Materials and Methods

Samples

• Soil samples (one from outside of The Diner and the other from Nyumburu Amphitheater)
• Apple cider (obtained from roadside stand of a local farmer)
• Strawberry Kiwi Lemonade (Turkey Hill brand)
• Rotting apples (one from The Diner that had been in the fridge for a while and the other from the Co-op)

Media Used

• 1.0 g (NH4)2SO4
• 0.9 g KH2PO4
• 0.25 g MgSO4*7H2O
• 0.1 g K2HPO4
• 0.02 g FeCl3*6H2O
• 200.0 mL Ethanol solution

  Add ethanol to distilled water and bring volume to 200.0 mL. Mix thoroughly. Filter sterilize.

Add all components, except ethanol solution, to distilled water and bring volume to 800.0 mL. Mix thoroughly. Distribute into test tubes in 4.0 mL volumes. Autoclave for 15 minutes at 15 psi pressure. Cool to 25 degress Celsius. Aseptically add 1.0 mL ethanol solution to each tube. Mix thoroughly.

• 20.0 g Agar
• 20.0 g Glucose
• 20.0 g CaCO3
• 10.0 g Yeast extract

Add all ingredients to distilled water and bring the volume to 1.0 L. Mix thoroughly. Gently heat and bring to boiling. Autoclave 30 minutes at 10 psi pressure. Cool to 48 degrees Celsius. Mix thoroughly. Pour into sterile petri dishes.

Procedure

1. Innoculate samples in two tubes of Hoyer's medium and two plates of YGC medium. Incubate at 30 degrees Celsius for 48 hours.
2. Observe the Hoyer's tubes. If anything grew in them, aseptically streak for isolation on the YGC medium. Incubate at 30 degrees Celsius for 48 hours.
3. Observe colony morphologies. If there are distinct types of colonies on the plates, take an isolated colony of each bacteria colony and streak for isolation on the YGC plates. Incubate at 30 degrees Celsius for 48 hours. If there is only one type of colony morphology, continue on to the tests. (NOTE: Each lab period before performing any tests, always be sure to re-streak for isolation as to assure the continuance of the bacteria line.)
4. Perform these simple stains and tests to verify the identity of bacteria.
• Make a wet mount to determine motility.
• Make a Gram stain of the bacteria. This will also allow the cell shape to be seen easily.
• Make an endospore stain. Try to use an older colony to ensure that endospores would have formed if they were going to.
• Perform the catalase test.
• Perform the oxidase test.
5. Using as straight a needle as can be found, perform a motility stab. Incubate for 48 hours at 30 degrees Celsius. Observe the results. It may be helpful to hold the tube up to the light to confirm growth away from the stab.
6. Obtain two glucose carbohydrate fermentation tubes per sample and inoculate with the bacteria. Label one tube fermentative and the other oxidative. For the fermentative tube, carefully pour mineral oil until it forms a layer about 1 cm thick. Incubate for 48 hours at 30 degrees Celsius. Observe the results.
7. Obtain two Durham tubes per sample and inoculate with bacteria. Carefully pour mineral oil into each tube until a layer about 1 cm thick is formed. Incubate for 24 hours at 30 degress Celsius. Observe the results.