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
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