Week 6: Endospore Test Results

Welcome back to Week 6 of identifying our Soil Microbe!

Last week, Katie thought she may have been able to determine our soil microbe - this week, I will use the tests we did in last week's lab to determine if Katie was right, or if we need to keep testing to find our soil microbe.

This week in lab, we used two different methods to determine if our soil microbe contained endospores or not. Endospores are structures found inside the cell of some bacteria, usually Gram-positive Bacillus and Clostridium (Bauman 2014), that make cells more durable and resistant to death in unfavorable conditions as well as the potential to increase pathogenicity. Microbes have evolved to form endospores as a way to become more resistant to different methods of killing these pathogens and harmful microbiota and to increase their virulence to spread more rapidly.

For our lab, we used the Schaeffer-Fulton method of endospore staining to try and determine if and where in the cell our unknown soil microbe's endospore was located. To do this, we prepared 3 smears: our unknown microbe, a positive, endospore-forming control (Bacillus megaterium), and a negative, non-endospore-forming control (Escherichia coli). We heat fixed our smears, stained them with malachite green and then safranin, and observed them under the oil immersion objective lens of our microscope. When a microbe contains an endospore, it stains green while the rest of the cell stains the safranin, or pink, color. If there is not an endospore, the green will not be present, and the cells will all appear pink. However, the results of this staining method are frequently either inconclusive, or not strong enough to make a determination based on this one technique. Our results were completely inconclusive, and we were not even able to attain sufficient images of our microbe's cells to include as an example.

Because of the inconclusiveness of this staining procedure, we also used a heat shocking technique to determine if endospores form. Due to the high resistance of endospores, many microbes that contain them can be more resistant to higher temperatures. This means that, even after being put under high temperatures that would kill most normal microbes, endospore-forming microbes will still grow under these conditions. To determine if our microbe did, in fact, contain an endospore, we inoculated tubes containing TSB, a liquid growth medium for microbes, with each of our unknown soil microbe, our same positive control, and our negative control. We inoculated 2 tubes with each microbe, and heat shocked one tube of each microbe in an 80°C water bath for 10 minutes. After 4 days I came back to the lab to see if our microbe had grown under heat shock or not. Our microbe, as shown in the pictures below, grew in both the heat shocked and the regular tube, indicating that it is endospore-forming. The negative control, as you can see, is very clear and not cloudy at all in the heat shocked tube, but has bacteria growing in the tube incubated at room temperature. The B. megaterium, which is what we compared our unknown soil microbe against, also has growth in both tubes, therefore confirming that the similarity of our soil microbe to the positive control is due to our microbe being endospore-forming.

Our unknown soil microbe. Heat shocked culture is on the left.

Our negative control, E. coli. Heat shocked culture is on the left.

Our positive control, B. megaterium. Heat shocked culture is on the right.

Using the dichotomous key from before, we can conclude that as Katie said, we more than likely have Pseudomonas aeuroginosa.

See you next week!

Patricia