Anaerobe bacteriology - C.perfringens (Feb/10/2011 )

this problem combined with the discussion on your previous problem sounds like you are not very experienced working in a microbiology lab? But please correct me if I am wrong.
Can you ask your supervisor or somebody in your lab to help you with your technique? People know that you need help when you start a thesis and experiments using methods you have not used before and are prepared to help; because everybody wants you to get proper data, and the experiment running. If you do not ask, it will be the other way round: a lot of time is lost, the experiment is not running and usually this includes you sitting in the lab at weekends doing the experiments (again).
Please do not get me wrong now, all of us here are happy to help you, but as your problem seems to persist for quite some time now, I really think that you should talk to an experienced microbiologist in your lab (or a nearby microbiology lab) and ask for help and advice. Some problems - especially when it comes down to lab techniques - can be understood and explained best directly from (wo)man to (wo)man. Finding somebody at your lab who can help you will save you a lot of (unnecessary) trouble for your PhD.