respected sir/madam i am doing research on clostridum difficle. The problem i am faing is sometimes the bacteria grow sometimes it does not grow but i am performing the same procedure. The procedure is explained below:
1. i bought the bacteia from CICC (clostriduim difficle) strain ID :CICC22951 and stored the bacteria at -80 degree in milk
2. Than after thawing the bacteria i cultured the 100ul bacteria of colomibia blood agar
3. the condition is 37 degreee anaerobic environment for 48 hours
4: after 48 hours i obsereved the results sometimes it grows and sometimes it not grows
5. when sometimes the bacteria grows than i pick one colony in the broth ( brucela broth with vitaminK1 and Hemin ) the same happens in broth that sometimes bacteria grows sometimes not.
can anyone please guide me?
Welcome to anaerobic bacterial research. You'll learn to love it. I promise. A few suggestions:
1) Do you pre-reduce your agar and broth before you inoculate it with bacteria? Removing the oxygen from the media can sometimes help with growth and storing the media anaerobically the night before (or even a few hours before) you inoculate can help get consistent growth.
2) When I work with C. difficile it seems to prefer Brain Heart Infusion (BHI) supplemented with 0.1% L-Cysteine and 5 mg/ml yeast extract over other types of media. Could you give that a try?
3) Although C. difficile might not grow it doesn't mean it isn't there. It could be present as a spore. Adding sodium taurocholate to the medium will allow the spores to germinate.
If I think of more suggestions I will let you know.
Sir thanks for your kind reply. I will do experiment according to your kind instructions and will let you know. Thanks for your time
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