Hi guys, I am here again with mycobacteria question. for plating organ homogenate from SPF mouse, we have to add antibiotic combination to the 7H11 agar. the combination I used for BalB/c mice from Charles River is polymycinB 5.5ug/ml. carbenicillin 50ug/ml, amphotericinB 4ug/ml, trimethoprim 2ug/ml and cycloheximide 100ug/ml.
Yet when I tried the combination on another mouse strain from a different company, I noticed some small colonies showing up after 3 to 5 days incubation. the colony grow very slow, with no color and looks like a tiny water drop on the plate. Normally, there are one or two colonies on one plate.
I do not know what that is but I want to avoid it. So I am thinking about increasing the antibiotic concentration. From Gibco selectable 7H11 agar, I get a different combination: polymycinB 33ug/ml (200U/ml), carbenicillin 50ug/ml, amphotericinB 10ug/ml, trimethoprim 20ug/ml.
Have anyone tried that?
Hi Wenxi,
Hope your Mtb work is coming along well. I don't have a direct answer for your question as we don't routinely use any of those drugs in our plates for organ homogenates but I will tell you what we do as it works for us.
Most people in our lab routinely use 7H10 plates for plating organ homogenates with no added antibiotics. We pour the plates in a class II biosafety cabinet and you can (but we don't routinely) incubate the plates overnight at 37 to verify that they are sterile (at least for fast growing microbes). I often use 7H11 without any antibiotics. It is a little richer than 7H10 and so may better support growth coming out of the mouse and has a decent amount of malachite green that can inhibit many other bugs. We very rarely encounter any contamination. There are a few things that may contribute to this. First is the mice are housed in micro isolator cages in a rack that draws air in through a filter and out through a house HEPA filter. When we harvest the organs, the mice are thoroughly wiped with EtOH to kill anything on the skin/hair and the organs are removed very carefully with EtOH-cleaned scissors and forceps to avoid touching any non-sterile surface.
Whatever you end up using in the plates I would be sure to check that it doesn't inhibit growth of your infecting strain prior to using them for an animal experiment. It is definitely annoying to find whatever it was that you saw on your plates, but I think you will want to balance your efforts to get rid of it with drugs with a consideration of where it could be coming from and whether or not it inhibits the growth of your infecting strain - you may find that adding larger amounts of the drugs could confound your results more than having a couple colonies of a non-Mtb inhibiting contaminant.
Best wishes,
Brian
Hi Brian, thank you again for your great answer. You point out the issue I worry about as well. even it is claimed that some antibiotics like polymycinB do not inhibit mycobacterial growth, I still need some clear evidence on my hand it is OK to increase the antibiotic concentration. Another thing is that in my animal facility, we do not use mouse cages with HEPA filter. In addition, animals I am working with are all SPF level instead of gem-free level, so there should be bacteria or fungi in the lung at least.
I fully understand it is critically important not to introduce any contamination in the organ harvest. Another thing is that animals I am working with are all SPF level instead of gem-free level, so there should be bacteria or fungi in the lung at least, which is also confirmed when I called Charles River. I did some pre-experiment and tried the 7H11 plates without antibiotic combination, the contamination was gross. So I guess there is no other choice for me other than using antibiotic combination.
I also asked my friend who works with mtb-infected monkey model in Chicago, they just use the pre-made selectable 7H11 agar from BD.
One issue which may be relevant is that I am using 7H11 agar prepared by adding casein into 7H10 agar. So it is not the real 7H11 agar which contains malachite green.
Right now I keep doing the monitoring to those suspicious colonies. they rarely grow, which makes them more and more like particles from mouse tissue...
anyway, I think I will try the plates with higher antibiotic concentration and compare the CFU output from my current plates.
A couple of notes to clarify. We also use SPF mice and I have rarely had contamination on 7H10 or 7H11 without antimicrobials. 7H10 (BD Difco) does have some malachite green but not as much as 7H11, which is also richer because of the casein, so you may want to consider adding more malachite green as well as the casein. 7H9 doesn't have malachite green so if that was used I would imagine it would be less selective for Mtb. I would second your guess that what you are seeing might be particles from the mouse tissue, I have definitely seen that. A good course is to do as you have and incubate the plates longer to verify that what you observe are not atypical very slowly growing Mtb.
Hi Wenxi,
I have worked with C57/BL6J and C3HeB/FeJ that we got from Jackson Labs, BALB/c from National Cancer Institute, and various KO mice on the C57/BL6 background from the Sanger Center in the UK. We breed the unusual KO mice in the lab as needed. For Mtb infections in each of these mice we use 7H10 or 7H11 for plating organ homogenates with no added antimicrobials and very rarely have issues with contamination.
a small update about the suspicious contamination.
The colonies I mentioned in the question turn up to be tissue debris. they dried out on the surface of the plates during 14 days incubation. I guess it is because I incubated the plates in an incubator with water tray so that the tissue debris dried out slower which made them suspicious.
So, it is safe. Thanks, Brian, I will try plates without antibiotics next time on this mouse strain.
Wen
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