Bacteria eat by secreting enzymes into their surroundings to digest the food, and then they ingest the digested particles. If the bacteria are on an agar plate with the correct nutrients, they will multiply and form colonies. If the bacteria do not consume the nutrients which are in their agar plate, then they will not multiply. Thus, by determining which agar plates the bacteria form colonies on, we can determine which nutrients the bacteria eat.
These experiments are for hydrolytic (digestive) enzymes, to determine if our bacteria digest starch, casein, gelatin, fats, and/or DNA. (Other future tests will determine carbohydrate utilization, amino acid degradation, respiration, and other miscellaneous properties of the bacteria.)
Starch Hydrolysis:
Materials:
Starch agar plate
Gram's iodine
Pure culture of our bacteria from broth culture
Steps:
1. Labeled the bottom of the starch agar plate with our group number and type of test.
2. Used aseptic technique to inoculate the starch agar plate from the broth culture.3. Let it incubate at 35 degrees for 48 hours.
4. Flooded agar surface with Gram's iodine, and waited 30-60 seconds to let a purple color develop
where starch was present.
5. Observed result.
6. Disposed of agar plate properly.
Results:
Casein Hydrolysis:
Materials:
Skim mile agar plate
Pure culture of our bacteria from broth culture
Steps:
1. Labeled the bottom of the skim milk agar plate with our group number and type of test.
2. Used aseptic technique to inoculate the skim milk plate from our bacteria's broth culture.
3. Let in incubate at 35 degrees for 48 hours.
4. Observed the plate for a clear zone around the inoculated streak, since this would indicate a positive test.
5. Disposed of the agar plate properly.
Results:
Discussion: Our bacteria is negative for casein, because there is not a clear rim around our bacteria.
Gelatin Hydrolysis:
Materials:
Nutrient gelatin deep tube
Pure culture of our bacteria from broth culture
Steps:
1. Labeled the nutrient gelatin deep tube with our group number and type of test.
2. Used aseptic technique to inoculate the nutrient gelatin deep tube. We used the inoculation needle and stabbed the gelatin.
3. Inoculated the tube at 35 degrees for 48 hours.
4. Placed tubes in the refrigerator for 15 minutes. Then we checked for liquidification by slightly tilting the tube. Liquidification would indicate a positive result.
5. Discarded the tubes properly.
Results:
Discussion: Our bacteria is negative for gelatin, because our liquid gelatin solidified after 15 minutes of refrigeration. This solidification means that our bacteria did not hydrolyze the gelatin that solidifies the medium.
Fat Hydrolysis:
Materials:
Tributyrin agar plate
Pure culture of our bacteria from broth culture
Steps:
1. Labeled the bottom of the tributyrin agar plate with our group number and type of test.
2. Used aseptic technique to inoculate the tributyrin agar plate from the broth culture.
3. Let it incubate at 35 degrees for 24 hours.
4. Examined the plate for a clear area around the bacterial streak. A clear zone would be a positive result for fat hydrolysis.
5. Discarded the plate properly.
Results:
Discussion: Our bacteria is positive for fat hydrolysis, because the area around our bacteria is clear.
DNA Hydrolysis:
Materials:
DNA agar plate
Pure culture of our bacteria from broth culture
1N HCL in a bottle with a dropper
Steps:
1. Labeled the bottom of the DNA agar plate with our group number and type of test.
2. Used aseptic technique to inoculate the DNA agar plate from the broth culture.3. Let it incubate at 35 degrees for 24 hours.
4. After incubation, we flooded a portion of the plate with 1N HCl and examined the plate for a clear area around the bacterial growth. A clear area would be a positive result for DNA hydrolysis.
5. Disposed of the plate properly.
Results:
Discussion of results: Our bacteria is negative for DNA hydrolysis, because addition of HCl did not indicate a clear area around the bacterial growth.
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