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.

Sunday, May 1, 2011

Fearless fungi

I had to realize, we had an extended break so there was no class this week either. Therefore, I'll tell you about an exciting story I've read. My flatmate wants to be a mycologist (a biologist whose main focus is on fungi) and he drew my attention to a phenomenon which is in connection with microbiology. As this discipline deals with almost anything that is on the 'small scale' there are many fungi within our scope.

Fruit body of the Amylostereum areulatumSource.
If you have read the article about the autumn leaves on Small Things Considered, this topic will not be totally new. What I intend to present is another way of symbiosis between an animal and a tiny organism. There is a certain wasp called the Sirex noctilio  which has much to do with fungi, namely the Amylostereum areolatum. As all insects, this Sirex goes through metamorphosis, it has clearly distinctive life stages in which the animals differ completely in their appearance. First they lay their eggs which later develop to larvae (just think of a caterpillar) vaguely said 'after a while' these larvae create a layer around themselves and form a pupa. As the animal inside undergoes many structural changes, this berrylike thing hatches and the adult wasp, moth, etc. reveals itself. We call the adult insects imagos. 

Tunnels made by the larvae. Source.
So, the imagos of this species lay their eggs with a special tool, an ovipositor (an egglaying tube) into a tree's trunk. There is a special sack in the imago's (females only) body which is connected to the tube where the eggs run out of the insect's body. This sack is the so called mycetangium containing fungi cells. As the eggs 'roll out' into the tree trunk, these fungi attach to them. There are many cases when the imagos create tunnels inside the tree, but now it is different. It is the  Sirex larvae that make their way inside the tree. However, they require the presence of the Amylostereum because they cannot digest cellulose and lignin (both are components of the plant cell wall). This shouldn't be new for you, just consider herbivorous animals. They also have many microorganisms dwelling in their guts, in order to digest cellulose. Furthermore, we don't need to go that far! We humans have a plethora of bacteria within our intestinal tract for similar kinds of purposes. So! Here, it is the other way around, these digestion helper guys are outside (!) the animal. As the larva crawls its way in the tree, it spreads the fungi onto the tunnels' walls. As the fungi begins the degradation of the tree tissue the tunnels become full of accessible food for the larvae.

It is interesting that the imagos have nothing to do with fungi during their lifetime, so every female must gain a fungi population within their body in their larval stage. So, during the pupal stage and until egg laying they keep their fungi fellows in their mycetangium (which produces some essential mucus for keeping the fungi alive). 

Sirex noctilio. Source.
But why is this good for the fungi?? The tree bark serves as a fence for the plant in protecting it from diseases. This ovipositor tube of the insect is a perfect tool for delivering not just the eggs but the fungi straight into the inner part of the tree trunk. Moreover, these tunnels serve as an ideal habitat for the Amylosterem, lacking any competitor fungi species. Think back to our lab practice, there were many times when we had the goal to create populations of only ONE certain microbe species. Basically that is what the Sirex does for the Amylostereum; they are clever, aren't they? 
A. A Sirex imago
B. This is a part of the wasp's abdomen,
containing the mycetangium (round bag) and the outer 'stick'
is the ovipositor which is pierced into the tree trunk.
C. The mycetangium from a closer look. The inner vesicle contains
and produces the mucus, in the outer part you can see fungi
D. Fungi cells in themselves.
This picture is from the book: Erzsébet Jakucs and László Vajna: