22.2: Exercise

Ecological Succession of Bacteria in Milk

Milk is a highly nutritious food containing carbohydrates (lactose, or milk sugar), proteins (casein, or curd), and lipids (butterfat). This high level of nutrition makes milk an excellent medium for the growth of bacteria. Pasteurizing milk does not sterilize it (sterilizing kills all bacteria) but merely destroys pathogenic bacteria, leaving many bacteria that can multiply and these bacteria will begin to grow and bring about milk spoilage. Biologists have discovered that as milk ages, changing conditions in the milk bring about a predictable, orderly succession of microorganism communities.

In this laboratory exercise, you will observe successional patterns in several types of milk. You will record changes in the environmental conditions of the types of milk as they age. These changes are a result of changes in the bacterial communities. Here are some of the major bacteria found at various stages.

Stages of Milk Succession

1. Psuedomonas and Achromobacter (gram-negative rods) digest butterfat and give milk a putrid smell
2. Lactobacillus (gram-positive rod) and Streptococcus (gram-positive coccus) ferment lactose to lactic acid and acetic acid.
3. Acidity sours milk and converts casein to curd.
4. Yeast (fungi) thrive in acidic conditions and metabolize the acids into non-acidic compounds.
5. Bacillus metabolize proteins into ammonia products and raise the milk’s pH. Spoiled milk odor is very noticeable at this stage.

QUESTIONS

1. What are some advantages to using bacteria as opposed to plants in this experiment?
2. What factors might speed or delay a successional process? Apply your example to succession in milk.

Locate the milk samples available in the lab. Take a look at the samples and form two conditions you would like to study. Develop a short design for studying the first condition, state the dependent and independent variable, control and experimental group, the hypothesis and any variables which have been controlled. Then develop a short design for studying the second condition, state the dependent and independent variable, control and experimental group, the hypothesis and any variables which have been controlled.

Just as in any of our experiments, you must use effective scientific method. Develop hypotheses (at least 2—one with different milk types and one with either temperature or time as an independent variable) that you can test in the process of the milk community succession.

We will be performing a gram stain on our milk samples. Remember from our microbiology section that bacteria can be either gram negative (pink) or gram positive (purple). We will also look at these bacteria under a microscope to identify their shapes. Recall that bacteria can be cocci, bacilli, or spirillum.

QUESTIONS

1. What hypotheses are you testing? List both here.
2. What information led you to ask these hypotheses?
3. Make predictions about your hypotheses. i.e. How will you know if the data supports or refutes your hypotheses?
4. Be sure to identify the variables you will test, and those you will control for each experiment.
5. Prepare a table for data collection. You will be recording the pH, smell, consistency, and bacteria shapes and colors present in your milk samples.

On each lab bench are several small beakers. You will obtain a sample of the milk samples you need for your experimental design and test the pH, color, consistency, smell and other characteristics of each sample. For each milk sample:

1. Using the Vernier, take the pH of each flask. Record your results.
2. Record the color, odor (sour, putrid), and consistency (coagulation slight, moderate, chunky) for the milk in each flask.
3. Perform Gram Staining as outlined below. Note: We will be using chemicals and open flames. Exercise caution when performing this portion of the lab.

Materials

• Microscope slide
• Bunsen burner and tubing
• Crystal violet (primary stain)
• Iodine solution/Gram’s Iodine (mordant that fixes crystal violet to cell wall)
• Decolorizer (e.g. ethanol)
• Safranin (secondary stain)
• Water (preferably in a squirt bottle)

Procedure

1. Make a slide with your milk sample to be stained. Heat fix the sample to the slide by carefully passing the slide with a drop or small piece of sample on it through a Bunsen burner three times.
2. Add the primary stain (crystal violet) to the sample/slide and let sit for 15 seconds. Rinse slide with a gentle stream of water for a maximum of 5 seconds to remove unbound crystal violet.
3. Add Gram’s iodine for 15 seconds – this is an agent that fixes the crystal violet to the bacterial cell wall.
4. Rinse sample/slide with acetone or alcohol for ~3 seconds and rinse with a gentle stream of water. The alcohol will decolorize the sample if it is Gram negative, removing the crystal violet. However, if the alcohol remains on the sample for too long, it may also decolorize Gram positive cells.
5. Add the secondary stain, safranin, to the slide and incubate for 15 seconds. Wash with a gentle stream of water for a maximum of 5 seconds. If the bacteria is Gram positive, it will retain the primary stain (crystal violet) and not take the secondary stain (safranin), causing it to look violet/purple under a microscope. If the bacteria is Gram negative, it will lose the primary stain and take the secondary stain, causing it to appear red when viewed under a microscope.

QUESTIONS

1. Describe the changing sequence of organisms and corresponding environmental changes during succession in the milk samples. Which bacteria are in each of your milk samples?
2. Describe the changing sequence of organisms and corresponding environmental changes during succession in chocolate milk. Do the results of your investigation match your hypothesis?
3. Compare succession in one or more types milk. Propose reasons for differences.
4. Propose another experiment to test the environmental factors and/or organisms changing in your proposed scenario for milk succession.
5. How could you improve your test of the hypotheses? Be specific!
6. Identify what happened to the pH of the milk as time passed.
7. Infer what the change in pH means about the populations of microorganisms in the milk.

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LICENSES AND ATTRIBUTIONS

CC LICENSED CONTENT, ORIGINAL

• Community Ecology Lab. Authored by: Dr. William Edwards. Provided by: Niagara University. Located at: Niagara.edu. License: CC BY: Attribution

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• Biology 102 Labs. Authored by: Lynette Hauser & Dr. James Holden. Provided by: Tidewater Community Colleg. Located at: http://www.tcc.edu/. License: Public Domain: No Known Copyright