Hi everyone !Today, I went on an ATV with Martin in order to collect soil samples for my microbiology experiment. We left the station at 9.30, and we drove for twenty minutes towards an old river bed. We arrived at 9.50 at the following coordinates : 12S0518323 UTH 4250634. In the river bed, I collected 5 samples of various soils and types of rocks. For each sample, I measured soil's temperature and took some pictures, for further identification with the help of a geologist.We left the old river bed after exploring it during 30 minutes, and we took back the ATV. We stopped on the way back to MDRS for another sampling at 10.57. We arrived home (sweet home) at 11.15.
Immediately, I grinded the samples in a mortar and riddled them in a geology sieve. I weighed 20gr. of each and stored them in falcons numbered from n°1 to n°6. After this, I put 1gr. of each soil in 6 eppendorfs, and I added 1ml PBS (Phosphate Buffer Saline). I shook vigorously these mix, and then centrifugated them gently. I took 200µl from each supernatant and spread it on LB-Petri dishes with a spreader. By this way, I obtained non diluted extracts, but it is possible that the bacteria's concentration will be to high on the dishes (and I won't be able to isolate pure cultures from the bacterial layer). I decided to dilute from 1 to 10-5 the first supernatant of each sample. In order to do this, I realized 5 successive dilutions by taking 100µl of the previous eppendorf, and putting it in 900µl PBS. I spread 200µl from each samples on Petri dishes (6 dilutions of each sample, so 36 dishes in all). I put them overnight in the incubator at 30°Celsius, which is the optimal growth temperature for Bacillus thuringiensis, the bacteria on which I work in my lab in Belgium. I hope that it will allow the growth of many other bacteria. If not, I will try other temperatures (37°C, 25°C...)
(04/15/2015)Bad news for today : no bacteria has grown on my LB media. When I opened the incubator, I realized that it was very cold. The real temperature seems far below the one I choose on the switch yesterday evening. I realized some tests with a thermometer, and actually I was right : when you put 30°C, you have a temperature of about 12°C (as cold as in a fridge : this temperature inhibits bacteria's growth). I switched to more than 45°C, and I checked on the thermometer : I obtained a real temperature of about 32°C, which seemed nice to me. I also tried the little incubator, which is working good despite the fact that you have to press the switches with a knife or another thin object. I let the bacteria grow at this temperature. I hope that I will have more results tomorrow, in order to begin identification and characterization (gram staining, catalase and oxydase tests, colorations and biochemicals tests...)
If no results tomorrow morning, I will begin to think about what did not work. Maybe the extraction of bacteria has failed because they were contained in the soil's solid fraction and not in the supernatant after the centrifugation. But the protocol that I read clearly indicated that the extraction was possible with the supernatant. Another explication is simply that there are no bacteria (or far less bacteria) in these desert's soils which are clearly less favourable to life than 'fertile' soil samples. Or maybe the bacteria are not cultivable, or do not grow on LB medium, despite the fact that it is an 'universal' growth medium. Finally, maybe the bacteria just need more time. Next days will help us to confirm these hypothesis !
Have a nice evening dear earthlings! Any suggestion for my experiment is still welcome!