Colony counting method of Clostridium perfringens

Use the culture medium conducive to the recovery of Clostridium as the diluent to prepare a serial dilution of the food to be tested. Serial dilutions were counted on yolk-free tryptone peptone iron citrate cuprate serine agar Shahidi and Ferguson polymyxin kanamycin sulfite agar or neoberry blood agar. For SICA and SFA agar, either the method of adding 1 mL of diluent and pouring the plates (10 mL medium), or surface inoculation on pre-dried or inverted plates. Neomycin blood agar plates should be poured in advance, dried and then inoculated on the surface. All plates were incubated at 35 ° C or 37 ° C for 24 hours on MCA and SFA agar. The colonies of suspected Clostridium perfringens were black (sulfite reduction produced iron sulfide precipitation). On neomycin blood (bovine, equine or human blood) agar, the colonies of Clostridium perfringens produce a complete hemolytic zone of the stenosis (formation of toxins), and a non-constrictive Select the suspicious Clostridium perfringens colonies from the complete hemolysis zone, puncture and inoculate into a nitrate peptone water test tube containing 0.3% agar, or inoculate on Willis lactose yolk milk agar, inject a few drops of gas beforehand Clostridium capsulatum spore cocoon sprayed half of each plate, dried, and streaked on the entire plate. Suspicious suspicions can also be streaked on blood agar plates.

The nitrate agar test tube and Willis and fiobbs medium were incubated at 37 ° C for 24 h. After the blood agar plate was placed in aerobic culture at 37 ° C for 24 hours, observe the growth of bacteria in nitrate agar (referring to the movement of the bacteria), and test the amount of nitrate reduction. Select the strain grown on Willis / Hobbs medium for leather Microscopic examination after Lan's staining. Clostridium perfringens is Gram-positive, non-motile, and can reduce nitrate. Verification by the following experiments: ① Observe the colony on the WmWHobbs medium-Clostridium perfringens in the part of the plate without antitoxin, there is a circle of mist around the colony, and there is no mist in the area with antitoxin. In addition, the bacteria can ferment lactose ② in blood agar aerobic culture, the bacteria does not grow.

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