I received several strains of bacteria from another lab far away. However, one of the samples appears to have high fungal contamination. When I grow it in broth, I see lots of fungal contamination. It would be difficult and time consuming to have them send it again, and I worry a new sample would still be contaminated. I am confident it's not my reagents that are contaminated (uninoculated media showed no growth, and other strains they sent were fine).
Is there a good way to recover my bacteria from the fungus?
0.45 um filters? Addition of cyclohexamide?
You can re-isolated on solid medium with anti-fungal agent. Streak on solid media and check growing colony, transfer to new media and purify.
Hi,
I faced fungal contamination in the past.
I would recommend a single colony isolation method. First of all, you can make an isolation streak of bacteria on an agar plate.
After incubation, you will see different colonies: bacterial and fungal. If you can not identify colonies by eyes - prepare several small tubes with 50 -100 ul of sterile PBS, transfer colonies of interest into the tubes ( one colony - one tube). Examine each tube by microscopy. Make an isolation streak using the tube with pure bacterial biomass.
You can streak on bacterial medium supplemented with antifungal cycloexamide in a slightly bigger concentration than normally used.
If you want to take an extra mile, I would suggest to plate serial dilutions of the culture on the supplemented bacterial medium from the beginning or after the initial streaking, in order to be absolutely sure that you pick a pure bacterial colony for further storing. Pure from any fungal hyphae and maybe other bacterial contaminants (supposing you know the type of colony that the bacterium of interest forms).
All of the answers above are suggesting the same thing, isolation and recovery of a bacterial colony. I would encourage you to dilute the culture you are taking the organisms from with additional broth, this then makes it easier to have proper isolation where the bacterial colony may be far from any contaminant (and yes, you would need to do multiple isolation plates at the same time). Using a media that does not support fungal growth, or a fungal inhibitor, should also help. And be very aware of the warning of Phil Geis .-. that the bacteria growing in the culture may itself be a contaminant. You will need to grow it (once isolated) and make sure it is the organism you should have received. Using a selective medium during the isolation may be helpful, as some can be inhibitory to fungi, and it will give you some indication as to whether your bacteria is what it should be. For example, if it is supposed to be Staph aureus, us a mannitol salt agar for the isolation, as only salt tolerant bacteria will grow and if it is able to ferment the mannitol that will help increase the likelihood it is the correct bacteria.
You can use any antifungal agents on solid media and pick up isolated colony on the very next day. As fungi being slow grower won't appear on solid media next day.
you can use antifungal and isolate a single colony of the bacteria and use the streak method. the first plate that you culture take another colony and culture again with this method. Do this many times maybe three time and grow the plate in 28ºC for 24 hr.
Ramírez Piña José Emilio .-. What is needed is isolation of the bacteria, and not cross streaking to determine if either has antimicrobial action on the other. Also, since we have not been told which bacterial strain is the one Gen is trying to isolate, we do not know if 25 or 28, or 30 or 37 is the appropriate temperature to incubate. Also Shanu Khandelval .-. many fungi are able to grow rather quickly and to grow over the bacterial colonies, and this might be the reason Gen has not been able to isolate the bacteria yet.
Streak the stock on agar that your strain grow faster than the contaminated fungi. Grow them at the temperature preferred for your strains. Keep your eyes on the plates and pick the single colones of your strains once they show up and streak to new plates. You probably need to do this for a few rounds to remove the contamination. You may add anti-fungal antibiotics but make sure they don't inhibit growth of your strains.
passage after passage. the spores will remain. may try all four classes of fungicides in your culture media, 4-5 passages should do
Anilinopyrimidines
Benzimidazoles
Dicarboximides
Dithiocarbamates
You can use antifungal agent like cycloexamide or nistatine in low concentration. After 24 or 48 hour incubation time , select bacterial colony and isolate .
Thank you for all the answers. Ultimately, picking from a plate proved surprisingly inadequate (even though I saw little evidence of fungal contamination on the plate). However, picking from a plate in combination with amphotericin B seems to have removed any visible fungal growth, and the bacteria now grows robustly (as normal).
I do worry fungal contamination could re-emerge as I grow the bacteria in selection-free media, however, I have not seen any indication of that yet.
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