I prepared 500ml of McCoy's Medium from the powder using autoclaved water and then filtered it using the filter pump.Then i kept 10ml of the same filtered medium for testing by adding 10% FBS and 1x Antibiotic/Antimycotic solution to 10ml medium in a petridish and incubated the plate at 37 degree celcius.Next day when I checked the plate,there was nothing visible but under the inverted microscope it showed some black patches.Can anyone help me about how to remove the contamination from my prepared 500ml McCoy's 5A medium.It is urgent.
Note: I haven't added FBS and Antibiotic/Antimycotic solution to 500ml medium till now.
It may be easier to add the water to the powder and any other reagents that are not heat sensitive and then do the autoclave step. This would eliminate the need for filtering and is much more likely to yield a sterile broth.
If your current batch of medium is contaminated I would simply throw it away and start again. If that is not an option for you then try to re-filter the contaminated medium with a .22um filter of some sort (PES syringe filters work well for me). This may remove whatever is growing.
I hope I am understanding your question and answering it correctly.
You could try to find out where the contamination comes from (the FBS; AB solution or the medium itself) by incubating different combinations where 1/2 components are left out and see what happens.
But as David said, if it is urgent, you can also resterilize everything: the FBS and AB solution by .22 filter and the medium by autoclaving. I would also go over every other step, make sure no contamination can occur there: is every material/equipment you use (e.g. the petri dishes) kept sterile and do you work in a flow cabinet?
Thank you for your valuable answers.
Dear Mam,
Yes,I work in a flow cabinet and I do prepare the medium in flow cabinet only.
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