The solid medium used for Legionella app is BCYE, which has charcoal inside. Looking up the literature I only found it was first used to replace starch in the medium. But still couldn't get the answer the role it plays.
Hi Xiao,
I have been under the impression that the role of activated charcoal is to remove toxic metabolites from the medium during the slow growth process. Not aware of formal literature, but there is some more information here.
https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/BCYEAgar.htm
Article Charcoal-Yeast Extract Agar: Primary Isolation Mediumfor Leg...
It also could bind CO2 and has a role into the superficial tension.
I have reviewed the literature. I believe that the supposed need for CO2 is not true. And this has also been my own practical experience.
Also, as far as I could tell no one has actually fully elucidated the role of charcoal. There was a research group that found that so long as the yeast extract was filtered sterilized in lieu of autoclaving, legionella would grow well in the resulting yeast extract broth. I tried making BCYE agar with filtered sterilized yeast extract and it didn't work. However I have been using buffered yeast extract broth w/o charcoal in shake flasks and that works great.
The same group noted that if they autoclaved the yeast extract broth with some perforated baggie of charcoal that legionella would grow in the broth that was removed from bottle. But autoclaving without any charcoal resulted in poor growth.
It clearly has something to do with autoclaving, but all the supposed explanations that float around are not based on empiracal evidence so far as I could find.
James Folsom James, would you please share the composition of the buffered yeast extract broth w/o charcoal that you used? I need to cultivate Legionella in shake flasks and would greatly appreciate your help.
The growth of bacteria is often enhanced by addition of carbon materials such as graphite or activated charcoal to the growth medium. Check the below link and you will get your answer.
https://jb.asm.org/content/jb/177/3/688.full.pdf
Hi Dina Raafat,
BYE Broth
Add 5 g of Yeast Extract to 450 mL of water and filter sterilize
Then add Oxoid SR0110C supplement
Final volume 500 mL
Growth should be excellent, but takes about 40 to 50 hours. You will probably want to use some means of containing the aerosol that will be generated by the shaking.
Ristroph, J. D., Hedlund, K. W. and Allen, R. G. (1980) ‘Liquid Medium for Growth of Legionella pneumophila’, Journal of Clinical Microbiology, 11(1), pp. 19–21.
Hi James Folsom ,
Thank you so much for sharing your recipe. I used the same formula, once with and once without charcoal, and it grew in both after around 48 hrs. The only difference is, I autoclaved the medium. Is there a reason why you chose to filter sterilize it instead?
What means do you use for containing the aerosol? And if I want to collect the cells at the early stationary phase, do you know how many hours I would need to wait?
Hi Dina,
Based on the citation I provided, I never tried autoclaving it. So that is interesting.
I bought some plastic flasks that had vented (0.2 micron) closures.
I've found that the various Legionella strains may exhibit vastly different specific growth rates. You should construct a growth curve of either OD or Cell count vs Time to determine this.
Hi James,
Thank you very much for the quick response.
Would you please share a link of the flasks you used? You mean like these culture flasks? I've never used them before, but it seems like a good (but rather expensive ;-)) option.
https://www.sigmaaldrich.com/catalog/product/sigma/cls431282?lang=de®ion=DE
I will construct a growth curve to see when I can harvest the cells anyways. I read the paper, and I think I can anticipate how the growth might proceed. It definitely is quite different from the bacteria I've been working with so far!
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