I have collected soil and water samples from Karnataka Estuaries and isolated bacteria from it. I have maintained all of them in nutrient agar slants with 2% salt conc. Some of the bacterial isolates were orange in colour in the beginning but after sub-culturing for 5th time a few bacteria have lost their colour. What could be the reason and what should I do, so that the bacteria retains its colour?
They are probably missing some nutritional molecule that is present in their natural environment. Either that or their colour could be a result of some stress present in their natural environment, but it is not present in the lab conditions.
Try replacing distilled water with the estuary water and prepare the media. Culture the isolates on this media it may be possible for the bacteria to produce pigment.
Pigmentation can protect bacterial cells against harmful radiation, which the cells can experience in their natural environment but not when kept in dark in the lab. So, if you keep the bacteria in the dark they might turn off / tune down the production of the pigment . . .
Yes Sir, I want to retain the pigmentation of the bacteria. Gram staining of these organisms was carried out and all of them were found to be gram negative cocci.(Four bacteria are diplococcus and one is streptococcus). And every time I have sub-cultured these in Nutrient agar with addition of 2% salt in it.
If the pigmentation is a photo-oxidative stress response, stressing the bacteria out with UV irradiation and oxidative agents could "wake them up to show their true colors". The response can be quite rapid (within 24 hours) and the test quite simple . . .
Thank you Martin, but I have already tried with exposure to UV. The bacteria's shows a good tolerating response to UV but it did not help in retaining their pigmentation.
We have good experience of color retaining by putting Petri dishes in the fridge for several days after incubation and growth for 48h at 30C. Besides the role as protection against UV radiation, pigments might be also an anti-freezing agent (?).
Thank you Helmut, I have also experienced such cases with mostly some of the terrestrial bacteria when keeping the plates after 24hrs of incubation in fridge for a period of a week. Even incubating the plates at 37 C for 4 days pigmentation has appeared in a few cases too. But In case of Halophiles, its not working.
Some pigment producing strains retains it by adding glycerol in to the nutrient agar medium as they're already growing on it. You ca try it by 1% of glycerol
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