I have been trying to start some cultures of thermophilic cyanobacteria (in the temperature range of 60-68°C), from environmental samples and from stock cultures, both of which have been stored in a walk-in fridge for 3-4 years. The samples and stock cultures are green in the fridge, but when I try to set them up in the incubator (in 75mL of BG11 medium in flasks, at source pH, under continuous illumination), they photobleach within 3 days. At first, I just set the inoculated flasks into the incubator and raised the temperature with the flasks in it. Then, I've tried several methods to keep the transition more gradual: (1) keeping the inoculated flasks in room temperature in the dark for 1-2 hours prior to moving them to the illuminated incubator and raising the temperature of the incubator while they're in it, (2) covering the flasks, putting them in the incubator, and raising the temperature 10 degrees every hour until they're at the goal temperature, (3) keeping the inoculated flasks in room temperature in the dark for 1 hour, then moving them to the incubator while covered so that they remain in the dark, then raising the temperature 10 degrees every hour, and uncovering them when the temperature reaches 50 degrees.
Could anyone offer me any advice on how to successfully get these cultures growing? Thanks in advance!
I think the approach you are taking of adapting the culture is correct, but it may be too quickly. Try to adapt the ligth (and temperature) depending of you doubling time, if you cyanobactia grow very slowly then you will have to do this adaptation slower, doing changes in light and temperature but also doing transfers. Were you able to tack if your bacteria was growing in the first ours and then stop in the third day? or did they ever grow and photobleach in the third day?
Isabel Seguro Because they are filamentous, I couldn't use OD to track growth directly. It appeared that they were photobleaching steadily, appearing lighter each day, but there could have been growth that was not visible to the naked eye. Perhaps I could do a stain to assess viability after being raised to culture temperature?
you can also check for contamination doing agar plates and incubating in darkness, cyanobacteria will grow only with light but other bacteria that may have contaminated your culture will grow without light. Other things may produce the bleaching, the media for example, the pH of the media need to be the right one for those cyanobacteria, trace metals in higher concentrations, that could also kill your culture. Good luck