Hemolytic Analysis

Hemolytic Analysis

     In our experiment this week, we conducted hemolysis blood agar tests on our soil microbes and controls. This tests specifically determines a bacteria's ability to lyse red blood cells by the production of metabolites, called hemolysins. Hemolysins are lipids and proteins that cause the lysis of red blood cells by either forming pores in the phospholipid bilayers, or hydrolyzing phospholipids in the cell membrane. There are three different forms that constitute hemolysis. The first is Alpha-hemolysis. This occurs when bacteria reduce the hemoglobin from red bloods cells into methemoglobin. In blood agar, this result is exemplified by a dark black color, or "bruising", surrounding bacterial colonies. Beta-hemolysis is considered "true" lysis of the cells. The bacterial colonies and their surroundings are colored a light yellow color when held up to transmitted flight indicating complete degradation of red blood cells. Gamma-hemolysis indicates there is no reaction with the blood cells at all. There is no bruising or degradation.
     When bacteria have hemolytic capabilities, it can potentially heighten their virulency. Being hemolytic means having the capability to lyse red blood cells, one of the primary components of our cardiovascular system. If hemolytic bacteria were to become pathogenic and infect a human, then they would continually lyse blood cells which would initiate different symptoms and could potentially be lethal.
     We would not expect our soil microbe to be hemolytic because this ability is usually seen in pathogenic bacteria as a virulence factor. Because this bacteria may thrive more in soil and not in a living being it would drake sense that it doesn't have the need to produce hemolysins necessary to lyse red blood cells.
     The results from our blood agar tests showed that our soil microbe is alpha-hemolytic. There was bruising around the colonies in the blood agar, but when held up to the light there there was no complete lysis of blood cels in medium. This suggests that our microbe possesses some form of hemolysin metabolite that reduces the hemoglobin from the blood cells. Our control microbes produced the expected results and gave a great comparison for our soil microbe. This test gives some contradiction in determining the identity of our microbe. Corynebacterium and Lactobacillus genuses were the ideal candidates for our soil microbe, however, our blood agar results indicate that we must have some kind of Streptoccocus. 

S. aureus

S. epiderdimis

Unknown Soil Microbe

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. 

http://textbookofbacteriology.net/pathogenesis_4.html

http://www.microbelibrary.org/component/resource/laboratory-test/2885-blood-agar-plates-and-hemolysis-protocols

Subtracting the Nitrogen?

Bloggers,

Back again to entertain everyone while dropping some knowledge on this week's blog post. This week in lab, we tested if our unknown soil microbe reduced nitrogen to any form. We inoculated our positive control, negative control, and our unknown microbe into a test tube. The positive turned a dark red as seen in the picture below. I couldn't get the picture to rotate, so the positive control is the bottom tube. After ~48 hours, I checked our soil microbe, which is very slow growing, to see if it also turned red. Well, it didn't, but after I added reagent A and reagent B to it, it turned red as seen in the top test tube. This shows that our soil microbe reduces nitrogen to nitrite, hey that's pretty neat.

Nitrogen is an essential to life because of the two macromolecules, proteins and nucleic acids (Vivian et al. 1999). Nitrogen is removed from the environment by nitrification, which plays a vital role in the nitrogen cycle, agriculture, and public health implications. Nitrate reduction performed by an array of bacteria also help in biological processes, accounting for man than 10,000 megatons of inorganic nitrogen transformed every year (Vivian et al. 1999). This shows how important, not only nitrogen is, but the reduction process itself which plays a huge role in everyday processes.


Last week, we tested motility in our unknown microbe which showed to be non-motile. But what is involved in motility? There are four cell structures involved in motility; centrioles, flagella, cilia and basal bodies (Baumann 2014). The two major structures include flagella, which are located on the outer surface and uses whip-like movements to move, and cilia uses a rowing motion to sweep across the surface (Baumann 2014).  Over the years, microbes have evolved to be nitrate reducers in order to increase their chance of survival (Cole 1996). Cole states that bacteria can reduce nitrate when the environment is changing when oxygen and nitrogen are scarce, which is essential for survival.  Other microbes haven't evolved to be nitrate reducers such as not having the ability to do so and other reasons, but can still acquire nitrogen through a process call nitrogen assimilation (Xu et al. 2012). According to one source, non-nitrate reducing bacteria get this nitrogen in a mutualistic relationship with a plant ( Brundage 2015).


Hopefully this posts states how important nitrogen is, along with nitrate reduction and putting nitrogen into a useful source. So, lets try to identify our microbe after this nitrate reduction test, which was positive. After all of the tests we have run so far, I believe our unknown soil microbe is Lactobacillus or Corynebacterium. All of our tests have pointed in this direction, and like I stated in my blog post two weeks ago, Lactobacillus seems to be the favorite to win it, but we still have work to do. According to our dichotomous key, a non-motile and nitrate reducing microbe along with all of our other tests are pointing us in this direction. So, we will see where it takes us and hopefully we are correct!


See you next week,
Michael Cowan

http://jb.asm.org/content/181/21/6573.full
http://femsle.oxfordjournals.org/content/136/1/1.abstract
Xu, G.; Fan, X.; Miller, A. J. (2012). "Plant Nitrogen Assimilation and Use Efficiency". Annual Review of Plant Biology 63: 153–182. doi:10.1146/annurev-arplant-042811-105532. PMID 22224450.  edit

Motile, Agile, Hostile (Updated)

Motility Testing 

     This past week we conducted motility tests on our particular soil microbes in order to further describe their identity. Motility allows microbes to move towards desired environments. Some stimuli that induce motility in microbes may be light, chemicals, food, and oxygen. Motility can come in a variety of different ways. Microbes may use actin filaments from whiting the cytoplasm to form actin tails on the external surface of the organism to move (M. Goldberg, 2001). Also, flagella are a common form of motility structures that allow microorganisms to change directions and move in desired directions (Kearns, 2010). Microbes that have evolved to equip motility into their lifestyle use it in many advantageous ways. Not only is motility useful in moving towards a desired environment that presents food and light, but some studies have revealed that motility can increase overall fitness in microbes by allowing a source of escape from undesirable conditions, including phages in bacteria (Taylor and Buckling, 2013). However, some microbes may utilize being non-motile for a variety of different reasons.  Pseudomonas aeruginosa is one such microbe responsible for causing pneumonia. It is much more prevalent in patents with cystic fibrosis. Research has shown that nonmotile phenotypes of this bacteria seem to be harder to get rid of because they are more resistant to phagocytosis by immune cells and conserve more enemy than that of strains of motile pathogens (Mahenthiralingam et al., 1994). So cells that are non motile may conserve more energy to put into certain virulence factors that would make them more pathogenic.
     The motility test of choice that we employed was the soft agar test. Inoculated needles are used to sample bacteria from culture and are then inserted into a tube containing soft agar. The needle punctures the agar until right above the butt of the tube. The tubes are left to incubate for 72 hours and then microbial growth is observed. If a strain grows outwards farther than from the point of inoculation throughout the agar, then it is deemed motile. If growth is observed only at the points of inoculation, then the strain is deemed non-motile. Our motility tests seemed to be inconclusive. Bacteria growth was observed from E. coli, but even this strain did not show motile growth that is has previously been identified with. Both our soil microbe and B. megaterium did not produce the desired microbial growth. We will conduct our motility tests again and update our findings. Once we confirm whether our soil microbe is motile or non-motile, we will then continue to further determine the true identity of our sample strain.

Update (4-12-15)

     The soft agar tests were ran again for the unknown soil microbe. Our results showed that the soil microbe is non-motile. Microbial growth did not disperse in medium from the inoculation point, so this implicates the bacteria does not have motility. From this information we may hypothesize that Corynebacterium is the genus that our microbe is categorized under. For instance, like the genus corynebacterium, our soil microbe is catalase negative, non-acid fast, and non-motile. Another possible genus could be lactobacillus, but this is less likely because lactobacillus are anaerobic microbes.

Goldberg, M. B. (2001). Actin-based motility of intracellular microbial pathogens. Microbiology and molecular biology reviews : MMBR, 65(4), 595-626, table of contents.

Kearns, D. B. (2010). A field guide to bacterial swarming motility. Nature reviews. Microbiology, 8(9), 634-644.

Mahenthiralingam, E., Campbell, M. E., & Speert, D. P. (1994). Nonmotility and phagocytic resistance of Pseudomonas   aeruginosa isolates from chronically colonized patients with cystic fibrosis. Infection and Immunity, 62(2), 596-605.

Taylor, T. B. and Buckling, A. (2013), Bacterial motility confers fitness advantage in the presence of phages. Journal of Evolutionary Biology, 26:   2154–2160. doi: 10.1111/jeb.12214