Microbes in Everyday Life; Pure Culture Project; Hand Washing; Light Microscopy Lab 2 T oday s lab looks at the results of microbial diversity and continues the project of working toward producing a pure culture. The effectiveness of hand washing is also examined. New activities include microscopy with viewing and measuring the sizes of bacterial cells. Exercise 1: Microbes in Everyday Life Objective To identify the diversity of microbes in the environment (soil, leaves, etc.) and on fomites; or on the human body (skin swabs). Analysis (A) T-Soy Agar Plate (This is an individual activity) 1. Obtain your individual agar plate from the back of the lab. 2. Examine the plate (DO NOT OPEN THE LID!) and observe the bacterial and/or fungal colonies growing. 3. Draw a representative sample of Streak Plate of Environmental/Body Specimen the colonies on your plate. How can you tell a bacterial colony from a fungal colony? (B) T-Soy Broth (This is a lab group activity) 1. Obtain your group s broth tubes from the back of the lab. 2. Examine the broths. Is there any growth in the C tube? Is there any growth in the E tube? Contrast the usefulness of a T-soy agar plate culture versus a T-soy broth culture. 11
Pure Culture Project Part II: Making a Subculture Objective To identify a bacterial colony and transfer it to another growth medium. Materials Needed Individual streak plate 1 T-soy agar slant (per student) Subculture: Refers to the transfer of microbes from one culture medium to another. Procedure 1. Draw a representative sample of the colonies present on your streak plate. 2. By consulting with Dr. P, use your streak-plate to subculture one of the three bacterial species [Serratia marcescens, Micrococcus luteus, or Escherichia coli] onto a sterile T-soy agar slant using aseptic technique. 4-Way Streak Plate The bacterial species I am going to try to pure culture is: 3. To subculture the identified bacterial species: a. Sterilize your transfer loop in the burner flame until the loop turns orange and then let the loop cool for about 15 seconds. b. Using your streak plate, gently scrape up the identified colony with your sterile transfer loop. c. Immediately streak the loop over the agar surface of the T-soy agar slant. d. Label the tube and place your slant in the test tube rack on the 22 C tray. 12
Experiment: The Effectiveness of Hand Washing Hypothesis: Hand washing will decrease the number of bacterial cells on the hand surface. Analysis Obtain your group s hand washing T-soy agar plates. Observe the plate(s) and make representative drawings in the diagrams below. Discuss with your lab partners how effective hand washing was at reducing the number of bacteria on the hand surface. Be ready to give a short one minute report to the class. Handwashing plate 1 Handwashing plate 2 Handwashing plate 3 13
Exercise 2: Examination of Stained Bacterial Cell Preparations with the Light Microscope A. HOW TO USE THE LIGHT MICROSCOPE: Identifying the parts of the light microscope. You will be using a light microscope in some of the labs this semester. This instrument contains several essential features. Watch the first part of the video [How to Use the Light Microscope] and label the essential parts of the microscope on the drawing below. Par focal: Important Terms Artifact: Calculating Total Magnification Objective Lens Ocular Lens Total Magnification 10X (low power) 100X (oil immersion) 10X 10X 14
B. HOW TO USE THE LIGHT MICROSCOPE: Preparing the light microscope for use. Before observing specimens in the light microscope, make sure the following have been completed: Watch part 2 of the video [How to Use the Light Microscope] and follow along with the information written below. My Microscope Number 1. Obtain your light microscope from the cabinet. Always pick up your microscope correctly; grasp it firmly with two hands, one hand under the base and the other on the arm. 2. Place the microscope on your lab bench, away from the edge. The microscope is positioned so you can look comfortably (sitting or standing) through the oculars (eyepieces). 3. Record its number. You will use this same microscope whenever you need to use a microscope in lab 4. Use the coarse-focusing knob to lower the stage. The low-power (10X) objective is in place above the stage. 5. Use lens paper to first clean the condenser lens and then the 10X and 40X lenses. Finally use the lens paper to clean the 100X (oil-immersion) lens. 6. Plug in the power cord and turn on the microscope light ("I" on switch). Rotate the dial to its highest setting (6 - brightest). 7. Adjust the distance between oculars to match the distance between your eyes. You should see a single, circular microscope field. C. HOW TO USE THE LIGHT MICROSCOPE: Observations with the light microscope. Once the microscope is set up, you are ready to observe specimens. Here are the steps you should follow: 8. Place your specimen slide (specimen on top side) on the mechanical stage. 9. With the course-focusing knob, raise the stage all the way up. Use the mechanical stage knobs to position the specimen over the center of the condenser lens. The iris diaphragm is shut as far as possible. 10. Look through the oculars and slowly rotate the coarse-focusing knob until the specimen comes into focus. Use the fine-focusing knob to bring the image into clear, sharp focus. If necessary, move the specimen into the center of the field. 11. To use the oil-immersion (100X) objective, rotate the 10X lens so you are between the 10X and 100X lenses. Add a drop of immersion oil to the slide and rotate the 100X lens into position. The lens will touch the oil. Open the iris diaphragm and focus on the specimen using only the fine-focusing knob. 15
D. HOW TO USE THE LIGHT MICROSCOPE: Observation of prepared slides. Using focusing steps 8-11 outlined above, observe and draw representative examples of each of the following microbes in the boxes on the next page. Materials Needed Each Styrofoam box contains a prepared slide of: Streptococcus pneumoniae: A bacterial pathogen transmitted to the respiratory system. In the lungs, it may cause pneumonia. Neisseria gonorrhoeae: A bacterial pathogen transmitted through sexual contact. In the reproductive system, it may cause gonorrhea, a common sexually transmitted infection (STI). Clostridium botulinum: A bacterial pathogen transmitted in improperly prepared food. In the nervous system, the toxin causes botulism, a rare type of food poisoning. Rhodospirillum rubrum: A nonpathogenic, photosynthetic bacterial species found in aquatic environments (ponds, mud, and sewage). Refers to cell shape. Procedure To observe each prepared slide: 1. Make sure the prepared slide is clean and dry before placing it on the microscope stage. Find the organism using the 10X objective lens. 2. When under the 100X oil immersion lens (1,000X total magnification), in the space provided in each box: a. Record the organism s morphology (rod, sphere, or wavy spiral) Watch the video [How to Use the Light Microscope] on measuring cell size and follow along with the information written below. b. Measure the cell size (in micrometers; µm) At 1,000X, each division (tic mark) on the ocular micrometer (measuring ruler) = 1µm (micrometer) 3. When finished with the slide, rotate the 10X objective lens into position, remove the slide, and clean it with soap and water. 4. If you are going to look at another specimen, leave the microscope light on and repeat steps 8 11. E. HOW TO USE THE LIGHT MICROSCOPE: Putting the microscope away. Before putting the microscope away: Make sure the low-power objective is in position and the stage is lowered completely. Turn off the microscope light (O on switch), turn the dial to 1, and unplug the power cord. Wipe off the 10X and then the 100X objectives with lens paper. 16
Observations of Prepared Slides Cell size: Streptococcus pneumoniae Cell size: Neisseria gonorrhoeae Clostridium botulinum Rhodospirillum rubrum Cell size: Cell size: 17
LAB 2 REVIEW QUESTIONS Some of these questions could be part of a lab quiz. 1. What is the purpose for each of the following parts of the light microscope? a. Course and fine adjustment knobs b. Iris diaphragm (contained in condenser lens) c. Mechanical stage d. Objective lenses e. Oculars (eyepieces) 2. A classmate missed the lab explaining the use of the microscope. Now, she is having some difficulty using the instrument. Identify how you would attempt to help the student correct the following problems. a. Insufficient light (microscope field appears dark) when viewing the specimen under oil immersion (100X objective). b. Artifacts are seen in the microscope field. 18