AS/NZS 3896:1998

1 - AS/NZS 3896:1998 WATERS-EXAMINATION FOR LEGIONELLAE INCLUDING LEGIONELLA PNEUMOPHILA
4 - PREFACE
5 - CONTENTS
6 - 1 SCOPE
6 - 2 REFERENCED DOCUMENTS
6 - 3 PRINCIPLE
6 - 4 SAFETY PRECAUTIONS
6 - 5 CULTURE MEDIA, REAGENTS AND REFERENCE CULTURES
6 - 5.1 Culture media
6 - 5.1.1 Buffered charcoal yeast extract agar with MWY selective supplement (MWY) or Buffered charcoal yeast extract agar...
7 - 5.1.2 Buffered charcoal yeast extract agar with BMPA selective supplement (BMPAa).
7 - 5.1.3 Buffered charcoal yeast extract agar (BCYEa).
7 - 5.1.4 Blood agar (BA).
7 - 5.1.5 Diluent: sterile 0.1% peptone or sterile quarter strength Ringer's solution.
7 - 5.2 Reagents
7 - 5.2.1 HCl/KCl buffer
7 - 5.2.2 Gram stain reagents
7 - 5.2.3 Latex agglutination reagents
7 - 5.2.4 Optional reagents for other identification tests
7 - 5.3 Reference organisms
7 - 5.3.1 Positive culture
7 - 5.3.2 Negative culture
7 - 5.3.3 Use of reference cultures
7 - 6 APPARATUS
7 - 6.1 Vortex mixer.
7 - 6.2 Water bath at 50 ±1°C.
7 - 6.3 Long-wave (360 ±20 nm) ultraviolet lamp.
7 - 6.4 Plate (or dissecting) microscope 10-20 × magnification.
7 - 6.5 Suitably calibrated micropipette capable of delivering the required volumes.
7 - 7 SAMPLE STORAGE
8 - 8 PROCEDURE
8 - 8.1 General
8 - 8.2 Isolation of Legionella-like organisms (LLOs)
8 - 8.2.1 Isolation procedure
8 - 8.2.2 Direct sample (untreated)
8 - 8.2.3 Heat-treated sample
8 - 8.2.4 Acid-treated sample
8 - 8.3 Incubation
8 - 8.4 Examination
9 - 8.5 Confirmation of legionellae
9 - 8.5.1 Procedure
9 - 8.5.2 Interpretation
9 - 8.6 Confirmation of L. pneumophila
10 - 8.7 Other tests for legionellae
10 - 9 CALCULATIONS
10 - 10 TEST REPORT
10 - 11 BIBLIOGRAPHY
12 - APPENDIX A - FLOW CHART FOR LEGIONELLAE TESTING
13 - APPENDIX B - CULTURE MEDIA
13 - B1 SCOPE
13 - B2 CHARCOAL YEAST EXTRACT AGAR BASE (CYE)
13 - B3 BUFFERED CHARCOAL YEAST EXTRACT AGAR (BCYEa)
13 - B3.1 Formulation
13 - B3.2 Growth supplement
13 - B4 BUFFERED CHARCOAL YEAST EXTRACT AGAR WITH BMPA SELECTIVE SUPPLEMENT (BMPAa)
13 - B4.1 Formulation
14 - B4.2 BMPA selective supplement
14 - B5 BUFFERED CHARCOAL YEAST EXTRACT AGAR WITH MWY SELECTIVE SUPPLEMENT (MWY)
14 - B5.1 Formulation
14 - B5.2 MWY selective supplement
14 - B6 BUFFERED CHARCOAL YEAST EXTRACT AGAR WITH GVPC SELECTIVE SUPPLEMENT (GVPC)
14 - B6.1 Formulation
14 - B6.2 GVPC selective supplement
15 - B7 TRYPTICASE SOYA + 5% BLOOD AGAR (BA)
15 - B8 PREPARATION OF ACID BUFFER
15 - B9 PEPTONE SOLUTION, 0.1%*
15 - B10 RINGER'S SOLUTION, FULL STRENGTH
15 - B10.1 Formulation
15 - B10.2 Ringer's solution, quarter strength
16 - APPENDIX C - FINAL READING SUMMARY
18 - AMENDMENT CONTROL SHEET

Specifies a method for detecting and estimating the number of Legionella pneumophila and some other legionellae in water. In this revision the biocide reduction step has been deleted as the dilution factors within this method, i.e. 0.1 mL onto agar plates, are sufficient to overcome any residual antimicrobial activity within the sample submitted for testing.

This Standard sets out a method for detecting and estimating the number of Legionella pneumophila and a range of other legionellae in water.NOTES:A flow chart of the procedure is shown in Appendix A. This Method will identify the species L. pneumophila but not all other legionellae (see Preface). Conditions that favour the isolation of L. pneumophila do not necessarily apply to some other legionellae. A step is included (see Note in Clause 8.2.4) to enhance the sensitivity of the method for such species. This Standard describes the testing of a sample of water as delivered to the laboratory and provides some information regarding sample size and delivery conditions. In samples submitted for testing, the dilution factors within this method i.e. 0.1 mL onto agar plates are sufficient to overcome any residual antimicrobial activity within the sample.

First published in Australia as AS 3896-1991.
Jointly revised and designated AS/NZS 3896:1998.