LABORATORY 2 Microbial Diversity; Effectiveness of Hand Washing; Pure Culture Subculture; Light Microscopy Today 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. The objectives of today's laboratory period are to: 1. Discover the diversity of bacteria and fungi in the environment and on our bodies. 2. Use the 4-way streak plate from a mixed culture to pure culture one of the bacterial isolates. 3. Evaluate the effectiveness of hand washing. 4. Master the use of the light microscope. Activity 1: Discovering the Diversity of Invisible Microbes Obtain your individual agar plate and group broth tubes from the back of the lab. 1. Examine the plate (DO NOT OPEN THE LID!) and observe the bacterial and/or fungal colonies growing. Streak Plate of Environmental/Body Specimen Draw a representative sample of the colonies on your plate. How can you tell a bacterial colony from a fungal colony? 10
2. Examine your group s broth tubes. Is the control tube still sterile? How do you know it is/is not sterile? Contrast the usefulness of a T-soy agar plate culture versus a T-soy broth culture. Project 1: Making a Pure Culture (Part II) 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. Subculture: Refers to the transfer of microbes from one culture medium to another. Materials Needed Individual streak plate 1 T-soy agar slant (per student) 4-Way Streak Plate Draw a representative sample of the colonies present on your streak plate. The bacterial species I am going to try to pure culture is: Procedure To subculture the identified bacterial species: 1. Sterilize your transfer loop in the burner flame until the loop turns orange and then let the loop cool for about 15-20 seconds. 2. Using your streak plate, gently scrape up the identified colony with your sterile transfer loop. 3. Immediately streak the loop over the agar surface of the T-soy agar slant. 4. Label the tube and place your slant in the test tube rack on the 22 C incubation tray. 11
Activity 2: The Effectiveness of Hand Washing Obtain your group s hand washing T-soy agar plates and determine how effective hand washing was at reducing the number of bacteria on one s hands. Be ready to give a short one minute report to the class. Activity 3: Examination of Stained Bacterial Cell Preparations with the Light Microscope A. Essential Features of the Light Microscope. You will be using a light microscope in some of the labs this semester. This instrument contains several essential features. Label the essential features of the microscope on the drawing below. Par focal: Working distance: Artifact: 12
Calculating Total Magnification Objective Lens Ocular Lens Total Magnification 10X (low power) 40X (high power) 100X (oil immersion) 10X 10X 10X B. Focusing with the Microscope. To observe specimens in the light microscope, follow the steps listed below. 1. Remove the 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. Move it to a position in which you can look comfortably through the oculars (eyepieces). 3. Make sure the low-power (10X) objective is in place over the hole in the stage. Use the coarse adjustment knob to lower the stage so it is as low as it will go. 4. Turn on the microscope light ("I" on switch) and turn the dial to 6 (brightest). Continued on next page 13
5. Place your specimen slide on the stage and carefully secure it in the mechanical stage. Using the mechanical stage knobs, adjust the position of the sample so that it is over the center of the condenser lens. Make sure the low-power (10X) objective is in position. 6. With the course adjustment knob, raise the stage all the way up. Look through the oculars and use the coarse adjustment knob to focus on the specimen. You will not have to turn the knob very far. When the specimen is in view, use the fine adjustment knob to bring it clear, sharp focus. If necessary, move the specimen into the center of the field. 7. To use the oil-immersion (100X) objective, move the 100X lens into the viewing position. Add a drop of immersion oil to the slide under the objective lens. You should focus on the specimen using only the fine adjustment knob. 8. While at 1,000X total magnification, make all drawings, size measurements, and any other notes in your lab manual. Measuring size: at 1,000X, each division (tic mark) on the scale bar = 1µm (micrometer) 0 10 20 30 40 50 60 70 80 90 100 If you are going to look at another specimen, leave the microscope light on, return to the low-power objective, and lower the stage before removing the slide. Repeat steps 5 through 8. 11. When you are finished using the microscope for the lab period: Make sure the low-power (10X) objective is in position. Lower the stage completely. Turn off the microscope light (O on switch) and turn the dial to 1. Put the microscope in the proper cabinet location. C. Observation of Prepared Slides. Using the focusing steps outlined above, observe and draw representative examples of each of the following bacterial species in the boxes below. Materials Needed Prepared slides of: Micrococcus luteus: A normal bacterial member of our skin microbiome. Neisseria gonorrhoeae: The causative bacterial agent of gonorrhea, a sexually transmitted disease in humans. Rhodospirillum rubrum: A photosynthetic bacterial species found in aquatic environments (ponds, mud, and sewage). Candida albicans: A fungus that grows both as yeast (oval-shaped) cells and as filaments; causative agent of candidiasis ( yeast infections ) in humans. 14
Procedure To observe each prepared slide: 1. Make sure the prepared slide is clean and dry before placing it on the microscope stage. 2. Examine the slide following the focusing steps outlined above. Under 1,000X total magnification (oil immersion), record (in the table provided): a. Morphology (rod, sphere, or wavy spiral) b. Relative size (in micrometers; µm) Morphology: Refers to cell shape. Observations of Prepared Slides Micrococcus luteus Morphology: Neisseria gonorrhoeae Morphology: Rhodospirillum rubrum Morphology: Candida albicans Morphology of oval cells: 15
LIGHT MICROSCOPE REVIEW QUESTIONS 1. What is the purpose for each of the following parts of the light microscope? a. Course and fine focusing 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 and, as such, is having some difficulties using the instrument. Identify how you would attempt to help the student correct the following problems. a. Inability to bring the specimen into sharp focus. b. Insufficient light while viewing the specimen under 1,000X (oil immersion). c. Artifacts in the microscope field. 16