Teacher Notes

Bacteria in Milk

Super Value Laboratory Kit

Materials Included In Kit

Powdered milk, 450 g
Resazurin, 1%, 40 mL
Test tubes, 13 x 100 mm, 90

Additional Materials Required

Milk storage containers, covered, 3
Test tube rack
Water bath (shared)

Prelab Preparation

The milk provided in this kit is powdered, dry milk. It will need to be reconstituted with water at least two days prior to the laboratory. Place the dry milk (approximately 90 g) in 1 L of distilled water. Mix the milk solution thoroughly until it all stays in solution and it “looks” like regular milk again.

Divide the milk into three covered containers, label them clearly, and treat them as follows:

  1. Container A: Cover and place in refrigerator until about one hour before use.
  2. Container B: Cover and place in warm area (room temperature) until use time.
  3. Container C: Boil the milk for several minutes in a heat-resistant container (beaker or pan) and then pour the milk into a covered container and place it in a refrigerator until one hour before use.

Safety Precautions

Once food-grade item have entered the laboratory, they are considered chemicals and should not be consumed. Resazurin is a permanent stain and is not easily removed from skin or clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron.


Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All solutions from this activity can be disposed of down the drain with volumes of water according to Flinn Suggested Disposal Method #26b.

Teacher Tips

  • Enough materials are provided in this Super Value Kit for 5 classes of 30 students each, working in pairs (75 total student groups). The laboratory can be reasonably completed in one 50-minute class period.
  • A water bath at 37 °C causes the bacteria to become active and makes the results of this lab very predictable. The refrigerated milk bacteria will start to grow more quickly when incubated. If a water bath is not available, each student can make a temporary water bath with hot tap water, a beaker, and a monitoring thermometer. Either way, the incubation period is critical for producing the expected results.
  • This activity is very basic and is a good introductory activity for a consumer chemistry/biology course. The activity provides good practice in stating and formulating predictions.
  • This activity can serve as a starting point for many additional investigations. With simple materials students can easily design other experiments to determine what variables can affect the rapid growth of microbes in milk. The resazurin test provides a quick and easy general indication of bacterial activity. Test whole milk versus skim milk, one brand versus another, or aged milk versus fresh milk.
  • If more rigorous microbiology is a part of the curriculum, the study of the succession of bacteria that inhabit milk over a period of time can be made. Samples can be taken at various times over a several day period and microscope slides can be made and bacteria studied. (This will require the use of specific staining techniques and the use of a microscope with 1000X capability.) The pH shift in the milk as it ages can also be studied.
  • Population estimates of bacteria in milk can be obtained by growing milk cultures on Petri dishes or Petrifilm™. More quantitative results can be secured if these microbiological tools are available.

Correlation to Next Generation Science Standards (NGSS)

Science & Engineering Practices

Analyzing and interpreting data
Planning and carrying out investigations
Constructing explanations and designing solutions
Developing and using models

Disciplinary Core Ideas

MS-LS1.C: Organization for Matter and Energy Flow in Organisms
MS-LS2.B: Cycle of Matter and Energy Transfer in Ecosystems
MS-PS3.D: Energy in Chemical Processes and Everyday Life
HS-LS1.C: Organization for Matter and Energy Flow in Organisms
HS-LS2.B: Cycle of Matter and Energy Transfer in Ecosystems
HS-PS1.A: Structure and Properties of Matter
HS-PS1.B: Chemical Reactions

Crosscutting Concepts

Cause and effect
Structure and function
Energy and matter

Performance Expectations

MS-LS2-3: Develop a model to describe the cycling of matter and flow of energy among living and nonliving parts of an ecosystem.
MS-LS1-7: Develop a model to describe how food is rearranged through chemical reactions forming new molecules that support growth and/or release energy as this matter moves through an organism
HS-LS1-7: Use a model to illustrate that cellular respiration is a chemical process whereby the bonds of food molecules and oxygen molecules are broken and the bonds in new compounds are formed, resulting in a net transfer of energy.
HS-LS2-4: Use mathematical representations to support claims for the cycling of matter and flow of energy among organisms in an ecosystem.
HS-PS1-5: Apply scientific principles and evidence to provide an explanation about the effects of changing the temperature or concentration of the reacting particles on the rate at which a reaction occurs.

Answers to Questions

  1. Make a prediction about the amount of bacterial activity in the various milk treatments by making a simple bar graph showing your predicted color of the resazurin dye.
  2. Explain your predicted color for each test tube. This lab provides the opportunity to model the parts of a hypothesis. If the students design their prediction and scientific explanation for Part A as a class, that will serve as their model for the predictions for Parts B and C. The conclusion can be treated in the same way.

    Tube A: Predictions will vary.
    Tube B: Predictions will vary.
    Tube C: Predictions will vary.

  3. Record your actual results in a bar graph.
  4. Compare the graphs in 1 and 3. Explain any differences between the actual results and your predictions.

    The predictions and actual results may be similar or disparate depending upon the student’s predictions.

  5. Why were the test tubes incubated for 30 minutes?

    The tubes were incubated to speed up the growth of any bacteria that might be present, especially in the refrigerated specimens.

  6. Which test tube(s) acted as a control in this experiment? Explain.

    The boiled and then refrigerated tube (Tube C) demonstrates what will occur with minimal bacteria present. The boiling should kill nearly all of the bacteria in the milk.

  7. Evaluate this experiment. What things were done to make this a fair test? What could be done to improve this lab?

    Some things students have included in the “fair test” part of this answer are: the test tubes were the same size; the test tubes were in the same water bath for the same amount of time; the amount of resazurin added was the same for each test tube; the milk was aged for the same amount of time. To improve the lab, students suggested: measure the amount of milk that goes in each test tube; let the bacteria grow longer than 30 minutes; have a color comparison chart so there are no color discrepancies.


Thanks to Jeannette Miller, Schalmont High School, Schenectady, NY, for this activity.

Student Pages

Bacteria in Milk


Are you willing to share your milk with bacteria? Leave your milk unrefrigerated for a while and the bacteria will consume it faster than you want them to!


  • Oxidation–reduction
  • Indicators


Milk has often been described as the “near-perfect” food for growing humans. It is probably the most nutritionally complete food that is found in nature. Since milk is the only food young mammals usually consume in the weeks following birth, it is important for their early development that milk is so nutritionally complete. Mammals’ milk contains vitamins, minerals, proteins, carbohydrates, and lipids, all essential for early growth and development. A “typical” chemical composition of milk is somewhat difficult to pinpoint. In an individual cow, for example, the actual content varies from breed to breed, herd to herd, and even month to month.

Since milk is near “perfect” as a food source it is logical that microbes would also thrive on milk. In fact, they do! Milk provides a potentially ideal growth media for many pathogens. Hence, contamination of milk by any pathogen can produce a very serious health problem. Contamination of milk due to unsanitary handling or processing has caused many milk-borne epidemics of human disease. Undesirable microbes can be introduced into milk not only by diseased cows, but more frequently during processing by dirty hands, unclean or contaminated utensils, flies, polluted water supplies, etc.

Milk that is sold commercially is carefully handled and treated from the cow to the supermarket shelf in order to keep the bacteria population under control. Testing of milk for microbes and the careful processing of milk are crucial to good public health. Dairy products derived from milk are manufactured with the controlled use of microbes. Specific microbes produce tasty cheeses and other dairy products.

One chemical test for microbial activity in milk will be used in this activity. The test is based upon a simple principle— bacteria in milk use up oxygen as they grow and multiply. The amount of bacteria in the milk affects the amount of oxygen used; oxygen concentration is therefore a relative reflection of the bacteria population size.

To measure the amount of oxygen in the milk, an oxidation–reduction indicator will be used. Oxygen is a good oxidizing agent and will oxidize an indicator, changing its color. Resazurin is an ideal indicator for oxygen. Resazurin is blue in its completely oxidized state. Upon reduction it goes through a series of color shade changes from purple-red to pink to colorless, as illustrated in Figure 1. The color continuum allows for a more quantitative comparison than is possible with other indicators that exist in only two color forms.

Tests with resazurin solution can be interpreted as follows:


Resazurin 1% solution, 3 drops
Milk, refrigerated for 2 days, 5 mL
Milk, standing at room temperature for 2 days, 5 mL
Milk, boiled and then refrigerated for 2 days, 5 mL
Test tube rack
Test tubes, 13 × 100 mm, 3
Water bath

Safety Precautions

Once food-grade item have entered the laboratory, they are considered chemicals and should not be consumed. Resazurin is a permanent stain and is not easily removed from skin or clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron.


  1. Label three test tubes A, B, C and include a code to identify your group’s test tubes.
  2. Place the appropriate milk solutions in each test tube as follows:
  3. Place the test tubes into a 37 °C water bath to incubate for 30 minutes.
  4. During the incubation period complete questions 1 and 2 on the Bacteria in Milk Worksheet.
  5. After the 30-minute incubation period, add one drop of resazurin to the top of the milk in each test tube. Wait for 2 minutes for the dye to diffuse into the milk in each test tube. Do not stir.
  6. Record the color of each test tube on the worksheet. Compare your predictions and actual results and complete the Bacteria in Milk Worksheet.

Student Worksheet PDF


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