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Selective and differential media for Clostridium perfringens
International Journal of Food Microbiology, 2 (1985) 89-98 Elsevier
89
JFM 00050
Selective and differential media for Clostridium
perfringens G.C. Mead Agricultural and Food Research Council, Food Research Institute, Colney Lane, Norwich NR4 7UA. Norfolk, U.K. (Received 15 September 1984; accepted 14 January 1985)
Many of the selective media which have been developed for isolating Clostridium perfringens from foods are based upon the use of a test for sulphite reduction as the main differential criterion. The advantages and limitations of such media are considered in the present paper and. on the basis of a number of comparative studies, it is concluded that the tryptose-sulphite-cycloserine media, with and without the addition of egg yolk, are probably the best of those currently available for the purpose of enumeration. Although media containing D-cycloserine appear to be more effective than most other selective media for recovering both vegetative cells and spores damaged by exposure to cold-storage conditions or certain sub-lethal heat treatments, there appears to be scope for developing appropriate resuscitation procedures. There is also a need to increase the specificity of media for C. perfringens, possibly by incorporating more critical differential tests. Key words: Clostridium perfringens: Isolation; Enumeration; Media
Introduction Although Clostridium perfringens appears to have been known as a cause of human food poisoning since the end of the last century (Klein, 1895), it is only in relatively recent years that effective methods have been developed for enumerating the organism and applied to routine food analysis. Any selective medium used for this purpose must be capable of isolating C. perfringens when present as only a minor component of the contaminating microflora of the food, a very different situation from that occurring in samples of suspect food or faeces associated with food-poisoning outbreaks where the organism can reach levels of several million per gram and may even be predominant. The ingestion of food which is heavily contaminated with an enterotoxin-producing strain of (7. perfringens usually results in a mild form of gastro-enteritis and the strains responsible for this condition invariably belong to type A, as do the majority of strains isolated from normal foods (Fruin, 1978). The organisms are of two main kinds, one of which produces only small amounts of alpha toxin, variable amounts of kappa and no theta toxin; these strains are either non-haemolytic or alpha-haemolytic on horse-blood agar and produce spores which can withstand heating at 100°C 0168-1605/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
90 for 30 min. By contrast, the 'classical' strains, which are more common, produce abundant alpha, kappa and theta toxins and are beta-haemolytic; in this case. the spores are readily inactivated by heating at IO0°C. Media devised for the isolation and enumeration of C. perfringens must be able to isolate both 'heat-resistant' and 'heat sensitive' strains with equal facility; however, the heat-resistant type is usually more sensitive to certain of the selective agents used in isolation media, particularly neomycin (Spencer, 1969). A further problem arises with some strains in that only a small proportion of the spores will germinate without prior heat activation (Barnes et al., 1963). The purpose of this paper is to consider the relative merits of those media which have been developed more recently for isolating C. perfringens and in some cases are used now in the routine monitoring of foods in different countries.
Development of selective isolation media
With most of the available media, differentiation of C perfringens is based on the well-known blackening reaction which arises from the ability of the organism to reduce sulphite to sulphide and, in the presence of an appropriate iron salt, results in the precipitation of black ferrous sulphide around individual colonies. In the case of a liquid medium containing the sulphite-iron system blackening occurs throughout the medium. The ability to reduce sulphite is not restricted to C. perfringens and is a property of most other clostridia, as well as certain facultative anaerobes such as Proteus spp. However, the reaction is of differential value if the necessary ingredients are included in a medium containing appropriate selective agents. The fact that numerous attempts have been made to develop a medium which is specific for C. perfringens suggests that this objective is not easily met. Table I lists most of the sulphite-containing media which have appeared in the literature since 1962 and includes both solid media used for plating purposes and liquid media which are sometimes used when only low numbers of C. perfringens are expected in a particular food. In general, the solid media represent progressive attempts to increase both selectivity and productivity. The sulfite-polymyxin-sulfadiazine (SPS) medium of Angelotti et al. (1962) was said to provide quantitative recovery of C perfring(ens from foods as well as ensuring reduced interference from facultative anaerobes, although it was recognised that the medium also permitted growth and black-colony formation by a wide range of other clostridial species. In consequence, SPS medium has been advocated for the isolation of clostridia as a whole (Gibbs and Freame, 1965). Subsequent experience has confirmed the low specificity of the medium in which various facuitative anaerobes do, in fact, grow uninhibited; moreover, selectivity can be markedly affected by the introduction of food material from the test sample (Shahidi and Ferguson, 1971). A further problem is that SPS permits poor growth of some strains of C. perfringens, with recovery being sometimes < 1%, especially with certain batches of medium, whether derived from the dehydrated,
Medium
Selective agents
Further differentiation/isolation
none
D-cycloserine
none
DRCM
PEM a
LS
subculture into sulphiteiron agar gas from lactose
subculture onto lactose-egg yolk-milk agar
subculture into SPS
none
polymyxin B + oleandomycin + sulphadiazine
neomycin + azide
none
egg-yolk reaction
egg-yolk reaction
none
none
D-cycloseri ne
polymyxin B + sulphadiazine polymyxin B+ neomycin polymyxin B + kanamycin D-cycloserine
TYD-C a
TSC minus EY OPSPA
TSC
SFP
TSN
SPS
a Sulphite present only in second-stage medium
Liquid
Solid
Sulphite-containing media for enumerating ('lostridium perfrmgens in foods
TABLE I
Incubation (°C)
46
46
37
37
37
37
35
35
46
37
Recommended method
Reference
Green and Litsky ( i 966 ) Gibbs (1973)
Angelotti et al. (1962) Marshall et al. (1965) Shahidi and Ferguson (1971 ) Harmon et al. (1971 a) Hauschild and Hilsheimer (1974a) Handford (1974)
multiple tube (MPN) + surface plate multiple tube (MPN) Debevere (I 979) plate method not specified multiple tube (MPN) Beerens et al. (1982)
multiple tube (MPN)
surface plate or deep-agar tube surface plate + overlay surface plate+ overlay pour plate + overlay pour plate + overlay
pour plate
92 complete medium, or prepared from separate ingredients (Shahidi and Ferguson, 1971: Hauschild and Hilsheimer, 1974a). Some of the criticisms made of SPS medium also apply to trypticase-sulphiteneomycin agar (TSN) which includes polymyxin and was aimed at increasing selectivity without impairing the recovery of C. perfringens (Marshall et al., 1965). Although TSN has not been widely used in food analysis, there is evidence that, while this medium is more selective than SPS, it still allows the growth of many facultative anaerobes (Harmon et al., 1971b); also, the medium is inhibitory to some strains of C. perfringens, possibly due to the use of 46°C as an incubation temperature (Handford, 1974). The introduction of SFP medium containing kanamycin, polymyxin and egg yolk by Shahidi and Ferguson (1971) overcame the problem of C. perfringens inhibition, partly by the substitution of sulphite by metabisulphite, but failed to provide adequate suppression of facultative anaerobes such as coliforms, enterococci and certain bacilli, some of which can produce egg-yolk reactions on this medium (Hauschild and Hilsheimer, 1974b). The inclusion of egg yolk to differentiate lecithinase-positive C. perfringens from other sulphite-reducers could be seen as a disadvantage since, following overnight incubation, not all strains of C. perfringens produce characteristic halos in the medium and hence may be overlooked (Hauschild and Hilsheimer, 1974a; Debevere, 1979). Also, it should be noted that some uncommon, lecithinase-negative strains of C. perfringens can also cause food poisoning (Pinegar and Stringer, 1977) and would escape detection on SFP, Another medium containing egg yolk is tryptose-sulphite-cycloserine agar (TSC) (Harmon et al., 1971a). This and the egg yolk-free modification introduced by Hauschild and Hilsheimer (1974a, b) are probably the best selective plating media currently available for C. perfringens and TSC was adopted by the Association of Official Analytical Chemists as official first action (Harmon, 1976). In both cases, the use of 400/~g/ml D-cycloserine as the selective agent gave high recoveries of C. perfringens whilst inhibiting most of the facultative anaerobes which cause interference in other media. In collaborative studies initiated by the International Commission on Microbiological Specifications for Foods, comparisons of different media for isolating C. perfringens from both foods and faeces showed that TSC and TSC minus egg yolk (EY) gave the best results (Hauschild et al., 1977, 1979). However, the remaining major problem with these media, as with the other agar media listed in Table I, is that certain other clostridia can also grow and produce colonies that are indistinguishable from C. perfringens. Some of these, e . g . C . paraperfringens and C. sardiniensis, are physiologically similar to C. perfringens and hence necessitate the use of appropriate confirmatory tests. The differential criteria in current use involve tests for nitrate reduction, motility, lactose fermentation and gelatin liquefaction; their efficacy has been discussed by Harmon and Kautter (1978) and will not be considered here. The comparative studies described by Hauschild et al. (1977, 1979) did not include the OPSPA medium of Handford (1974) but, nevertheless, this medium is favoured in the U.K. for use in food analysis. The medium, which contains oleandomycin, polymyxin and sulphadiazine, is otherwise similar to TSC minus EY
93 TABLE II Composition of TSC and OPSPA media Basal medium (g / l)
Tryptose (Difco), 15 Soytone (Difco). 5 Yeast extract (Difco), 5 Sodium metabisulphite, 1 Ferric ammonium citrate, 1 Agar 20 pH 7.6 I
I TSC
OPSPA
Egg-yolk emulsion 8% D-Cycloserine 400 #g/ml
Oleandomycin phosphate 0.5 # g / m l Polymyxin B sulphate 10 i.u./ml Sulphadiazine 100 # g / m l
T S C minus E Y
o-Cycloserine 400 # g / m l
(Table II); it supports good growth of most strains of C. perfringens but is more specific for this organism than TSC minus EY (Table III) or, presumably, TSC. The main disadvantage of OPSPA is that some facultative anaerobes, especially enterococci, grow readily, forming small, white colonies. Table I includes a number of liquid media which are favoured by some workers, especially for enriching small numbers of C. perfringens. The disadvantages of liquid media are that for counting purposes the most probable number (MPN) method must be used and this is both more laborious and less accurate than the plating method (Gibbs and Freame, 1965). Of the liquid media listed in Table I, all except
TABLE 111 Growth of clostridia other than Clostridium perfringens on TSC minus EY and OPSPA media incubated at 37°C (data from various sources)
C. • C. C. C. C. C. C. C. C. C. C. C.
absonum bifermentans cadavaris celatum difficile glycolicum paraperfringens perenne sardiniensis sordellii sporogenes tertium
TSC minus EY
OPSPA
+ + + + + + + + + v +
+ + + + + ±
+, growth and blackening; - , no growth; +, weak reaction; v, strain variation.
94 lactose-sulphite medium (LS) require subculture into or onto a second medium and hence are even more laborious and therefore less attractive for routine use. Little information is available concerning the effectiveness of either the TYD-C or PEM methods but the differential reinforced ciostridial medium (DRCM) procedure involving subsequent plating on lactose-egg yolk-milk agar (Gibbs, 1973) was found by Adams and Mead (1980) to give lower counts than either TSC minus EY or OPSPA when used for enumerating C. perfringens on processed chicken carcasses. The LS medium of Beerens et ai. (1982) has yet to be tested widely but is said to be virtually specific for C. perfringens because of its restricted nutrient composition and the use of 46°C for incubation. No selective agents are added and the sole diagnostic criterion, apart from sulphite-reduction, is the production of gas from lactose.
Influence of incubation conditions
As suggested by Professor Mossel (this symposium), the use of 46°C as an incubation temperature for increasing the specificity of C. perfringens isolation media deserves further consideration. Although Handford (1974) reported that some strains were inhibited at 46°C on TSN, Mossel and Pouw (1973) successfully Used this temperature in conjunction with a sulphite-cycloserine medium and found no adverse effect on recovery of the required organisms. However, results obtained by Mossel and Pouw (1973) with food, animal feed and water samples show that false positive colonies still occurred and it is difficult to see how growth conditions could be manipulated to exclude the organisms most closely related to C. perfringens such as C. paraperfringens which grows and produces similar reactions even in LS medium (Beerens et al., 1982). All the plating media in Table I require anaerobic incubation but the composition of the gaseous environment used has varied from one study to another. For example, Harmon et al. (1971a) used a nitrogen atmosphere for TSC while Handford (1974) incubated OPSPA under hydrogen + 5% carbon dioxide. It was concluded by Mead et al. (1982) that the nature of the gaseous environment is not critical for isolating C. perfringens provided that the degree of anaerobiosis is adequate; C. perfringens is, of course, a relatively aerotolerant anaerobe. However, reducing conditions in the medium are more critical for effective blackening due to sulphite-reduction (Mead, 1969) and hence both surface-inoculated plates and pour-plates are normally overlaid with sterile medium before incubation. The inconvenience of needing anaerobic jars in some situations has led to the development of a variety of deep-agar techniques which are simpler to use whilst still providing the conditions necessary for growth and blackening. Some of the available systems have been reviewed by Gibbs and Freame (1965); others are listed by Mead et al. (1982).
95 Recovery of stressed or damaged cells and damaged spores Most studies aimed at developing improved selective media for C perfringens have involved the use of vigorous, pure cultures of both the organism being sought and others likely to cause interference. Generally, much less attention has been given to the recovery of organisms subjected to treatments such as those used in food processing, which may cause microbial stress or damage. During cold storage, cells of C perfringens rapidly lose viability and spores are also affected but to a lesser extent (Canada et al., 1964). Thawing of frozen meats at low temperatures causes a further reduction in the numbers of vegetative cells (Trakulchang and Kraft, 1977). The influence of various selective agents on the recovery of non-stressed, refrigerated and frozen cells of two strains of C. perfringens was investigated by Apiluktivongsa and Walker (1980). Although the presence of sodium metabisulphite had little effect on cold-stressed cells of either strain, the recovery of one, which was a non-haemolytic, heat-resistant type, was slightly impaired in the presence of D-cycloserine, kanamycin, oleandomycin or polymyxin. Both of the test stains were adversely affected by sulphadiazine and neomycin: inhibitory effects were most pronounced for frozen cells and colonies were often smaller than usual. Media containing o-cycloserine consistently yielded the best recoveries of cold-stressed cells. In another study, Traci and Duncan (1974) showed that up to 75% of viable cold-shocked cells were damaged, as demonstrated by holding late exponential-phase cells at 10°C. Repair of damage occurred in both a complex medium and in 0.1% peptone but not in plain water. The effect of subjecting spores of C. perfringens to heating at ultrahigh temperatures for short periods and then plating on different selective media was studied by Barach et al. (1974). TSC and SFP media gave higher recoveries than either SPS or TSN and this was partly attributed to the presence of lysozyme in the egg-yolk component of these media and its influence on the germination of damaged spores. However, addition of lysozyme to SPS or TSN did not improve the recovery of heat-damaged spores because the selective agents present interfered with the action of the lysozyme. On their own, both D-cycloserine and sulphadiazine only slightly reduced the recovery of heated spores. Stress or damage effects can also be observed with selective plating media when used in the examination of retail foods. Adams and Mead (1980) examined various further-processed poultry products and found that TSC minus EY and OPSPA consistently yielded unusually small colonies of C perfringens ( < 1 mm diam.), due, presumably, to the effects of heat treatment a n d / o r subsequent freezing and cold storage of the products.
Recent and future trends in isolation media for C. perfringens Apart from the LS medium of Beerens et al. (1982), more recent developments in selective media have tended to move away from the use of sulphite-reduction as the main differential criterion for clostridial growth. Another medium, RPM, devised by
96 Erickson and Deibel (1978), contains polymyxin and neomycin and makes use of the characteristic ' s t o r m y clot' reaction of C. perfringens in litmus milk, coupled with incubation at 4 6 - 4 8 ° C . With 85 food samples, the medium yielded 71% of positives by comparison with only 14% on TSC and the new m e d i u m also gave higher counts of C. perfringens. It was suggested that the greater sensitivity of R P M was due to the stimulation of spore germination, an obvious advantage with an organism producing spores which do not always germinate fully without prior activation. The superiority of RPM, by comparison with TSC media and PEM, for isolating C. perfringens from herbs and spices was demonstrated by de Boer and Boot (1981). The only obvious disadvantage of R P M for enumerating C. perfringens is the need to use the laborious and less accurate M P N method. The same drawback applies to the iron-milk m e d i u m of St. John et al. (1982) which contains no selective inhibitors and for specificity depends upon an incubation temperature of 45°C. Despite the high ~electivity of R P M , confirmatory tests are still needed and it is difficult to imagine any medium which would obviate the necessity for further tests to distinguish C. perfringens from the other, physiologically similar clostridia. An alternative would be to incorporate more specific differential tests in the primary isolation medium. Such an a p p r o a c h was adopted by Bisson and Cabelli (1979) with a medium containing polymyxin and D-cycloserine and incubated at 45°C. This m e d i u m utilized the following differential properties: (a) fermentation of sucrose (b) production of acid phosphatase and (c) the absence of fl-D-glucosidase activity. Although the m e d i u m was devised for the enumeration of C. perfringens in water and hence involves a membrane-filtration procedure, an application to foods appears to be possible. Moreover, the use of m e m b r a n e filters could facilitate the incorporation of a resuscitation procedure for cells or spores which m a y have been d a m a g e d during food processing since it would permit a preliminary period of incubation on a non-selective medium.
References
Adams, B.W. and G.C. Mead, 1980. Comparison of media and methods for counting Clostridium perfringens in poultry meat and further-processed products. J. Hyg. 84, 151-158. Angelotti, R., H.E. Hall, M.J. Foter and K.H. Lewis, 1962. Quantitation of Clostridium perfringens in foods. Appl. Microbiol. 10, 193-199. Apiluktivongsa, P. and H.W. Walker, 1980. Influence of selective agents on recovery of cold-stressed cells of Clostridium perfringens. J. Food Sci. 45, 574-576. Barach, J.T., D.M. Adams and M.L. Speck, 1974. Recovery of heated Clostridium perfringens type A spores on selective media. Appl. Microbiol. 28, 793-797. Barnes, E.M., J.E. Despaul and M. Ingram, 1963. The behaviour of a food poisoning strain of Clostridium welchii in beef. J. Appl. Bacteriol. 26, 415-427. Beerens, H., C. Romond, C. Lepage and J. Criquelion, 1982. A liquid medium for the enumeration of Clostridium perfringens in food and faeces. In: Isolation and identification methods for food poisoning organisms, edited by J.E.L. Corry, D. Roberts and F.A. Skinner, Academic Press, London, pp. 137-149. Bisson, J.W. and V.J. Cabelli, 1979. Membrane filter enumeration method for Clostridium perfringens. Appl. Environ. Microbiol. 37, 55-66.
97 Canada, J.C., D.H. Strong and L.G. Scott, 1964. Response of Clostridium perfringens spores and vegetative cells to temperature variation. Appl. Microbiol. 12, 273-276. Debevere, J.M., 1979. A simple method for the isolation and determination of Clostridium perfringens. Eur. J. Appl. Microbiol. Biotechnol. 6, 409-414. de Boer, E. and E. Boot, 1981. Comparison of media for the isolation of Clostridium perfringens from spices and herbs. De Ware (n)-Chemicus 11, 98-104. Erickson, J.E. and R.H. Deibel, 1978. New medium for rapid screening and enumeration of Clostridium perfringens in foods. Appl. Environ. Microbiol. 36, 567-571. Fruin, J.T., 1978. Types of Clostridium perfringens isolated from selected foods. J. Food Protect. 41, 768-769. Gibbs, P.A., 1973. The detection of Clostridium welchii in the differential reinforced clostridial medium technique. J. Appl. Bacteriol. 36, 23-33. Gibbs, B.M. and B. Freame, 1965. Methods for the recovery of clostridia from foods. J. Appl. Bacteriol. 28, 95-111. Green, J.H. and W. Litsky, 1966. A new medium and 'mimic' MPN method for Clostridium perfringens isolation and enumeration. J. Food Sci. 31,610-614. Handford, P.M., 1974. A new medium for the detection and enumeration of Clostridium perfringens in foods. J. Appl. Bacterio]. 37, 559-570. Harmon, S.M., 1976. Collaborative study of an improved method for the enumeration and confirmation of Clostridium perfringens in foods. J. Assoc. Off. Anal. Chem. 59, 606-612. Harmon, S.M., D.A. Kautter and J.T. Peeler, 1971a. Improved medium for enumeration of Clostridium perfringens. Appl. Microbiol. 22, 688-692. Harmon, S.M., D.A. Kautter and J.T. Peeler, 1971b. Comparison of media for the enumeration of Clostridium perfringens. Appl. Microbiol. 21,922-927. Harmon, S.M. and D.A. Kautter, 1978. Media for confirming Clostridium perfringens from foods and faeces. J. Food Protect. 41,626-630. Hauschild, A.H.W. and R. Hitsheimer, 1974a. Evaluation and modifications of media for enumeration of Clostridium perfringens. Appl. Microbiol. 27, 78-82. Hauschild, A.H.W. and R. Hilsheimer, 1974b. Enumeration of food-borne Clostridium perfringens in egg yolk-free tryptose-sulphite-cycloserineagar. Appl. Microbiol. 27, 521-526. Hauschild, A.H.W., R.J. Gilbert, S.M. Harmon, M.F. O'Keefe and R. Vahlefeld, 1977. ICMSF methods studies. VIII. Comparative study for the enumeration of Clostridium perfringens in foods. Can. J. Microbiol. 23, 884-892. Hauschild, A.H.W., P. Desmarchelier, R.J. Gilbert, S.M. Harmon and R. Vahlefeld, 1979. ICMSF methods studies. XII. Comparative study for the enumeration of Clostridium perfringens in faeces. Can. J. Microbiol. 25, 953-963. Klein, E., 1895. Uber einen pathogen anaeroben Darmbacillus, Bacillus enteritidis sporogens. Zentralbl. Bakteriol. Parasitenk. lnfektionskr. Hyg. Abt. I. Orig. 18, 737-742. Marshall, R.S., J.F. Steenbergen and L.S. McClung, 1965. Rapid technique for the enumeration of Clostridium perfringens.. App]. Microbiol. 13, 559-563. Mead, G.C., 1969. The use of sulphite-containing media in the isolation of Clostridium welchii. J. Appl. Bacteriol. 32, 358-361. Mead, G.C., B.W. Adams, T.A. Roberts and J.L. Smart, 1982. Isolation and enumeration of Clostridium perfringens. In: Isolation and identification methods for food poisoning organisms, edited by J.E.L. Corry, D. Roberts and F.A. Skinner, Academic Press, London, pp. 99-110. Mossel, D.A.A. and H. Pouw, 1973. Studies on the suitability of sulphite cycloserine agar for the enumeration of Clostridium perfringens in food and water. Zentralbl. Bakteriol. Parasitenk. lnfektionskr. Hyg. Abt. i. Orig. A 223, 559-561. Pinegar, J.A. and M.F. Stringer. 1977. Outbreaks of food poisoning attributed to ]ecithinase-negative C/ostridium welchii. J. Clin. Pathol. 30, 491-492. Shahidi, S.A. and A.R. Ferguson, 1971. New quantitative, qualitative and confirmatory media for rapid analysis of food for Clostridium perfringens. Appl. Microbiol. 21, 500-506. Spencer, R., 1969. Neomycin-containing media in the isolation of Clostridium botulinum and food poisoning strains of Clostridium welehii. J. Appl. Bacteriol. 32, 170-174.
98 St. John, W.D., J.R. Matthews and M.M. Wekell, 1982. Use of iron-milk medium for enumeration of Clostridium perfringens. J. Assoc. Off. Anal. Chem. 65, 1129-1133. Traci, P.A. and C.L. Duncan, 1974. Cold shock lethality and injury in Clostridium perfrmgens. Appl. Microbiol. 28, 815-821. Trakulchang. S.P. and A.A. Kraft, 1977, Survival of Clostridi~'m perfringens in refrigerated and frozen meat and poultry items. J. Food Sci. 42, 518-521.
89
JFM 00050
Selective and differential media for Clostridium
perfringens G.C. Mead Agricultural and Food Research Council, Food Research Institute, Colney Lane, Norwich NR4 7UA. Norfolk, U.K. (Received 15 September 1984; accepted 14 January 1985)
Many of the selective media which have been developed for isolating Clostridium perfringens from foods are based upon the use of a test for sulphite reduction as the main differential criterion. The advantages and limitations of such media are considered in the present paper and. on the basis of a number of comparative studies, it is concluded that the tryptose-sulphite-cycloserine media, with and without the addition of egg yolk, are probably the best of those currently available for the purpose of enumeration. Although media containing D-cycloserine appear to be more effective than most other selective media for recovering both vegetative cells and spores damaged by exposure to cold-storage conditions or certain sub-lethal heat treatments, there appears to be scope for developing appropriate resuscitation procedures. There is also a need to increase the specificity of media for C. perfringens, possibly by incorporating more critical differential tests. Key words: Clostridium perfringens: Isolation; Enumeration; Media
Introduction Although Clostridium perfringens appears to have been known as a cause of human food poisoning since the end of the last century (Klein, 1895), it is only in relatively recent years that effective methods have been developed for enumerating the organism and applied to routine food analysis. Any selective medium used for this purpose must be capable of isolating C. perfringens when present as only a minor component of the contaminating microflora of the food, a very different situation from that occurring in samples of suspect food or faeces associated with food-poisoning outbreaks where the organism can reach levels of several million per gram and may even be predominant. The ingestion of food which is heavily contaminated with an enterotoxin-producing strain of (7. perfringens usually results in a mild form of gastro-enteritis and the strains responsible for this condition invariably belong to type A, as do the majority of strains isolated from normal foods (Fruin, 1978). The organisms are of two main kinds, one of which produces only small amounts of alpha toxin, variable amounts of kappa and no theta toxin; these strains are either non-haemolytic or alpha-haemolytic on horse-blood agar and produce spores which can withstand heating at 100°C 0168-1605/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
90 for 30 min. By contrast, the 'classical' strains, which are more common, produce abundant alpha, kappa and theta toxins and are beta-haemolytic; in this case. the spores are readily inactivated by heating at IO0°C. Media devised for the isolation and enumeration of C. perfringens must be able to isolate both 'heat-resistant' and 'heat sensitive' strains with equal facility; however, the heat-resistant type is usually more sensitive to certain of the selective agents used in isolation media, particularly neomycin (Spencer, 1969). A further problem arises with some strains in that only a small proportion of the spores will germinate without prior heat activation (Barnes et al., 1963). The purpose of this paper is to consider the relative merits of those media which have been developed more recently for isolating C. perfringens and in some cases are used now in the routine monitoring of foods in different countries.
Development of selective isolation media
With most of the available media, differentiation of C perfringens is based on the well-known blackening reaction which arises from the ability of the organism to reduce sulphite to sulphide and, in the presence of an appropriate iron salt, results in the precipitation of black ferrous sulphide around individual colonies. In the case of a liquid medium containing the sulphite-iron system blackening occurs throughout the medium. The ability to reduce sulphite is not restricted to C. perfringens and is a property of most other clostridia, as well as certain facultative anaerobes such as Proteus spp. However, the reaction is of differential value if the necessary ingredients are included in a medium containing appropriate selective agents. The fact that numerous attempts have been made to develop a medium which is specific for C. perfringens suggests that this objective is not easily met. Table I lists most of the sulphite-containing media which have appeared in the literature since 1962 and includes both solid media used for plating purposes and liquid media which are sometimes used when only low numbers of C. perfringens are expected in a particular food. In general, the solid media represent progressive attempts to increase both selectivity and productivity. The sulfite-polymyxin-sulfadiazine (SPS) medium of Angelotti et al. (1962) was said to provide quantitative recovery of C perfring(ens from foods as well as ensuring reduced interference from facultative anaerobes, although it was recognised that the medium also permitted growth and black-colony formation by a wide range of other clostridial species. In consequence, SPS medium has been advocated for the isolation of clostridia as a whole (Gibbs and Freame, 1965). Subsequent experience has confirmed the low specificity of the medium in which various facuitative anaerobes do, in fact, grow uninhibited; moreover, selectivity can be markedly affected by the introduction of food material from the test sample (Shahidi and Ferguson, 1971). A further problem is that SPS permits poor growth of some strains of C. perfringens, with recovery being sometimes < 1%, especially with certain batches of medium, whether derived from the dehydrated,
Medium
Selective agents
Further differentiation/isolation
none
D-cycloserine
none
DRCM
PEM a
LS
subculture into sulphiteiron agar gas from lactose
subculture onto lactose-egg yolk-milk agar
subculture into SPS
none
polymyxin B + oleandomycin + sulphadiazine
neomycin + azide
none
egg-yolk reaction
egg-yolk reaction
none
none
D-cycloseri ne
polymyxin B + sulphadiazine polymyxin B+ neomycin polymyxin B + kanamycin D-cycloserine
TYD-C a
TSC minus EY OPSPA
TSC
SFP
TSN
SPS
a Sulphite present only in second-stage medium
Liquid
Solid
Sulphite-containing media for enumerating ('lostridium perfrmgens in foods
TABLE I
Incubation (°C)
46
46
37
37
37
37
35
35
46
37
Recommended method
Reference
Green and Litsky ( i 966 ) Gibbs (1973)
Angelotti et al. (1962) Marshall et al. (1965) Shahidi and Ferguson (1971 ) Harmon et al. (1971 a) Hauschild and Hilsheimer (1974a) Handford (1974)
multiple tube (MPN) + surface plate multiple tube (MPN) Debevere (I 979) plate method not specified multiple tube (MPN) Beerens et al. (1982)
multiple tube (MPN)
surface plate or deep-agar tube surface plate + overlay surface plate+ overlay pour plate + overlay pour plate + overlay
pour plate
92 complete medium, or prepared from separate ingredients (Shahidi and Ferguson, 1971: Hauschild and Hilsheimer, 1974a). Some of the criticisms made of SPS medium also apply to trypticase-sulphiteneomycin agar (TSN) which includes polymyxin and was aimed at increasing selectivity without impairing the recovery of C. perfringens (Marshall et al., 1965). Although TSN has not been widely used in food analysis, there is evidence that, while this medium is more selective than SPS, it still allows the growth of many facultative anaerobes (Harmon et al., 1971b); also, the medium is inhibitory to some strains of C. perfringens, possibly due to the use of 46°C as an incubation temperature (Handford, 1974). The introduction of SFP medium containing kanamycin, polymyxin and egg yolk by Shahidi and Ferguson (1971) overcame the problem of C. perfringens inhibition, partly by the substitution of sulphite by metabisulphite, but failed to provide adequate suppression of facultative anaerobes such as coliforms, enterococci and certain bacilli, some of which can produce egg-yolk reactions on this medium (Hauschild and Hilsheimer, 1974b). The inclusion of egg yolk to differentiate lecithinase-positive C. perfringens from other sulphite-reducers could be seen as a disadvantage since, following overnight incubation, not all strains of C. perfringens produce characteristic halos in the medium and hence may be overlooked (Hauschild and Hilsheimer, 1974a; Debevere, 1979). Also, it should be noted that some uncommon, lecithinase-negative strains of C. perfringens can also cause food poisoning (Pinegar and Stringer, 1977) and would escape detection on SFP, Another medium containing egg yolk is tryptose-sulphite-cycloserine agar (TSC) (Harmon et al., 1971a). This and the egg yolk-free modification introduced by Hauschild and Hilsheimer (1974a, b) are probably the best selective plating media currently available for C. perfringens and TSC was adopted by the Association of Official Analytical Chemists as official first action (Harmon, 1976). In both cases, the use of 400/~g/ml D-cycloserine as the selective agent gave high recoveries of C. perfringens whilst inhibiting most of the facultative anaerobes which cause interference in other media. In collaborative studies initiated by the International Commission on Microbiological Specifications for Foods, comparisons of different media for isolating C. perfringens from both foods and faeces showed that TSC and TSC minus egg yolk (EY) gave the best results (Hauschild et al., 1977, 1979). However, the remaining major problem with these media, as with the other agar media listed in Table I, is that certain other clostridia can also grow and produce colonies that are indistinguishable from C. perfringens. Some of these, e . g . C . paraperfringens and C. sardiniensis, are physiologically similar to C. perfringens and hence necessitate the use of appropriate confirmatory tests. The differential criteria in current use involve tests for nitrate reduction, motility, lactose fermentation and gelatin liquefaction; their efficacy has been discussed by Harmon and Kautter (1978) and will not be considered here. The comparative studies described by Hauschild et al. (1977, 1979) did not include the OPSPA medium of Handford (1974) but, nevertheless, this medium is favoured in the U.K. for use in food analysis. The medium, which contains oleandomycin, polymyxin and sulphadiazine, is otherwise similar to TSC minus EY
93 TABLE II Composition of TSC and OPSPA media Basal medium (g / l)
Tryptose (Difco), 15 Soytone (Difco). 5 Yeast extract (Difco), 5 Sodium metabisulphite, 1 Ferric ammonium citrate, 1 Agar 20 pH 7.6 I
I TSC
OPSPA
Egg-yolk emulsion 8% D-Cycloserine 400 #g/ml
Oleandomycin phosphate 0.5 # g / m l Polymyxin B sulphate 10 i.u./ml Sulphadiazine 100 # g / m l
T S C minus E Y
o-Cycloserine 400 # g / m l
(Table II); it supports good growth of most strains of C. perfringens but is more specific for this organism than TSC minus EY (Table III) or, presumably, TSC. The main disadvantage of OPSPA is that some facultative anaerobes, especially enterococci, grow readily, forming small, white colonies. Table I includes a number of liquid media which are favoured by some workers, especially for enriching small numbers of C. perfringens. The disadvantages of liquid media are that for counting purposes the most probable number (MPN) method must be used and this is both more laborious and less accurate than the plating method (Gibbs and Freame, 1965). Of the liquid media listed in Table I, all except
TABLE 111 Growth of clostridia other than Clostridium perfringens on TSC minus EY and OPSPA media incubated at 37°C (data from various sources)
C. • C. C. C. C. C. C. C. C. C. C. C.
absonum bifermentans cadavaris celatum difficile glycolicum paraperfringens perenne sardiniensis sordellii sporogenes tertium
TSC minus EY
OPSPA
+ + + + + + + + + v +
+ + + + + ±
+, growth and blackening; - , no growth; +, weak reaction; v, strain variation.
94 lactose-sulphite medium (LS) require subculture into or onto a second medium and hence are even more laborious and therefore less attractive for routine use. Little information is available concerning the effectiveness of either the TYD-C or PEM methods but the differential reinforced ciostridial medium (DRCM) procedure involving subsequent plating on lactose-egg yolk-milk agar (Gibbs, 1973) was found by Adams and Mead (1980) to give lower counts than either TSC minus EY or OPSPA when used for enumerating C. perfringens on processed chicken carcasses. The LS medium of Beerens et ai. (1982) has yet to be tested widely but is said to be virtually specific for C. perfringens because of its restricted nutrient composition and the use of 46°C for incubation. No selective agents are added and the sole diagnostic criterion, apart from sulphite-reduction, is the production of gas from lactose.
Influence of incubation conditions
As suggested by Professor Mossel (this symposium), the use of 46°C as an incubation temperature for increasing the specificity of C. perfringens isolation media deserves further consideration. Although Handford (1974) reported that some strains were inhibited at 46°C on TSN, Mossel and Pouw (1973) successfully Used this temperature in conjunction with a sulphite-cycloserine medium and found no adverse effect on recovery of the required organisms. However, results obtained by Mossel and Pouw (1973) with food, animal feed and water samples show that false positive colonies still occurred and it is difficult to see how growth conditions could be manipulated to exclude the organisms most closely related to C. perfringens such as C. paraperfringens which grows and produces similar reactions even in LS medium (Beerens et al., 1982). All the plating media in Table I require anaerobic incubation but the composition of the gaseous environment used has varied from one study to another. For example, Harmon et al. (1971a) used a nitrogen atmosphere for TSC while Handford (1974) incubated OPSPA under hydrogen + 5% carbon dioxide. It was concluded by Mead et al. (1982) that the nature of the gaseous environment is not critical for isolating C. perfringens provided that the degree of anaerobiosis is adequate; C. perfringens is, of course, a relatively aerotolerant anaerobe. However, reducing conditions in the medium are more critical for effective blackening due to sulphite-reduction (Mead, 1969) and hence both surface-inoculated plates and pour-plates are normally overlaid with sterile medium before incubation. The inconvenience of needing anaerobic jars in some situations has led to the development of a variety of deep-agar techniques which are simpler to use whilst still providing the conditions necessary for growth and blackening. Some of the available systems have been reviewed by Gibbs and Freame (1965); others are listed by Mead et al. (1982).
95 Recovery of stressed or damaged cells and damaged spores Most studies aimed at developing improved selective media for C perfringens have involved the use of vigorous, pure cultures of both the organism being sought and others likely to cause interference. Generally, much less attention has been given to the recovery of organisms subjected to treatments such as those used in food processing, which may cause microbial stress or damage. During cold storage, cells of C perfringens rapidly lose viability and spores are also affected but to a lesser extent (Canada et al., 1964). Thawing of frozen meats at low temperatures causes a further reduction in the numbers of vegetative cells (Trakulchang and Kraft, 1977). The influence of various selective agents on the recovery of non-stressed, refrigerated and frozen cells of two strains of C. perfringens was investigated by Apiluktivongsa and Walker (1980). Although the presence of sodium metabisulphite had little effect on cold-stressed cells of either strain, the recovery of one, which was a non-haemolytic, heat-resistant type, was slightly impaired in the presence of D-cycloserine, kanamycin, oleandomycin or polymyxin. Both of the test stains were adversely affected by sulphadiazine and neomycin: inhibitory effects were most pronounced for frozen cells and colonies were often smaller than usual. Media containing o-cycloserine consistently yielded the best recoveries of cold-stressed cells. In another study, Traci and Duncan (1974) showed that up to 75% of viable cold-shocked cells were damaged, as demonstrated by holding late exponential-phase cells at 10°C. Repair of damage occurred in both a complex medium and in 0.1% peptone but not in plain water. The effect of subjecting spores of C. perfringens to heating at ultrahigh temperatures for short periods and then plating on different selective media was studied by Barach et al. (1974). TSC and SFP media gave higher recoveries than either SPS or TSN and this was partly attributed to the presence of lysozyme in the egg-yolk component of these media and its influence on the germination of damaged spores. However, addition of lysozyme to SPS or TSN did not improve the recovery of heat-damaged spores because the selective agents present interfered with the action of the lysozyme. On their own, both D-cycloserine and sulphadiazine only slightly reduced the recovery of heated spores. Stress or damage effects can also be observed with selective plating media when used in the examination of retail foods. Adams and Mead (1980) examined various further-processed poultry products and found that TSC minus EY and OPSPA consistently yielded unusually small colonies of C perfringens ( < 1 mm diam.), due, presumably, to the effects of heat treatment a n d / o r subsequent freezing and cold storage of the products.
Recent and future trends in isolation media for C. perfringens Apart from the LS medium of Beerens et al. (1982), more recent developments in selective media have tended to move away from the use of sulphite-reduction as the main differential criterion for clostridial growth. Another medium, RPM, devised by
96 Erickson and Deibel (1978), contains polymyxin and neomycin and makes use of the characteristic ' s t o r m y clot' reaction of C. perfringens in litmus milk, coupled with incubation at 4 6 - 4 8 ° C . With 85 food samples, the medium yielded 71% of positives by comparison with only 14% on TSC and the new m e d i u m also gave higher counts of C. perfringens. It was suggested that the greater sensitivity of R P M was due to the stimulation of spore germination, an obvious advantage with an organism producing spores which do not always germinate fully without prior activation. The superiority of RPM, by comparison with TSC media and PEM, for isolating C. perfringens from herbs and spices was demonstrated by de Boer and Boot (1981). The only obvious disadvantage of R P M for enumerating C. perfringens is the need to use the laborious and less accurate M P N method. The same drawback applies to the iron-milk m e d i u m of St. John et al. (1982) which contains no selective inhibitors and for specificity depends upon an incubation temperature of 45°C. Despite the high ~electivity of R P M , confirmatory tests are still needed and it is difficult to imagine any medium which would obviate the necessity for further tests to distinguish C. perfringens from the other, physiologically similar clostridia. An alternative would be to incorporate more specific differential tests in the primary isolation medium. Such an a p p r o a c h was adopted by Bisson and Cabelli (1979) with a medium containing polymyxin and D-cycloserine and incubated at 45°C. This m e d i u m utilized the following differential properties: (a) fermentation of sucrose (b) production of acid phosphatase and (c) the absence of fl-D-glucosidase activity. Although the m e d i u m was devised for the enumeration of C. perfringens in water and hence involves a membrane-filtration procedure, an application to foods appears to be possible. Moreover, the use of m e m b r a n e filters could facilitate the incorporation of a resuscitation procedure for cells or spores which m a y have been d a m a g e d during food processing since it would permit a preliminary period of incubation on a non-selective medium.
References
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