Hello and welcome back, once again, to D⁴!! This past week Dylan and I ran an experiment that dealt with the determination of fastidious organisms.

 

Fastidious organism- is an organism that is only capable 

to growth and sustainability when their diet includes

 very specific nutrients.

Fastidious microorganism- (a more specialize term for the 

field of microbiology) describes an organism that only 

grows in a culture medium that contains that organism’s 

specific nutrient needs.

        To test to see if the controls and the unknown that we have been using throughout the duration of the semester are indeed fastidious, we acquired blood agar plates for inoculation with our microbes. Blood agar plates contain a variety of general nutrients and also contain 5% sheep blood, which gives them their vibrant RED color. When working with blood agar plates, there are some microbes that can produce hemolysins.

Hemolysins- different proteins and lipids that cause the lysis of red blood cells by the destruction of the cell membranes of the red blood cells.

        So a few questions that you may want answers to in order to 1.) better understand the project and 2.) to put it into perspective as to why we are even conducting this experiment are:

1.) How do bacteria lyse red blood cells?

• To start lysis is the compromising of cell's integrity by breaking down it's membrane.
•  In order to breakdown the cell membrane hemolysin is needed, and as previously stated hemolysin is a mix of proteins and lipids that cause the destruction of red blood cell membranes.
• This hemolysin either lyses the erythrocytes by hydrolyzing the bilayer's phospholipids or by forming pores in the phospholipid bilayer.

2.) What is the difference between alpha, beta, and gamma hemolysis?

• Alpha- is a type of hemolysis where the red blood cells are reduced, but not completely eradicated. This reduction causes a greenish brown color to appear in the medium.
• Beta- unlike alpha, is the absolute lysis of all present red blood cells. When looking at the plate after incubation, beta hemolysis can be indicated by clear zones or "windows" in the medium that surround that bacterial colonies.
• Gamma- is hemolysis that produces no hemolysis. No reaction is present after the incubation period has occurred.

3.) Would you expect hemolytic microbes to be more or less virulent than non-hemolytic microbes? why?

• I would say that hemolytic microbes are more virulent than non-hemolytic microbes because iron has been known to be a limiting factor in growth rates of numerous pathogenic bacteria.
• The reason that iron plays a large role in virulence is because RBCs are very rich in heme that contains iron, and when these RBCs lyse the iron rich heme is released into the surroundings; this heme is now free for the bacteria in the area of lysis.
• Luckily, there are numerous cases where hemolysis does not put human health at severe risk, but when the heme released from lysis teams up with other virulence factors, human lives are put at extremely high risk.
• One specific outcome that occurs due to hemolysis that makes me believe that hemolytic microbes are more virulent than non-helytic microbe is hemolytic anemia. Hemolytic anemia is the destruction of erythrocytes and then the very quick removal of those erythrocytes from the bloodstream entirely.
• This is a huge problem because the removal of these erythrocytes at such high speeds is causing a decline in RBCs because the bone marrow can't produce new erythrocytes quickly enough to meet the demands of the decline. This can lead to many issues like fatigue, overall body aches, arrhythmias (irregular heart beats), or even an enlarged heart which will eventually lead to heart failure.

4.) Would you expect a "typical" soil microbe to be capable of hemolysis?  why or why not?

• We do not think that a typical soil microbe would be capable of hemolysis because in the lab we incubated our three samples in an incubator that was set to 37 degrees Celsius.
• 37 degrees Celsius is not a temperature that these microbes normally grow at, so being incubated at this temperature definitely had an effect on the capabilities of these microbes to produce hemolytic effects.

          The procedure for this experiment had a very simple set up, and very few steps to follow in order to determine if the microbes being used were in fact fastidious. First three blood agar plates were obtained; one was labeled S. aureus, one was labeled S. epidermis, and one was labeled unknown. The three plates were T-streaked with one of the three microbes and then incubated at 37 degrees Celsius for 48 hours (maybe longer for very slow growers). After the incubation period the plates were checked on and read to determine the results of the test. In order to correctly read the plates, they must be held up to a light source, so that the light is shining through the plate from behind. This method will ensure the best reading of the blood agar plates. Below are pictures of our plates with and without the light source behind them.

Left:S. epiderimis Middle: S. aureus Right:Uknown.

Left: S. epiderimis Middle: S. aureus Right:Uknown.

       The results of our test show that S. epidermis is an alpha hemolytic species of microbe, the S. aureus is a beta hemolytic species and microbe, and that out unknown is a gamma hemolytic species. Although there is a bit of confusion with our two controls, our unknown is 100% a gamma hemolytic species. As for the S. epidermis (which we believe to be alpha) it can be seen that there is brownish coloration around the colonies as was expected, but there is also one colony on the bottom right that appears to be a beta colony. When exposed to the light source you can very easily see through the medium, but for the most part we are confident that the S. epidermis is alpha. Then for the S. aureus (which we believe to be beta) the medium has definitely become transparent as expected but the perimeter of the plate and a few spots on the middle appear to have the greenish brown characteristic of the alpha, but overall this species is clearly beta. 

      To continue on with Dylan's "What the heck is this microbe?" section, we will delve one step further into classifying WHAT THE HECK this microbe may be. As Dylan stated last week, so far we have conducted...

• Pleomorphic (between cocci and bacillus).
• Gram stain results were inconclusive (a perfect color between our + and - controls).
• Not acid fast.
• No catalase activity.
• Ferments carbohydrates via aerobic and anaerobic metabolism.
• Endospore former.
• No Motility.
• No nitrate reduction abilities.
• Hemolysis test.

      Unfortunately, it seems as though our microbe wants to stay mysterious because putting a name on this guy has been quite the struggle. Although we are starting to lean towards the genus Streptomyces, there are sources like http://terpconnect.umd.edu/~asmith/ALSACE/Discussion.html that yield different results than the ones Dylan and I have seen in lab.

      So, once again we have taken a small step forward in identifying that our microbe is a gamma hemolytic species, but until further experiments are conducted, it seems as though we are still stuck without a verdict. Streptomyces is looking promising, so hopefully our next experiment points us even further in that direction!!

Works Cited
Bhakdi S, Mackman N, Menestrina G, Gray L, Hugo F, Seeger W, Holland IB (June 1988). "The hemolysin of Escherichia coli". Eur. J. Epidemiol. 4 (2): 135–43. doi:10.1007/BF00144740. PMID 3042445.

Chalmeau J, Monina N, Shin J, Vieu C, Noireaux V (January 2011). "α-Hemolysin pore formation into a supported phospholipid bilayer using cell-free expression". Biochim. Biophys. Acta 1808 (1): 271–8. doi:10.1016/j.bbamem.2010.07.027. PMID 20692229

 Sritharan M (July 2006). "Iron and bacterial virulence". Indian J Med Microbiol24 (3): 163–4. PMID 16912433.

 "What Is Hemolytic Anemia? - NHLBI, NIH". United States National Institutes of Health. 2011-04-01. Retrieved 2012-11-24.