Experiment 3: Selection and Differentiation of Gram-Negative Bacteria from Liquid Samples

Water is often screened for the presence of coliform bacteria as an indicator of fecal contamination. Contaminated water can transmit a number of human diseases, including cholera, salmonella, dysentery, shigella, and many others. Coliform bacteria are Gram-negative rods, many of which can ferment lactose (the same sugar found in milk). MacConkey agar is a selective and differential media that contains a pH indicator dye (neutral red), lactose, bile salts, and crystal violet. The bile salts and crystal violet select for Gram-negative bacteria by inhibiting the growth of Gram-positive organisms. The lactose and neutral red demonstrate which bacteria can ferment lactose as lactose fermentation produces acidic compounds that lower the pH of the media and turn the lactose fermenting colonies red (neutral red is colorless at pH above 6.8 and red at pH less than 6.8). In this experiment, you will collect liquid samples and test them for the presence of coliform bacteria using MacConkey agar plates.

Procedure

1. Prepare three MacConkey agar plates according to Steps 1 - 5 as listed in Lab 9 – Experiment 1.
2. Use your 15 mL conical tubes to collect 3 water-based liquid samples from different sources. These can come from a faucet in your home, a water fountain, a flowing stream (be sure to not collect any of the sediment at the bottom of the stream), iced tea from a restaurant, etc. Collect approximately 8-10 mL of each sample.
3. Use a permanent marker to label each tube with the sample type and location.
4. Label each MacConkey agar dish with the same information you put on the collection tubes (i.e., each petri dish should have the same information as each corresponding test tube).
5. Use a pipette to transfer approximately 4 drops of each sample onto the corresponding petri dish.
6. Use a disposable spreader to gently spread the liquid sample over the agar surface. Use a new spreader for each sample/plate combination.
7. Allow to air dry. This should take approximately 5 - 10 minutes.
8. Cover the plates with the petri dish lid and incubate them in a warm location (not to exceed 37.7 °C or 100 °F) for 1 - 2 days; or, until well defined, isolated bacterial colonies appear.
9. Examine the plates and record the bacterial growth and color of the colonies in Table 5.
10. When you are finished with the experiment, flood the plates with the 10% bleach solution, incubate them for 20 minutes, and then pour the bleach down the sink with running water.
11. Seal the plates with Parafilm™ and dispose of them in the trash.