AS 5013.14.2:2009 pdf free download – Food microbiology Method 14.2: General procedures and techniques—Colony count-Membrane filtration method

AS 5013.14.2:2009 pdf free download – Food microbiology Method 14.2: General procedures and techniques—Colony count-Membrane filtration method.
Where a solid medium is used, the plates should be prepared as follows:
(a) Pour sufficient volume of molten medium into a Petri dish to give a depth of approximately 5 mm. Allow to set.
(b) As soon as the medium has set. remove excess moisture from the plates by one of the following methods:
(i) Incubate the plates open with the internal surface of the base facing downwards and with the base resting on the lid, for the minimum time necessary to obtain plates free from condensate. e.g. at 37°C for about 2 h or at 45°C for about I h. Do not dry plates at temperatures above 45°C.
(ii) Incubate at 37°C for 16 h in the inverted position with the lids on. If the plates are not then free of condensate. open them and incubate as described in Step (i) above until dry.
(iii) Other suitable time/temperature regimes. For example, on bench over night. NOTE: In humid climates either extend drying time or place a tray of desiccant, such as silica gel, in the base of the drying oven.
7 FILTRATION PROCEDURE
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 say. 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 thai 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 he 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.
(f) Remove any liquid medium in the Petri dish in excess of that required to saturate the filter pad.
NOTE: A sterile Pasteur pipetie 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 upssards. 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 aoid excessive drying of the membrane filter.
8 INCUBATION
The dishes shall he incubated as follows:
(a) Transfer the Pctri 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 dishes in such a maniier that overcrowding is avoided and there is no 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.
9 STAINING, CoUNTING AND IDENTIFICATION (Sec Note 1)
Where the medium that has been used does not give rise to a good contrast for the colonies developed, staining of the membrane ma be needed. If this is the case, stain the membrane by gently flooding the surface sith a 0.01 percent aqueous solution of malachite green oxalate, and after 5 s to 6 s contact. pouring off the excess dye.
NOTES:
I If required, subculturing of colonies should be carried out before any staining operation.
2 Colonies normally remain unstained, and the filter area not covered by colonies is stained a light green.
Using a tally counter, count the presumptively identified colonies and confirm their identification. Where spreaders occur, count each as a single coIon provided that the outer edge of each spreader can be defined.