There is a paucity of information in similar studies to compare the results of the present study, though enterotoxin production is sporulation specific. Moreover, sporulation of C. perfringens is extremely poor in invitro compared with invivo (De Jong et al., 2002, Hsieh and Labbe, 2007). Handful of laboratory media has been developed to maximize invitro sporulation of this bacterium. Thus, sporulation of broad spectrum of C. perfringens strains seemed dependent on sporulation promoting compounds which may facilitate the resting stage than the active vegetative stage (De Jong et al., 2002; Hsieh and Labbe, 2007).Favourable pH and inoculation levels are also equally important in this regard (De Jong et al., 2002). Amino acids, minerals and a slowly fermentable carbohydrate as a source of carbon (C) are the basic ingredients in any sporulation medium (Meyer and Tholazan., 1999; De Jong et al., 2002). In the present study raffinose, starch and soluble starch were in cooperated as carbohydrate source in MDS, SB and DS respectively. None of the sporulation media were contained easily fermentable glucose as the carbohydrate source. This finding is on par with previous findings and recommendations (De Jong et al., 2002; Hsieh and Labbe, 2007). Raffinose was identified as the most efficient slowly fermentable carbohydrate source superior (Hsieh and Labbe, 2007) not only to soluble starch but insoluble starch also. Hence, modified Duncan and Strong medium (MDS) with raffinose resulted in a considerably higher proportion of sporulating isolates, which was statistically significant when compared with conventional Duncan and Strong medium which did not contain raffinose. Furthermore, soluble starch in DS medium was better than insoluble starch in SB medium. Similar findings were obtained in the same study mentioned earlier (De Jong et al., 2002; Hsieh and Labbe, 2007). However, De Jong and colleagues also found that some strains of C. perfringens sporulated poorly in the presence of starch, whereas with other strains sporulation was promoted (De Jong et al., 2002). Strain specificity in sporulation was reported as an inherent limitation in previous studies (De Jong et al., 2002), thus inability of sporulating of 38.46% of C. perfringens meat curry isolates (29.79% of chicken and 51.61 of beef) mainly due to strain specify, rather effectiveness of slowly fermentable carbohydrate source.
Rapidly metabolizable carbohydrates such as glucose and maltose are vigorously fermented by vegetative cells to produce acid with consequent lowering of pH. Hence, rapidly metabolizable carbohydrates repress the sporulation process by enhancing the active vegetative growth. The pH range (pH 6.0 to 8.0) for sporulation is narrower than for growth. Most laboratory media for sporulation specify an initial pH of 7.5 or higher (Labbe 1989). This pH was ideal for sporulation of many strains but not for all (De Jong et al., 2002). This seems one of the reasons for least performance of DS compared with other two media.
It is reasonable to assume that considerable number of heat resistant spores which survived during cooking of meat curries did not sporulate in any sporulation media. This means sporulation media may not have been optimal.
It is therefore concluded that for induction of sporulation in laboratory conditions is a cumbersome task which essentially need more than one medium Also based on the evidence of the present study the superiority of raffinose containing media in comparison to starch containing media is declared. Although these media are especially designed to support sporulation of strains isolated from foods and stools of patients involved in food poisoning, several strains of C. perfringens still do not sporulate in any laboratory media. Addition of theophylline to DS medium was shown improved results of sporulation (De Jong et al., 2002). Moreover, another sporulation medium: .peptone bile theophylline medium was appeared as the most promising sporulation medium tested as peptone bile theophylline starch medium yielded highest spore numbers (2.5 3 105 /ml) (De Jong et al., 2002). Hence, inability of including afore mentioned medium in the present study or at least improving DS medium by addition of theophylline and controlling inoculum levels preparing the inoculum (overnight or log – phase culture) and the amount of inoculum (1% or 5%), were the other limitation of the present study.
Hence further improvements to overcome the limitations stated here are highly recommended to optimize sporulation of strains of C. perfringens in laboratory media.