Materials Included In Kit

Additional Materials Required

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:

Container A: Cover and place in refrigerator until about one hour before use.
Container B: Cover and place in warm area (room temperature) until use time.
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.

Disposal

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 kit for 30 students working in pairs. 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.

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 the following bar graph:

4. Compare the graphs in Questions 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.

References

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

Introduction

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!

Concepts

• Oxidation–reduction

• Indicators

Background

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:

Materials

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.

Procedure

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.
7. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10336_Student1.pdf