As a part of one of my R&D projects, I had to examine the effect of a set of industrial co-products on the growth of a single microalgal species.
I had to prepare (6) flasks containing 100 ml of media from rather contaminated industrial co-product samples. Despite autoclaving the media, the culture always seems to be contaminated.
Upon examining the samples under the microscope it turned out that the bacterial cells were moving, however, when plated on the equivalent agar media, they were unable to grow, and only the microalgal species showed growth.
The bacterial contaminant (a single species) seems to originating from the sample, rather than being introduced during inoculation (it is evident that the bacterial species in the original sample is the same as the one in the 100 ml culture)
My questions/queries:
- How come the bacterial cells manage to move (look viable), but cannot grow on agar media? (The agar media consists of the same ingredients as the original media from the liquid culture) I have heard of some theory that some bacterial cells exhibit ''twitching'' after death. Does anyone know anything about this phenomenon?
-Having plated them, it seems that the bacteria are not viable, but their cells seem to be in-tact ( I have tried using trypan blue stain to examine the viability of the bacteria but the cell walls seem to be in-tact and the cells do not stain blue). How come the autoclave does not disrupt the bacterial cells? Is it possible that spores survive? Why wont they grow on agar?
I have repeated this experiment 4 times and the outcome is always the same.
Provided the bacteria are dead, they shouldn't impair to growth of the microalgae, however it is very difficult to perform cell counts in the presence of a contaminant and other detritus. Cell counts is the only method I have been able to use to estimate cell density, due to the nature of the co-products (very dense solutions, with plenty of detritus).
Is there any way to disrupt the bacterial cells (lyse them) other than autoclaving, which clearly does not seem to be working in my case?
Thank you in advance for the replies.
If the autoclave works properly, all cells should be dead. Are you sure you're not observing Brownian motion? Also, check the paper by Lunau et al. 2005 for some ideas on how to separate cells from environmental debris.
If the autoclave works properly, all cells should be dead. Are you sure you're not observing Brownian motion? Also, check the paper by Lunau et al. 2005 for some ideas on how to separate cells from environmental debris.
Jakub Grzesiak Thank you for your reply. Yes, I think you are right, and it might be brownian motion. This would explain why they do not grow on media. I will upload a video of it, and perhaps then, someone will be able to confirm what it is. Also, thank you for recommending a paper - I will have a look at it.
PS. I have attached a photo of it, although I know it will not be nearly as helpful as a video.
It is also worth checking if the microalgal inoculum is bacteria free.
Jakub Grzesiak As previously, thank you for your reply. The inoculum seemed to be contamination-free. It has been plated after inoculating the liquid media and turned out to be a pure culture. If the inoculum was contaminated, or the contamination was introduced post-autoclaving, wouldn't the bacteria grow normally on agar media?
If it wasn't plated on proper agar media for bacteria (I suggest R2A or 10% TSA) then growth might not occur. I remember having to add some antibiotics to an algal culture growing in bg11 medium to get it bacteria-free.
Hi, Olivia Szulikowska,
do you find any change ordifference in morphologies of the bacteria before and after autoclave?
Dear Olivia,
it is possible that the inoculum you used was contaminated. In fact we can culture only small precentage of all bacteria (around 1%). The number is even smaller when the medium is unsuitable for the growth. Other factor is that some environmental species live in syntrophic relation and they cannot be grown separately. Other possibility is that some strains that we culture on plates lose their ability to be grown on culture media when released to other environment. Of course you should check your autoclave. Maybe the device is broken. These small long cells from the figure look alike Bacillus sp. it can produce spores that can survive heating up to 100 degrees, so if your autoclave is not working properly, there is a chance that they survive. Just in case you may also aeseptically filter your medium and then check again.
Good luck!
Dear Olivia,
I agree with the suggestions above. I would definitely test your autoclave (maybe there's another autoclave around which you could use?)
If the issues continue, you could try LIVE/DEAD staining with SYTO 9 and propidium iodide? This stains living cells green and dead cells red. See for example
https://www.thermofisher.com/uk/en/home/brands/molecular-probes/key-molecular-probes-products/live-dead-viability-brand-page.html#prd
Good luck!
Many microorganisms are thermophylles and thermoresistant (e.g. in gheyser of Island) and standard autoclavation is not efficient. Tray one "super-autoclavation" (220-240 C 1 h) or the gamma-irradiation, if is possible.
Hi Olivia,
I seem to have a similar problem as yours. I am currently trying to make a sputum medium of which a key component is mucin(a glycoprotein from porcine stomach). But after i autoclave the mucin, there are stil bacteria present. At first i also doubted wether they were alive, but if i put a sample (of pure disolved and autoclaved mucin) in the incubator and check it again after 24h, a lot of growth can be observed. Yet i am (like you) unable to culture it on TSA, in LB broth or on blood agar plates and i have tried it anaerobically as well as aerobically and microaeropihlic. So, i am wondering if you eventually found an answer?
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