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

Week 4: Acid-Fast Staining

Welcome back!

This week in lab, we worked on determining if our mysterious soil microbe was acid-fast or not. Acid-fast staining is a technique used to test for waxy cell walls in bacteria - it is similar to gram-staining because it involves using dyes to stain the cells, but the dyes are different and have different affects on the cells. If a bacteria in acid-fast staining stains a pink-ish color, the bacteria is said to be acid-fast, meaning it has a very waxy cell wall, and these are considered to be the micobacteria. If a bacteria in acid-fast staining stains a purple or blue color, the bacteria is said to be non-acid-fast, meaning it does not have a waxy cell wall.

We tested our soil microbe against some controls, which were all non-acid-fast. For our non-acid-fast control, Katie and I used B. megaterium, which is also a Gram-positive control that we used in last week's Gram-staining procedure. We expected our control to be a blue/purple color, and we compared the result of our control to our soil microbe and determined which type of acid-fast bacteria we assumed we had. An image of our soil microbe under the oil-immersion lens of the microscope is shown below:

As you can tell from this image, our microbe is clearly very non-acid-fast. The purple/dark blue color indicates to us that it is very non-acid-fast and thus has a thin waxy cell wall.

Using a dichotomous key, we can start to classify our microbe. I used a key provided by the professor, and since our microbe was Gram-negative, the next step on the dichotomous key is to determine if it is an anaerobic or aerobic, or facultatively anaerobic microbe. Since we have not done an experiment to determine this yet, we will have to wait to continue to classify our organism. Stay tuned until next week to see how we classify our microbe further!

Patricia