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Unknown Lab Report

Kelly Downar

Spring 2015



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Identifying a specific bacterium from a wide variety of bacteria is important in trying to help a sick patient take the correct preventative measures needed. It is essential in knowing the right bacteria causing the problem, so that the person can be treated in the correct way and the correct antibiotics are administered. In this particular study an unknown was given by the professor. The procedures taken to figure out, and isolate the two separate bacteria were taught thus far in Microbiology lab class. Doing specific tests on the two bacteria’s will give you the ultimate answer at the end as to what the bacteria is, so correct measures can be taken. Once the correct identity of the bacteria is know, more information on the specific bacteria can take place.

Materials and Methods

Mr. Snaric gave each student a specific unknown labeled with different numbers on them that contained both specimen. Unknown labeled 104 was given to me in lab by the instructor. Numbers of different methods were used to identify the specific bacteria given. The procedures following the various different methods can be found in our lab manual.

The first procedure that took place was to take the mixed culture of bacteria, and do a streak plate. The streak plate is done on a nutrient agar plate to try and isolate a pure culture of each unknown. (pg.10 Lab Manual) Alternate number 4 and 6 were given by the professor due to overgrowth on the original streak plate. Separate streak plates are done on the nutrient agar using the quadrant streak method. This would get the bacteria to grow as isolated pure colonies in order to run tests on them.

Once growth appeared for my alternates it was then time to figure out which alternate number was gram negative, and which was gram positive. In order to figure this out you have to do a Gram stain. This is where a small scrap of each sample of bacteria, is placed on the slide using all the specific dyes. The dyes used in order are Gram Crystal Violet, Gram Iodine, Gram Decolorizer, and Gram Safarin. The exact procedure followed is in the lab manual. (pg.67 Lab Manual) Once this is done the microscope is used to identify the color and shape, which will tell  whether the bacteria is gram positive or negative.

Each bacterium is now isolated and alternate number 4 is gram negative, and other tests are now needed to identify the exact bacteria.  The next procedure performed on alternate 4 was the Simmons Citrate. A citrate agar slant is inoculated with the bacteria on the surface of the test tube. Once that is done it is inoculated, and the results are checked the next day. This is used to determine if the bacteria that produce the enzyme citrase breakdown citrate. (pg.36 Lab Manual)  The next procedure performed on alternate 4 was the Urea test. For this urea tubes are used that contain a broth known as urea. The citrate agar slants is inoculated, and then incubate it. If the ph indictor turns a bright magenta color then it indicates the breakdown of urea by the particular bacteria. (pg.38 Lab Manual) The last procedure was the indole and Hydrogen Sulfide test. This was done with the Sim tube that is a liquid medium. The Sim tube is inoculated, and then incubated.  The “I” in Sim stands for indole. To test for indole you add Kovac’s reactant as stated in the lab procedure. This reactant brings indole to the top of the test tube. (pg.36 Lab Manual)  The “S” in Sim stands for sulfur reduction. The Sim tube is inoculated, and then incubated to find the results a day later. These tests gave the answer to which bacteria alternate number 4 was.

The other alternate given was number 8. From doing the Gram stain it was found that is it positive, and is rod shaped. For the gram stain you follow the procedures in the lab manual , and then look at the results under the microscope.(pg.67 Lab Manual) Knowing that it gave two options of bacteria to go with, and run tests on. The first procedure was the Casein, and a milk agar was used for this. It is to be inoculate with the bacteria, and then incubated. Clearing around the inoculated area was to be observed, as stated in the lab manual. (pg.28 Lab Manual) The next procedure was Methyl Red which the MR-VP test tubes are used. It is a broth based test tube that essential is used to test whether or not glucose is fermented. (pg.34 Lab Manual)  These were the only procedures done for this because I had it narrowed down to two from the beginning, so not as many test needed to be run. This gave the answer to alternative number 8.


All the following tests were performed on Alternate 4:

  • Simmons Citrate
  • Urea
  • Indole
  • Hydrogen Sulfide

All the following tests were performed on Alternate 8:

  • Casein
  • Methyl red



Simmion Citrate test- Purpose: Is the ammonium phosphate in the test tube broken down? – If so then it produces a blue color on the surface, otherwise if not it stays its normal green color.

Urea test- Purpose: Does the enzyme urease break down urea? If so then the broth turns magenta pink, otherwise it stays the same color it normally is.

Indole test- Purpose: Does the enzyme trytophanase degrade the amino acid tryptophan into indole? If so then when the Kovac’s Reagent is added it will bring the indole to the surface of the test tube producing a red color making the result positive, otherwise no red color is produced.

Hydrogen Sulfide test- Purpose: Does the hydrogen sulfide react with ferrous sulfate to produce a black precipitate? If black is produced in the tube then the test would be positive, otherwise if not then a negative result.

Casein test- Purpose: Does the bacteria produce the enzyme casease which hydrolyzes the milk protein casein? If so and there is clearing around the bacteria then it indicates a positive result, otherwise would be negative.

Methyl Red- Purpose: Is a mixture of acids produced as a result of glucose fermentation? If so the broth will turn red indicating a positive result, otherwise it will be a negative result.

Discussion/ Conclusion

The only problem encountered was in the beginning when given the initial number 104. When a streak plate was done on 104 to try and isolate the two different bacteria’s there was always overgrowth. They were never able to isolate on their own, so two different alternate numbers were given by the professor.

Alternate number 4 from doing a Gram stain was figured out to be gram negative rods. First the Simmons Citrate test was performed it did not turn blue on the surface, so I knew the results were negative. The citrate test tube stayed it’s normal green color. Next was the Urea test, which came back negative because it didn’t turn the bright magenta color. The urea stayed the color it normally is in the test tube. With those two tests being done it narrowed it down to two options, which were Proteus vulgaris and Escherichia Coli. Next were the Hydrogen sulfide and indole test. The indole test gave me a positive result because the surface of the test tube turned red. The Hydrogen sulfide test on the other hand was negative because it didn’t turn the test tube a black color. Proteus vulgaris would be positive for both urea and hydrogen sulfide. Leaving to believe that alternate number 4 gram negative rod is E.coli.

Alternate number 8 when a Gram stain was done came out to be gram positive rod. That being known it only left two options Bacillus cereus or Bacillus subtilis. The first test was the Casein , which came out to be a positive result because there was clearing around the bacteria. Next was the Methyl Red test this also gave a positive result showing that it does produce a mixture of acids as a result of glucose fermentation, which turns the broth a red color. This concluded that alternate number 8 gram positive rod is Bacillus cereus because both these tests were positive. If both the test were negative then it would have been Bacillus subtilis.


Background Information on Alternate number 8

Bacillus cereus is a large gram positive, rod shaped bacteria that produce toxins and is capable of producing endospores.B. cereus is mesophilic, growing optimally at temperatures between 20°C and 40°C, and is capable of adapting to a wide range of environmental conditions”, as stated in Bacillus cereus article. There are two illnesses that this particular bacterium can cause. “The first illness gives you diarrhea, whereas the second one gives you vomiting on top of diarrhea”, as stated in Bacillus cereus. The duration of this illness can last up to 24 hours at the most. As in most illness you are to get rest and drink lots of fluids, just like it is essential to do with this illness. This bacterium is found in many foods especially certain foods that sit out for long periods of time at room temperature. Their main habitat is soil. “Many sources of outbreak for B. cereus is said to be rice, meat loaf, turkey loaf, and many vegetables.” (Bacillus Species)



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