Identifying Bacteria in Lake Michigan Angela Fuller, Faith Wittmus, Chelsea Burns, Chris Nelson, and Deborah Tobiason

Department of Biology, Carthage College, 2001 Alford Park Drive, Kenosha, WI 53140

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Introduction Lake Michigan is one of the Great Lakes whose microbes drive transformation and

cycling of biological elements. Its bacteria also have a large impact on elemental fluxes

and water quality by way of their unique metabolisms (2).

The five most common bacterial phyla in Lake Michigan are Proteobacteria, the

Cytophaga-Flavobacterium-Bacteroides group, Actinobacteria, and Verrucomicrobia (3).

The three most common pathogenic bacteria in freshwater lakes are Vibrio cholera,

Escherichia coli, and Pseudomaonas aeruginosa (2). All of these bacterium should be

carefully considered when looking at samples of unknown bacteria from freshwater lakes.

Common methods used when analyzing bacteria from freshwater lakes include fluorescent

labeling, PCR, and metagenomic profiling (2).

The objectives of this lab are to identify and/or discover bacterial species in Lake

Michigan and gain a better understanding of common microbiology protocols by

performing identification tests.

Sampling Methods

• Two 50 mL water samples (Table 1) were collected on 10/18/12 at 12:30 pm from

Lake Michigan at latitude: 42.623◦ and longitude: -87.819◦

• Samples stored on ice until plated on TSA (Tryptic Soy Agar) an hour late

Plating Methods

• Aseptic technique (1) was used on 10/18/12

• Glass spreader was dipped in ethanol and then flamed for sterilization

• 400 µL of each sample was placed onto an accordingly labeled TSA plate

• Two plates per sample (total of four)

• One plate of samples 1 and 2 was stored at room temperature (25o C)

• One plate of samples 1 and 2 was stored in 37o C incubator

• Inverted plates were stored

Isolation of Unknown Bacteria

From sample 2 incubated at 37o C for 24 hours, a large white colony was taken and

streaked on two TSA plates:

• one plate was kept at room temperature

• one plate was incubated at 37o C

From sample 2 at room temperature that was grown for 96 hours, a clear colony was

taken and streaked on two TSA plates:

• one plate was kept at room temperature

• one plate was incubated at 37o C

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PCR Analysis Both bacterial colonies were analyzed via PCR and sent off for sequencing

•PCR analysis with BLAST on WAC discovered a

98% match with Bacillus stratosphericus •Bacillus stratosphericus is a gram-positive, motile, rod-shaped bacterium that is

normally found in the stratosphere but is brought down to the ground by atmospheric

cycling

•CAF was not able to be analyzed because PCR analysis returned no results

Possible causes:

• ineffectiveness of primers and/or polymerase used

• cell wall thickness or a greater susceptibility to heat

Isolated Bacteria CAF is a gram negative rod, signifying that it has a thin peptidoglycan layer in its cell

membrane. Original cultures of the bacteria were killed during growth when the

temperature of the shaker was changed from 25o C to 37o C, which contradicted procedure

and consequently resulted in no further testing because it did not survive the temperature

change.

WAC was determined to be a Gram positive rod from the results of a gram stain (Figure

3), signifying that it has a thick peptidoglycan layer and an outer cell membrane. Through

the OF-glucose test, it was determined that this microbe is an obligate aerobe. This is

known because the bacteria did not grow when the tested culture was covered with

mineral oil. The result of the OF-glucose test was supported by the results of the

Thioglycolate test because there was white growth at the top of the culture. These results

both showed that the bacteria only grew at the uppermost layer of the media, indicating a

requirement for oxygen in order to grow. The Catalase test also strengthens the argument

that WAC is aerobic due to the positive result, the presence of bubbles when exposed to

hydrogen peroxide, showing that the bacteria can convert hydrogen peroxide into water

and gaseous oxygen.

Through the MRVP test, it was determined that WAC is able to perform mixed acid

fermentation in order to lower the pH of its surroundings and cannot produce acetonin.

Upon completion of the MRVP tests, the culture was exposed to Kovacs oxidase reagent.

There was no color change of the culture, signifying that WAC is oxidase negative.

The results of the MIO test showed that WAC is not motile because there was growth

present in the media after incubation, but there were no signs of the bacteria dispersing

throughout the media.

Both bacteria had a negative result for the oxidase test, indicating that they do not use

cytochrome c oxidase; however, they may be able to use a different cytochrome to

transfer electrons to oxygen (1).

Abstract Lake Michigan is a diverse ecosystem that supports a variety of microbes. Two samples were

collected and two microbes were isolated. Multiple identification tests were run to determine the

identity of these bacteria. The first unknown bacteria, CAF, was determined to be inconclusive due to

complications with incubation and PCR. WAC was determined to be a close relative to Bacillus

stratosphericus, having 98% genetic consistency. Other factors, such as being Gram positive, having

the ability to produce catalase, and endospore formation capabilities were also consistent with

Bacillus stratosphericus. Despite these similarities an exact identity of WAC was unable to be

determined, which could mean it is a novel species.

Acknowledgements & References

We would like to thank Dr. Tobiason for her background knowledge in the field and

for being our Microbiology professor.

1.Leboffe, Michael J., and Burton E. Pierce. A Photographic Atlas for the Microbiology

Laboratory 4th Edition. Englewood, CO: Morton Publishing, 2011. 57-98.

2.Newton RJ, Jones SE, Eiler A, McMahon KD, Bertilsson S. 2011. A guide to the natural

history of freshwater lake bacteria. Microbiology Molecular Biology Reviewed. 75:14–49.

3.Zwart, G., Crump, B. C., Kamst-van Agterveld, M. P., Hagen, F., & Han, S. (2002).

Typical freshwater bacteria: an analysis of available 16S rRNA gene sequences from

plankton of lakes and rivers. Aquatic Microbial Ecology, 141-155.

4.Merrifield, Frederic.The Bergess's Manual. London: C. Howorth and Sons, 1854.

Site #1 Site #2

Distance from shore 2 m 2 m

Depth below surface 0 cm 20 cm

Temperature of water (oF) 50.5 50.5

Environment Rocks, no plants,

sandy sediment

Rocks, no plants, sandy

sediment

Sky conditions overcast overcast

Weather and

Air temperature

48o F, 70% humidity,

light drizzle, SSW 5-10

mph wind

48o F, 70% humidity, light

drizzle, SSW 5-10 mph

wind

White Colony (WAC) Clear Colony (CAF)

Gram stain Purple G+ Rod Pink G- Rod (small)

Oxidase test Negative Negative

Catalase test Positive Positive

Glucose test (MR)

Positive --

Glucose test (VP)

Negative --

Thioglycolate test

Positive (white growth on top)

--

OF-glucose test With mineral oil: negative Without Mineral oil: positive

--

MIO test Growth without motility Growth with motility

Endospore stain Positive --

Figure 1: CAF growth at 25°C

Figure 2: WAC growth at 25°C

Figure 3: WAC cells at 1000x

magnification (oil immersion) after

Gram Stain, showing Gram positive

result

Table 1: Conditions of Sites for Unknown Samples

Results and Discussion

Table 2: Results of Classification Tests of WAC and CAF