Micro December 2018

• A few strains of bacteria can produce β-glucuronidase such as Shigella, Salmonella, Enterobacter, Citrobacter and Klebsiella and will produce blue to blue-green colonies on the 3M Petrifilm Rapid E. coli /Coliform Count Plate. • Foods with high sugar content may increase the potential for gas production from

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non-coliform Enterobacteriaceae

D. Sample Preparation

1. Use appropriate sterile diluents (BPBD or PPS). Do not use diluents containing citrate, bisulfate, or thiosulfate with the 3M Petrifilm Rapid E. coli/ Coliform

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Count Plates, as they can inhibit growth.

2. For food samples, prepare test portion (10, 11, or 50g) or equivalent ratio of sample to diluent to create a 1:10 dilution as appropriate to the sample being tested.

3. For environmental surface samples, add 25 mL of the appropriate diluent to each

sponge sample.

4. Blend or homogenize sample as appropriate.

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5. For optimal growth and recovery of microorganisms in acidic products (< pH 5), adjust the pH of the sample suspension to greater than pH 5. For acidic products,

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adjust with pH 1N NaOH.

6. Remove all required plates and allow to come to ambient temperature (20-25 o C).

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5. Retract the top film to fully expose the culture medium and dispense 1.0 mL of

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sample onto the center of the plate.

5. Reapply the cover by rolling down the film. Place the 3M Petrifilm Flat Spreader on the center of the plate and press gently to allow sample to spread evenly over the medium causing gel to form. Let the plate sit undisturbed for at least 1 minute. 6. Incubate the plates at 30 ± 1 o C, 32 ± 1 o C, 35 ± 1 o C, 37 ± 1 o C or 42 ± 1 o C for 18-24

hours.

7. Enumerate all blue to blue-green colonies with or without gas regardless of size or

intensity of color as E. coli.

8. Interpretation of non- E. coli coliform colonies varies by reference method.

a. The United States Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 4 defines coliforms as Gram negative rods, which produce acid and gas from lactose during metabolic fermentation. Enumerate red colonies which are closely associated with entrapped gas within one colony diameter of the colony. Colonies not associated with gas (a distance greater than one colony diameter between colony and gas bubble) are not counted as coliforms. The total coliform count consists of both the red colonies with gas and blue colonies with and without gas, countable range is 1-100 CFU.

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