Anita Rong

MICROBIO 233

Unknown Project Report

Introduction

The bacteria around us have one thing in common: they’re microscopic and unable to be seen by the naked eye. They, however, also have a lot of different characteristics that set them apart from each other. They vary in shapes, grouping, as well as how they react to different food sources. We can take advantage of these unique characteristics by using them to identify unknown bacteria. In this experiment, we will be using a variety of tests as well as our knowledge of how certain bacteria will react to these tests to try to identify two unknown species. In order to do so, we created a flow chart of tests to perform in order to accurately isolate our unknowns. This flow chart will keep us organized and prevent us from administering any unnecessary tests, thus identifying our unknowns in the most efficient manner possible.

This project will allow us to utilize the various skills we’ve obtained over the course of the semester along. The reasoning skills are important in the field of science. It allows people to produce and follow a method that will lead them to their ultimate goal.

The two bacteria identified were
Bacillus cereus and
Salmonella typhimurium.
B. cereus is a gram-positive rod. It was first isolated around 1969 from a blood sample of a man with a case of fatal pneumonia. It is naturally found in the soil, and it is an anaerobic bacterium that grows best between 20 and 40 degrees Celsius, classifying it as mesophilic. Furthermore, the bacteria is known to produce endospores (Vilain et al 2006). Despite it being a soil bacterium,
B. cereus has been known to cause food poisoning and diarrhea via toxins that it releases. The bacteria can sometimes be found on starches including rice, vegetables, and meat. There are approximately 64,000 incidences of
B. cereus related food poisoning cases per year (Osborne 2013).

S. typhimurium on the other hand is a gram-negative rod. It was first discovered in 1885 by Dr. Daniel Salmon and his research assistant Theobald Smith. The bacteria can be found in the gastrointestinal tracts of animals and people who are infected (USDA 2013). It is a facultative anaerobe that grows best around 40 degrees Celsius, making it mesophilic.
S. typhimurium has been known to cause Salmonellosis, which includes the symptoms of nausea, stomach pain, diarrhea, and fever. It can be contracted by eating food contaminated with the bacterium that is not cooked thoroughly (UNL Food).

Methods/Materials

Nonspecific Materials

· Bunsen burner

· Inoculating loops

· Unknown Sample 5A + 5B

· Wax pencil

Gram Stain Materials

· Light microscope

· Glass slides

· Immersion oil

· Crystal violet stain

· Iodine solution

· Ethyl alcohol

· Safranin stain

Methyl-Red (MR) Test

· MR Tubes

· Methyl red reagent

SIM Test (Indole)

· SIM tube

· Kovac’s reagent

Casease Test

· Casease agar plate

Results

Gram Stain

To start off the experiment with our unknown sample, we first performed a gram stain. The gram stain allows us to visualize the shape of the bacterium as well as determine whether it is gram positive or negative. A gram positive bacteria contains a thick peptidoglycan layer while gram negative bacteria have an outer membrane surrounding the cell wall. We are able to differentiate between positive and negative by viewing the color of the bacteria under a light microscope. Gram positive bacteria have a thick wall that absorbs the crystalline violet stain best, thus appearing purple. The gram negative bacteria, on the other hand, have a thin cell wall, so they are more likely to take up the safranin stain after the violet is decolorized. Gram negative species appear pink. By performing a gram stain, we are able to first identify some basic characteristics of the unknown bacteria. With this information, we can build off by performing other tests to confirm the identity. For 5A, it was discovered that it was a gram positive bacilli that stained purple, as shown below in Figure 1A. Sample 5A was a gram negative bacilli, staining pink as evident in Figure 1B.

Figure 1A. Gram (+) Rods for Sample 5A. Figure 2A. Gram (-) Rods for Sample 5B.

Methyl Red (MR) Test

The Methyl Red test is designed to determine whether or not a species can perform mixed acid fermentation. If they are capable of it, the bacteria will produce acidic products as a result of fermentation. The acidity can be detected after inoculating a loopful of the unknown species into a MR tube and allowing it to incubate at 37℃ for about 48 hours. After incubation, the methyl red reagent is added to the test tube. The reagent allows us to determine the pH of the contents of the tube. If the tube takes up a red color, that is indicative of a positive mixed acid fermentation result due to a low pH. For species 5A, we can see in figure 2A that the tube (middle) turned a deep pink. Although it was hard to decipher, it was confirmed to be a positive result. A MR negative tube can be seen on the left of 5A for comparison. Species 5B also yielded a MR positive result (right tube) and turned red.

From the results of the MR test, we confirmed the identity of sample 5A. By following the flowchart designed in class, a gram (+) rod that is also MR (+) are characteristics that are indicative of
B. cereus. Sample 5B also yielded a MR (+) result, but because there were still multiple possibilities behind a gram (-) and MR (+) result, further tests needed to be done before isolating the unknown identity.

Figure 2A. Left to right: MR negative tube, sample 5A, sample 5B. Sample 5A and 5B

Yielded positive results, compared to a negative test result.

SIM Test (Indole)

The SIM test can actually test for 3 different results: sulfur reduction, production of indole, and motility. The test is one tube in which the different results can be inferred after inoculating and incubating at 37℃ for 48 hours. A positive result for sulfur reduction can be inferred if the tube is black in color. This is a sign of sulfur being released to create a black precipitate in the tube. Indole production is done when the bacteria is capable of breaking down the amino acid tryptophan. In order to detect the indole, Kovac’s reagent needs to be added to the tube. The presence of a red band indicates a positive result for the indole test. Finally, motility of a bacteria can be determined by seeing if there is growth in the tube outside of the area of inoculation. If positive, it usually appears cloudy.

Since the identity of sample 5B was not found, the indole test was done to further help narrow down the possibilities. After incubation, the tube was positive for sulfur reduction, as shown in Figure 3 by the deep black color. To determine if it tested positive for indole, Kovac’s reagent was added to the tube. As seen in figure 3, the tube presented with a negative result: no red band was visible at the top of the tube. According to the flowchart in class, there were still two possible identities. A final test needed to be done to finally identify the unknown 5B species.

Figure 3. Sample 5B was positive for sulfur reduction but negative for indole production.

Casease Test

The final test to be performed was the casease test. This test is performed by using a plate made of skim milk agar. Casein is the protein in milk that produces the white color. The purpose of this test is to determine whether or not a bacteria has the casease enzyme. If it does have the enzyme, the species will be able to breakdown the casein for further metabolization. A positive result will yield a plate that has a clear zone where the bacteria was streaked. A negative result shows as still milky white and opaque, indicating that the bacteria was unable to break down the protein.

As shown in Figure 4, the left side of the plate demonstrates a clear zone around the bacteria. This is a positive result for casease activity in the bacteria. On the left, there is no clearing and the area is still opaque. That is a negative result for the presence of casease activity. Sample 5B appeared as the left side of the plate in Figure 4: no clear zone, which means a negative result for casease. From this result, we can firmly identify sample 5B as
Salmonella typhimurium.

Figure 4. Left side indicates a casease positive result, while the right shows casease negative.

In the case of 5B, it appeared like the right side, indicating casease negative.

Discussion

After a series of tests, sample 5A was identified to be
B. cereus and sample 5B was shown to be
S. typhimurium. By following the flowchart, we confirmed that
B. cereus was a gram positive rod that is capable of mixed acid fermentation. We also see that
S. typhimurium is a gram negative rod that is also capable of mixed acid fermentation as well as sulfur reduction. We also learned that is unable to break down tryptophan or casein.

Overall, the experiment went very well. All the tests were conclusive and produced the expected results (when confirmed with Professor Manow). There appeared to be no sign of contamination in the sample since the gram stains were consistent with only one species present. Also, the tests did not produce any incorrect or surprising results. If I were to recreate the experiment again, one thing I would change would be to try to perform the tests in a shorter time frame. Although my results were as expected, I wonder if the results for the MR test would be stronger (a deeper red instead of a pink) for sample 5A if the reagent was added immediately after 48 hours of incubation instead of waiting a week.

The unknown species project was a great way for us to utilize our knowledge of all the different techniques and tests learned in this class. Furthermore, it allowed us to design the most efficient method to follow to get to the results in the shortest amount of time. By solving the puzzle, we demonstrated that we have learned to work independently to successfully carry out a project from start to finish.

References

Albrecht, J. (n.d.). Salmonella. Retrieved from University of Nebraska-Lincoln website:

https://food.unl.edu/salmonella

Osborne, N. (2013, October 7). Fact Sheet on Bacillus cereus. Retrieved from 

     https://www.thermofisher.com/blog/food/fact-sheet-on-bacillus-cereus/ 

United States Department of Agriculture. (2013, August 7). Salmonella Questions and Answers. 

Retrieved from 
            https://www.fsis.usda.gov/wps/portal/fsis/topics/food-safety-  education/ 
        

get-answers/food-safety-fact-sheets/foodborne-illness-and-disease/salmonella-questions-and-answers/ 

Vilain, S. (2006). Analysis of the Life Cycle of the Soil Saprophyte Bacillus cereus in Liquid Soil 

     Extract and in Soil. 
            Applied Environmental Microbiology, 
            72. https://doi.org/10.          
        

     11.1128/AEM.03076-05