Materials Included In Kit

Additional Materials Required

Prelab Preparation

This kit of materials assumes teacher familiarity with basic microbiological techniques. A working knowledge of sterilization (with an autoclave or pressure cooker), sterile techniques for transferring cultures, and preparation of liquid cultures from agar slant cultures is assumed. If these techniques are not known, a basic microbiology laboratory manual should be consulted.

Detailed instructions for the use of E. coli/Coliform Petrifilm Plates are included in the kit materials. Read the directions carefully before preparing the demonstration materials. This kit of materials is designed to be done in two parts: a teacher demonstration/discussion followed by student-designed experiments.

Four demonstration Petrifilm Plates should be set up, examined, discussed and analyzed as a class before students design their own experiments. The four plates to be set up are:

Plate 1: Control plate, sterile water or bottled water only. Neither water source should have coliform bacteria!

Plate 2: E. coli inoculated, positive results with large number of colonies from a 1:10 dilution.

Plate 3: E. coli inoculated, positive results with smaller number of colonies from a 1:1000 dilution.

Plate 4: Environmental sample, with positive results.

Techniques for the preparation of each plate are briefly outlined as follows:

Plate 1: Follow the directions for inoculating 3M^® Petrifilm™ E. coli/Coliform Count Plate using the directions provided in the kit. Water can be sterilized in test tubes, flasks or other capped containers. Several sterile pipets are provided in the kit for sterile transfers. Follow the directions carefully for inoculation, spreading, and incubation of the E. coli/Coliform plates.

Plate 2: Prepare a suspension of E. coli in a sterile broth or water solution. To do this, pour about 5 mL of sterile liquid (broth or water) into a sterile test tube. Using either a sterile inoculating loop or sterile swab, take a sample of bacteria from the slant culture. Make a lightly clouded suspension of the bacteria in the sterile liquid. Use a 1:10 dilution (1 mL of E. coli solution to 9 mL of sterile water or broth) of this sample to inoculate plate 2.

Plate 3: Divide the solution used to make plate 2 two more times (1:10) to yield a 1:1000 dilution. Inoculate plate 3 with this 1:1000 dilution.

Plate 4: Set up a serial diluted plate (1:100) from a place in your environment that you feel would be of interest to your students. Two suggested possibilities are:

1. 1. Add 1 drop of E. coli original suspension to a sample of coffee “half and half.” (The small containers you get in a restaurant.)
   2. Use sterile instruments to chop up soil or other solid material. Take a measured sample and add it to 100 mL of sterile liquid. Mix and let sit for 20–30 minutes to allow the material to settle. Pour a 1-mL test sample of the supernatent liquid into a sterile test tube. This supernatent liquid can be plated to test for coliforms.

Note: Coliform bacteria like a slightly alkaline environment. Sterile water tends to absorb CO₂ and becomes slightly acidic. Bottled water tends to have a pH of 7.5–8.0. Try to maintain sterile water in this pH range for better results. Sterile phosphate buffers can be used if available.

Coliform bacteria prefer to grow at 42 °C. Pathogens also do well at this temperature. Coliform bacteria will grow slower at lower temperatures, 25–30 °C, but will give similar results. Therefore, growing the coliforms at this lower temperature decreases the chance of growing unwanted pathogens.

Incubate the four demonstration plates until you have desired results. Plates can then be kept in a freezer indefinitely and reused for many classes in the future. Store in a zipper-lock bag.

Tape the four demonstration plates shut so that students cannot open them. Have students examine the four demonstration plates and make note of their observations. Discuss the treatment of each plate and the results illustrated. Explain the use of Petrifilm Plates, inoculation and spreading procedures. Discuss coliforms and the “coliform test” provided by Petrifilm Plates.

Safety Precautions

There is always the potential, when dealing with microorganisms, for contamination by pathogens. The proper disposal and handling of microbiological material is critical. Always use sterile techniques when handling cultures and encourage such behavior in your students. Set a good example for sterile technique when handling Petrifilm Plates. Be sure to tape all plates shut so they cannot be opened. Be sure to sterilize work areas before and after handling microbes and have students wash their hands before leaving the laboratory area. Microorganisms are opportunistic and compete with us for nourishment—no food should ever be allowed in the laboratory.

Disposal

To be safe, all bacterial cultures taken from wild and all recombinant bacteria should be assumed pathogenic and destroyed before thrown in trash. Liquid cultures can be killed by adding bleach to reach a 10% concentration. For example, add 10 mL of household bleach to 100 mL of culture. Swabs should never be allowed to touch the lab bench and should be immediately disposed of in glass jars with either 10% household bleach or 70% ethanol. Keep these jars on all student lab benches so that they are convenient for students. The best way to dispose of bacterial cultures is to pressure sterilize them in a heat stable biohazard bag. This is done in a standard pressure cooker or autoclave. The biohazard bag then can be discarded into the trash. Using Petrifilm™ Plates reduces the volume of disposable material. If sterilizing equipment is not available, an alternative is to soak the materials in bleach solution. Saturate agar plates, slants, or Petrifilm Plates with 10% household bleach. Let them sit overnight in the solution, pour off the excess, and dispose of them in the trash.

Utilize community resources to help with disposal procedures and “piggy-backing” activities. Consider contacting the following for assistance with disposing of bacterial cultures.

• School nurse
• University laboratories
• Research labs
• Hospitals

Lab Hints

• It is easy to count coliform colonies on Petrifilm E. coli/Coliform Count Plates. An indicator dye in the plate colors all coliform colonies red, and the top film traps gas produced by the coliforms. Coliforms produce red colonies which have gas bubbles. Non-coliform colonies are red but do not have gas bubbles.
• The E. coli/Coliform Count Plates also contain a glucouronidase indicator dye. Glucouronidase, which is produced by most E. coli, reacts with the indicator dye to form a blue precipitate around the colony. Most E. coli also produce gas which is trapped by the top film. Count the blue colonies that have gas bubbles to get an E. coli count. Count all red and blue colonies with gas bubbles to obtain a total coliform count.

Teacher Tips

• Store unopened E. coli/Coliform Count Plate foil pouches at or below 46 °F (8 °C). Use before expiration date on package. To seal an opened package, fold end over and tape shut. Keep resealed package at ≤25 °C (≤77 °F) and ≤50% relative humidity. Do not refrigerate opened packages. For best results, use Petrifilm Plates within one month after opening.

References

Special thanks to John Fedors, San Diego, CA, for the development of this activity.

Petrifilm materials are a 3M^® manufactured product and are to be used for non-commercial research and development purposes only.

Introduction

Water that looks very clear may contain microorganisms that could be a health hazard. Food that looks edible may actually be harboring dangerous pathogens. How are these items tested to see if they are safe from harmful microorganisms?

Concepts

• Indicator organisms

• Sterile technique
• Serial dilution

Background

A number of significant disease organisms are water borne. Even water that looks clear and pure may be sufficiently contaminated with pathogenic microorganisms to be a health hazard. Some means of testing is necessary to ensure that drinking water is safe. It is usually not practical to examine water directly for all pathogenic organisms. In practice, indicator organisms are used instead. These are organisms, usually associated with the intestinal tract, whose presence in water indicates that the water has received contamination of an intestinal origin. The most widely used indicator organisms are the organisms in the coliform group. This group is defined in bacteriology as all the aerobic and facultatively anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria that ferment lactose with gas formation within 48 hours at 42 °C. This is an operational definition and not a taxonomic one. The coliform group includes a variety of organisms, including the ever present E. coli.

Organisms in the coliform group are suitable as indicators because they are common inhabitants of the intestinal tract in both humans and other warm-blooded animals in large numbers. When excreted into the water, the coliforms eventually die, but slower than pathogenic bacteria that behave similarly during the water purification process. Thus, it is likely that if coliforms are found in a water sample, the water has received fecal contamination, not undergone purification, and is unsafe for drinking purposes. The presence of coliform organisms in food or water serves as an indicator (an index) of contamination and implies the potential presence of pathogens.

It is easier and cheaper to demonstrate the presence of coliforms than pathogenic organisms. Thus, the use of coliform tests is a quick and easy way to infer the presence of pathogens. Positive coliform tests are used as a basis for doing more expensive and extensive test procedures.

The incubation time and temperature for coliform testing can vary with more sophisticated methods. The suggested methods for 3M^® Petrifilm™ Plates are widely accepted. Petrifilm Plates have been used in the food industry and have been approved for use by international food monitoring agencies.

Materials

Safety Precautions

There is always the potential, when dealing with microorganisms, for contamination by pathogens. Always follow sterile techniques illustrated by your teacher. Tape your Petrifilm plates shut after inoculation and do not reopen them. Dispose of them as directed by your teacher.

Procedure

1. Form a research group to investigate a potential source of coliforms. Note: Do not sample the inside surface of toilets.
2. Discuss your selection of materials and area to be explored with your teacher.
3. Review your procedure and be sure to include controls.
4. Secure Petrifilm Plates and other needed materials.
5. Collect samples and inoculate the Petrifilm Plates. Tape the plates shut and do not open them again for the rest of the experiment.
6. Incubate plates and make observations after 24, 48 and 72 hours.
7. Write a complete laboratory report for your exploration. Include: (a) statement of your hypotheses; (b) description of your procedures and experimental design (including any safety precautions you followed); (c) record of your data and observations; (d) interpretation of your results; (e) indications of complications from your results or further activities to try.
8. Share your results with other investigators in your class.
9. Discuss the following questions as a class:

• • What role does coliform bacteria perform in nature?
  • Why are coliform bacteria used as indicator organisms?
  • How might we be aware of the presence of coliforms?
  • What precautions should food manufacturers take?
  • Since these organisms are so common, how are they kept in check?