I have some marine isolates that were freeze dried. To reactivate the cells, the pellet was rehydrated with saline and immediately transferred to Zobell marine broth. After 24h of incubation, the culture grew quite well and even produced pigment. And the same when they were transferred to new media twice. But whenever I tried to plate them in Zobell marine agar (same composition plus the agar), no growth occurred, even after 5 days of incubation. What could possibly be the reason for this?
Do you know which species and the source?
Could you make an Overlay Agar and incubate the agar plates for 2-3 days?
What is the concentration OD in the liquid culture?
You should test different temperatures, and also microaerophilic and anaerobic conditions if you do not know more details about the cultures.
http://archimer.ifremer.fr/doc/00035/14631/11950.pdf
Wow, that's quite a helpful paper, thanks!
We still don't know much about those marine isolates, as sequencing of 16S rDNA is still in progress. All microbes have been isolated and cutured under aerobic conditions, so I don't know if testing microaerophilic or anaerobic conditions would suit here? But temperature and that agar overlay tip are something I am going to try.
Still, I just can't figure out why in the world just by adding agar to the media they will stop growing... =/
Anyway, thank you very much for your help!
You could reduce the agar to a minimum and prepare a semisolid medium in Petridishes.
As in Msrv Medium for Salmonella detection.
http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM1112&org=124&c=UK&lang=EN
Only 2.7 g Agar/ l is included in this formula.
During incubation you should not put the Petri dish bottom up.
Liquid growth is often more or less not a real aerobic condition, so maybe they would prefer a small amount of extra CO2.
You could also have a look at the liquid culture were is the growth actually happening, are the bacteria concentrating their growth to the middle or bottom?
Good luck Bea.
Since growth is made under agitation (500 rpm), it is hard to notice. But when I let the broth settle down, sometimes the cells precipitate in the bottom, and sometimes they remain suspended along the broth column, depending on the bacterial culture.
Well, it won't hurt to try it. Thanks again!
Try the broth-both with and without agitation-this is usually a 'gentler' re-introduction from freeze drying. Unfortunately freeze drying (good for long term storage) does not suit all isolates well and they may no longer be viable.
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