Microorganism Lab Report

Purpose:

To identify an unknown microorganism by performing a series of biochemical tests on a pure bacterial culture.

Materials and Methods:

1. Carbohydrate Utilization: Two culture tubes, phenol red lactose broth and phenol red sucrose broth, were each inoculated with one loopful of organism 3 from a broth culture. The broths were incubated at 37°C. After 24-48 hours, the mediums were examined. A positive test result is indicated by a change from the red broth to a yellow broth. A change from red broth to orange broth, or no change in color, is indicated as a negative result. A gas bubble produced in the Durham tube is indicated as a positive result for gas. A negative result for gas is indicated by no gas in the Durham tube.

2. Indole Production: A 1% tryptone broth was inoculated with one loopful of organism 3 from the broth culture. The tube was incubated at 37°C for 24-48 hours. After adding 5 drops of Kovac’s reagent, a yellow/green or red ring will appear at the top of the broth in the tube. The yellow/green ring around the top indicates a negative test result, while a red ring indicates a positive test result.

3. Urea Hydrolysis: One tube of urea broth was inoculated with one loopful of organism 3 from the broth culture. Before the experiment, the broth is a yellow color. The urea broth was incubated at 37°C for 24-48 hours. A color change from yellow to red indicates a positive test result. A change from yellow to orange, or no color change at all, indicates a negative result.

4. Starch Hydrolysis: A starch agar plate was inoculated with one loopful of organism 3 from the culture broth. The agar was inoculated with one straight line in the middle of the plate. The plate was incubated at 37°C for 24-48 hours. After incubation, iodine is added to cover the plate. A clear zone formed around the growth on the plate is considered a positive result. No clear zone around the growth is considered a negative test result.

5. Sulfide Production: A disposable inoculating needle was rolled in organism 3 on the slant growth. The needle was inserted straight into one tube of SIM media, and pulled straight out. The tube was incubated at 37°C for 24-48 hours. A positive test result will show black precipitate in the medium. A negative test result will show very little or no black precipitate in the medium.

6. Motility: A disposable inoculating needle was rolled in organism 3 on the slant growth. The needle was inserted into one tube of motility test medium. The tube was incubated at 37°C for 24-48 hours. Abundant red growth in the tube indicates a positive result for motility. Red growth strictly where the needle was inoculated results in a negative result for motility.

7. Casein Hydrolysis: One skim milk agar plate was inoculated with one loopful of organism 3 in a straight line across the plate. The plate was incubated at 37°C for 24-48 hours. A clearing around the growth on the plate indicated a positive test result. No clearing around the growth is a negative test result.

8. Catalase Production: One nutrient agar plate was streaked with organism 3. The plate was incubated at 37°C for 24-48 hours. After incubation, 3% hydrogen peroxide is dropped onto a single colony of bacteria. The formation of bubbles indicates a positive test result, while no formation of bubbles indicates a negative test result.

Results:

1. Carbohydrate Utilization: In the phenol red lactose broth, the color changed from red to yellow, indicating a positive test result. No gas bubbles were seen in the Durham tube, indicating a negative test result. In the phenol red sucrose broth, the color did not change at all and there was no gas in the Durham tube. Both of these results are negative test results.

2. Indole Production: When Kovac’s reagent was added to the 1% tryptone broth, the color of the ring at the top of the tube was green. This is a negative test result.

3. Urea Hydrolysis: In the urea broth, the color started as yellow when the experiment started. After incubation, the color was still yellow. This is a negative test result.

4. Starch Hydrolysis: After incubation, the starch agar plate was covered in iodine. There was no clear zone around the growth on the plate, and no color change. This means the test result is negative.

5. Sulfide Production: The SIM media did not show any growth of organism 3. There was no black color in the medium. This is a negative test result.

6. Motility: Organism 3 did move a little bit in the motility broth. It only moved in one direction, slightly away from the space where the needle was inoculated. This did not move enough to be considered motile, resulting in a negative test.

7.

Casein Hydrolysis: The skim milk agar plate did not have any clearing around the growth. This is a negative test result.

8. Catalase Production: When 3% hydrogen peroxide was added to an isolated colony, no bubbles formed. This is a negative test result.

Discussion:

1. Carbohydrate Utilization: The test result for the phenol red lactose broth was positive for acid and negative for gas. The positive result for the acid means that the organism is able to ferment the particular sugar lactose. The Durham tube did have gas in it which means the organism produced gas in the lactose broth. The rest result for the phenol red sucrose broth was negative for both acid and gas. This means the organism was not able to ferment in sucrose. The organism was also not able to produce gas in the sucrose broth.

2. Indole Production: The result for this test was negative. This means the organism does not produce tryptophanase. The organism is not able to split the amino acid tryptophan into indole and pyruvic acid. There was no indole produced from the breakdown of tryptophan.

3. Urea Hydrolysis: This test result was negative. There was no accumulation of sufficient ammonia from the hydrolysis of urea. An alkaline environment was not created by organism

3.

4. Starch Hydrolysis: The result for starch hydrolysis was negative. This means that after the iodine was covering the bacteria, there was no clearing around the growth. This bacteria does not produce the enzyme amylase. The starch is not broken down into single units of glucose that can be used in cellular respiration or energy production.

5. Sulfide Production: The negative test result for sulfide production means that no hydrogen sulfide was produced. There was no reaction with ferrous salts to form black metal sulfides, which indicates a positive test result.

6. Motility: The negative test result for motility shows that organism 3 is not a motile organism. Organism 3 does not have any flagellum on them. Flagella help the bacteria move toward nutrients and other beneficial elements and move away from toxic substances and environments.

7. Casein Hydrolysis: The negative test result for this test shows that organism 3 was not able to produce the extracellular enzyme caseinase, which breaks down casein into smaller soluble units.

8. Catalase Production: The result for the catalase production is negative. A negative test result means that catalase was not produced by organism 3. The organism is anaerobic because it did not produce bubbles to protect itself from the hydrogen peroxide added to it.

Conclusion:

The unknown organism number 3 appears to be Staphylococcus epi. The table of positive and negative results that we made had two columns of same results as my unknown bacteria. The gram stains were also the same for Enterococcus faecalis and Staphylococcus epi. After researching both gram stains, the Staphylococcus epi gram stain was most similar to the way organism 3 looked like under the microscope. Enterococcus faecalis had some pink bacteria stained behind the gram positive cells. This did not appear in my gram stain of organism 3. Therefore, I have concluded that organism 3 must be Staphylococcus epi.