Using a Microwave to Prepare Bacterial Media for Inquiry-Based Experiments Judith A
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Illinois Math and Science Academy DigitalCommons@IMSA Staff ubP lications & Research Student Inquiry and Research (SIR) Fall 2014 Watts Cooking: Using a Microwave to Prepare Bacterial Media for Inquiry-Based Experiments Judith A. Scheppler Illinois Mathematics and Science Academy, [email protected] Follow this and additional works at: http://digitalcommons.imsa.edu/sir_staffpr Part of the Education Commons, and the Microbiology Commons Recommended Citation Scheppler, Judith A., "Watts ooC king: Using a Microwave to Prepare Bacterial Media for Inquiry-Based Experiments" (2014). Staff Publications & Research. 2. http://digitalcommons.imsa.edu/sir_staffpr/2 This Article is brought to you for free and open access by the Student Inquiry and Research (SIR) at DigitalCommons@IMSA. It has been accepted for inclusion in Staff ubP lications & Research by an authorized administrator of DigitalCommons@IMSA. For more information, please contact [email protected], [email protected]. Articles Watts Cooking: Using a Microwave to Prepare Bacterial Media for Inquiry-Based Experiments Judith A. Scheppler Illinois Mathematics and Science Academy Abstract depend on the knowledge base students bring to Microbiology provides an excellent opportunity this experience. to capture student interest, encourage explora- tion, and to begin the development of research Preparation and Sterilization of Luria-Ber- skills. With a low power microwave, similar to tani Agar for Bacterial Growth the type found in homes, and a short list of ma- Bacteria media plates will need to be prepared terials easily obtainable and/or found in many at least one day in advance of carrying out any biology laboratories, you can begin to open this experiment. The following procedure should be exciting world to your life science and biology used to prepare sterile Luria-Bertani (LB) agar classes. Microwaves are available at very rea- media, in a microwave, for use in experiments sonable prices, and can substitute for a much with bacteria. Other kinds of bacterial media more expensive laboratory autoclave. Your stu- may be prepared in a similar manner. Luria- dents can choose and design inquiry investiga- Bertani broth is made with the same formulation, tions as well as learn basic laboratory techniques. leaving out the agar; the agar (similar to gelatin in Jello-O) serves to make the media semi-solid. Bacterial Culture Students across all grade ranges and ability levels Materials are naturally fascinated by the growth of microor- - sterile swabs (Q-tips, though not sterile, are ganisms, especially those growing in their envi- clean and work fine) ronment. However, in order to observe and study - balance bacteria, growth media, or food that the bacteria - microwave need to thrive, must be prepared and sterilized. - hot pads In a research or clinical laboratory this is done by - 1 ml pipetting device and 1 ml pipettes heating the media to 121 ºC and 15 psi pressure, - four sterile plastic petri dishes per student group a process known as autoclaving. This kills both - one 250 ml flask or bottle per group (make vegetative and spore forms of microbes and is the sure it isn’t too tall for the microwave) most effective method of sterilization. However, - wax marking pencil or permanent marker an autoclave (figure 1) can be very costly and is - tryptone not always available in schools with increasingly - yeast extract tight science budgets. Purchasing prepared me- - sodium chloride dia plates can be costly. Using a low power mi- - agar crowave provides an inexpensive alternative for - solution of 0.1 N NaOH preparing sterile media to be used in investiga- - distilled water tions of bacterial growth most often performed - beaker or tray containing 10% chlorine bleach in schools. The depth of these investigations will for disposal of used swabs and Petri dishes Fall 2014 13 Figure 1. Left: A large industrial size autoclave; cost $10,000+ plus plumbing and electrical work required. Middle: A small table-top autoclave; cost ~$2000 - $5000 or more. Right: A household microwave; cost $100 or less. Optional materials for Sabouraud’s agar ingredients. The media should boil vigorously - peptone for about 5 seconds, but microwaving for too - dextrose long will boil the solution over. Once done, the solution should appear clear with no media gran- Procedure ules visible. If the media is not clear, then con- 1) Obtain four sterile Petri dishes and label the tinue microwaving in short 10-15 second bursts bottom with your name, date, and “LB,” then until all components are dissolved (figure 2). turn the plates right side up. This identifies who - This procedure was worked out for a 1000 or made the plates, how old they are, and what me- 1100 watt microwave; microwave ovens of lesser dia is contained in the Petri dish. The Petri dishes power will require slightly longer times. are already sterile, so do not take the lids off the 5) Once all of the ingredients have dissolved, mi- plates until the media is poured into the bottom. crowave the solution for 15-20 seconds to bring 2) Weigh out the following ingredients for “LB” it to boiling. The solution should boil vigorously and place all of them into a 250ml flask or bottle: again, but not boil over. 1 gram of tryptone 6) Bring the media to boiling two more times by 0.5 gram of yeast extract repeating step 5 twice. 1 gram of NaC1 - Be very careful because the bottle is very hot. 1.5 grams of agar Media may superheat. This means that it may boil up when agitated, even after the initial roll- - Use a container that holds two to three times the ing boil has stopped. Allow the bottle to stay in volume of media you wish to make so that the the microwave for a few minutes before handling media can boil vigorously without boiling over. to reduce the danger associated with superheat- Make sure your contained will fit in the micro- ing. Use hot pads when handling the media from wave. the microwave. 7) Place the bottle of LB agar media in a water 3) Add 100 ml of distilled water and 1 ml of 0.1 bath, adjusted to 56 ºC, and allow the media to N NaOH. Do not put a lid on the container. cool. Loosely cover the bottle with its lid or a piece of foil. This temperature will prevent the Students should be assisted with the following media from solidifying, but allow it to be han- steps dled more safely and easily. Petri dishes may 4) Microwave the media for 60-90 seconds to be poured immediately after microwaving, with bring the solution to boiling and to dissolve the close and careful supervision, but it is best to 14 ISTA Spectrum, Volume 40, Number 2 This prevents any moisture, which may have condensed in the lid when the warm media was poured, from falling into the bacteria and con- taminating them or disrupting colony formation. Most bacteria like to grow in warm places. Usu- ally 37 ºC, which is also body temperature, is perfect. Obtaining this temperature, however, requires an incubator that many classrooms may not have. A very viable alternative is to grow the bacteria at room temperature, usually about 25 ºC. The bacteria will still grow, just more slowly than they would at 37 ºC. Try to pick one of the warmer locations in the room, such as away from the windows in the winter. Avoid placing the Pe- Figure 2. Left: Flask with poorly dissolved agar; note the cloudiness of the liquid and the powder granules tri dishes directly onto a radiator or heating vent, at the bottom. Right: Flask with agar that has been however, as the metal may get hot enough when properly heated in a microwave to thoroughly dissolve in direct contact with the plastic Petri dish to the agar. Note that the liquid is clear. melt it and the agar. Growth is usually assessed after 24 hours, but plates may need to incubate for 48 hours or longer if incubation temperatures cool the media to avoid student burns and melt- are less than 37 ºC. ing plates. 8) Pour the liquid LB agar media into the bottom An Inexpensive Incubator of the four petri dishes. Pour slowly and stop A heating pad and Styrofoam container can be when the media has covered the bottom of the used to make an inexpensive incubator. Place a plate (about 20-25 ml). Be sure to work in an heating pad in the bottom of a plastic or Styro- aseptic manner. This means do not leave the Pe- foam bin, then put four jars in each corner and tri dishes open on the lab table and replace the put another bin on top of the jars. Put the plates covers as quickly as possible. in the upper bin, and cover your incubator with 9) Allow the plates to sit at room temperature un- a lid. til the agar has solidified. Do not move or tilt the plates until the media has hardened. Plates may be stored at room temperature for several days before use. Plates should be stored inverted, with the solidified agar media in the “top” so that con- densation (a potential source of contamination) can’t fall onto the agar (figure 3). For longer storage, place the inverted agar plates in a plastic bag to prevent dehydration of the media and store them stacked in the refrigerator. Plates may be stored in the refrigerator for one month or longer. Figure 3. Left: A Petri dish turned upside down. Bacterial Incubation Right: An agar-filled Petri dish with the agar in the Once the media plates have been inoculated bottom; note the condensation on the lid of the dish.