Experiment 1: Assessing the Bacterial Load of Milk with Methylene Blue

Processed milk is pasteurized, that is, heated to a specific temperature for a specified amount of time, then cooled. This process reduces the amount of microorganisms present to non-disease causing levels. However, pasteurization does not kill all microorganisms in milk. The off-putting odor of expired milk is due to the growth and metabolism of bacteria. In this experiment, you will qualitatively assess the presence of bacteria in milk using the methylene blue reductase test. Methylene blue is a dye that is normally blue but turns colorless when it acted upon by bacteria (it is reduced, or it gains electrons, through the aerobic electron transport system). Methylene blue is essentially an oxygen sensor: the blue color disappears when the oxygen in the system is used. Aerobic bacteria metabolize oxygen; therefore, the faster the color changes, the more bacteria are present. This is a time course experiment, meaning that you will set up samples at different times then perform the assay on all samples at once. Please read the Procedure section carefully before beginning the experiment.

Procedure

1. Use a permanent marker to label one of the tubes as “4 Hour Sample”.
2. Add 10 mL fresh refrigerated milk to the tube with a sterile transfer pipette. Place this tube at room temperature but out of direct sunlight.
3. After 1 hour has passed, use a permanent marker to label another tube as “3 Hour Sample”.
4. Add 10 mL fresh refrigerated milk to the tube with a sterile transfer pipette. Place this tube at room temperature but out of direct sunlight, preferably in the same location as tube 1.
5. After an additional 2 hours have passed, use a permanent marker to label another tube as “1 Hour Sample”.
6. Add 10 mL fresh refrigerated milk to the tube with a sterile transfer pipette. Place this tube at room temperature but out of direct sunlight, preferably in the same location as tube 1 and 2.
7. When 4 hours have passed since you began the experiment, label the final tube as “0 Hour Sample”.
8. Add 10 mL fresh refrigerated milk to the tube with a sterile transfer pipette. Place this tube at room temperature but out of direct sunlight, preferably in the same location as tube 1, 2, and 3. Prepare this sample immediately before performing the methylene blue reductase assay.
9. Collect all the tubes and add 1 mL of the methylene blue solution with a sterile transfer pipette to each tube. Caution: Methylene blue dye can stain clothing, furniture, carpet, etc. Wear gloves and a lab apron when using and add the dye with the experiment set up on the benchcoat pad.
10. Tightly screw the cap on each tube and invert the tubes several times to mix. The samples should all turn blue. Record the starting time in Table 1.
11. Examine the tubes for a blue to white color change at 30 minute intervals. Record the time when samples turn white in Table 1. Calculate how long it took for each sample to change from blue to white.