AS 5013.14.2:2009 – Food microbiology Method 14.2: General procedures and techniques-Colony count—Membrane filtration method.
(b) Using sterile forceps. centre a membrane lilter on the membrane support with the grid-marked side upwards.
(c) Place the funnel in position and having lightened it. turn off the vacuum.
(d) Pour a measured volume of sample into the filler funnel. Gently apply vacuum and gradually increase it to that recommended by the supplier of (he membrane. Where no such value is indicated, adjust the vacuum to about —40 kPa.
NOTE: When the volume to bc liltcrcd 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.
(c) When the level of the samplc has fallen to within about 6 mm of the membrane. reduce the vacuum to approsimately —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 filler pad.
NOTE: A sterile Pasteur pipette is convenient for this operation.
(g) lmmediatel’ filtration has ceased, turn oft’ 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: Whetc 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 escessive dry ing of the membrane filter.
The dishes shall be incubated as follows:
(a) Transfer the Petri dishes to the incubator Place the plates in either an upright or an inverted position, according to the instructions for the organisms under test.
(b) Distribute the dishcs in such a manner that overcrowding is avoided and there is flo contact with the sides of the incubator,
(c) Incubate the dishes at the temperature and for the period specified for the organism to be estimated.
Where the medium that has been used does not give rise to a good contrast for the colonies developed, staining of the membrane may be needed. If this is the case, stain the membrane by gently tlooding the surface with a 0.01 percent aqueous solution of malachite green oalate. and after 5 a to 6s contact, pouring off the escess dye.
I If required. subeLilluring of colonies should be carried out bcforc any staining operation
2 Colonies norrnall remain unstained, and the f.ltcr area not covered by colonies is stained a light green
Using a tally counter, coLint the presumptively identified colonies and confirm their identification. Where spreaders occur, count each as a single colony provided that the outer edge of each spreader can be defined.
If the count is greater than 80. repeat the test where possible. using either a smaller volume or a dilution designed to produce a count in the range of 20 to 80.
Ironi the actual count, the number of organisms per unit soluine or per unit mass of the sample shall be calculated taking dilution factors into account.
Where tests hae been carried out on to ditTerent volumes or dilutions (see Clause 7.1) and each membrane has a count ithin the range 20 to O. the Po counts shall be calculated separately and the mean of the to reported as the result (see AS 5013.14).
The report shall conlain the foIIosing information:
(a) Reference to this Australian Standard, i.e. AS 50 13.14.2.
(b) The number, and identity if confirmed, of colony-forming units (CFUs) per unit solume or per unit mass sample. stating that the count sas determined by the membrane filtration method.
(c) The presence of spreading organisms, if encountered.
td) The membrane filters used and the supplier’s specification of pore size.
(e) The culture medium used,
(f) The conditions of incubation.
(g) Details of confirmation used.