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The Procedure(s)

Field

  1. Open the Epicollect project ASM SU18 Bacteria. Record important identification information, such as Location, Field Number, and Date and Time.

  2. Use the “water grabber” to collect a water sample.

  3. Take the water temperature by holding the tip of the thermometer in the water until the temperature reading stabilizes (note: the temperature should be taken directly in the water body. If you are unable to reach the water surface, take the temperature of the sample in the water grabber immediately after collected).

  4. Transfer the water sample to the large clear jar.

  5. Record the water color by holding the jar against a white background (e.g. a piece of paper). Use the color terms listed on pg. 5 of this packet.

  6. Follow the instructions for the turbidity measurements (both tube and disk) and record in Epicollect.

  7. Start the chemical tests (full procedures can be found here), with each person in the group doing at least one of the chemical test. Be sure to use the correct labeled vial for each test. Prioritize doing the tests that take the longest first. Tests are listed below in order from longest to shortest. Be sure to collect results in Epicollect as tests are completed.

  8. Thoroughly rinse all of the chemical vials and caps with distilled water before and after each test

Lab

Diluting the Sample

  1. Label three beakers with a sharpie, each beaker should have a labeling of either 10-2,10-4, 10-6 respectively.

  2. Order your beakers from left to right, from least to greatest dilution. (10-2 should be first, 10-6 should be last)

  3. Fill each beaker with approximately 99 mL of water, the closer to 99 mL the better.

  4. After filling each beaker, using a new sterile pipette, transfer EXACTLY 1 mL of water from the sampling location vial labeled with the appropriate field number to the beaker labeled 10-2.

  5. Repeat step 4, transferring 1 mL of 10-2 dilution water to 10-4 dilution water with a sterile pipette.

  6. Repeat step 4, transferring 1 mL of 10-4 dilution water to 10-6 dilution water with a sterile pipette.

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Preparing the Growth Plates

  1. Before starting the plating procedure, be sure to clearly label the bottom (write in small letters along the edge) of each plate with the following information:

    1. Field number

    2. Date

    3. Dilution

    4. Initials

    5. Incubation Temperature

  2. Using a sterile pipette, drop 0.1 mL of the chosen dilution into the agar plate. Avoid removing the cover of the plate for too long, as this may contaminate the sample.

  3. Sterilize the glass rod by dipping it in 71% isopropyl alcohol. Use a lighter to burn off the excess liquid.

  4. Use the sterilized glass rod to spread the sample across the surface of the plate. Cover with the lid

  5. Allow the sample to incubate upside-down at room temperature for about a week.

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Bacteria Analysis

  1. Count the number of bacteria colonies found on the plate.

    1. If the number is less than 25, note TFTC (too few to count).

    2. If the number is more than 250, note TMTC (too many to count).

  2. Use a microscope to describe the morphology of all colonies found on the plate, using the following descriptions:

    1. Label (use A for the most numerous colony, B for the next numerous, etc.)

    2. Form (shape of bacteria)

    3. Color

    4. Elevation

    5. Margin

    6. Texture

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