Prokaryotic Diversity! and Ecological Succession in Milk Name INTRODUCTION Milk is a highly nutritious food containing carbohydrates (lactose), proteins (casein or curd), and lipids (butterfat). is high level of nutrition makes milk an excellent medium for the growth of bacteria. Pasteurizing milk does not sterilize it (which would kill all bacteria) but merely destroys pathogenic bacteria, leaving many others that will multiply very slowly at refrigerated temperatures. At room temperature, however, 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. Community succession is a phenomenon observed in the organizational hierarchy of all living organisms, from bacterial communities in milk to animal and plant communities in a maturing deciduous forest. In each example, as one community grows, it modifies the environment, and a different community develops as a result. In this laboratory exercise, you will work in teams of 3-4 and observe successional patterns in two types of milk: plain, whole milk and chocolate milk that has been supplemented with sucrose and chocolate. You will record changes in the environmental conditions of the two types of milk as they age. Below, note certain observations scientists have made about milk bacteria and their environment: 1. Lactobacillus (gram-positive rod) and Streptococcus (gram-positive coccus) survive pasteurization. 2. Lactobacillus and Streptococcus ferment lactose to lactate and acetic acid. 3. is acidic environment causes casein to solidify, or curdle. 4. Two bacteria commonly found in soil and water, Pseudomonas and Achromobacter (both gram-negative rods), digest butterfats and give milk a putrid smell. 5. Yeasts and molds (both fungi) grow well in acidic environments. On each lab bench are flasks of plain whole milk and chocolate milk. One flask of each has been kept under refrigeration. One flask of each has been at room temperature for 24 hours, one for 4 days, and one for 8 days. On each bench, there are also SDA (Saboraud dextrose agar) plate cultures of each of the milk treatments. SCENARIO Propose a scenario (the hypothesis) for bacterial succession in each type of milk: 1
PROCEDURE: OBSERVATION OF THE MILK FLASKS AND AGAR PLATES 1. Using the ph paper provided, take the ph of each flask. Record your results in Tables 1 and 2. 2. Observe the odor (sour, putrid), color, and consistency (coagulation slight, moderate, chunky) for the milk in each flask. Record your results in Tables 1 and 2. 3. Using the SDA plates, observe and describe bacterial/fungal colonies in each age and type of milk. Record the shape, margin, and texture of the colonies in Tables 1 and 2. Refer to the figure below: PROCEDURE: PREPARATION OF GRAM STAINS FROM AGAR PLATES PART 1 PREPARATION OF THE SMEAR At your table there should be slides labeled Fresh, 24-hr, 4-day, or 8-day from either whole milk or chocolate milk. Each sample was taken from the corresponding agar plate developed from each milk flask. For each slide, the following steps have been done for you in order to fix the bacteria to the slide: 1. e slide was cleaned using alcohol. 2. e sample was spread on the slide and allowed to air-dry. 2
3. e slide was gently passed through the flame of a Bunsen burner several times. is process facilitates the adherence of the bacteria to the slide so that they will not wash off when you stain. PART 2 THE GRAM STAIN 1. Place the slides on the staining rack. 2. Cover the smears with the primary dye, crystal violet, and let it stand for 30 sec. 3. Rinse the slides with water for 5 sec. 4. Apply the mordant, Gram s Iodine, by covering the smears and let it stand for 1 min. 5. Rinse the slides with water for 5 sec. 6. Decolorize with ethanol for 15-30 sec. Add the ethanol slowly until the crystal violet no longer floats up from the slide. Be careful not to decolorize for too long. 7. Rinse the slides with water for 5 sec. 8. Cover the smears with the counter stain, safranin, and let stand for 1 min. 9. Rinse the slides with water for 5 sec. 10. Place the slide within the pad of bibulous paper and blot dry, gently patting. Do not rub the slide as this will remove some of the bacteria. Repeat this process with your other sample. OBSERVING YOUR SLIDE USING THE OIL IMMERSION MICROSCOPE 1. Place slide on the stage of the microscope. 2. View your specimen under the microscope under each of the objective lenses (4x, 10x, and 40x). 3. Move the 40x objective lens out of position so that no objective lenses are over the specimen. 4. Place a drop of oil directly on the slide where you were viewing the specimen. 5. Move the 100x oil immersion objective lens into position. 6. Use the fine adjustment knob to bring your specimen into focus. 7. When you have viewed the slide, rotate the nosepiece so that the lowest power objective lens is over the slide. Move the coarse adjustment knob to raise the objective lenses and then remove the slide. 8. Record your observations in Tables 1 and 2. 9. Place another slide on the stage and repeat steps 1-7. 10. Pool you data with that of the others in your group, so that you can clearly observe a gram stain made from the samples in each of the four flasks from both types of milk. NOTE: DO NOT PUT YOUR MICROSCOPE AWAY UNLESS I HAVE CHECKED IT FOR ANY REMAINING OIL! 3
Age of Milk Refrigerated 24-hr 4-day 8-day Table 1. Physical features and microorganism communities of aged plain whole milk. Flask Characteristics Characteristics of Organisms Present Bacterial Characteristics ph Consistency Odor Colony Characteristics (see Figure) Gram +/- Cell shape Colony Shape Colony Margin Surface Texture Yeast/Fungi Present? 4
Age of Milk Refrigerated 24-hr 4-day Table 2. Physical features and microorganism communities of aged chocolate milk. Flask Characteristics Characteristics of Organisms Present Bacterial Characteristics ph Consistency Odor Colony Characteristics (see Figure) Gram +/- Cell shape Colony Shape Colony Margin Surface Texture 8-day Yeast/Fungi Present? 5
DISCUSSION 1. Describe the changing sequence of organisms and corresponding environmental changes during succession in plain milk. Do the results of your investigation match your hypothesis? 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 plain and chocolate milk. Propose reasons for differences. 6