Endospore Testing & Verification

To further examine our bacterium, we used Endospore Testing to look at and compare our unknown bacterium to the positive and negative controls, Bacillus and E. coli, respectively. The endospore stain helps us to recognize the presence of bacterial endospores, which is only found in a very few species of bacteria. It also helps with determining the size of the bacteria, as well as morphology and the location of the endospore within the parent cell (Lab Handout). Endospores are resistant to heat, desiccation, chemicals toxic to vegetative cells, as well as UV light (Lab Handout). Endospores may serve beneficial to bacteria as they could escape certain environmental conditions in order to survive (Nicholson, 2002). They can survive severe harsh environments and are easily spread. Specifically, environmental structures they have adapted to become resistant to are stated above: heat, desiccation, chemicals, pH changes, and UV light. Other bacterium may have not formed endospores in order to have a moderation of bacteria when high numbers are present. They are resting structures and are only used when nutrient supply is depleted. Therefore, the Schaeffer-Fulton method was used to help us determine whether our unknown bacterium has or lacks endospores. The endospores we are looking for appear to be green surrounded by a pink cytoplasm.

Bacillus - Positive Control

After we stained our bacterium with Schaeffer-Fulton, we observed our slides with oil immersion and compared the three slides. Pictures of our slides are below. Our positive control, Bacillus, presented endospores. The endospores are the green colored bacteria on the picture, surrounded by the pink cytoplasm.

Our negative control, E. coli, had no endospores as we found only the pink cytoplasm and no green endospore. The image that we found is consistent with what we expected from our negative control. This control was compared to our unknown to help us determine more about our bacterium.

E. Coli - Negative Control

Our unknown bacterium appears to have a green endospore with light pink cytoplasm surrounding. However, the cytoplasm surrounding the endospore is not as prominent as what was found in the positive control. The pink bacteria surrounding the endospore are rod-shaped and only have a few bacteria present. The green center appears to trigger an idea that our unknown bacterium is endospore-positive.

Unknown Bacterium

Unknown Bacterium

Unknown Bacterium

In order to verify our results from our Endospore Stain using the Schaeffer-Fulton method, we tested each control and our unknown by observing what happened when the bacterium was “heat shocked”. As previously stated, endospores are resistant to high temperatures, desiccation, as well as radiation. We used the heat shock method to verify whether or not our bacterium did or did not have endospores. Samples were placed in an 80 °C water bath for 10 minutes and then incubated for 24 hours at room temperature. Our results are shown in pictures below. Our positive control, Bacillus, had bacteria in the medium without heat shock. After the heat shock, bacteria were still present in the tube. Serving as a positive control, we expected their to be bacteria still present after heat shock, solidifying the idea that endospores were present and could withstand the heat in the heat bath. Our negative control, E. coli, did not have bacteria present after the overnight incubation at room temperature. The medium was not clear, indicating amounts of bacteria in the tube. The heat shock for the negative control killed the bacteria, observed by a clear medium. Our unknown bacterium presented bacteria without the heat shock. The heat shock cleared up the bacteria, with no bacterial particles or murky medium shown. 

	No Heat Shock	Heat Shock	Endospore?
Bacillus – Positive Control	+	+	Endospore Positive
E. Coli – Negative Control	+	-	Endospore Negative
Unknown Bacterium	+	-	Endospore Negative

+ = Bacteria present

-  = Bacteria not present

Bacillus - Positive Control: Heat Shock

Bacillus - Positive Control: No Heat Shock

E. Coli - Negative Control: Heat Shock

E. Coli - Negative Control: No Heat Shock

Unknown Bacterium: Heat Shock

Unknown Bacterium: No Heat Shock

These results conflicted with our original findings after our initial endospore-staining test. After the original endospore staining test, we thought that our bacteria was endospore positive, like our positive control, Bacillus. However, our verification test using heat shock lead us in a different direction. The media, TSB, that the bacteria were living in, cleared up after the heat shock, showing that the bacteria were not resistant to heat. Some bacterium may not be producing endospores due to the number of already viable bacterium presented with endospores in a certain environment (Schaeffer et al., 1965).

According to the Dichotomous Key, endospore bacterium that was not produced leads us to believe that our unknown bacterium could possibly be Lactobacillus spp. We now know that since our bacterium did not produce endospores, it eliminates Clostridium spp. These observations and further testing have helped us eliminate several bacteria, but also have continued to add other bacterium to our possibility list. Next week, we will continue our search into what our bacterium exactly could be.  

Nicholson, W. L. (2002) Roles of Bacillus endospores in the environment. Cellular and Molecular Life Sciences. 59(3), 410-416.

Schaeffer, P., J. Millet, J. Aubert. (1965) Catabolic Repression of Bacterial Sporulation. Proc Natl Acad Sci USA. 54(3), 704-711.