The Survey of growth and toxigenesis of Clostridium botulinum type A under the effect of multivarient pH, salt, temperature and time of storage in B.H.I model

In this study, growth (visible turbidity) and toxigenesis of Clostridium botulinum type A with the size level of inoculum 4×104 cfu/ml was surveyed with interference of Intrinsic and extrinsic parameters, such as pH, salt, temperature and storage time in B.H.I broth and results evaluated with completely randomized, ANOVA, in SAS system.So, this survey was performed during three steps. The fist step was sporogenesis of bacteria in agar media (EYA , BHI). After three consecutive passages of lyophilized bacteria (provided from University of California Davis), under anaerobic conditions, transferred to (EYA,BHI) agar. Sporogenesis started from 4th day till 15th day, that maximum free spores were observed (More than %90) using of continuing wet lam and Malaschite Green staining , through video microscope, every day.Then the colonies of plates were washed aseptically, separately and gathered with phosphate buffer gell dilutor and cold centrifuged 5000-10000/min. At last spore count got in one mililiter of suspension. In the second step, the effect of multifactorial parameter combination such as pH (5.5 and 6.5); salt concentration (0.5, 3 , 6%), temperature (15 , 25 , 35oC) and storage time (2,4,8,16,32 days) in anaerobic condition with inoculom size level 4×104 spore/ml were cultured in to the B.H.I broth, for the time of turbidity and toxigenesis.In the third step, toxigenesis confirmed with USDA instruction and mouse bioassay injection. The growth and toxigenesis concluded a shorter period of time in 35oC in contrast with 25oC and in %0.5 with %3 salts cocentration. Also in growth and toigenesis of bacteria observed a substitional effect between salt and temprature (25oC and %0.5 salt with 35oC and %3salt)The results of present study reveals the salt : (%6) pH : (5.5) and temperature : (15oC) with the inoculum level (4×104 cfu/ml)inhibits the growth and toxigenesis of Clostridium botulinum type A in contrast with salt (0.5 and 3)% , pH: 6.5 and temperature (25 and 35)oC in 32 days.