EXPERIMENT. Introduction: Microbiology- Rules for Success

Transcription

1 EXPERIMENT Introduction: Microbiology- Rules for Success Hands-On Labs, Inc. Version MB-01 Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise. Experiment Summary: This section prepares you for successful microbiology experiments. You will review the time allocations for each experiment. You will also be provided with the process for pouring agar plates, the process for incubating, a series of helpful hints, and tools for success. 1 Hands-On Labs, Inc.

2 Objectives Upon completion of this laboratory, you will be able to: Review time allocations for each microbiology laboratory. Describe the process for pouring agar plates. Identify a location for incubating cultures. Review helpful hints and tools for success with microbiology experiments. Time Allocation: 30 minutes 2 Hands-On Labs, Inc.

3 Materials No materials required for this experiment. 3 Hands-On Labs, Inc.

4 Background Time Allocations For each microbiology laboratory, you will complete a background section, a procedures section, and answer a series of open-ended questions. The active time required to complete these activities averages three hours per experiment. Many experiments also require active cultures, poured agar plates, and lengthy incubations. It is essential that you manage your time appropriately. Please review the experiment time allocations in Table 1 prior to starting any experiment. Note: Although general time allocations are provided at the beginning of each laboratory, regularly reference the details below to properly plan. It is recommended that you print a copy of this table and post it for easy reference. Experiment Table 1. Time Allocations for Microbiology Experiments. Active Culture* Plate Pouring** Active Time Incubation Microscopy for Microbiology - Use None None 3.5 hours None and Function Aseptic Technique and Culturing None 1 hour 3 hours 3 to 6 days Microbes Bacterial Enumeration and Plate None 1 hour 3 hours hours Counts Bacterial Morphology and Staining hours None 3 hours None Techniques Antibiotic Sensitivity-Kirby Bauer hours 1 hour 3 hours hours Diffusion Test Biochemical Testing for Microbial hours None 3 hours hours Identification Methyl Red, Voges- Proskauer, and Catalase Testing Biochemical Testing for Microbial hours None 3 hours hours Identification Carbohydrate Fermentation Testing Bacteria Identification through hours None 3 hours hours Functional Media Motility Testing Environmental Influences on hours None 3 hours hours Microbial Growth Salt Tolerance Testing Fomite Transmission None 1 hour 3 hours hours Food Safety 5-7 days 1 hour 3 hours hours *Cultures can be maintained and used for multiple experiments. **Plates may be poured in advance and used for future experiments. 4 Hands-On Labs, Inc.

5 How to Pour Agar Plates The directions for pouring agar plates below, also included in the Appendices folder, will be referenced in each experiment where agar plates are required. Plates may be poured several days or weeks in advance if stored properly. Follow the detailed instructions provided below and/or watch the instructional video included on the Digital Courseware platform to learn how to prepare agar plates. The entire process takes approximately 1 hour, so please plan your time accordingly. 1. Gather and/or locate the following items: a large cooking pot (at least 8-inches deep), stove, access to tap water, test tube rack, roll of paper towels, isopropyl alcohol, oven mitt, permanent marker, pair of safety gloves, coffee mug, test tube clamp, agar tubes listed in experiment step, and Petri dishes listed in experiment step. 2. Use the permanent marker to label the bottom of each Petri-dish with the type of agar it will hold. Do not open the Petri dishes. See Figure 1. Note that the label says NA, which stands for nutrient agar. Figure 1. Labeled Petri dishes. Label the outside of the bottom (smaller) side of each Petri dish with the type of agar. 3. Place the test tube rack in the middle of a large cooking pot on a stovetop burner. Then carefully place the agar tubes into the test tube rack. 4. Add tap water into the large cooking pot until the water level is approximately 1 mm higher than the agar in the tube. The water level must be higher than the agar to ensure that the agar will melt consistently. See Figure Hands-On Labs, Inc.

6 Figure 2. Cover agar line with tap water. The picture on the left shows that the agar line is above the water line. There is not enough water. The picture on the right shows that the water line is approximately 1mm higher than the agar in both tubes. This is the correct amount of water. 5. Carefully loosen the caps of the agar tubes to allow air to escape from the tubes as they are heated. Gently tilt the caps on the tubes so they provide coverage while allowing air to escape from the tubes. See Figure 3. Figure 3. Loosen the caps on the tubes. Carefully loosening the cap of an agar tube as demonstrated in the left picture. The caps of each agar tube are tilted to cover the agar tube while allowing air to escape as demonstrated in the picture on the right. Note that the water level is above the agar line of each tube as shown 6. Turn the stove on and allow the water to come to a rolling boil. The temperature of the water must be above 90 C in order for the agar to melt uniformly. 7. Monitor the agar tubes as the water comes to a rolling boil. Periodically lift the agar tubes from the water to monitor the melting process. Use an oven mitt or paper towels as necessary to protect your hands from the heat of the glass agar tubes, without removing the caps. See Figure Hands-On Labs, Inc.

7 Figure 4. Carefully lift the agar tubes from the hot water to monitor the melting process. 8. Continue monitoring the agar tubes until the agar is completely melted (in liquid form). The agar will appear translucent when it is completely melted. See Figure 5. If the agar has not melted after 10 minutes in boiling water, place a lid on the pot for 2 minutes before rechecking the agar. Continue to check at 2 minute intervals until the agar is completely melted. Figure 5. Melted agar is in liquid form on right and is translucent while the solid agar on the left is cloudy. 7 Hands-On Labs, Inc.

8 9. When the agar is completely melted, turn off the burner, and carefully remove each agar tube. Place the tubes into a coffee mug filled with hot water. See Figure 6. Figure 6. Melted agar tubes in a coffee mug. 10. Pour approximately 1 tablespoon of alcohol onto your working surface and use a paper towel to spread the alcohol across the surface. This will create a sterile working surface to pour the plates. 11. Working quickly, place the labeled Petri dish onto the prepared working surface with the labeled side (bottom) of the plate is touching the surface. Remove the corresponding melted agar tube from the coffee mug, unscrew the lid and place it to the side of the Petri dish. Lift the lid of the Petri dish up, just enough to allow for the pouring of the agar. Gently pour half the contents of the agar tube into the Petri dish. See Figure 7. Note: Use the test tube clamp as necessary to hold the hot agar tube. Note: If the agar from the tube has begun to solidify during pouring, simply hold the agar tube in the still hot water for approximately 1 minute until the agar has returned to a liquid form. 8 Hands-On Labs, Inc.

9 Figure 7. Quickly pouring the melted agar into the labeled Petri dish. 12. Once the agar is poured it should appear as a smooth surface in the Petri dish. If the agar appears slightly lumpy in the dish, it will still work for the experimentation. You will become more proficient with practice. 13. Place the lid of the Petri dish over the poured plate, and leave a slight opening to allow the heat to escape from the plate. See Figure 8. Figure 8. Poured plate. Lid of Petri dish is gently tented over the cooling agar. 9 Hands-On Labs, Inc.

10 14. Allow the poured agar plates to sit, untouched, until they are completely solidified in the Petri dishes. When the agar has completely solidified, the plates can be inverted and the agar will remain in the plates. Plates should remain in the inverted position until inoculated. See Figure 9. Figure 9. Poured and cooled agar plate. 15. When the plates are cooled they are ready to be used in the experiment. The entire process takes approximately 1 hour. Note: Poured plates may be stored in a sealed plastic bag and refrigerated for several weeks before use. Make sure to store the plates inverted (agar side up). See Figure 10. Figure 10. Plates ready for cold storage Hands-On Labs, Inc.

11 Helpful Hints 1. You will be incubating cultures for many of the experiments. Locate a warm area, 21 o C-25 o C, in your home away from drafts and direct light. An empty cabinet is ideal. If cultures must be incubated on a counter or table, make sure to cover with a box and keep secure from children and pets. 2. There are 30 pairs of safety gloves included in the Microbiology kit, allotting two pairs per experiment and 8 extra pairs to be used as necessary. If you require additional nitrile gloves, they may be purchased from a variety of stores and online retailers. 3. Always review the time allocations before beginning a laboratory to ensure that you are managing your time appropriately. 4. Always use aseptic (sterile) technique as instructed in the procedural steps. 5. Always follow safety rules and wear gloves, goggles, and a face mask when working with bacteria. 6. At the end of each exercise, read closely as to whether you should save the samples for future experiments or bleach and dispose of the samples. 7. When beginning an experiment, always wipe down your workspace with a 10% bleach solution or bleach-based cleaning product. 8. After completing an experiment, always wipe down your workspace with a 10% bleach solution or bleach-based cleaning product. Questions A. Approximately how many hours should you allocate for an experiment that requires an active culture, pouring plates, active experimenting, and incubating microbes? B. What is the proper water level for immersing agar tubes in a pot for heating? C. List three characteristics of an ideal incubating site for experimental cultures Hands-On Labs, Inc.