(04/16/2015)
When I woke up this morning, I immediately ran to the incubator, and at first sight I knew it was going to be a good day ! Many bacteria had grown in the little incubator. It contained only non-diluted supernatants from the six soil's samples. Many CFU (Colony Forming Units) were present, so numerous that it was impossible to estimate them. In view of their number, it is probably not due to a contamination. I observed several type of bacteria at first glance. I think there are also many mycetes/fungi, recognized by their characteristic surface. However it has to be confirmed.
Despite these good news, the big incubator was very cold again this morning (9°C). This temperature inhibited the growth inside the 36 Petri dishes. I think it stopped to work during the night, because it was still at 32°C yesterday evening. I can not trust it for the moment ; I will try to check the evolution of the temperature during all the night by taking pictures of the thermometer inside the incubator at regular intervals. In the meantime, I will only use the little incubator, which seems reliable.
(addendum: I just have learnt that the big incubator was in fact... an oven! I will not use it anymore :p )
The first thing I did this morning was to count all the CFU obtained in every dishes. I then chose 15 various colonies, and I tried to isolate them by spreading them on new LB media. I hope I will obtain a maximum of different bacteria. I named them by a number (representing the soil sample where they come from) followed by a Greek letter (to differenciate them) :
- 1α, 1β, 1γ, 1δ, 1ε
- 2α, 2β
- 3α, 3β, 3γ
- 4α, 4β
- 5α
- 6α, 6β
We will see tomorrow if the isolation succeded ; if lucky enough, I will obtain pure cultures of various desert's martian bacteria !
This afternoon, I tested the protocols that I am going to use tomorrow morning (if I obtain pure cultures of course). I first tried to realize a Gram staining on several colonies chosen at random. The aim was not to identify them (it would be useless because these are not pure cultures), but to see if the protocols are working. My first try did not work : I did not see anything on the microscope slides except a big purple stain. I think I put too much water on the slide, and I did not let enough time for the action of the decolorizer (ethanol). But my second try was succesful ! I chose a yellow colony from sample n°3, and at the end I obtained a beautiful microscope slide where I could easily see that the bacteria were rod-shaped and gram-negative (pink coloration). A nice picture of these bacteria is available in the pictures of the day sent in this capcom.
Here is my protocol for the Gram staining :
- put a drop of water on a microscope slide
- take a colony with a toothpick and put it in the drop
- let it dry and pass it quickly 3 times above the flame
- put the slide 1 minute in crystal violet
- rinse it with water
- put the slide 1 minute in iodide
- rinse it with water
- put the slide 5 seconds in ethanol (decolorizer)
- rinse it with water
- put the slide 1 minute in safranin
- rinse it with water
- dry it, cover the stain with a micro cover slide
- put a drop of paraffin oil
- observe it with the microscope at G=100x
I also sterilized 250ml LB-agar, 250 ml liquid LB and 250ml distilled water with the pressure cooker.
Finally, I tried two biochemical tests with random colonies : the catalase test and the oxydase test. These tests are very easy and you know the answer very quickly. In the catalase test, you put a drop of hydrogen peroxyde (H2O2) on a microscope slide, then you take a colony with a toothpick and you put it in the drop. If there are bubbles (gas liberation), the bacteria possesses the catalase enzyme (catalase positive), and is able to transform hydrogen peroxyde in water and oxygen:
2H2O2 ----catalase----> 2H2O + O2 (gas liberation)
In the oxydase test, you just pick a colony with a toothpick (and not with any metallic object, because it creates false positives!) and you put it on the top of the oxydase test, a little piece of paper. If the top turns to blue, then your bacteria is oxydase positive, and it possesses the cytochrome C oxydase enzyme. This enzyme play an important role in a variety of redox reactions.
Tomorrow, I will use these tests on pure cultures obtained after a night of incubation. It will allow to characterize them with a biochemical approach, and it will help for their further identification.
Can't wait to see what kind of martian bacteria I will discover in the next few days ! Maybe an unknown one ?