Monday, March 21, 2011

Hold your breath!

This gorgeous creature is a Clostridium species. There are about 100 types of them.
Some living freely, some being pathogenic. This one in the picture is the Clostridium difficile,
which starts thriving in your gut after you used antibiotics to kill all the others. Then this guy
realizes that the place is free, then swings into motion, which you usually realize
by having antibiotic-associated diarrhea. Source.

This post aspires to give a brief overview on my first encounter with anaerobe microbes. Basically any organism, which doesn’t require oxygen for living is called anaerobic. There are certain categories, for example obligate and facultative anaerobes. Apparently, the obligate guys made up stricter rules for themselves. They love being without oxygen so much, that if they come across any amount of oxygen, they stop growing and finally give up living. The facultative ones are cleverer, (this is of course an exaggeration, there are instances when being obligate is more adaptive for one) they are happy growing without oxygen but don’t mind its prevalence in their neighborhood (for a certain level).

Schematic structure of an endospore. These
several layers are responsible for
saving the DNA from UV light and high
temperatures, for instance. Source.
The subject of our experiment was a genus (this is just a taxonomic category, which contains very similar species) called Clostridium sp. These microbes fall into the obligate category, also having a special feature: they produce endospores. This characteristic was the one we needed for selection, I’ll explain you why. Endospores are very inventive tools for surviving harsh environmental conditions, lack of food, draught, chemicals, UV light, extreme temperatures, etc. The endosporal life stage might be compared to the hibernation of let’s say grizzly bears. The metabolic activity (all biochemical reactions that function to produce energy from food and to use energy for any kind of processes, for instance motion) of bears changes and slows down, in contrast the these endosporic microbes go completely dormant, by arresting all metabolic pathways. Sporulation occurs relatively quickly, it involves secretion and formation of several layers, coats around the cell plus the inner structural changes, for instance DNA condensation (it gets more dense, packed into a smaller volume). Eight hours are sufficient for a bacterial cell to develop an endospore. Due to this completely dormant, inactive phase it is not only one winter, as in the case of bears, that such kind of organisms can survive.

This is the hot bath, where we
eliminated everybody
except for Clostridiums.
Now, a little history: in 1947 Clostridium endospores were put into a sterile medium and weren’t opened until 1981. It didn’t take more than 12 hours for the bacteria to reactivate themselves and start cheerfully thriving, 34 years later! If that won’t be enough for you, there are further, much bolder claims. In the 1990s a 25-40 million years old bee, preserved in amber was found. In itself that wouldn’t be exceptional at all, but some scientists isolated endospores from this oldish bee’s gut. Believe it or not, they were able to bring those bacteria back to life, by simply putting them into appropriate culturing medium!

This is and anaerobic 'booth'. This elephant trunk like thing
is where you put your hands and start operating inside that
box. It keeps oxygen out, so is perfect for experiments
done with anaerobic bacteria.
Our task in class was to selectively grow this famous Clostridium. Its name has a Greek origin, it refers to its rod like shape. First, we needed to eliminate all other species from our bacteria culture. We did it by placing our Clostridium suspensions into a hot bath, they stayed there for 10 minutes at 80 Celsius degrees. That process yielded us a suspension full of dead microbes, except for those who bore endospores. (Endospores can survive even 150 Celsius degrees). After that we used a syringe to place a little amount of this suspension to the bottom of an empty Petri dish (no agar!). We melted some agar/culturing medium solution and poured it over our samples. After mixing them, we placed the top of the Petri onto the still warm and soluble agar upside down, so it stuck to it. This tightly fitting construction, called the ‘Brewer plate’ was able to expel ‘all’ oxygen particles, letting our dear little Clostridiums to start reactivating. I can’t wait for Tuesday to see what they ended up with! 
As I promised, here is my little Elsa. You can observe her better if you enlarge
the picture. She is that white cream like guy on the surface of the yellow agar.
If you are careful enough you can notice how she started going deeper into the agar,
it is like little roots going down from the indented surface.

1 comment:

  1. Your writing is interesting, but your really make it shine with the photos. Terriffffic!