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Unknown Number 124 

Caroline Marshak

April 5, 2015

Jay Snaric

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Spring 2015



There are very many reasons to know about microorgansims, a few of which have been shown in the microbiology lecture class. These materials were then transferred to the lab portion and studied by several students to learn more about the significant effects that bacteria can have on people. It is important to know differences between microorganisms, especially in the health industry, to help diagnose and treat illnesses. There are some illnesses that are talked about everyday in the US, such as Staphlococcus (staph) infections and Streptococcus (strep) throat infections but there are hundreds of microbacterium that kill human beings everyday which are not as obvious. On the other hand, microbacteria are very important for a normal, healthy body, which can make it difficult to tell the difference between the good and bad. In the microbiology class at Meramec, students applied the information that was studied throughout the year to identify 2 unknown bacterium. To do so, the unknown organisms were placed in numerous environments and put through different tests to see what grew and how it grew. This information was written down and by the process of elimination, the two microbacterium could be identified.


A vial with 2 unknown specimen labeled #124 was given out by the lab instructor. Procedures learned during the lab portion of this class were performed on each bacterium to figure out what they are. These methods were followed as stated in the lab manual by McDonald et. Al (1).

The first procedure performed was to streak the unknowns on to a nutrient agar plate. To isolate the unknowns, the quadrant streak method was used, described in the lab manual on page 10 (1). Once the plate was streaked with the bacteria in zigzag lines on the edge of the plate, it was placed into the incubator at 37°C. Two days prior to incubation, the plate was removed and 2 distinct colonies were present which were then isolated on to 2 separate plates using the quadrant streak method. These two plates were incubated for another two days.

The second procedure that needed to be done was gram staining. To do this, a series of reagents (seen on table 1) were flooded onto slides for an allotted amount of time (1). After the stains were complete, the slides were viewed under the microscope to then decide which were Gram- and Gram+ and the shapes of each. After looking through the microscope, it was obvious that there were Gram- rods on one slide and Gram+ rods on the second slide, which lead to the next test.

Since the Gram- shapes were rods, no organisms on the unknown chart could be eliminated. To narrow down the organisms, the Simmon’s Citrate test was performed. After inoculating the citrate tube with unknown Gram-, the tube was placed in the incubator. Two days later, the tube was removed and it was still green, indicating a negative test. This left Escherichia coli, Pseudomonas aeruginosa, and Proteus vulgaris as possible organisms. Professor Bohra substituted as lab instructor on this day and stated that Pseudomonas aeruginosa glows green, which was not the case for either unknown organism given. Pseudomonas aeruginosa was then eliminated from the unknown chart.

To determine which of the two left was the unknown Gram- organism, a nitrate test and H2S test were done.

The unknown Gram- was inoculated in the nitrate broth and stabbed into the Kligler tube, then incubated for 2 days. The following class, the Kligler tube was black, indicating a reduction of sulfur (a positive result). Nitrate reagents A and B were added to the Gram- Nitrate broth and there was no color change. Zinc was added and the color still remained the same, which ended up as a false negative. The tests used and results of unknown Gram- are shown in Table 1.


The following tests were performed on the unknown Gram-.

  1. Simmon’s Citrate
  2. Kligler (H2S)
  3. Methyl Red
  4. Nitrate


Since the Gram+ bacteria were shown as rods on the gram stain, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis could be eliminated, leaving Bacillus cereus and Bacillus subtillis as possible organisms. To determine which of these two organisms was in tube #124, the methyl red test was used along with the casein test.

The unknown Gram+ was inoculated onto a milk agar plate then incubated for 2 days. The following class, there was a clearing where the bacteria was placed, indicating the presence of the enzyme casease (a positive result). To make sure that the test’s results were correct, a methyl red test was done.

An MRVP tube was inoculated with the unknown Gram+ and incubated until the following class. When class returned, 10 drops of the methyl red reagent was placed into the tube and it immediately turned red at the top, indicating the presence of a mixture of acids (a positive result).

The tests used and results of unknown Gram+ are shown in Table 2.

The following tests were performed on the unknown Gram+

1.Methyl Red

2. Casein


Table 1: Gram- Physiological Results

Gram Stain Crystal violet, Iodine, Alcohol, Safranin None Pink Rods Gram negative rods Gram negative bacteria
Simmon’s Citrate Citrate Slant 37°C No color change Negative Organism is unable to use citrate as a carbon source
Kligler (H2S) Kligler iron tube 37°C Black appearance in slant Positive Organism is able to reduce sulfur
Nitrate Test Nitrate tubes None No red color after adding Nitrate A & B. No color change after zinc addition False Negative Organism cannot reduce Nitrate




Table 2: Gram+ Physiological  Results

Gram Stain Crystal violet, Iodine, Alcohol, Safranin None Purple rods Gram Positive rods Gram positive bacteria
Methyl Red MRVP 37°C Red color at the top of the tube Positive Organism is able to produce acid from glucose fermentation
Casein Milk Agar plate 37°C Clearing where bacteria was inoculated Positive Organism is able to produce the enzyme casease


NOTE: Flowcharts were removed due to formatting issues.


The test results for the unknown Gram- bacteria showed that the specimen was Proteus vulgaris. The conclusion was made by three important tests. The first was the Simmon’s Citrate test which came back green, meaning that it was unable to use citrate as a carbon source. The second test that was done was the Kligler test for H2S, which came back as a positive result. The third test was Nitrate, which came back as a false negative. Perhaps when trying to inoculate the broth with the Gram- bacteria, the inoculating loop was still too hot and killed the Gram- cells, which would make the broth then, without bacteria. With these three tests the unknown Gram- bacteria has been identified as Proteus vulgaris.

The test results for the unknown Gram+ bacteria showed that the specimen was Bacillus cereus. The conclusion was made by doing two tests. The first test that was done was Methyl red, which came back positive, as the broth turned red after adding MRVP reagent. To be sure that the results were correct, the Casein test was done on a milk agar plate. After incubation there was a clearing around the bacteria, which is a positive test. With these tests, the unknown Gram+ bacteria has been identified as Bacillus cereus. 

Bacillus cereus is a facultative aerobic bacterium that is highly pathogenic to humans if not handled properly. This specimen is a soil-derived drug, which makes it easy to get into foods form the ground. It produces toxins which can result in diarrhea, nausea and vomiting. Bacillus cereus is often found in rice dishes, but can be found in most types of food that have sat out too long at room temperature, as well (2). It can also be found in foods that are not cooked to the optimum temperatures, which allows the bacteria to continue to thrive. Some strains of this bacteria can have a positive effect for animals, acting as a probiotic. This is because Bacillus cereus competes with other bacteria of the intestine, such as salmonella. Often times, animals will be given food with a B. cereus additive so that the animal does not get salmonella in the gut (3) This helps the animal grow as well as helping to protect the humans who eat it. This bacterial strain can be doubled in less than 30 minutes at room temperature. B. cereus can produce protective endospores which help the bacterial strain live on, even if it is inactive (4).





  1. Virginia, McDonald. Lab Manual for General Microbiology (2011). Print.
  1. Bacillus Cereus.” Bacillus Cereus. Web. 29 Apr. 2015. <>.
  1. “Bacillus Cereus.” Wikipedia. Wikimedia Foundation. Web. 30 Apr.
  2. <>.
  3. “Bacillus Cereus.” – MicrobeWiki. Web. 29 Apr. 2015. <>.


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