Welcome back to Dylan and Devin Discovering Dirt (D^4). This week we preformed a stain that Devin and I have been looking forward to for a few weeks now, the Endospore Stain.
Before we get into our experiment there are some questions that need to be answered.
- What are endospores?
- What are the benefits of endospores?
- Why might some microbes have evolved to form endospores?
- Why might some microbes have evolved to NOT form endospores?
- Endospore- a dormant, tough, non-reproductive structure produced by a small number of bacteria from the Firmicute family.
http://www.sciencedaily.com/articles/e/endospore.h
 |
| Above the process of endospore formation can be see. Figure credit: http://academic.pgcc.edu/~kroberts/Lecture/Chapter%203/03-22_Sporulation_L.jpg |
2. Endospores can be advantageous for bacterial cells. As seen above, endospore formation results in a DNA surrounded by a cell membrane and a tough spore coat. This spore coat results in resistance to heat (>100C), radiation, acids, bases, alcohol, and desiccation. As you could imagine, the previously mentioned environmental factors are account for a great number of cell deaths, so having a mechanism for combating these environmental changes can be very advantageous. Microbiologytext.com even reports that endospores from 25-40 million year old amber have been germinated in medium.
You can read more about that, and more on endospores here:
http://www.microbiologytext.com/index.php?module=book&func=displayarticle&art_id=69
3. The trait of endospore formation is undoubtedly a shining example of evolution. Due to the beneficial nature of endospore formation, it is feasible that an obligate bacteria that evolved this trait was a lone survivor of a change in environment at which point the trait became successful. Endospore formation would allow for the survival of bacteria that would otherwise be selective in the environments in which they could persist.
4. In contrast, some bacteria are less sensitive to changes in environmental changes, in which case the formation of a vegetative endospore could be a disadvantage. Furthermore, there are instances in which an environmental change will persist for a short period of time, then subside. In this case, it may be more advantageous to remain in a non-endospore state and utilize resources that are now available due to the vegetation of endospore-forming bacteria. In another way of thinking, the "Endospore trait" may not be seen in all bacteria because their are other methods of resisting environmental stress.
Now that we know a bit more about Endospores lets dive into this weeks experiment.
Devin and I preformed an Endospore Stain as well as an Endospore Growth Assay! An endospore stain results in endospores showing up as a green, and the cytoplasm of the cell as pink.
Condensed Procedure:
Endospore Staining
- Prepare a smear of unknown, Endospore positive, Endospore negative bacteria on respective slides.
- Flood slides with malachite green, while suspending the slides over a steaming hot beaker of water.
- Rinse with water.
- Counterstain with aqueous safranin
- Examine under microscope.
 |
| Example of successful Endospore stain of Endospore forming bacteria. Photo Credit: http://academic.missouriwestern.edu/jcbaker/bio251sec01/DSC02875.JPG |
| Results of endospore staining our unknown soil microbe. Taken in lab March 24, 2015. |
As can be seen above our bacteria has a rather peculiar appearance. This week we obtained a stain with the least amount of contamination of any stain we have attempted. Interestingly, we since preforming the gram stain, we were predicting our bacteria was an endospore former, due to it's pleomorphic shapes, and unusual white circles in every cell. To our surprise the results from this endospore stain suggest our bacteria is actually not and endospore forming bacteria. This can be seen by the lack of green color in the above stain. For further assurance of the status of our bacteria we also preformed an Endospore Growth Assay.
Endospore Growth Assay
- Six 2ml tubes of tryptic soy broth were inoculated with one 3 different bacteria.
- Bacillus (Positive control)
- E.coli (Negative control)
- Our unknown microbe)
- Each bacteria were separated into two treatments:
- Heat Shock
- Non-Heat Shock
- Heat-Shock bacteria were incubated in a 80C heat bath for 10 minutes.
- After heat shocking all treatments were left to sit for 4 days to measure growth.
Results.
 | ||||
Above the results of the Endospore Growth Assay can be seen. From left to right the treatments are: Positive control (Bacillus ....), Heat Shock Bacillus, Negative control E.coli, Heat shocked E.coli,, unknown control, unknown Heat shock.
As seen in the gloriously blurry picture above (adding pictures to BlogSpot requires a master of witchcraft. Seriously.) Our positive control, a species of Bacillus showed precipitate in both the control and Heat shock trials (as expected for an endospore former). In excellent micro-lab fashion, our negative control E.coli also showed precipitate across both the control and heat shock trials. This was unexpected as E.coli is a known non endospore former. There must have been a failure in our technique via either contamination with an endospore former, or we were unsuccessful in killing the E.coli in the heat shock sample. Even more intriguing is the results of our unknown bacteria. Our bacteria showed precipitate in both the control and the heat shock treatments. This leads to further confusion as to whether our methodology was compromised, and to whether our bacteria is actually an Endospore Former.
The Dirty Truth About Our Microbe!
As Devin discussed last week, we now know quite a bit about our soil microbe
"Thus far we have conducted five experiments to further our knowledge of our unknown soil microbe. We have done a Gram stain to determine if our microbe is gram positive or gram negative, we have tested to see if our microbe was acid or non-acid fast, we have tested for catalase activity, we have tested to see if our microbe is aerobic or aerobic, and now we have determined if our microbe is an endospore former.
We have determined so far, that our bacteria is neither gram positive or gram negative, but rather a bacteria called endospore-forming bacteria, which is a mix of bacilli and cocci shaped bacteria. We have also determined that our bacteria is non-acid fast due to the color it appeared after staining. There was also no sign of bubble formation during the catalase test which means that our bacteria has no catalase activity. Finally, we tested to see if the bacteria was an aerobic or anaerobic bacteria, and the results stated that there was activity of both types of metabolism. "
After this week we are once again left to interpret our results lightly. We found that our stain showed our microbe in the best clarity of any stain thus far, but it did not suggest we are working with an endospore former. Conversely our Endospore Growth Assay provided evidence that our microbe does a dandy job of surviving high temperatures through endospore formation. Since our results thus far have been a bit inconclusive in a few areas; I set out on a quest to the vast expanses of the internet to gain some more knowledge on our pleomorphic bacteria.
My quest for dirty knowledge was fruitful (Thanks Dr. Hanson) as there may be a potential order of bacteria that fits with our unknown microbe: Actinomycetales. More specifically the genus Streptomycetes. Members of the Actinomycetes Order:
Our mystery microbe aligns with these characteristics in these ways nearly identically. (Depending on your interpretation of some of our results.)
In comparing the picture of Streptomyces to our unknown microbe taken on day one, there is a strong argument to be made that there is a similarity. Both display similar pleomorphic shapes, with a mysterious round white center. Further investigations need to be done to confirm this information, but I believe we are on the right track. Streptomyces are reported as having no flagella, so next week we will preform a motility stain which will help in supporting or ending our case for the unknown soil microbe being Streptomyces.
Join us next time as Devin Breaks it down and discusses Motility and our Flagella Stain! Is our mystery microbe flagellin?
|
No comments:
Post a Comment