I have been culturing anaerobic bacterial strains using the same standard methodology for the past few years (liquid cultures with overnight oxygen removal and incubation in an anaerobic glove box) but recently, the strain that I'm studying has not been growing properly. Growth is suddenly much slower and it seems like the cells stop growing and die rapidly at what should normally be late stationary phase. Additionally, the culture morphology appears different with a cursory glance, but I'm not sure if this is due to debris from cell death or changes induced in the cells themselves.
This strain has always been difficult to culture so cell density did tend to fluctuate a bit between replicates, but growth curve time points were at least consistent. I have also tested an additional anaerobic strain that tends to grow more rapidly, and although growth was mostly normal, it was also slightly delayed and lower than previously observed.
Assuming that nothing has changed with my culture preparation process, what could be some potential causes of these issues? I have tried new stock cultures, prolonged oxygen removal, checking culture conditions (oxygen sensors, oxygen removal catalysts, incubation temperature, gas tanks, etc.), new lots of media, and using a different culturing method (Hungate tubes), but all of them yield the same results. Any comments or suggestions would be much appreciated!
You don’t say what kind of anaerobe. Methanogen? SRB? Heterotrophic? Maybe an oxygen scavenger in the medium, like dithionite or something milder; like ascorbate? Trace metals and all growth factors the same as usual? Do you need to poise the Eh at any optimal level?
Jim C Philp Thank you for your suggestions, and I apologize for the late reply. Actually, it is an uncharacterized strain, so we have been using a very rich, complex medium (GAM) to culture it. Overnight oxygen removal along with L-cysteine HCl in the medium have been sufficient for creating anoxic conditions until now, so I'm inclined to believe that oxygen contamination is not the issue.
In terms of growth conditions, nothing has changed so I'm quite stumped. The strain used to grow just fine in the medium without any adjustment or addition of additional growth factors.
Traci L Testerman It is true that I did not test the accuracy of the oxygen sensor very rigorously, so that is definitely something that I may look into. Unfortunately, I previously tried culturing my strain both inside and outside the glovebox, but results were similar. I did also wonder if the stock was the issue, but I tried multiple, previously unused stocks without success.
At this point, I am starting to wonder if the medium formulation hasn't suddenly been changed by the manufacturer! It seems like almost every possible point of failure has been looked into already.
About pyrogallic acid + NaOH as oxygen scavenger ― check my post at:
https://www.researchgate.net/post/Can-somebody-recommend-a-method-for-making-48-96-well-plate-anaerobic
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