Are you sensitive or resistant?

Bloggers,

Back at it this week with discussing whether or not our soil microbe is resistant or sensitive to certain antibiotics. First, the antibiotics that we tested were; 1-erythronycin, 2- cabernicillin, 3- ampicillin, and 4- tetracyclin. The numbers are associated to where each antibiotic was placed in the pictures below.

The first picture is of S.Aureus, which as seen in the photo is sensitive to antibiotic 1 and 4, which are erythronycin and tetracyclin . S. Aureus is resistant to drugs  2 and 3, which are cabernicillin and ampicillin. This is shown by how there is no growth around numbers 1 and 4, showing that the drug inhibited the growth of this bacteria.



This next picture is of S.epiderdimas, and is a clear example of how all four of the antibiotics inhibited the growth of this bacteria, showing that the organism is very sensitive to the drugs used. Once again, there is no growth around the disks (drugs) which clarifies that it is sensitive.



This next picture is of E.coli, and is another clear example of how this organism is sensitive to the drugs used. Once again, there is no growth around the disks (drugs) which clarifies that it is sensitive. The results from E. coli look almost identical to S. Epiderdimas.



Lastly, this picture is of our unknown soil microbe which shows that our microbe is resistant to all four of this antibiotics. Now, it makes me a little cautious to definitely come to this conclusion due to the fact that our soil microbe is a very slow growing bacteria. Our protocol stated to check our results 24-48 hours after we inoculated it, and this is the picture from after 48 hours. Although I followed the protocol designed, I think with 3-4 days could have given us a better indicator of whether our microbe was resistant or sensitive. But, the results shows our microbe is resistant to all four antibiotics, and even with 48 hrs. to grow, I think we would have seen, at least, a little inhibition if it was sensitive.




Our unknown microbe was resistant to all four of the drugs, S. Aureus was resistant to two and sensitive to two, while both S. epiderdimas and E. coli was sensitive to all of the drugs. Since the other organisms tended to be sensitive to all four of the drugs, that is one reason why I am a little skeptical about our microbe being resistant to all four of the drugs. Once again, it could be due to the fact that our microbe was very slow growing.

So, last week we threw Streptococcus back into the equation with determining what our soil microbe could be due to the blood agar test. We are still not 100% sure which bacteria our unknown is, but according to ours results, I would narrow it down to Lactobacillus, Corynebacterium, or Streptococcus. I think the next test done should be determining whether or not each bacteria is anaerobic, because this would eliminate either Lactobacillus or Corynebacterium from the decision with that data. Then, we could narrow the options down to two and run another test which would determine which soil microbe is ours.


Last time blogging :((((((

M&M Microbiology

 

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

Endospores!!

Fellow Bloggers,

This week in lab, we experimented to see if our microbe formed endospores. We used an endospore stain with a growth assay to detect the presence of endospores. As stated in the lab handout, endospores are highly durable and are resistant to killing by multiple factors such as heat and drying out. Our methods involved inoculating two tubes with three different bacteria; Bacillus (positive control), E. coli (negative control) and our control bacteria. After using good aseptic technique and transferring the bacteria into tubes with tryptic soy broth, we transferred one tube from each group to a water bath of 80 degrees C to heat shock the bacteria.


        After the heat shock, we determined that our unknown microbe does not appear to form endospores. When I went back to look at our microbe, our sample was still clear which means that no endospores were formed. The clear sample after the heat shock tells us that our unknown microbe was resistant to 10 minutes of heat shock at 80 degrees C. As Heather points out, there was zero cloudiness in our control microbe.

Bacillus (positive control)

       Next, we stained our microbes to provide a way to recognize whether or not there were endospores in the bacteria. This also allows us to determine the size, morphology and location of the endospores. We used a method called Schaeffer-Fulton to look for endospores, which stains the endospores a different color that the microbe we were looking at. Once again, we prepared a slide with three different microbes ( positive, negative, and unknown) and flooded the slides with malachite green and then counterstained it with aqueous safranin.
        Once ready, the slides were put under the microscope to look for the staining of the endospores. For B. mega (positive control), it was very clear that the endospores had been stained which is really neat to look at. The E. coli (negative control) didn't have any endospores so the stains looked different than B. mega. Our unknown microbe came back as endospore positive, which conflicts with our results from the heat shock. The first picture below shows the endospore staining, which resembles what B. mega (2nd picture below) looked like under the microscope. Once again, these results conflicted with the previous heat shock test we also performed.

Soil Microbe

B. mega



The picture below shows E. coli (negative control) where there are no endospores. As you can tell, the dye put onto the microscopic slides aren't as bunched up as the two pictures above. This shows that our soil microbe, in my opinion, is positive for producing endospores.

          Endospores are very resistant asexual spores with extremely thick walls that develop inside of some bacteria cells. These endospores are produced by a small number of bacteria from the Firmicute family and are commonly found in soil and water where the lifespan is extremely high. The primary function of endospores is to ensure that the bacteria will survive even through very rough environmental conditions. Endospores can survive extremely high heat, desiccation, radiations, and harsh chemicals.

One reason that may explain this is the fact that endospores contain four layers; core, cortex, coat, and exosporium. Scientists have been able to discover endospores from bees trapped in amber that are 25-40 million years old...WOW! That is absolutely crazy to think that one thing can survive that many years. The benefits of endospores include that these bacteria can survive extremely harsh conditions, and enable a bacteria to spread very easily. Some microbes have evolved to form endospores by surviving through these harsh conditions that the bacteria that keep surviving, keep producing endospores because these help the bacteria survive longer, which would be a huge benefit to these bacteria. This would allow the producing endospore bacterium to survive.  Some microbes have evolved to not form endospores because the bacteria just cannot produce them or cannot survive long enough for the endospores to grow inside of the bacterium.


      According to the tests that we have run so far, I think our unknown microbe is Lactobacillus which is contradicting to what we thought last week. Lactobacillus does not produce endospores along with being catalase negative which agreed with our test that we ran a few weeks ago. All of the dichotomous key steps have led us to this microbe, but we are working hard to figure out what exactly it is!


Stay Tuned!!


Over and out,
M&M Microbiology




http://www.sciencedaily.com/articles/e/endospore.htm


http://www.microbiologytext.com/index.php?module=book&func=displayarticle&art_id=69


http://www.faqs.org/espionage/An-Ba/Bacterial-Biology.html

Catalase and Sugar Iron Test

    This week our endeavor in determining the identity of our soil microbe continued as we employed two new microbial techniques, the catalase and sugar iron test. Catalase is an enzyme that breaks hydrogen peroxide into water and oxygen. Many kinds of bacteria have evolved to produce catalase because hydrogen peroxide can be deadly within a cell. The catalase test determines whether or not a strain of bacteria produces catalase by exposing a colony of bacteria on a microscope slide to hydrogen peroxide and observing whether or not oxidization, or bubbling, occurs. Microbes that are catalase-positive are deemed Micrococcaceae and catalase-negative are deemed Streptococcaceae. Our particular soil microbe did not express any catalase activity. After employing the catalase test on our microbe many times, we continued to not observe any bubbling or activity. This suggests that our microbe is under the catalase-negative Streptococcus group. 

   

    The triple sugar iron test determines carbohydrate fermentation and hydrogen sulfide production. The agar gel changes color in presence of different fermenting abilities and gas production allowing the researcher to differentiate a microbes decease of their abilities. One of our controls acted as planned. The E. Coli tube reaction was over acid, indicating fermentation, and produced a yellowish color in the agar. However, our P. vulgarism  control tube did not produce the desired reaction. In the butt of the tube, the bacteria did not produce stable-acid end products which produces a black precipitate at the bottom of the tube. Our unknown soil microbe appears to be a glucose, sucrose, or lactose fermenter because it tinted the entire tube a yellowish color similar to our control subject, E. Coli. There was no precipitate at the bottom of the tube indicating there was no hydrogen sulfide production. This means our soil microbe has carbohydrate fermenting abilities. Below are pictures of our control groups and soil microbe.

     According to our tests this week, our soil microbe should be Streptococcaceae and have the ability to ferment different carbohydrates. It was catalase-negative and did not produce hydrogen sulfide or gas. I believe this indicates that this bacteria is under the genus Enterococcus faecalis or Streptoccocus spp. This is because the microbe was a facultative anaerobe because it utilized the glycolosis pathway anaerobically and aerobically meaning it is specific to air conditions. These two groups are also very likely because our bacteria is gram positive and cocci-shaped. However, this does contradict previous hypotheses that our bacteria could be under the genus  Listeria monocytogenes because of its non-acid fast characteristic. 

             

Are you (ab)staining from (fast) acid?

Fellow bloggers;

This week in lab we used certain techniques to determine if our soil microbe along with another sample bacteria was an acid-fast or non-acid fast organism. An acid- fast organism contains a waxy-like cell wall, that with the techniques used, will turn a certain color to determine if the organism is acid-fast or non-acid fast. The primary stain used works by penetrating and sticks to the cell wall turning the organism blue (non-acid fast) or purple (acid-fast). An acid-fast microorganism has a cell wall with a high lipid content that allows the wall to bind to carbol-fuchsin (discussed later). Acid-fast microbes belong to the genus of Nocardia spp. or Mycobacterium spp.

Our methods included transferring our microbe to a microscope slide and flooding the microbe with carbol-fuchsin and methylene blue. These are two major dyes that are used in acid-fast staining techniques. Once we created the slides, we used oil immersion to increase the resolution on the micro scope. The first slide we looked at was our control microbe that we had saved from our soil sample.

This is our control microbe from our soil sample, which turned out to be non-acid fast due to the blue color of the microbes. Through the dichotomous key provided, I believe this sample is from the genus of listeria monocytogenes bacteria due to the overall shape, as well as the bacteria being non-acid fast. Also, this bacteria was characterized this way because of the very small, circular shape which looks similar to the genus of listeria monocytogenes.
                                         

This was our second microbe that we tested in the experiment, which also turned out to be non-acid fast. This was determined due to the blue coloring of the organism. This bacteria was from a sample of B. subtius , which turned out to have strains of bacteria unlike our first microbe tested. I believe this microbe is from the Actinomyces spp. due to the long strain-like structure seen in this picture.

 

There are a few conflicts in this blog due to the lack of tests that haven't been run yet. Although these are all educated guesses on which are based off of a dichotomous key, this week in lab will help us further investigate which genus our microbe belongs to with determining whether it is catalase positive or catalase negative. This will help us in the future to really narrow down which microbe we have been working with in lab.

 

See you next week,

M&M Microbiology