AS 5013.14.2:2009 pdf free download – Food microbiology
7.1 General
The optimum volume of liquid to be used will depend upon the amount of undissolved solids in the sample and the expected count. If the expected count is high, suitable dilutions of the sample should be made (see Note). Where the expected count is uncertain, it is recommended that two determinations be made using two different volumes. In this way, the probability that at least one determination will be within the range 20 to 80 will be increased. NOTE: The optimum number of colonies on the filter is about 50 and the volume of sample filtered should be such that the number of colonies to be counted on the membrane is not greater than 80. For example, samples expected to contain less than 80 organisms per 100 mL require the filtration of at least 100 mL of sample for each test.
7.2 Procedure
The procedure shall be as follows: NOTE: Where a pre-assembled filtration unit is used, Steps (a) to (c) below are not required.
(a) Assemble the funnel base in the filter flask and connect the flask to the source of vacuum. Apply a slight vacuum.
(b) Using sterile forceps, centre a membrane filter on the membrane support with the grid-marked side upwards.
(c) Place the funnel in position and having tightened it, turn off the vacuum.
(d) Pour a measured volume of sample into the filter funnel. Gently apply vacuum and gradually increase it to that recommended by the supplier of the membrane. Where no such value is indicated, adjust the vacuum to about −40 kPa. NOTE: When the volume to be filtered is less than 10 mL, add at least 20 mL of sterile diluent to the funnel before addition of the sample to aid uniform dispersion of the bacteria over the entire surface of the membrane during filtration.
(e) When the level of the sample has fallen to within about 6 mm of the membrane, reduce the vacuum to approximately −10 kPa. Rinse the sides of the funnel with 20 mL to 30 mL of diluent, added from the graduated cylinder used to measure the volume of the sample.
(f) Remove any liquid medium in the Petri dish in excess of that required to saturate the filter pad. NOTE: A sterile Pasteur pipette is convenient for this operation.
(g) Immediately filtration has ceased, turn off the vacuum at the control tap, disconnect the funnel and remove the membrane using sterile forceps. Roll the membrane grid- marked side upwards, on the filter pad or on the solid medium, taking care to avoid entrapping air bubbles between the membrane and the substrate. Replace the lid on the Petri dish. NOTE: Where there is no control tap directly under the funnel, it may be necessary to release the vacuum just before the completion of filtration, to avoid excessive drying of the membrane filter.