Publication No. 10199
Hide and Seek: A Coliform Test
Student Laboratory Kit
Materials Included In Kit
3M® Petrifilm™ E. coli/Coliform Count Plates, 25
Additional Materials Required
(for each student group)
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.
Plate 1: Control plate, sterile water or bottled water only. Neither water source should have coliform bacteria!
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.
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.
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.
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.
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.
Special thanks to John Fedors, San Diego, CA, for the development of this activity.
Hide and Seek: A Coliform Test
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?
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.
(per student group)
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.