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

Gram Would Be Proud

     In our project this week, we began the process of determining the identity and makeup of our chosen soil microbe. In order to have the basic skills required to do this we used the Gram-staining technique to determine whether our microbe was Gram (-) or Gram (+) in three control groups of bacteria. The reason this information is useful to is because it gave us a better idea of what broader category of bacteria our microbe is identified in. But, it also plays a significant role in the medical field when determining how to treat an infection or sickness caused by bacteria. 

     Gram (+) bacteria have thicker cell walls made of peptidoglycan than absorb crystal violet stain very well and make the bacteria appear purple in color. Gram (-) bacteria have a thinner cell wall of peptidoglycan that is sandwiched between two layers of cell membrane. This structure allows Gram (-) bacteria to absorb the crystal violet initially, then after washing and counterstaining, the bacteria appear to be a pink color. The reason this is important in the treatment of bacterial infections is because Gram (-) bacteria are much harder to combat and develop a much higher resistance to antibiotics because of the cellular structure. The outer membrane of Gram (-) bacteria can prove useful when infecting hosts because it provides more protection from autoimmune bodies or antibiotics. This makes it more deadly and harder to treat in humans or animals. 

     In order to use this technique to identify our microbe, we first stained three other control microbes that resembled different Gram identities. Each one had already been previously determined so it could give us an idea of what each type of bacteria would look like before we tested our unknown soil microbe. 

                                             Gram-Stain Micrograph of control B. megaterium

     Then, we Gram-stained our unknown microbe and determined its shape and Gram-identity. Our group's microbe appeared to be of a diplococcous shape and stained purple meaning it was Gram (+). We recorded this result, but then did further testing to confirm our results from the Gram Stain. We used McConkey agar which is selective for Gram (-) bacteria because it contains a high concentration of salt which deters Gram (+) bacteria from growing. After allowing this agar to grow at 30 degrees Celsius for five days, we found that there was no growth of our unknown bacteria on the agar plate. This result was similar to the responsiveness of B. megaterium which is also a Gram (+) bacteria. So, the result of our McConkey agar confirmed our initial hypothesis that our unknown microbe is Gram (+).