Austin McLean

10/12/19
SBL 223 02

Title: Using endospore stains to observe different strains of bacteria.

Introduction: The purpose of this experiment was to successfully create endospore stains of
Bacillus megaterium (B. meg) and Escherichia coli (E. coli) bacteria strains. These stains allow
endospores to be observed within the cells of bacteria. By using an endospore stain, cell bodies
and endospores can be differentiated between each other within the bacterium. B. meg is a gram-
positive bacterium that forms endospores, while E. coli is a gram-negative bacterium that does
not form endospores. It is important to consider this when making predictions about the results of
an experiment. Gram positive bacteria cells are much more likely to contain endospores than
gram negative bacteria cells. The production of endospores form in response to a lack of
nutrients that commonly occurs in gram-positive bacteria. As a result of this, gram-positive
bacteria are much more tolerant of harsh environments. (Cornell University, 2019) Endospores
are very difficult to kill, so learning about them is important. One way that endospores can be
stained is with malachite green. Malachite green binds only with the endospores and does not
bind with the cell bodies. Heat must be applied so that the malachite green can penetrate the
thick walls of the endospore, but once that process is finished, the stain will remain within the
endospore. (Keating, 2016) The study of these forms of bacteria is relevant because they present
issues in society, but they also have some applications. Bacillus is commonly used as a source
for antibacterial drugs and is sensitive to a series of antibiotics. (Canada, 2018) On the other
hand, there have been instances where certain strains of E. coli have developed strong antibiotic
resistance. (Reinthaler, 2003) E. coli is generally harmless and usually resides in our intestine,
but some strains of E. coli can be harmful and threatening to our health. I Hypothesize that the
stain of B. meg will contain a moderate number of endospores while the stain of E. coli contains
little to no signs of endospores. Improper conduction of the experiment is the only way for
endospores to appear in E. coli.

Materials and Methods:

The bench was sterilized with disinfectant, and a sterile field was created with the Bunsen
burner. Two slides were then prepared for the stain by adding one drop of water to each slide
with an inoculation loop. The loop was then sterilized through flaming with the Bunsen burner.
Using the inoculation loop, a sample of B. meg was transferred to the water of one slide, and E.
coli was transferred to the water of the other slide, while also flaming the loop in between each
application. After the slides had been given time to dry, they were then heat-fixed by running
each slide over the flame 3 times. Each slide was then placed in its own petri dish and each heat
fixed sample was covered by a by a small section of paper towel, each piece just barely covering
the sample on the slide. This led into the staining process in which each slide was flooded with
malachite green so that both slides were completely cover. After staining, the slides were placed
into an incubator at for 20 minutes at 55℃. After the incubation process had been completed, the
sections of paper towels were then removed from the slides. The slides were subsequently
washed for roughly 30 seconds so that any of the green malachite that has not stained a spore can
be removed. Once the green malachite had been removed, slides were placed in a staining tray
and were counterstained with safranin for 1 minute. After 1 minute had passed, slides were
rinsed with water and dried. Now that the staining process was complete, slides were observed
under light microscope.

Results:
Figure 1: Escherichia coli at 400x magnificatio

Figure 2: Bacillus megaterium at 1000x magnification with immersion oil

Discussion: Based on our observations, I accept my hypothesis regarding the experiment. In

figure 2, we can clearly see that there are multiple endospores the bacterium, and they are
indicated with the malachite green. At the same time, we can look at figure one and clearly
notice an absence of any malachite green, indicating that there are no endospores in the
bacterium. The use of immersion oil to observe B. meg provided the detail necessary to really
differentiate between the endospores stained with malachite green, and the rest of the cell parts
that were stained with safranin. I do notice a small spot on the E. coli slide that looks slightly like
malachite green among the safranin stained cells, but this is likely a product of not rinsing the
malachite green off completely.

References:

Reinthaler, F. Posch, J. Feierl, G. Wüst, G. Haas, D. Ruckenbauer, G. Marth, E. (2003).

Antibiotic resistance of E. coli in sewage and sludge. Water Research. Retrieved from
https://www.sciencedirect.com/science/article/abs/pii/S0043135402005699

Cornell University. Bacterial Endospores. (n.d.). Retrieved from

https://micro.cornell.edu/research/epulopiscium/bacterial-endospores/.
Keating, S. (2016). Microbiology: the laboratory experience. New York: W.W. Norton &
Company.

Canada, H. (2018, June 20). Government of Canada. Retrieved from

https://www.canada.ca/en/health-canada/services/chemical-substances/fact-sheets/chemicals-
glance/bacillus-megaterium.html.