3M® Petrifilm™ E. coli/Coliform Count Plates

Introduction

The Petrifilm™ plate ready-made media system allows for easy counting of coliform colonies. An indicator dye in the plate colors all colonies red, and the top film traps gas produced by the coliform. In addition, the E. coli/Coliform Count Plate contains a dye that produces a blue precipitate around E. coli colonies. By counting all colonies, both red and blue, a total coliform count can be taken.

Materials

Safety Precautions

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. Use Petrifilm plates within one month after opening.

Disposal

After use, Petrifilm™ E. coli/Coliform Count Plates will contain viable microbes. Do not open the plates unnecessarily. Use sterile techniques when handling them. Before disposal, plates should be soaked in a 10% bleach solution or autoclaved. They should then be disposed of in the normal garbage disposal channels. Set a good example for sterile technique for students when handling Petrifilm plates. Be sure to sterilize work areas before and after handling microbes and have students wash their hands before leaving the laboratory area.

Procedure

Caution: This activity requires the use of hazardous components and/or has the potential for hazardous reactions. Be sure to review the Safety Precautions and Disposal sections and use sterile techniques when using these materials.

1. Place the Petrifilm E. coli/Coliform Count Plate on a flat surface (see Figure 1).

2. Lift the top film, hold a pipet perpendicular to the plate, and carefully dispense 1 mL of sample onto the center of the bottom film (see Figure 2).

3. Carefully roll the top film down to avoid trapping air bubbles. Do not let the top film drop (see Figure 3).

4. With the flat side down, place the Petrifilm spreader on the top of the film over the inoculum (see Figure 4).

5. Gently apply pressure on the spreader to distribute the inoculum over a circular area. Simply apply a gentle downward pressure to the spreader. Do not twist or slide the spreader (see Figure 5).

6. Lift the spreader. Wait at least one minute for the gel to solidify (see Figure 6).

7. Incubate Petrifilm E. coli/Coliform Count Plates with the clear side up in stacks of up to 20. To minimize moisture loss inside the incubator, add a glass culture dish containing water. Incubate the plates for 24–48 hours at 42 °C.
8. Coliform counts can now be determined using a standard colony counter or other magnified light source.

Discussion

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.

In addition, the E. coli/Coliform Count Plate contains a glucuronidase indicator dye. Glucuronidase, 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.

References

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