% bile. About 75% of the strains form
capsules, predominantly composed of polysaccharides.
They are sporeforming but spores are rarely seen in vivo
or in vitro. When present they are oval, central or subter-
minal and distend the sporangia. According to ICMSF
(1996) they rarely sporulate in culture but spores are
produced readily in the intestine. According to Bates
(1997) the optimal pH for growth of C. perfringens is
6.0–7.0 and the minimum is 5.5.
The characteristics most used for C. perfringens
identification in food analysis is the sulfite reduction
(positive), motility (negative), lactose fermentation
(positive), gelatin hydrolysis (positive), and nitrate
7007TS-DASILVA-Book.indb 129
Harmon, 2001). However, Rainey et al. (2009) reports
11 to 39% of strain negative for nitrate reduction.
One of the most striking characteristics of C. perfrin-
gens is the ability to grow actively at high temperatures:
maximum of 50°C, optimal between 43 and 47°C,
generation time of only 10 min at 45°C (Bates, 1997).
C. perfringens also ferments milk rapidly at 45°C, pro-
ducing a typical fermentation within 18 h (stormy
fermentation), due to the production of an acid curd
and subsequent disruption of the curd by vigorous gas
formation (Labbe, 2001, Rhodehamel and Harmon,
2001).
At low temperatures, on the contrary, vegetative
cells of C. perfringens are very sensitive. ICMSF (1996)
reports a minimum growth temperature of 12ºC and
survival of only 6% of cells after 14 days in meat stored
at minus 23°C. They die rapidly between zero and 10°C
and storage for only a few days under refrigeration or at
freezing temperatures may lead to a reduction of three
to five logarithmic cycles in plate counts. They are also
relatively sensitive to NaCl, grow well at concentrations
of up to 2% but not at 6.5% NaCl. The minimum
water activity is 0.93.
C. perfringens is capable of producing a series of toxins
and, based on their capacity to produce the four most
lethal toxins (alpha, beta, epsilon and iota), the strains
are classified into five types: A, B, C, D and E (Rainey
et al., 2009), as shown in Table 12.1.
The alpha toxin is a phospholipase C that hydrolyzes
lecithin (lecithinase), and is not associated with food-
borne diseases. It causes gas gangrene (myonecrosis),
an infection of muscle-tissue in animals and humans
(Rainey et al., 2009). According to ICMSF (1996)
the production of the alpha toxin can be observed in
media that contain egg yolk, on which the colonies
are surrounded by a turbid precipitation halo, typical
of lecithinase activity. Inhibition of the activity of the
11/26/2012 11:15:29 AM
 130 Microbiological examination methods of food and water
Table 12.1 Classification of C. perfringens into types based on the
production of the alpha, beta, epsilon, and iota toxins
(Rainey et al., 2009).
C. perfringens type Toxins
Type A alpha
Type B alpha, beta, epsilon
Type C alpha, beta
Type D alpha, epsilon
Type E alpha, iota
alpha toxin by its antitoxin, on the same culture media
(Nagler’s reaction), is a diagnostic test used for the con-
firmation of C. perfringens.
12.1.2 Epidemiology
Only types A and C of C. perfringens have been associ-
ated with diseases transmitted by foods, type A causing
a common and relatively mild sickness and type C a rare
but more severe illness (Bates, 1997).
12.1.2.1 C. perfringens type A food
poisoning
The disease is the result of the action of an enterotoxin
(Clostridium perfringens enterotoxin - CPE) produced
in the intestine of the host after the ingestion of a high
number of cells. The production of CPE is associated
with the in vivo spore formation (Bates, 1997) and
causes an intestinal disorder characterized by abdomi-
nal cramps and diarrhea. The incubation period is from
eight to 22 hours, with the symptoms lasting for about
24 hours (in the elderly or sick people they may last for
up to two weeks) (FDA/CFSAN, 2009).
In outbreaks, the diagnosis is confirmed by the detec-
tion of C. perfringens in suspected foods (counts ≥105/g)
or in the feces of the patients (counts ≥106/g). The
detection of CPE in feces of patients also confirms the
diagnosis. It is important to highlight, however, that a
high number of cells may not be detected in foods, since
C. perfringens loses viability if the product is stored under
refrigeration or frozen for prolonged periods of time. In
these cases, the preparation of smears of the food sam-
ple followed by Gram-staining may help to confirm the
presence of C. perfringens, the presence of which is evi-
denced by high numbers of typically large rods.
C. perfringens is widely distributed in nature, soil,
dust and vegetation. It is also part of the normal flora of
7007TS-DASILVA-Book.indb 130
the intestinal tract of man and animals. Counts of about
103–104/g (dry weight) in the feces of healthy human
adults are normal. When there is clinical involvement,
counts are much higher, however, it is important to
stress that in healthy elderly persons, it is common to
find high number of spores.
The presence of small numbers of C. perfringens is
not uncommon in raw meats, poultry, dehydrated soups
and sauces, raw vegetables, and spices. Spores that sur-
vive cooking may germinate and grow rapidly in foods
that are inadequately refrigerated after cooking (Rhode-
hamel and Harmon, 2001). Meats, meat products, and
gravy are the foods most frequently implicated (FDA/
CFSAN, 2009).
12.1.2.2 C. perfringens type C necrotic
enteritis
The ingestion of foods contaminated with high num-
bers of C. perfringens type C cells causes necrotic enteri-
tis, a very serious disease caused by the beta toxin, which
results in necrosis of the intestine, sepsis or septicemia
and, often, death. According to Bates (1997), the first
cases occurred during the Second World War, due to
the consumption of canned meat. In 1966–1967 new
cases were reported in New Guinea, due to the con-
sumption of contaminated pork meat. The disease was
called the “pigbel” syndrome. The beta toxin is sensi-
tive to trypsin, but, in New Guinea, the population is
more vulnerable as a result of the high consumption
of sweet potatoes. Sweet potatoes contain an inhibi-
tor of trypsin which predisposes patients to the dis-
ease, particularly children and adolescents. Hunger
and malnutrition also reduce the level of trypsin in the
gastrointestinal tract, thereby increasing susceptibility
to the beta toxin. This has probably been one of the
factors involved in the necrotic enteritis cases occurred
during the Second World War. Sporadic cases have
also been reported in Uganda, Malaysia, and Indonesia
(Murrel, 1983).
12.1.3 Methods of analysis
There are several culture media available for the enu-
meration of C. perfringens in foods, such as Neomycin
Blood Agar, Cycloserine Blood Agar, Sulfite Polymyxin
Sulfadiazine Agar (SPS), Tryptone Sulfite Neomycin
Agar (TSN), Shahidi Ferguson Perfringens Agar (SFP),
Trypticase Soy Sheep Blood Agar (TSB), Oleandomy-
11/26/2012 11:15:29 AM

cin Polymyxin Sulfadiazine Perfringens Agar (OPSP)
and Tryptose Sulfite Cycloserine Agar (TSC), which
may be used added with or without egg yolk (Labbe,
2001).
The selectivity of these media results from the incor-
poration of one or more antibiotics, added to inhibit
several anaerobes and facultative anaerobes and, except
in the case of the media containing blood, the differen-
tial characteristic common to all the other media is the
presence of iron and sulfite. C. perfringens reduces sulfite
to sulfide which reacts with iron and precipitates in the
form of iron sulfide, producing black colonies. Among
these media, SPS and TSN are considered excessively
selective and little satisfactory, since they may inhibit
several strains of C. perfringens. Some strains may grow
on SPS, but without producing characteristically black
colonies. On the other hand, SFP, OPSP and Neomy-
cin Blood Agar are considered not selective enough and,
for that reason, more adequate for situations in which
C. perfringens constitutes the predominant microbiota
in the food to be analyzed. Cycloserine Blood Agar
seems adequate for the enumeration of C. perfringens,
however, the data available that would allow better eval-
uation of its performance are still limited, since it hasn’t
been routinely used in the microbiological examination
of foods (Labbe, 2001).
TSC Agar is the medium most frequently used for
the enumeration of C. perfringens by direct plating, con-
stituting at the same time an excellent alternative for the
enumeration of sulfite-reducing clostridia in general,
due to its ability to suppress the growth of practically
all facultative anaerobes that accompany clostridia in
foods. It is important to emphasize, however, that TSC
Agar (as well as the media used in the subsequent stages
of the analysis) allows the growth and toxin production
of C. botulinum, and for that reason, utmost care should
be taken when performing the tests, to ensure the safety
of both the analysts and the laboratory.
When used for enumerating C. perfringens, the TSC
medium may be supplemented with egg yolk to verify
the production of the alpha toxin (lecithinase). The
incorporation of egg yolk, however, does not represent
much of an advantage, since some facultative anaerobes
may produce a similar reaction. Furthermore, not all
C. perfringens strains produce a halo indicative of the
lecithinase reaction, thus making it impossible to disre-
gard and eliminate the colonies that are not surrounded
by a halo. The halo produced by a colony may also be
obscured by the halos of other colonies, making visuali-
zation of the reaction difficult.
7007TS-DASILVA-Book.indb 131
Clostridium perfringens 131
The colonies presumptive for C. perfringens on TSC
Agar should be confirmed by biochemical tests, with the
following characteristics being considered the most rec-
ommended for this purpose: motility, lactose fermenta-
tion, hydrolysis of gelatin and nitrate reduction to nitrite.
The characteristic stormy fermentation at 46°C is rec-
ommended by BAM/FDA (Rhodehamel & Harmon,
2001) as a presumptive test for C. perfringens. 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.
12.2 Plate count method APHA 2001
for Clostridium perfringens
in foods
Method of the American Public Health Association
(APHA), as described in Chapter 34 of the 4th Edition
of the Compendium of Methods for the Microbiological
Examination of Foods (Labbe, 2001).
12.2.1 Material required for analysis
Preparation of the sample and
serial dilutions
• Glycerol-Salt Solution Buffered (to treat samples
before storing at 55–60ºC)
• Diluent: 0.1% Peptone Water (PW) or Butterfield’s
Phosphate Buffer
• Dilution tubes containing 9 ml of 0.1% Peptone
Water (PW) or Butterfield’s Phosphate Buffer
• Observation: consult Annex 2.2 of Chapter 2 to
check on special cases in which either the type or
volume of diluent vary as a function of the sample
to be examined.
Presumptive counting
• Tryptose Sulfite Cycloserine (TSC) Agar
• Anaerobic jars
• Anaerobic atmosphere generation systems (Anaero-
gen from Oxoid, Anaerocult A from Merck, GasPak®
from BD Biosciences, or equivalent)
• Laboratory incubator set to 35–37°C
Confirmation
• Thioglycollate Medium (TGM fluid) (tubes)
• Iron Milk Medium Modified (tubes optional)
11/26/2012 11:15:29 AM
 132 Microbiological examination methods of food and water
• Lactose-Gelatin Medium (tubes)
• Motility-Nitrate Medium (tubes)
• Fermentation Medium for C. perfringens containing
1% salicin (tubes)
• Fermentation Medium for C. perfringens containing
1% reffinose (tubes)
• Nitrate Test Reagents (sulfanilic acid solution,
α-naphthol solution)
• 0.04% Bromthymol Blue Indicator
12.2.2 Procedure
A general flowchart for the enumeration of Clostrid-
ium perfringens in foods using the plate count method
APHA 2001 is shown in Figure 12.1.
Before starting activities, carefully read the guidelines
in Chapter 3, which deal with all details and measures
required for performing plate counts of microorgan-
isms, from dilution selection to calculating the results.
The procedure described below does not present
these details, as they are supposed to be known to the
analyst.
a) Storage of samples for analysis: The Compendium
recommends that samples intended to be used
for the enumeration of C. perfringens be analyzed
immediately. If impossible, the samples should
be refrigerated for the shortest possible time, but
should not be frozen. If necessary to store the sam-
ples for longer than 48 hours, treat then with Glyc-
erol-Salt Solution Buffered (in an amount required
to reach a final concentration of 10% of the sam-
ple) and freeze at temperatures between minus 55
and minus 60°C.
b) Preparation of the samples and inoculation: For
the preparation of the samples and serial dilutions
follow the procedures described in Chapter 2. Since
C. perfringens rarely sporulate in food, heat shock
for spore count is not recommended.
Select three appropriate dilutions of the sam-
ple and inoculate in Tryptose Sulfite Cycloserine
(TSC) Agar. Use the pour plating technique and,
after complete solidification of the medium, cover
the surface with a 5–10 ml thick layer of the same
medium. TSC containing egg yolk emulsion may
be used (spread plate technique) with an overlay of
TSC without egg yolk emulsion.
c) Incubation and colony counting: Incubate the
plates (upright position) at 35–37ºC/18–24 h in
7007TS-DASILVA-Book.indb 132
an anaerobic jar. To establish anaerobic conditions,
use anaerobic atmosphere generation systems
(Anaerogen from Oxoid, Anaerocult A from Merck,
GasPak® from BD Biosciences, or equivalent).
Select plates containing 20 to 200 colonies and
count the typical black colonies, which may be sur-
rounded by a zone of precipitate on the TSC con-
taining egg yolk.
Caution: TSC allows the growth (and toxin pro-
duction) of Clostridium botulinum and its colonies
cannot be distinguished from C. perfringens colo-
nies (Serrano & Junqueira, 1991). The plates and
all the cultures isolated during confirmation should
be handled with care.
d) Confirmation: Select five typical colonies for con-
firmation and if there are fewer than five, select
all. Transfer the suspect colonies to Thioglycol-
late Medium (TGM) tubes (before inoculation
exhaust oxygen from the tubes). Incubate the tubes
at 46°C/4 h (water bath) or at 35–37°C/18–20 h.
Check for purity (Gram stain) and purify if
contaminated (streak on TSC, incubate at
35–37ºC/18–24 h in anaerobic condition, transfer
a well isolated colony to TGM and incubate TGM
at 46°C/4 h or at 35–37°C/18–20 h). Use the TGM
cultures for confirmatory tests.
d.1) Lactose fermentation test and gelatin liq-
uefaction test: From each culture, inocu-
late a tube of Lactose-Gelatin Medium
by stabbing (exhaust oxygen from tubes
before inoculation). Incubate the tubes at
35–37°C/24–44 h. A color change from red
to yellow indicates a positive reaction for
lactose fermentation and the lack of color
change indicates a negative reaction. Refrig-
erate the tubes at 5°C/2 h and examine for
gelatin solidification. If the medium remains
liquid, the reaction is positive (gelatin liq-
uefied). If the medium solidifies (negative
result), incubate an additional 24 h at 35°C
and test again. C. perfringens strains ferment
lactose and liquefy gelatin.
d.2) Motility test and nitrate reduction test:
From each culture, inoculate a tube of Motil-
ity-Nitrate Medium by stabbing (exhaust
oxygen from tubes before inoculation). Incu-
bate the tubes at 35–37°C/24 h and check
for motility. C. perfringens is non-motile and
the growth will occur only along the line of
inoculum. To test for nitrate reduction to
11/26/2012 11:15:29 AM
 -1 -2 -3
10 10 10
Homogenization 1 ml 1 ml

9ml 9ml
PW PW

25g of sample + 1 ml 1 ml 1 ml
225ml of Peptone Water (PW)

TSC Tryptose Sulfite Cycloserine (TSC) Agar TSC

(pour plate with overlay)

Anaerobic
o
35-37 C/18-24h

Typical colonies (5)

Thioglycollate Medium (TGM)

35-37oC/18-20h or 46oC/4h (in water bath)

BIOCHEMICAL TESTS

If necessary

Fermentation Medium Fermentation Medium

for C. perfringens for C. perfringens
Motility Nitrate Medium Lactose Gelatin Medium with raffinose with salicin

o
35-37oC/24h o
35-37 C/24-44h 35-37 C/24h 35-37 C/24h
o

NITRATE (+) LACTOSE FERMENTATION (+) RAFFINOSE SALICIN (-)

MOTILITY (-) GELATIN LIQUEFACTION (+) (usually +)

Confirmed cultures

Clostridium perfingens
CFU/g

Figure 12.1 Scheme of analysis for the enumeration of Clostridium perfringens in foods using the plate count method APHA 2001 (Labbe,
2001).

7007TS-DASILVA-Book.indb 133 11/26/2012 11:15:29 AM

 134 Microbiological examination methods of food and water
nitrite, add 0.5 ml each of nitrate test rea-
gents (sulfanilic acid solution and α-naphthol
solution) to each culture. An orange color
indicates a positive reaction (nitrite present,
nitrate reduced to nitrite). If no color devel-
ops (nitrite absent), test for residual nitrate
by adding a small amount of zinc dust. An
orange color indicates a negative reaction
(nitrate is present, has not been reduced) and
the absence of color indicates a positive reac-
tion (nor nitrate or nitrite present, nitrate has
been completely reduced to N2). C. perfrin-
gens strains are positive for nitrate reduction.
d.3) Raffinose and salicin fermentation tests:
The raffinose and salicin fermentation tests
are required only for cultures that do not
liquefy gelatin within 44 h, or are atypical
in other respects. Use a 24 h TGM culture
for the tests. Inoculate 0.1 ml of TGM cul-
ture into a tube of Fermentation Medium
for C. perfringens containing 1% salicin and
0.1 ml into a tube of the same medium con-
taining 1% raffinose (exhaust oxygen from
tubes before inoculation). Incubate the tubes
at 35–37°C/24 h and check for acid produc-
tion. To test for acid, transfer 1 ml of the
culture to a test tube and add two drops of a
0.04% Bromthymol Blue Indicator. A yellow
color indicates that acid has been produced.
Reincubate negative cultures for an additional
48 h and retest for acid production. C. perf-
ringens strains usually ferment raffinose and
do not ferment salicin.
e) Calculation of results: Consider as C. perfringens
the cultures of non-motile Gram positive rods show-
ing lactose fermentation positive, gelatin liquefaction
positive, and nitrate reduction positive. If one of these
reactions is atypical, consider the results of raffinose
fermentation (usually positive), salicin fermentation
(usually negative) and “stormy fermentation” (posi-
tive) to consider the cultures as C. perfringens.
Calculate number of cells/g of sample based on
percentage of colonies tested that are confirmed as
C. perfringens.
Example 1: The presumptive count obtained with
10−4 dilution of sample was 65 (pour plate, 1 ml
inoculated). Four of five colonies tested were con-
firmed (80%). The number of C. perfringens cells/g
of food is 65 × 0.8 × 104 = 5.2 × 105 CFU/g.
7007TS-DASILVA-Book.indb 134
Example 2: The presumptive count obtained with
10−3 dilution of sample was 30 (spread plate, 0.1 ml
inoculated). Two of ten colonies tested were con-
firmed (20%). The number of C. perfringens cells/g
of food is 30 × 0.2 × 103 × 10 = 6.0 × 104 CFU/g
(dilution factor is tenfold higher than sample dilu-
tion because only 0.1 ml was tested).
12.3 Presence/absence method
APHA 2001 for Clostridium
perfringens in foods
Method of the American Public Health Association
(APHA), as described in Chapter 34 of the 4th Edition
of the Compendium of Methods for the Microbiological
Examination of Foods (Labbe, 2001). Not applicable to
water samples.
This method is recommended for foods likely to con-
tain a small population of injured cells.
Caution: All the steps of the presence/absence method
allow the growth (and toxin production) of Clostridium
botulinum, which cannot be easily distinguished from
C. perfringens. The tubes, plates, and cultures isolated
during examination should be handled with care.
12.3.1 Material required for analysis
Presumptive test
• Liver Broth
• Agar Plug (2% agar) sterile
• Tryptose Sulfite Cycloserine (TSC) Agar
• Anaerobic jars
• Anaerobic atmosphere generation systems (Anaero-
gen from Oxoid, Anaerocult A from Merck, GasPak®
from BD Biosciences, or equivalent)
• Laboratory incubator set to 35–37°C
Confirmation
• The same items required as for the plate count
method APHA 2001 (12.2.1)
12.3.2 Procedure
A general flowchart for the detection of Clostridium
perfringens in foods using the presence/absence method
APHA 2001 is shown in Figure 12.2.
11/26/2012 11:15:29 AM
 2g of sample

Liver Broth sealed with Agar Plug

35-37°C/20-24h

Tube with growth and gas

Streak a loopful
Tryptose Sulfite Cycloserine (TSC) Agar
35-37°C/18-24h (anaerobic)

Typical colonies

Thioglycollate Medium (TGM)

35-37oC/18-20h or 46oC/4h (in water bath)

BIOCHEMICAL TESTS

If necessary

Fermentation Medium Fermentation Medium

for C. perfringens for C. perfringens
Motility Nitrate Medium Lactose Gelatin Medium with raffinose with salicin

35-37oC/24h 35-37oC/24-44h 35-37oC/24h 35-37oC/24h

NITRATE (+) LACTOSE FERMENTATION (+) RAFFINOSE SALICIN (-)

MOTILITY (-) GELATIN LIQUEFACTION (+) (usually +)

Confirmed cultures

Clostridium perfingens
Presence in 2g

Figure 12.2 Scheme of analysis for the detection of Clostridium perfringens in foods using the presence/absence method APHA 2001 (Labbe,
2001).

7007TS-DASILVA-Book.indb 135 11/26/2012 11:15:29 AM

 136 Microbiological examination methods of food and water
Before starting activities, carefully read the guidelines
in Chapter 5, which deal with all details and measures
required for performing presence/absence tests. The pro-
cedure described below does not present these details, as
they are supposed to be known to the analyst.
a) Inoculation and incubation. Inoculate about 2 g
food sample into 15–20 ml of Liver Broth (before
inoculation exhaust oxygen from Liver Broth). Over-
lay the medium surface with Agar Plug (2% agar)
sterile. Incubate the tubes at 35–37°C/20–24 h and
examine for growth and gas production (agar plug
displacement).
b) Confirmation. From each tube showing growth
and gas production, streak the culture on Tryptose
Sulfite Cycloserine (TSC) Agar (with or without
egg yolk). Incubate the plates at 35–37°C/18–24 h
under anaerobic conditions. To establish anaero-
bic conditions, use anaerobic atmosphere genera-
tion systems (Anaerogen from Oxoid, Anaerocult
A from Merck, GasPak® from BD Biosciences, or
equivalent). Examine the plates for typical black
C. perfringens colonies. From each plate showing
growth, select at least one colony suspected to be
C. perfringens and continue the procedure for con-
firmation, as described in the plate count method
APHA 2001 (12.2.2.d). Report the result as C. per-
fringens presence or absence in 2 g of food.
12.4 References
Bates, J.R. (1997) Clostridium perfringens. In: Hocking, A.D. Arnold,
G., Jenson, I., Newton, K. and Sutherland, P. (eds.). Foodborne
Microorganisms of Public Health Significance. 5th edition. Chapter 13.
Sydney, Trenear Printing Service Pty Limited. pp. 407–428.
7007TS-DASILVA-Book.indb 136
Cato, E.P., George, W.L. & Finegold, S.M. (1986) Genus
Clostridium. In: Sneath, P.H.A., Mair, N.S., Sharpe, M.E. &
Holt, J.G. (eds.). Bergey’s Manual of Systematic Bacteriology, Vol. II.
Baltimore, Williams & Wilkins. pp. 1141–1200.
FDA/CFSAN (ed.) (2009) Foodborne Pathogenic Microorganisms
and Natural Toxins Handbook “Bad Bug Book”. [Online] College
Park, Food and Drug Administration, Center for Food Safety
& Applied Nutrition. Available from: http://www.fda.gov/
food/foodsafety/foodborneillness/foodborneillnessfoodbornepa-
thogensnaturaltoxins/badbugbook/default.htm [accessed 10th
October 2011].
ICMSF (International Commission on Microbiological Specifica-
tions for Foods) (1996) Microorganisms in Foods 5. Microbiologi-
cal Specifications of Food Pathogens. London, Blackie Academic &
Professional.
ICMSF (International Commission on Microbiological Specifica-
tions for Foods) (2002) Microorganisms in Foods 7. Microbiological
Testing in Food Safety Management. New York, Kluwer Academic/
Plenum Publishers.
Labbe, R.G. (2001) Clostridium perfringens. In: Downes, F.P. & Ito,
K. (eds.). Compendium of Methods for the Microbiological Exami-
nation of Foods. 4th edition. Washington, American Public Health
Association. Chapter 34, pp. 325–330.
Murrell, T.G.C. (1983). Pigbel in Papua New Guinea: An Ancient
Disease Rediscovered. International Journal of Epidemiology,
12(2), 211–214.
Rainey, F.A., Hollen, B.J. & Small, A. (2009) Genus I Clostridium
Prazmowski. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R.,
Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds.).
Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3.
New York, Springer. pp. 738–828.
Rhodehamel, E.J. & Harmon, S.M. (2001) Clostridium perfringens.
In: FDA (ed.) Bacteriological Analytical Manual, Chapter 12.
[Online] Silver Spring, Food and Drug Administration. Avail-
able from: http://www.fda.gov/Food/ScienceResearch/Laborato-
ryMethods/BacteriologicalAnalyticalManualBAM/default.htm
[accessed 10th October 2011].
Serrano, A.M., Junqueira, V.C.A. (1991) Crescimento de Clostrid-
ium botulinum em meios de cultura de Clostridium perfringens
em diferentes atmosferas anaeróbias e temperaturas de incubação.
Revista de Microbiologia, 22(2), 131–135.
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