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Endospore Lab Report - 2

The document summarizes an experiment where endospore stains were used to determine if Escherichia coli and Bacillus megaterium formed spores after exposure to stressful conditions. B. megaterium cultures showed faintly stained spores under 40x magnification and clearly stained spores under 100x magnification, confirming it can form endospores. In contrast, E. coli cultures did not show any endospores and only stained with the counterstain, consistent with knowledge that E. coli does not sporulate. The results demonstrate the importance of identifying bacterial species capable of sporulation for sterilization purposes.

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321 views

Endospore Lab Report - 2

The document summarizes an experiment where endospore stains were used to determine if Escherichia coli and Bacillus megaterium formed spores after exposure to stressful conditions. B. megaterium cultures showed faintly stained spores under 40x magnification and clearly stained spores under 100x magnification, confirming it can form endospores. In contrast, E. coli cultures did not show any endospores and only stained with the counterstain, consistent with knowledge that E. coli does not sporulate. The results demonstrate the importance of identifying bacterial species capable of sporulation for sterilization purposes.

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Haylee Stiffler

Creating an Endospore Stain of Escherichia coli and Bacillus megaterium

Introduction:

The purpose of this experiment was to perform an endospore stain to determine if cultures of

Escherichia coli and Bacillus megaterium formed spores after being in stressful conditions. An

endospore stain is a type of structural stain that is designed to highlight a particular feature of a

bacterial cell. In this case, endospores. Endospores are formed when certain species of bacteria

are under stressful conditions. Spores contain the DNA of a bacteria along with some enzymes,

all of which are stored in a protective coating which allows the bacteria to survive, potentially for

years, until they are in a proliferative environment (Aliabadi et al. 2018). Endospores are only

formed by certain types of bacteria, namely those of the Bacillus genre (Alibadi et al. 2018).

Knowing which bacterial species are capable of sporulating is particularly important when trying

to sterilize a surface, as spores are able to withstand some physical agents of disinfection, like

heat, as well as low or intermediate level chemical disinfectants (Alibadi et al. 2018). Due to the

current knowledge surrounding these bacterial species, it’s hypothesized that the B. megaterium

culture formed spores while the E. coli culture did not.

Materials and Methods:

Prior to starting this experiment pure cultures of E. coli and B. megaterium were placed in

stressful environments, specifically lowered temperatures, to see if they would sporulate. After

this was done two microscope slides were labeled, one for each of the cultures. A bacterial smear

was performed for both bacteria. After the smears had fully air-dried they were heat fixed by
passing each slide through a lit Bunsen burner three times. The slides were then placed onto the

lid of an agar plate. A paper towel was cut to fully cover each bacterial smear without hanging

over the edge of the slides. After the paper towels were placed over the smears they were

drowned in malachite green dye. The agar plate containing the covered slides was then incubated

at 55°C for 20 minutes. While being incubated the slides were checked regularly to ensure that

the paper towels were not drying out. After the incubation was complete the paper towels were

removed from the slides and the slides were washed with water for 30 seconds to decolorize the

samples. The slides were then blotted dry using bibulous paper. After they had been dried the

slides were flooded with the counterstain, safranin, which sat on the stains for 60 seconds. After

the 60 seconds had passed the slides were washed with water and blotted dry using bibulous

paper. At this point the slides were viewed using the microscope.

Results:

Figure 1: E. coli endospore stain viewed using a 10x microscope lens


Figure 2: E. coli endospore stain viewed using a 40x microscope lens

Figure 3: B. megaterium endospore stain viewed using a 10x microscope lens

Figure 4: B. megaterium endospore stain viewed using a 40x microscope lens


Figure 5: B. megaterium endospore stain viewed using a 100x microscope lens

Figure 6: B. megaterium endospore stain viewed using a 100x microscope lens

Discussion:

The results show that the E. coli sample did not form spores while the B. megaterium did, which

is what had been hypothesized. Spores are shown faintly stained by the malachite green dye in

the 40X magnification, seen in figure 4, and very clearly in the 100X magnifications with

immersion oil, seen in figures 5 and 6. The E. coli sample; however, only stained with the pink

safranin counterstain, meaning no spores were present. This is to be expected, as E. coli is known

not to sporulate while it’s known that B. megaterium does. The presence B. megaterium spores
became obvious when using higher magnifications and were especially clear when using the

100x lens with immersion oil. The spores were also shown throughout the culture, which is

evident considering how figures 5 and 6 show two different areas of the same slide, both using

100x magnification. Knowing this information is important when trying to sterilize surfaces.

Since spores are resistant to certain physical and chemical methods the possible presence of

spores should be taken into account when choosing how to sterilize a surface. This skill of

endospore staining can be used to determine if a bacterial species will form spores when this

information is unknown.

References:

Aliabadi Z, Foster J, Slonczewski, J. 2018. Microbiology: The Human Experience. New York

(NY): W. W. Norton & Company.

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