I am working with oral anaerobes. They grow well in the agar, in the 25 ml tube but they don't grow in the microtiter plates. I am using an anaerobic incubator. I am new in this field so any suggestion could be useful.
Thank you
I cannot answer by experience with anaerobic microplate culture, but would put into discussion the following. Prepare your microplates with a stock of your culture medium before adding your bacteria. Store the prepared microplate in an anaerobic jar (a jar may be better than an incubator as with a gaspack oxygen is reduced quickly) for at least one hour to have the media stock short of oxygen. Then, quickly, add your bacteria. In addition you may think about overlaying the wells with mineral oil. Put back the plate in anaerobic jar or incubator.
Did you make sure that within your incubator you have indeed anaerobic conditions? You may check with a culture of Pseudomonas aeruginosa, which should not grow as it is a non-fermenting, obligate aerob bug. Good luck, Oliver
Dear Oliver Nolte and Jan B. A. Arends,
thank you for your answers.
Yes, I meant an anaerobic work station but I think that you are right, I should check the conditions because I am quite sure that for example I have a problem with the humidity since the volume is not constant after a couple of days of culture.
The media that I am using is the FAB and since I am started only one month ago to work with bacteria I didn't try to adjust its composition.
I will also check if it is better to use a anaerobic jar (I stocked the media and the plates without bacteria in the anaerobic cabinet).
In any case thank you again for your suggestions and for the papers.
Kind regards,
Elisa Serra
Elisa,
Jan and Oliver pretty much summarized all the important factors. You may also try the AnaeroGen™ Atmosphere Generation System used in a recent paper. Judging from the video the posted in the paper, it seems pretty good for their micro-plate. You may find the link the paper below.
Also, many plates are made of plastic material that is highly permeable to oxygen, since they are mostly used for aerobes. There should be other types of plastic plates that are less permeable to oxygen, so you may want to get those. Glass plates would be ideal, but I am sure if those are commercially available.
Good luck!
http://www.sciencedirect.com/science/article/pii/S1075996413000802
Hello Elisa,
I know this was some time ago, but did you ever resolve this issue? I am having a similar problem growing ETBF and NTBF, where in 5 mL culture it will grow fine but once I transfer it to the plate I get little to no growth. I am also using an anaerobic chamber. The little growth I do get is in a dot formation, which I guess is indication that the bacteria is trying to get as far from oxygen as possible. How were you able to resolve this issue?
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