I set up 10 subcultures of MTBC from frozen aliquotes. Two grew well and 8 became cloudy after 7 days in the incubator. I thought it was bacterial contamination so, I repeated the subcultures. The same happened. I sis purity plates. They have been in the incubator for 2 weeks and there is nothing growing. Any thoughts? Is this a technical problem? Might it be the OADC? Can it be a bacterium or a fungus that grows in 7H9 but not in BHI?
To start with, I suggest a quick stain to see what is in there................
Thank you both.
Unfortunately, smear is not available in our facilities due to health and safety local regulations. That's why it was not done.
I inoculate 7H9 broth directly from frozen aliquotes. Solid medium is BHI, a richer medium to improve recovery of many bacteria.
Thank you Floris.
7H9 is only broth. A similar solid option is 7H10. I could do this. Just to clarify, I do not set up liquid culture alone from frozen aliquotes. At the moment of inoculation, I also set up a purity plate. In this set, I set up such plate and had no growth. When the media became cloudy on repeat, a set up a second purity plate.
The 'cloudiness' might be an anaerobe of microaerophile.
As others have pointed out, put it under a microscope and have a look. You don't necessarily have to stain - just put a (small) drop on a microscope slide and put a coverslip over it. If you're not used to microscopy of bacteria, it's worth practicing with a suspension of a colony - finding the right depth of focus can be quite fiddly. I often use the edge of a bubble to focus on.
Thank you all for your input. I will try the 7H10 option. I agree that microscopy would be the easiest and probably the most accurate. However, I work with XDR and MDR tuberculosis strains and I cannot due to health and safety regulations remove a slide from tha hood.
Floris, I will try with 7H10 but have you got any experience on cloudyness due to precipitation or other similar phenomenon? I take as a negative control the 2 strains that grew normally. All 10 strains were set up the same day using the same batch of 7H9 and OADC.
Dear Floris,
I do know what a negative control is. What I mean is that is this 10 strains inoculated at the same time, using the same materials and reagents, incubated in the sabe incubator within the same secondary containment, 10 showed signs of what I thought it was contamination and 8 did not. When repeating the trst, the same happenend. What type of information a tibe with no bactetia would have added that those two that grow normally were not providing?
Rather than experimenting with controls, I'll de-contaminate with oxalic and take it from there.
No doubt tips may have been contaminated but two different boxes, separated by three weeks in use, with the same two not being contaminated, I think that it is unlikely. Since I cannot assess contamination by microscopy, I will assume those stocks are contaminated, de-contaminate as per protocol and start again.
My boss suggested for me to explore the possibility of a chemical issue. I'll do that if this happens again.
As Andrew Jenkins suggested, this can be a fastidious microorganism.
Thank you again for your time and I'll incorporate some of your suggestions for the future!
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