Thursday, 03 August 2017 / Published in Uncategorized

Assessing the Bacterial Load of Milk with Methylene Blue

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 beforebeginning the experiment.

Materials:

4
Sterile screw-top tubes
8 Sterile, disposable transfer pipettes
0.05% Methylene blue solution
Stopwatch
Permanent marker

10
mL Graduated cylinder
*60 mL Fresh
refrigerated milk (whole, 2%, 1%, or fat-free)

*You must provide

Procedure

Use a permanent marker to label one of the tubes as
“4 Hour Sample”.
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.
After 1 hour has passed, use a permanent marker to
label another tube as “3 Hour Sample”.
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.
After an additional 2 hours have passed, use a
permanent marker to label another tube as “1 Hour Sample”.
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.
When 4 hours have passed since you began the
experiment, label the final tube as “0 Hour Sample”.
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.
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.
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.
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.

Table 1: Color Change Over Time
Milk Sample	Starting Time (Determined in Step 10)	Ending Time (Determined in Step 11)	Time Required for Color Change (End time-Start time)
Zero hour
1 hour
3 hour
4 hour

Which sample took the least amount of
time to become white? Why do you think this is so?

How does refrigeration affect the
amount of bacteria present in milk?

How does pasteurization affect the
amount of bacteria present in milk?

What differences, if any, would this
assay show if performed using raw (unpasteurized) milk?

What differences, if any, would this
assay show if performed using milk that was past its expiration date and had an
off-putting odor?

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