6.2.1 Have sterile diluent ready. Disinfect the surface of the work area.

6.2.2 Place the Petrifilm plate on a flat surface. Mark identifying sample information on film.

6.3.1 To ensure a representative analytical unit, agitate liquids until the contents are homogeneous. If the food sample is a solid, take representative portions from several locations within the sample. Prepare a 1:10 dilution in an appropriate (see 5.2) diluent. Mix thoroughly.

6.3.2 For acid products adjust pH of the diluted sample to 6.6 - 7.2 with 1N NaOH.

6.4.1 Lift the top film and carefully inoculate 1 mL of sample or diluted sample to the center of the bottom film. Either a pipette or a pipettor can be used for sample addition.

6.4.2 Replace top film. Carefully roll top film down to avoid entrapping air bubbles.

6.4.3 Distribute sample evenly using a downward pressure on the center of the plastic spreader, flat side down. Do not slide the spreader across the film. Leave plate undisturbed for at least 1 minute to permit the gel to solidify.

6.4.4 Return unused plates to foil pouch. Seal pouch by folding and taping the open end. Store resealed foil pouch in a cool dry place. Use plates within one month after opening pouch. Exposure of Petrifilm plates to temperatures above 25 EC and/or humidities >50% RH can affect the performance of the plates. Do not use plates that show orange or brown discoloration. Expiration date and lot number are Noted on each package of Petrifilm plates. The lot number is also Noted on individual test films.

6.4.5 Incubate plates with the clear side up in stacks not exceeding 20 units. Follow current industry standards for incubation temperature. Incubate plates 24 ± 2 hours. Examine for coliforms.

6.5.1 Count plates promptly after incubation period. If impossible to count at once, store plates in the freezer. This should be avoided as a routine practice.

6.5.2 Use a standard colony counter for counting purposes. A magnified-illuminator may also be used to facilitate counting.

6.5.3 The circular growth area is approximately 20 cm². Estimates can be made on plates containing more than 150 colonies by counting the number of colonies in one or more representative squares and determining the average number per square. Multiply the average number by 20 to determine the total count per plate.

6.5.4 Calculate the number of colonies per mL or g of sample from the number of colonies obtained on plates chosen at dilution levels which give a statistically significant result.