Saturday, May 14, 2011

Bacterial Butchery

Today, I’ll turn to some practical stuff which we in fact did during lab sessions. Our bacteria strains (in my case Elza) were to be examined with various tests. Now, I present you some of these.

There are many bacteria which synthesize and secrete so called exoenzymes. These are large protein molecules that have an essential purpose in appropriate bacterial metabolism. Microbes require a wide range of nutrition which is often hard to access for them. Most of these aliments are biopolymers, such as amilose and amylopectin (the components of starch), DNA, or casein (the main protein component of milk), fats, etc. These are called polymers, because they are constructed of lot (poly) of repeated molecular segments constituting to their enormous size. It is a bit sloppy comparison, but if you have ever been in a hurry and tried to eat something in seconds, you must have experienced how hard it is to send down huge chunks of food on your throat. It is kind of the same with bacteria, they can’t take up extreme sized molecules at once. Just as you better use a knife to slice up a cake before biting in it, they need these exoenzymes to do some of the cleaving job for them. The word exo- refers to the notion that these enzymes are transported into the ‘outer world’ and that the partial degradation of biopolymers are done outside the cells.

The result of a positive casease test. Those crosses are the
bacteria we inocluated on the Petri. That transparent area
is due to the degraded casein.
So, our first experiment was to test whether Elza secretes casease, an enzyme responsible for degrading casein (milk component). In our Petri dishes, we used a special medium, it had some agar in it to ensure the jelly-like consistency but the most important part was that it had some milk dissolved in it which creates a pale, white, blurry kind of appearance. What we did, was basically placing some bacteria (using our sterilized stick) onto the plate and incubating them at 28 Celsius for a week. In case if our cells secreted casease we have expected to see a transparent ring around them which would indicate the degradation of casein, the agent of the initial blurriness. However, this whole thing is a bit more complicated as we might get mislead if the casein degrades into para-casein. This degradation isn’t done by the bacterial casease, although it leads to the clearing of the Petri. We solved this problem by pouring a little mercury(II) chloride and hydrogen chloride solution onto the plate, which resulted in the precipitation (turning blurry again) of both casein and paracasein. Therefore, we could only see transparent areas where bacterial casease was present and degraded all forms of casein. In my case, there was no transparent skirt around Elza, so she isn’t capable of producing casease.

Agar dish containing blood. In the lower right corner you
can see an alpha hemolyis.
A second test I’d guide you through is not so ‘innocent’. It is done on agar Petri plates containing blood, which medium is commonly used for determining distinct microbe species. Some of them are able to degrade red blood cells (a process called hemolysis), some of them not. Those who have this skill can even do it in two different ways, called alpha and beta hemolysis. The alpha way of breaking down blood is also named ‘partial hemolysis’ as the red color of the blood doesn’t clear up totally, you can rather experience a brownish-greenish area around the bacteria. This is due to the peroxide production of those kinds of cells; the peroxide oxidizes the hemoglobin (the most important oxygen carrier molecule of red blood cells) and that gives the greenish color. The beta “hemlyzers” are more accurate, their exotoxin called streptolysin degrades red blood cells completely, hence results in a transparent circle around them. The process of the experiment was just placing bacteria on dishes and incubating them for a week. We tested E. coli, Streptococcus pyogenes, and all the unknown strains. For the S. pyogenes we observed beta hemolyis, the E. coli didn’t show any interest in degrading blood and there were a couple of the unknown strains who appeared to be doing alpha hemolysis.

Here are the skyscrapers of 'test tube city'  we used
for the plenty of other experiments in order to characterize our strains.



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