What is the Bacterial Content of Milk?

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Author: Cy Ashley Webb

Grade Level: Middle School; Type: Biology


Study the presence of aerobic bacteria in milk using methylene blue.

Research Question:

  • What is methylene blue and how does it work?
  • How do federal standards for the number of allowable organisms in milk relate to the time it takes for milk to regain it’s white color after methylene blue has been added?
  • Which has more bacteria: raw milk or pasteurized milk?

Methylene blue is a blue dye that reacts to the lack of dissolved oxygen by turning translucent if there is no dissolved oxygen in a sample. Methylene blue is a good indicator for the presence of aerobic bacteria in a sample because aerobic bacteria use up oxygen. In this experiment, students will use methylene blue to study the presence of aerobic bacteria in milk.


  • Access to a stove and big pot for sterilizing equipment
  • Two test tubes with rubber stoppers or caps
  • Test tube rack
  • Paper towels
  • Calibrated eye dropper or pipette
  • 10 ml graduated cylinder
  • Mason jar to use as a water bath
  • Refrigerated pasteurized milk
  • Fresh raw milk (optional)
  • Thermometer
  • Kitchen tongs
  • Hotplate
  • Methylene blue (available from a pharmacist or online)

Experimental Procedure #1:

  1. Before starting, make sure you have sufficient temperature control with your hotplate. Fill a mason jar half-full with tepid water. Put it on the hotplate and raise the temperature to 98 degrees. Make sure you can keep it at this temperature for several hours.
  2. Wash your hands. Cover the surfaces adjacent to your kitchen stove with paper towels. Place the test tube racks close to the stove on the paper towels.
  3. Fill the pot with water and bring to a boil. Uncap your test tubes. Using your kitchen tongs, carefully drop your graduated cylinder, test tubes and their caps in boiling water. Boil for one minute.
  4. Remove your test tubes and graduated cylinder from the water. Place the test tubes in the test tube rack.
  5. Measure 9 ml of milk in the graduated cylinder. Pour 9 ml milk into each test tube
  6. Using your calibrated eye dropper or pipette, measure 1 ml methylene blue. Place the methylene blue in one of the test tubes.
  7. Using the kitchen tongs remove the test tube caps from the pot, and cap the test tubes. The test tube with the methylene blue is your test sample. The test tube without the methylene blue is your control. Label each test tube.
  8. Pick up your test tube and gentle shake them so that the methylene blue is dissolved.
  9. Place each test tube in the water bath.
  10. Examine the test tubes every 15 minutes for two hours and every hour afterwards. Record your observations. If it takes more than 8 hours for the milk to turn white again, the quality of the milk is excellent. If it takes 5.5 to 8 hours for the milk to turn white, then there are less than 0.5 million organisms/ml of milk. If it takes 2 to 5.5 hours for the milk to regain color, then there are 0.5 to 4 million organisms/ml of milk. If it takes 20 minutes to 2 hours, then there are 4 to 20 million organisms/ml of milk. If it takes less than 20 minutes, then there are over 20 million organisms/ml.

Experimental Procedure #2: Repeat Experiment #1, using raw milk and pasteurized milk

Terms/Concepts: aerobic bacteria, oxygen consumption by bacterial, methylene blue; regulation of milk


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