Letters in Applied Microbiology 1986, 3, 77-79 GWGf013

A sporulation medium for Clostridium perfringens

K.D. PHILLIPS Anaerobe Reference Unit, Public Health Laboratory, Luton and Dunstable Hospital, Lewsey Road, Luton LU4 ODZ, U K

Received 1 1 July 1986 and accepted 14 July 1986

PHILLIPS, K . D . 1986. A sporulation medium for Chtr id ium perfringens. Letters in Applied Microbiology 3, 17-19.

A new solidified medium for inducing sporulation of Clostridiurn perfringens is described. The essential components of the medium are bile, bicarbonate and quin- oline. The medium induced significant sporulation in a11 of 100 strains of Ci. per- fringens isolated at random from human faecal specimens. The majority (94%) of strains sporulated profusely.

The development of a reliable medium for indu- cing in vitro sporulation of Clostridium per- fringens is a long established challenge in anaerobic bacteriology. Although spores of C1. perfringens are known to be readily formed in the gastrointestinal tract, they are rarely observed in growth on ordinary culture media. Indeed, the absence of spores in artificial culture is a characteristic feature of this organism (Willis 1977). Numerous attempts have been made to devise a sporulation medium for this species; most, however, have met with limited success. One widely used medium is that of Duncan & Strong (1968), other noteworthy examples include those of Robertson (1915- 1916), Ellner (1956), Sacks & Thompson (1977), Labbe & Nolan (1981) and Sacks (1982). Unfor- tunately not all strains of Cl. perfringens sporu- late either heavily or regularly in any of these media.

This paper describes a new medium which has been found to induce profuse sporulation of most intestinal isolates of CI. perfringens.

Materials and Methods

ISOLATION O F ORGANISMS

One hundred strains of Cl. perfringens were iso- lated from randomly selected human faecal specimens obtained from patients under investi- gation for other bacterial pathogens. All isolates

were constituents of the normal intestinal flora; none was implicated in C1. perfringens food poi- soning. The isolates were not toxicologically or serologically typed.

All strains were isolated by ethanol treatment of stools to select for spores (Koransky et al. 1978; Borriello & Honour 1981) followed by enrichment in glucose meat broth prior to puri- fication on Blood Agar base No. 2 (Oxoid Ltd, Basingstoke, Hampshire) supplemented with 5% v/v defibrinated horse blood (Lab m, Ford Lane, Salford). This blood agar was used as the base for the sporulation medium.

P R E P A R A T I O N O F THE S P O R U L A T I O N

MEDIUM

Blood Agar base No. 2 (Oxoid CM27) when reconstituted contains (g/l): proteose peptone, 15.0; liver digest, 2.5; yeast extract, 5.0; sodium chloride, 5.0; agar, 12.0. The sporulation medium was prepared by dissolving 39.5 g of dehydrated blood agar base and 10 g of desiccated ox bile (Oxoid L50) in 1 1 of distilled water prior to sterilization by autoclaving at 121°C for 15 min. When cooled to about 50°C, 5 g of sodium bicarbonate and 0.5 ml of quinoline (BDH Chemicals Ltd, Poole, England; Product No. 30012) were added and mixed to completely dissolve. Defibrinated horse blood (50 ml) was then added and the sporulation medium was dispensed in plastic

K . D. Phillips petri dishes in 25 ml amounts. The pH of the sporulation medium was 8.5.

Contamination of the medium by the use of unsterilized bicarbonate and quinoline was never encountered but, if deemed necessary, bicarbonate solution could be filter sterilized, and the quinoline added to the medium prior to autoclaving.

INOCULATION O F THE SPORULATION MEDIUM

Plates of sporulation medium were inoculated over their whole surface with individual strains of Cl. perfringens using dry swabs charged with growth from cultures on blood agar. Inoculated sporulation medium was then incubated in an anaerobic chamber containing an atmosphere of 80% nitrogen, 10% carbon dioxide and 10% hydrogen at 37°C for 48 h. Blood agar cultures of all strains were grown in parallel under iden- tical conditions.

SPORE COUNTS

For each strain, total growth was harvested ,from the surface of the sporulation medium and suspended in 10 ml of 50% v/v ethanol in saline ‘to kill vegetative cells. The suspension was homogenized by vortex mixing and allowed to stand at room temperature for 1 h. Ten-fold dilutions in saline of the ethanol/saline suspen- sion were plated on blood agar to obtain a total count of recoverable spores per plate of sporu- lation medium. Spore counts of cultures on blood agar were obtained in an identical manner. All cultures on sporulation medium and on blood agar were examined microscopi- cally using a specific spore stain.

Results and Discussion

The medium induced heavy sporulation in vir- tually all of 100 strains of Cl. perfringens (Table 1). As judged by microscopy of spore- stained preparations and by viable spore counts, 94 strains sporulated profusely. Only six strains showed scanty sporulation by microscopic examination, although spores were not difficult to detect. Moreover all six strains produced in excess of lo7 recoverable spores per culture plate of sporulation medium. No spores were detected microscopically in any of the 100

Table 1. Spore yields of 100 isolates of Clostridium Derfrinaens

~ ~ ~~

Total spores/25 ml medium No. of strains

> 109 59 > lo8 35 > 107 6

strains grown on blood agar. Viable counts revealed that some strains did produce low numbers of spores on blood agar, on average five or six logs lower than counts of parallel cultures on sporulation medium. As judged by colony size, all strains grew as well on the sporulation medium as they did on blood agar. It is known that an alkaline pH is favourable to the sporulation of C1. perfringens (Gibbs & Hirsch 1956). In the small intestine, the organism encounters an alkaline environment due to bicarbonate derived largely from pancre- atic fluid; some bicarbonate is also contributed by bile. It seemed feasible therefore to attempt to mimic conditions prevailing in the small intestine by the inclusion of bicarbonate and bile in a sporulation medium. Preliminary experiments indicated that addition of these two ingredients to blood agar promoted scanty sporulation of some isolates of C1. perfringens. However, a vast improvement in the sporu- lation frequency of all strains was obtained by the addition of quinoline.

The mechanism of action of quinoline is not known. However, metabolites of tryptophan containing the quinoline nucleus are known to be formed via the quinoline pathway (Stanier et al. 1951). Some possible role for tryptophan or its quinoline-related metabolites is suggested in sporogenesis of Cl. perfringens.

I thank Dr A.T. Willis for his support and encouragement of this work.

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