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Determination of Enterobacteriaceae
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7.3.3.3 In some instances it may be desirable to prepare the initial dilution on a percent basis in order to obtain a more accurate weight of the material to be tested than is provided for by the customarily employed dilution ration basis, i.e., a 10% solution (suspension) is represented by 10 g (mL) per 100 g (mL) of solution (suspension), whereas a 1:10 dilution is based on 10 g (mL) of product (solute) plus 90 g (mL) of diluent (solvent).
7.3.4 Check the pH of the food suspension. If the pH is outside the range of 5.5-7.6, adjust the pH to 7.0 with sterile 1N NaOH or HCl.
7.3.5 Prepare succeeding decimal dilutions as required, using a separate sterile pipette for making each transfer.
7.3.6 Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present.
7.4 Plating
7.4.1 Medium used is Violet Red Bile Glucose (VRBG) agar. Prepare according to manufacturer's directions.
7.4.2 Temper prepared melted agar in a water bath at 45°C ± 1E ensuring that the water level is 1 cm above the level of the medium in the bottles.
7.4.3 Mark clearly the bottom half of duplicate Petri plates, identifying sample, and dilution.
7.4.4 Agitate each dilution bottle to resuspend material that may have settled out during preparation.
7.4.5 Pipette 1 mL or 0.1 mL of the required dilutions to appropriately marked duplicate Petri plates.
7.4.6 In the case of products that tend to adhere to the bottom of the plates, add the inoculum to 1.0 mL of sterile diluent previously placed in the Petri plate.
7.4.7 Remove agar bottles from the water bath and remove excess water from the bottle exterior with a clean disposable towel.
7.4.8 Pour 10-15 mL of tempered agar into each plate, and mix by rotating and tilting. Plates should be poured within 15 min of preparation of the dilutions. Allow agar to solidify.
7.4.9 Pour a cover layer of approximately 10 mL of VRBG over all plate contents. Allow cover layer to solidify.
7.5 Incubation
Incubate plates in the inverted position at 35°C ± 0.5° for 48 ± 2 hr. Avoid crowding or excessive stacking of plates to permit rapid equilibration of plates with incubator temperature.
7.6 Counting Colonies
7.6.1 Count colonies promptly after the incubation period.
7.6.2 If possible, select plates with 20-200 colonies (including pinpoint colonies). If counts do not fall within this range select plates that fall nearest to the 20-200 range.
7.6.3 Count colonies using a colony counter and a tally register. Count the colonies that are rose-coloured and surrounded by a halo of purple precipitate.
7.6.4 Calculate the count per g of presumptive Enterobacteriaceae by multiplying the average count per plate x the dilution factor. If 0.l mL of the dilution is used, multiply x 10 to calculate the count per g(mL).
