AS 4276.3 pdf download

AS 4276.3 pdf download.Water microbiology
1  Scope and general
1.1  Scope
This Standard specifies methods for enumeration of heterotrophic microorganisms in water using the following techniques: (a) Pour plate. (b) Spread plate. (c) Spiral plate. (d) Membrane filtration. (e) Most probable number (MPN). These techniques are intended to measure the operational effectiveness of treatment processes and be used for the general examination of many types of water to evaluate and/or note changes in water quality. This document does not cover the comparison of results obtained from different techniques.
6  Quality assurance
6.1  General The laboratory shall have a clearly defined quality control system to ensure that the apparatus, culture media, reagents and technique are suitable for the test. NOTE  The use of controls and blanks, as appropriate, is part of the quality control system. 6.2  Performance testing of culture media The performance of the culture medium shall be tested in accordance with the method and criteria in AS 5140. Table 6.1 provides a summary of expected performance results. NOTE  Refer to AS 5140 for the following: (a) Definitions of productivity. (b) Preparation of the working cultures and test suspensions.6.3  Reference cultures 6.3.1  General The reference cultures are used in media quality control and analytical quality control procedures relevant to this method. The purpose of the reference cultures is to demonstrate and ensure that typical growth characteristics are exhibited by the cultures on the media used. As a minimum, the reference cultures shall be used to ensure that the method has been used properly. Each day that samples are processed by this method, suspensions of the reference cultures shall be submitted to the test procedure. If the reference cultures do not give appropriate results, then an investigation shall be undertaken. Test results shall be invalid unless the investigation reveals reasons for this failure that invalidate the quality control procedures (e.g. prepared suspensions shown not to contain target bacteria). 6.3.2  Positive culture Escherichia coli, WDCM 00090, WDCM 00012 or WDCM 00013 or a culture traceable to any of these strains. NOTE  World Data Centre for Microorganisms (WDCM).
7  Techniques
7.1  Pour plate 7.1.1  General This technique is commonly used for the enumeration of HCC when water samples may vary widely in microbial loads. There are limitations, however, when water samples contain particulates that can interfere with colony development or the counting process. NOTE  A flow diagram of the procedure is shown in Figure A.1. 7.1.2  Procedure The procedure for pour plate shall be in accordance with AS 4276.1.During the preparation of the pour plates the following precautions shall be taken: (a) Keep the lids of the plates in place except when they are partially lifted for adding the inoculum and the medium. (b) Do not exceed a time interval of 10 min between pipetting into the plate and pouring the medium. (c) Do not exceed a total time interval of 20 min between the preparation of the first dilution and the pouring of the last plate. 7.1.3  Inoculation and pouring of plates Plates shall be inoculated and poured as follows: (a) Prepare plates by adding 1 mL of sample or appropriate dilution to each plate. When dilution is necessary, plate out appropriate dilutions. (b) To each plate add molten agar, which has been cooled to 44 °C to 46 °C, to a depth of 3 mm to 5 mm. NOTE  Some organisms may suffer heat-shock above 47 °C. (c) Immediately after pouring, keep the plate horizontal and, taking care not to wet the lid, thoroughly mix the medium and the inoculum by to-and-fro movements, followed by circular clockwise movements, followed by to-and-fro movements at right-angles to the first set, followed by circular anti-clockwise movements. (d) Allow the plates to stand on a horizontal surface until the medium has set. 7.1.4  Incubation Incubation shall be carried out as described in AS 4276.1. The plates should be incubated at 36 °C ± 2 °C for 44 ± 4 h and/or 22 °C ± 2 °C for 68 ± 4 h, as required. For cooling tower waters, the incubation used shall be 36 °C ± 2 °C for 44 ± 4 h.