I have faced a great problem that my Actinomycetes isolates get contamination by gram negative bacteria many times. To face this problem I made isolation of them many times on ISP 4 agar containing rifampicin as antibacterial to get red of these gram negative contamination. I got pure colonies of Actinomycetes and reisolated them on new plates of ISP 4 agar. However after subculture of these isolates for production of antimicrobial activity, I found that contamination reappeared in some isolates. I need a trusted method to get red of gram negative contamination as many of them are difficult to be removed from mixed cultures and can grow even in presence of antibacterial agents in the media.
Please help me with solving this problem as it's a great challenge for me now to get pure Actinomycetes isolates without contamination.
Best regards
Thanks in advance
The simple way is to incorporate the rifanpicin in agar also. Should do optimization study to see the suitable concentration. Try to sterilize the environment you are working in. If is in bio-safety cabine, you can use formaldehyde (follow instruction)
Mild heat treatment of suspensions (65C for 10 minutes) will kill most G- bacteria, make dilutions and plate on M3 agar (low nutrient media) and let it grow at 30C. The G- that survive will not grow much in low nutrient agar, after 10 or 15 days you will be able to pick actinomycete colonies.
You can add to medium acid nalidixic 10 mg/L. and add it by filtration after finish to sterilise the medium
Try to use nalidixic acid at 20-50 ug/mL. or Using any antibiotics your actinomycetes strain is resistant to.
Antibiotics should be added only to mother plates for isolation of actinomycetes. Addition of antibiotic may not be a good practice while sub-culturing the isolates. You should not depend always on antibiotic which will consume your time. Check your working place for contamination. Along with the inoculated plates, incubate some uninoculated control plate. Appearance of bacteria in control plates could confirm contamination level at your working place (Air-flow hood or incubator). Based on that you can do some decontamination measures. Seal the plates partially with thinfilm or parafilm. For inoculations, just touch the top of spores of the actinomycetes colonies, do not touch base that normally in contact with other bacteria.
Try to kill the Gram negative contaminant with nalidixic acid. It may work.
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