Microbes Around Us
Student Laboratory Kit
Materials Included In Kit
Cellophane tape, 3 rolls
Nutrient agar, 25 g
Petri dishes, sterile, 60
Additional Materials Required
Autoclave or pressure cooker
Flasks, 500-mL, 2
Foam plugs, 2
The standard recipe for making 1 L of agar is 23 g of nutrient agar in 1 L of distilled water. One liter of liquid agar will be enough to make 50–60 plates if poured thinly into the bottom of each dish (just enough to cover the bottom).
Add the dry agar powder slowly to the water while heating and stirring to avoid clumping and burning. (Make it like you would gravy.) The agar should be brought to near boiling and the liquid should become a golden clear liquid with no lumps.
Put the liquid agar into a flask(s) and place a foam plug into the top of the flask. It is usually more convenient to put liquid media into smaller flasks and not fill them completely. This allows for easier pouring and less boiling over when in the pressure cooker or autoclave.
Autoclave the flasks of agar at 15 psi for 15 minutes. Be sure to follow the directions for using the autoclave or pressure cooker. If using a pressure cooker, allow it to cool slowly to prevent reboiling and “popping” the foam plugs.
Disinfect the work area (with 10% bleach solution or Lysol®) and place the sterile Petri dishes in a row along the outer edge of the table or counter. When the agar has cooled to 55–60 °C (at this temperature the flask can be safely touched but it will still feel warm) pour the liquid media into each Petri dish. Pick up the top of each Petri dish only long enough to allow the agar pouring. Lift the cover straight up to prevent microbes from falling into the dish. Replace the cover on each disk successively as all the dishes are poured. Make your pouring movements deliberate and create as little draft as possible. Do not breathe into the open dishes.
Allow the agar in the dishes to cool and harden without the dishes being disturbed. Use cellophane tape on opposite sides of each dish to tape the two halves of the dish together so they cannot fall apart. When completely cooled the dishes should be turned over, stacked and placed inside a refrigerator. Poured Petri dishes are always be stored “upside” down to prevent water droplets from dripping onto the agar surface.
This activity requires the use of equipment that may be hazardous if not used properly. Consult the operations manual for procedures related to the use of the autoclave or pressure cooker.
The activity requires the collection of microbes that may be pathogenic. Sterile procedures must be used before, during and after the activity to prevent the growth and spread of possible pathogenic microorganisms. Once the Petri dishes have been taped shut, they should not be opened again. Wash hands thoroughly with soap and water before leaving the laboratory. Please observe all normal laboratory safety guidelines.
Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures governing the disposal of Biological Waste, Type I, and review all federal, state and local regulations that may apply, before proceeding. Obtain a container large enough to hold all of the contaminated plates as well as those used by students. Ensure that all plates are taped shut and then use the container to collect the plates. Carefully pour a 10% bleach solution into the container until all the plates are covered and let them soak overnight. Carefully drain off the used bleach into a laboratory sink with the water running. Using gloves, place all the plates into a plastic garbage bag. Tie off the bag and then place this bag inside a second bag. Label the bag and dispose of in the dumpster yourself or warn the custodian to avoid tearing the bag while transferring it to the dumpster.
- This activity is a great first microbiological experiment because it involves little aseptic technique while dramatically illustrating the need for aseptic technique. Microbes are very likely to grow in all four quadrants of the Petri dishes. Since the “fresh” tape strip is exposed to the air, it is possible for it to be contaminated. Which locations will produce the greatest number or greatest diversity of organisms is difficult to predict. We encounter microbes where we least expect them. Surfaces that appear clean may be contaminated and dirty surfaces may not yield overwhelming numbers of microbes.
- Question 5 on the student record sheet can become your next laboratory activity and it lends itself to an open-ended, student-designed experiment. With some equipment (mostly more sterile Petri dishes) students can experiment with various methods/materials to determine if they can retard microbe growth. Controls become more of an issue in possible extension activities. Be sure to assist students in cleaning up their experimental designs. Results with many “bacteria-fighting products” will likely be surprising (i.e., they won’t work)!
Some authors have suggested a “kitchen microbiology” technique for basic microbiology if sterilizing equipment is not available. Refer to the kitchen microbiology article in the reference section.
- To avoid having to purchase more sterile dishes to test the effectiveness of anti-microbial products, you can have students do the following:
- After dividing the dish into four quadrants (Procedure 4), add the “samples” in one of two ways:
—Use four pieces of tape on 4 different surfaces (Procedures 4–8)
—Use four pieces of tape on the same surface
- Use filter paper and a paper punch to make disks from the paper. Punch the disks directly into clean, used Petri dishes containing distilled water and others containing solutions of anti-microbial chemicals.
- Set up controlled experiments like this:
—If tape from four different surfaces is applied to each quadrant, use only one kind of chemical. Use forceps to place a soaked filter paper disk from the distilled water inside one quadrant over top of the tape imprint. Then, place soaked disks of one chemical in the other three quadrants. Note: Shake excess liquid off the disks before adding them.
—If tape from only one surface is applied to each quadrant, use forceps to place a filter paper disk soaked in distilled water in one quadrant and then, disks from three different chemicals inside the other quadrants over top of the tape imprint.
- After incubation, determine each chemical’s effectiveness at killing each type of microorganism by measuring the diameter of the “zones of inhibition”—clear areas surrounding the filter paper disks.
- The diameters of the zones of inhibition for each chemical may be graphed for comparison.
Correlation to Next Generation Science Standards (NGSS)†
Science & Engineering Practices
Asking questions and defining problems
Planning and carrying out investigations
Developing and using models
Analyzing and interpreting data
Constructing explanations and designing solutions
Engaging in argument from evidence
Obtaining, evaluation, and communicating information
Disciplinary Core Ideas
MS-LS1.B: Growth and Development of Organisms
HS-LS1.B: Growth and Development of Organisms
Systems and system models
Cause and effect
MS-LS1-5. Construct a scientific explanation based on evidence for how environmental and genetic factors influence the growth of organisms.
HS-LS2-2. Use mathematical representations to support and revise explanations based on evidence about factors affecting biodiversity and populations in ecosystems of different scales.
Answers to Questions
- Were any of the surfaces free of microbes? Explain.
Probably not, since no surface, including the tape, was sterilized.
- Which location seemed to have the most microbes? Explain.
Student answers will vary.
- Which location seemed to have the greatest variety of microbes? Explain.
Student answers will vary.
- How could you rid these surfaces of microbes?
By using products designed to kill microorganisms.
- Design an experiment to test your answer to Question 4.
Inoculate microbes in the quadrants of a Petri dish, add specific antimicrobial products within each quadrant and note whether microbial growth occurs.
Kreuzer, H. and Massey, A. Recombinant DNA and Biotechnology, ASM Press, Washington, D.C., 1996.
Wilcoxson, C.; Shand, S. M.; Shand, R. F.; “Kitchen Microbiology”, The American Biology Teacher, Vol. 61, No. 1, January, 1999.