https://www.journalofdairyscience.org/article/S0022-0302(13)00152-5/pdf

Fill a test tube with RCA agar medium. Dip a needle into your turbid culture. Stab deep into the solid agar medium.

Hi To Chiu you say you left your plates aerobically at 2-3 hours prior to anaerobic conditions. Were your RCA plates pre-reduced prior to this i.e. did you incubate the RCA anaerobically overnight prior to inoculating them with your RCM cultures? If not, this can affect the growth of your Clostridium strains as there may still be residual oxygen present in your media.

I know for logistic reasons it is not always possible to work anaerobically so I get why your RCA plates were left aerobically for a while. However I would try and reduce that 2-3 hour time period and get the plates back under anaerobic storage as quickly as possible. Also, when plating out or streaking I would do the work as quickly as I can particularly when lifting the lid and exposing the agar to oxygen.

Finally, are you confident that what is growing in the RCM is actually your Clostridium strains and not something else? Has your RCM been pre-reduced as well prior to inoculation and incubated anaerobically? Are you able to check under the microscope to see what is growing?

Thank you all for your advice, I now stability prepare and plate Clostridium spores. I find that after heating the culture at 75 degrees celsius for 15 minutes, the plate count is stabler than without heating. And Columbia agar is a good alternative to reinforced clostridium agar. Thanks Neogen for its introduction of RCM. It is wasteful to anaerobically store the plates for only several hours; incubates the spores for an extra day looks good to me.

I hope my experience can help someone else.