AS 3896:2017 pdf download.Waters- – Examination for Legionella spp. including Legionella pneumophila.
3 PRINCIPLE
The sample of water is cultured on specified media directly as well as after heat and acid treatments. Presumptive Legionella colonies are taken through confirmation tests. Confirmed Legionella isolates can be speciated and serotyped as required.
4 SAFETY PRECAUTIONS
The safety precautions for microbiological laboratories, as described in AS/NZS 2243.3, shall be observed. Legione/la cultures on solid culture media can be handled safely by trained staff on the open bench in a conventional microbiology laboratory conforming to Containment Level 2. Infection is caused by inhalation of the microorganism and it is advisable therefore to assess all procedures for their ability to produce aerosols. If in doubt, carry out the work in a biological safety cabinet.
5 CULTURE MEDIA, REAGENTS AND REFERENCE CULTURES
5.1 Culture media
The culture media shall be as follows:
(a) Buffered charcoal yeast extract agar (BCYE).
(b) Buffered charcoal yeast extract agar with MWY selective supplement (MWY) or buffered charcoal yeast extract agar with GVPC selective supplement (GVPC).
(c) Buffered charcoal yeast extract agar with BMPA selective supplement (BMPA) or buffered charcoal yeast extract agar with selective supplements (BCYE+AB).
(d) Buffered charcoal yeast extract medium without L-cysteine (BCYE-Cys) or Blood Agar (BA).
(e) Diluent: sterile 0.1% peptone or sterile quarter strength Ringer’s solution.
NOTE: See Appendix B.
5.2 Reagents and identification systems
5.2.1 Acid buffer
Acid buffer shall be prepared using the formulation and procedure. NOTE: See Appendix B, Paragraph BI 1.
5.2.2 Specia tiQll/tVJ) ing system(s)
Speciation systems, using latex agglutination, direct or indirect immunofluorescence, mass spectrometric and molecular techniques are available for identifying known scrogroups and/or genotypes of L. pneumophila and other Legionella spp. The laboratory shall ensure that the system or systems used are suitable for the laboratory’s requirements for identification/typing. Fitness for purpose may be demonstrated by reference to the manufacturer’s validation data or to evaluation studies published in the scientific literature.
5.3 Reference cultures
5.3.1 Positive cultures
Two positive cultures (Legionella pneumophila and a non-pneuinophi!a sp.) shall be used. The following choice of the non-pneumophila reference culture [see Clause 5.3.1(b) or 5.3.1(c)J is left to the discretion of the laboratory:
(a) Legionella pneumophila serogroup 1 WDCM 00107 or a culture traceable to this strain. NOTE: WDCM—World Data Centre for Microorganisms.
(b) Legionella micdadei (synonym Tutlockia ,nicdadei) ATCC 33218 or CIP 103882 or NCTC 11371 or a culture traceable to one of these strains.
NOTES: 1 There is currently no WDCM culture reference for this strain. 2 ATCC—Arnerican Type Culture Collection. 3 CIP—Collection of Institut Pasteur.
4 NCTC—National Collection of Type Cultures.
(c) Legionella bozeinanae (synonym Fluoribacter bozeinanae) ATCC 33217 or NCTC 11368 or a culture traceable to one of these strains. NOTE: There is currently no WDCM culture reference for this strain.
5.3.2 Use of reference cultures
The reference cultures are used in analytical quality control procedures relevant to this method. The purpose of the reference cultures is to demonstrate and ensure that typical growth characteristics and test reactions are exhibited by the cultures on the media and in the tests used in this method.
As a minimum, the reference cultures shall be used to provide assurance that the method has been applied properly. Each day samples are processed by this method, suspensions of the positive reference cultures are submitted to the test procedure. Growth of the positive reference cultures obtained is taken through the procedure as far as any isolate obtained from the samples, including confirmation tests.
If the reference cultures do not give appropriate results, then an investigation shall be undertaken. Test results shall be regarded as 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.
5.4 Quality control systems
The laboratory should have a clearly defined quality control system to ensure that the apparatus, culture media, reagents and technique are suitable for the test. The use of positive controls is part of this system.AS 3896 pdf download.