Download EVALUATION OF DIFFERENT GROWTH MEDIA FOR THE

Presented at the WISA 2000 Biennial Conference, Sun City, South Africa, 28 May – 1 June 2000
EVALUATION OF DIFFERENT GROWTH MEDIA FOR THE RECOVERY OF
SULPHIDE REDUCING ANAEROBIC Clostridium perfringes FROM THE
ENVIRONMENT
N. Potgieter, E.Musie, R. Sono, C.L. Obi and P.J. du Toit
The Department of Microbiology, University of Venda,
Private Bag X 5050, Thohoyandou, 0920, South Africa
Abstract
Clostridium perfringes is a gram positive, spore forming, anaerobic bacterium, and its spores are
present in human and animal faecal matter in water and soil. The use of this organism as an
indicator of water quality has not received its full attention, even though the presence of
Clostridium perfringes in water can be considered conclusive evidence of faecal pollution. This
organism is also extremely resistant to the action of chemical and physical factors which are used
for the treatment of water. The objective of this study was to optimize and assess different
growth media for the detection of Clostridium perfringes from environmental samples Three
agar growth media, SFP (Bacto Perfringes agar base), OPSP (Oxoid perfringes agar) and BA
(blood agar), were compared for utilization in the membrane filtration counting of Clostridium
perfringes from different water sources in rural settlements. The results indicated that SFP agar
recovered significantly greater numbers of C.perfringes than OPSP and blood agar. API and
other identification test showed a significant number of false presumptive positive and negative
isolates on OPSP and blood agar.
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Introduction
Microbial monitoring of drinking water sources requires the use of microorgnisms as indicators
of contamination. The most commonly used indicator of faecal pollution of water sources is
Eschericia coli, but the use of Clostridium perfringes as a secondary indicator of contamination
of water sources and supplies, has recently begun to receive attention (Burger et al., 1984; Arion,
1994; Satory et al., 1998).
Clostridium perfringes occur widely in the environment and in human and animal faeces (Rood,
1991). The organism has been incriminated in food and water borne diseases especial gasgangrene and food poisoning in humans (Rood and Cole, 1991). Occurrence in aquatic
environments is attributable to faecal pollution (Cabelli, 1977). C. perfringes is endowed with
spores which are resistant to adverse environmental conditions and water treatment processes
(Grabow et al., 1978). Burger et al. (1984) reported that counts of the organism in waste and
polluted waters are usually greater than those of enteric viruses or pathogenic bacteria and that
the spores are highly resistant to disinfection and water treatment processes compared with other
organisms. This connotes that the absence of C. perfringes in treated water supplies is a possible
index of the absence of enteric viruses and bacterial pathogens (Bisson and Cabelli, 1980). The
presence of C. perfringes in water meant for human consumption therefore implies that the water
is not safe for consumption because it is an opportunistic pathogen.
In spite of the excellent features of C. perfringes as a primary candidate for assessing faecal
pollution or efficiency of water treatment procedures, its use as a primary indicator has not been
generally adopted because of lack of unity of opinions among researchers on the array of
available test methods (Grabow and Isaacson, 1978). However, recently, the European Union
(1998), proposed that a new microbiological standard for C. perfringes (0/100 ml) be adopted
in their directive on drinking water.
Several media have been described and employed for the enumeration of C. perfringes. These
include mCP medium (Bisson and Cabelli, 1979; Armon and Paymnet, 1988), Tryptose Sulphite
Cycloserine (TSC) agar (Satory et al., 1988), and Wilson and Blair Glucose Sulphite Iron (WB
) medium (Burger et al., 1984). In this study SFP (Bacto Perfringes agar base), OPSP (Oxoid
perfringes agar) and BA (blood agar) were compared for use in the membrane filtration
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enumeration of C. perfringes in water. Results obtained are expected to be of value in
determining the best media for the enumeration of C. perfringes in our environment.
Materials and methods
Growth media
SFP was prepared as specified by the manufacturers using Bacto SFP Agar base (Code 0811-018), Bacto egg yolk enrichment 50% (Code 3347-61-2), Bacto antimicrobial vial K (Code 333960-3) and Bacto antimicrobial vial P (Code 3268-60-8).
OPSP was prepared from Oxoid
Perfringes agar base (Oxoid CM543), OPSP-selective supplement A (Oxoid SR76) and OPSPselective supplement B (Oxoid SR77), according to the manufacturer=s instructions. Blood agar
plates were supplied by the Department of Medical Microbiology, University of Pretoria, South
Africa.
Membrane filtration tests
Aliquots (10 ml and 100 ml) from environmental water sources were filtered through 47 mm
Gelman GN-6 membrane filters (pore size 0,45 µm). The plates were incubated anaerobically
in gas jars using Oxoid anerobic gas generating kits. Incubation was at 37YC for 18 - 24 hours.
All tests were done in triplicate and results recorded as average values. All black colonies on
SFP and OPSP media and colonies with clear haemolysis on blood agar, were counted as
presumptive C. perfringes.
Identification of isolates
Colonies (50 from each media type) were picked from the membranes after incubation and
purified on the same medium. Isolates which were gram-positive rods, catalase positive,
obligatory anaerobic, and induced stormy fermentation in milk were confirmed as C. perfringes.
API tests were used as a final confirmation test of the isolates.
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Water samples
Environmental samples were collected from rural settlements in the Venda region of the Northern
Province over a period of six months. The sources included river water, boreholes and wells
used by the local population as a primary water source. Samples were collected and analysed
within 24 hours after collection.
Results
In tests on various environmental samples aimed at evaluating the recovery of naturally occurring
Clostridium perfringes, the counts were generally higher on SFP agar than on OPSP agar for
vegetative cells and spores (Table 1). Blood agar showed a high count of clear haemolysed
colonies for most of the water sources, but the confirmation tests indicated that only a low
percentage of these colonies (41%) were identified as Clostridium perfringes. API identification
of colonies from OPSP agar revealed that all typically black colonies were Clostridium
perfringes (96%), while the remaining 4% (grey colonies and white colonies with a black dot in
the middle) were other bacterium species. API identification of colonies from SFP agar revealed
that 99.8% of the typically black colonies were Clostridium perfringes, whereas the other 0,2%
were identified as other Clostridium bacteria.
Table 1.
Relative recovery of spores and vegetative cells of C.perfringes by SFP, OPSP
and BA media (counts/100 ml)
Source
Vegetative cells
Spores
OPSP
BA
SFP
OPSP
BA
SFP
River A
30
5056
443
2
20
0
River B
33
5950
943
10
10
5
River C
152
3382
942
8
16
8
Borehole A
6
502
28
5
45
10
Borehole B
10
910
88
0
55
4
Well A
44
5200
890
10
80
40
Well B
2
1278
82
0
10
2
Well C
120
1485
1158
6
2
18
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Discussion
The main objective of this study was to determine the efficiency of different agar mediums for
the selective recovery of C.perfringes from environmental samples. The results indicated that
SFP agar is a more specific and selective medium for the general recovery of C.perfringes from
water when present in the form of vegetative cells. There was not much difference between
OPSP and SFP medium for the selective recovery of spores from the same samples. Blood agar
(BA) proved to be inadequate as a selective medium because of the high false positive counts.
The high counts of C.perfringes present in different water sources in rural communities, indicates
that this organism poses a significant health risk for these communities who use these sources
to collect water for domestic purposes.
Future work will include a comparison of OPSP and SFP media with mCP and TSC medium.
Acknowledgements
We would like to thank the post graduate students in the Department of Microbiology for their
valuable assistance with this project.
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