I have done 2 different method to isolate mercury resistant bacteria from Soil.
1. Streak plate method: From my mix culture in Nutrient Broth, I got 28 colonies. Those 28 colonies were subcultured into Nutrient Agar which contains higher mercury concentration (HgCl2) from 10-100 ppm. When i did the gram staining procedure, I noticed that those colonies weren't pure enough because there's more than 1 cell morphologies (coccus, bacillus, and coccobacillus).
2. Using Nutrient broth which contains HgCl2 from 10-100 ppm to subcultured my mix culture was done as well. And the result is, when I subcultured the inoculum from NB contains 100 ppm HgCl2 into Nutrient Agar (100 ppm HgCl2), I got pure colony which is a gram negative coccobaccilus bacteria.
Any idea why my first method doesn't work?
I made a hypothesis that there's a chance that maybe the other bacteria are relying on the Mercury resistant bacteria that might have remove the mercury from the medium (Agar medium). It's the same principle as in satellite colonies.
But is it possible? Thanks for answering.
Hi, your first method seems to be a bit short on the purification site. I would definitely do a series of clean streaks on my plates to make sure that my colonies are pure (I normally perform at least three clean streak setups). I would also try to apply the 'dilution to extinction' method for the isolation of bacteria before even plating them. If you have a high cell density you might likely end up with two or more different bacteria forming something that looks like a pure culture on the first plate. Your hypothesis seems possible but to my knowledge rather unlikely as you do get pure cultures with your second method.
Greetings and good luck
HI there, sounds interesting project! I would think method 2 is more selective than method 1. Did you check your colonies in both methods for mercury resistance? Maybe you should try higher concs of Hgcl2 in method 1 and dilute as Bastian has suggested. Are you able to use "spiral plating"? Might be worth a try
Best wishes with your work
Another vote for diluting your broth culture going to your plates. It's also always an interesting game in where to put your selective media in your isolation process. Sometimes you have to play with it a bit to get optimal selection. Starting your isolation with the selective broth in the second method seems to work better for enriching your higher mercury tolerant bugs. When you use a non-selective medium, you generally will have to do that after applying some level of selection on the front end, or else you run the risk of your bug of interest being out-competed and more difficult to isolate. Good luck!
I guess i'll keep up with my result from the 2nd method...
Thanks for answering my question.
Isolation of desired bacteria from a mixed population is very easy. Among the streaking procedures quadrant streak method is the best. Flame the inoculation loop and cool it for 5 seconds before every streak. Gram variation occurs in old cultures. Always use fresh culture for staining
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