1、 Collection of SANS standards in electronic format (PDF) 1. Copyright This standard is available to staff members of companies that have subscribed to the complete collection of SANS standards in accordance with a formal copyright agreement. This document may reside on a CENTRAL FILE SERVER or INTRA
2、NET SYSTEM only. Unless specific permission has been granted, this document MAY NOT be sent or given to staff members from other companies or organizations. Doing so would constitute a VIOLATION of SABS copyright rules. 2. Indemnity The South African Bureau of Standards accepts no liability for any
3、damage whatsoever than may result from the use of this material or the information contain therein, irrespective of the cause and quantum thereof. ICS 07.100.20 ISBN 0-626-13975-9 SANS 10705-1:2002 Edition 1 ISO 10705-1:1995 Edition 1 SOUTH AFRICAN NATIONAL STANDARD Water quality Detection and enume
4、ration of bacteriophages Part 1: Enumeration of F-specific RNA bacteriophages This national standard is the identical implementation of ISO 10705-1:1995 and is adopted with the permission of the International Organization for Standardization. Published by Standards South Africa 1 dr lategan road gro
5、enkloof ! private bag x191 pretoria 0001 tel: 012 428 7911 fax: 012 344 1568 international code + 27 12 www.stansa.co.za Standards South Africa 2002 SANS 10705-1:2002 Edition 1 ISO 10705-1:1995 Edition 1 Table of changes Amdt No. Date Scope National foreword This South African standard was approved
6、by National Committee STANSA SC 5140.19B, Biological treatment of water, in accordance with procedures of Standards South Africa, in compliance with annex 3 of the WTO/TBT agreement. INTERNATIONAL STANDARD IS0 10705-I First edition 1995-08-01 Water quality - Detection and enumeration of bacteriophag
7、es - Part 1: Enumeration of F-specific RNA bacteriophages Qualit6 de Ieau - D6tection et dhnombrement des bactkiophages - Partie 1: Dhnombrement des bactkiophages ARN F spkifiques Reference number IS0 10705-I :I 995(E) IS0 10705=1:1995(E) Foreword IS0 (the International Organization for Standardizat
8、ion) is a worldwide federation of national standards bodies (IS0 member bodies). The work of preparing International Standards is normally carried out through IS0 technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be r
9、epresented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. IS0 collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Draft International Sta
10、ndards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. International Standard IS0 10705-l was prepared by Technical Committee lSO/TC 147, Water quality
11、 Subcommittee SC 4, Microbiological methods. IS0 10705 consists of the following parts, under the general title Water quality - Detection and enumeration of bacteriophages: - Part 7: Enumeration of F-specific RNA bacteriophages - Part 2: Enumeration of somatic coliphages Annex A forms an integral p
12、art of this part of IS0 10705. Annexes B and C are for information only. 0 IS0 1995 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permissi
13、on in writing from the publisher. International Organization for Standardization Case Postale 56 l CH-1211 Geneve 20 l Switzerland Printed in Switzerland ii INTERNATIONAL STANDARD 0 IS0 IS0 10705-1:1995(E) Water quality - Detection and enumeration of bacteriophages - Part 1: Enumeration of F-specifi
14、c RNA bacteriophages 1 Scope This part of IS0 10705 specifies a method for the detection and enumeration of F-specific ribonucleic acid (RNA) bacteriophages by incubating the sample with an appropriate host strain. The method can be applied to all kinds of water, sediments and sludges, where necessa
15、ry after dilution. In the case of low numbers, a preconcentration step may be necessary for which a separate part of IS0 10705 will be devel- oped. The method can also be applied to shellfish extracts. Depending on the relative abundance of F- specific RNA bacteriophages to background organisms, add
16、itional confirmatory tests may be necessary and are also specified in this part of IS0 10705. The presence of F-specific RNA bacteriophages in a water sample generally indicates pollution by wastewater contaminated by human or animal faeces. Their survival in the environment, removal by widely used
17、water treatment processes and concentration or retention by shellfish resembles that of foodborne and waterborne human enteric viruses, for example the enteroviruses, hepatitis A virus and rotaviruses. 2 Normative references The following standards contain provisions which, through reference in this
18、 text, constitute provisions of this part of IS0 10705. At the time of publication, the editions indicated were valid. All standards are subject to revision, and parties to agreements based on this part of IS0 10705 are encouraged to investi- gate the possibility of applying the most recent edi- tio
19、ns of the standards indicated below. Members of IEC and IS0 maintain registers of currently valid International Standards. IS0 3696: 1987, Water for analytical laboratory use - Specification and test methods. IS0 5667-l : 1980, Water quality - Sampling - Part I: Guidance on the design of sampling pr
20、o- grammes. IS0 5667-2: 1991, Water quality - Sampling - Part 2: Guidance on sampling techniques. IS0 5667-3: 1994, Water quality - Sampling - Part 3: Guidance on the preservation and handling of samples. IS0 6887: 1983, Microbiology - General guidance for the preparation of dilutions for microbiolo
21、gical exam- ina tion. I SO 8199: 1988, Water quality - General guide to the enumeration of micro-organisms by culture. 3 Definition For the purposes of this part of IS0 10705, the fol- lowing definition applies. 3.1 F-specific RNA bacteriophages: Bacterial vi- ruses which are capable of infecting a
22、specified host strain with F-pili or sex-pili to produce visible plaques (clearance zones) on a confluent lawn grown under appropriate culture conditions, whereas the infectious process is inhibited in the presence of a concentration 1 IS0 10705-1:1995(E) . 0 IS0 of 40 (occasionally 400) Clglml of R
23、Nase in the plating medium. 6.3 Reagents 6.3.1 RNase from bovine pancreas, specific ac- tivity approximately 50 units/mg (Kunitz). 4 Principle The sample is mixed with a small volume of semi- solid nutrient medium. A culture of host strain is added and plated on a solid nutrient medium. After this,
24、incubation and reading of plates for visible plaques takes place. Where necessary, simultaneous examination of parallel plates with added RNase for confirmation by differential counts is carried out. The results are expressed as the number concentration of plaque-forming particles (Cpfp) per unit of
25、 volume. 5 Safety precautions The host strain used is a Salntonella fyphimurium mutant of low pathogenicity and should be hand- led in accordance with the appropriate national or international safety procedures for this bac- terial species. F-specific RNA bacteriophages are non-pathogenic for man an
26、d animals, but are very resistant to drying. Appropriate precautions should therefore be taken to prevent cross- contamination of test materials, particularly when examining or handling cultures of high titre or when inoculating cultures of the host strains. Such procedures must be carried out in a
27、biohazard cabinet or a separate area of the lab- oratory. 6 Diluent, culture media and reagents 6.1 Basic materials Use ingredients of uniform quality and chemicals of analytical grade for the preparation of culture media and reagents, and follow the instructions given in an- nex A. For information
28、on storage see IS0 8199, ex- cept where indicated in this part of IS0 10705. Alternatively, use dehydrated complete media and follow strictly the manufacturers instructions. For the preparation of media, use glass-distilled water or deionized water free from substances which might inhibit bacterial
29、growth under the conditions of the test, and in accordance with IS0 3696. 6.2 Diluent For making sample dilutions, use peptone saline sol- ution as indicated in A.8. 6.3.2 Antibiotic discs, for susceptibility testing with nalidixic acid (130 pg; 9 mm) and kanamycin (100 pg; 9 mm). 6.3.3 Glycerol, 87
30、0 g/litre. 6.4 Microbiological reference cultures Salmonella typhimurium strain WG49, phage type 3 Nal (F 42 /ac:Tn5), NCTC 12484. Bacteriophage MS2, NCTC 12487 or ATCC 15597-Bl . Escherichia co/; K-12 Hfr from appropriate culture col- lection, e.g. NCTC 12486 or ATCC 23631. NOTE 1 The NCTC strains
31、are available from the National Collection of Type Cultures, 61 Colindale Avenue, London NW9 6HT, England. The ATCC strains are available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, U.S.A. 7 Apparatus and glassware Usual microbiological laboratory equipment,
32、 including 7.1 Hot-air oven for dry-heat sterilization and an autoclave. Apart from apparatus supplied sterile, glassware and other equipment shall be sterilized ac- cording to the instructions given in IS0 8199. 7.2 Incubator or water bath, thermostatically controlled at 37 “C + 1 “C. - 7.3 Incubat
33、or or water bath, thermostatically controlled at 37 “C +_ 1 “C and equipped with a rotary platform at 100 min- + 10 min-. - 7.4 Water bath, thermostatically controlled at 45 “C + 1 “C. - 7.5 Water bath or equivalent device, for melting agar media. 7.6 pH-meter. 7.7 Counting apparatus, with indirect,
34、 oblique light. 7.8 Deep freezer, thermostatically controlled at - 20 “C + 5 “C. - 2 0 IS0 7.9 Deep freezer, thermostatically controlled at - 7o”c+100c. - 7.10 Spectrometer, capable of holding 1 cm cuvettes or side-arm of nephelometric flasks (7.17) and equipped with a filter in the range 500 nm to
35、650 nm with a maximum bandwidth of + 10 nm. Usual sterile, microbiological laboratory glassware or disposable plastics ware according to IS0 8199 and including the following. 7.11 Petri dishes, of diameter 9 cm or 15 cm. 7.12 Graduated pipettes, of capacities 1 ml, 5 ml and 10 ml. 7.13 Glass bottles
36、 of suitable volumes. I 7.14 Culture tubes, with cap: 10705=1:1995(E) 7.15 Measuring cylinders, of suitable capacity. 7.16 Conical flasks, of capacity 250 ml to 300 ml, with cotton wool plugs or suitable alternatives. 7.17 Cuvettes, of optical path length 1 cm or nephelometric conical flasks, of ca
37、pacity 250 ml to 300 ml, with cylindrical side-arms which can be fitted to the spectrometer (7.10) and with cotton wool plugs or suitable alternatives. (See figure 1.) 7.18 Membrane filter units, for sterilization, pore size 0,2 pm. 7.19 Plastics vials, lidded, of capacity I,5 ml to 2 ml. Figure 1 -
38、 Nephelometric conical flasks for culturing the host strain IS0 10705-1:1995(E) 8 Sampling Take samples and deliver them to the laboratory in accordance with IS0 8199, IS0 5667-1, IS0 5667-2, and IS0 5667-3. 9 Preparation of test materials 9.1 Culturing and maintenance of host strains WG49 and E. co
39、/i K12 Hfr The culturing and maintenance of host strains in- volves several stages which are summarized in figure2. The figure also indicates the stages where quality control of the host culture is performed. REFERENCECULTURE (9.1.1) Overnight culture, add 20 % (V/V) glycerol STOCKCULTURE Store at -
40、 70 “C (9.1.1) 9.1.1 Preparation of stock cultures Rehydrate the contents of a lyophilized ampoule of the reference culture of the host strains in a small volume of TYGB (A.l) using a Pasteur pipette. Trans- fer the suspension to 50 ml of TYGB in a 300 ml conical flask (7.16). Incubate for 18 h incu
41、bate at 37 “C + 1 “C for 24 h plate count on TYGA (9.2) at 3 h: 7 to 40 x IO7 cfp/ml; lactose-negative colonies (plasmid segregation) 80 %. 10.2 Method for samples with high bacterial background flora Add nalidixic acid to ssTYGA (A.3) until a final con- centration of 100 pg/ml is obtained. NOTE 10
42、Nalidixic acid is stable when heated. It can ei- ther be added from a filter-sterilized solution (A.4) (02 ml/50 ml) after melting of ssTYGA or can be added to TYGA before autoclaving. 10 Procedure 10.3 Confirmatory test 10.1 Standard procedure Take one vial of working culture from the freezer and t
43、haw it at room temperature. Add 50 ml of TYGB (A.l) to a nephelometric conical flask (7.17), or plain conical flask (7.16). Adjust the spectrometer reading to 0 as described in 9.2 and prewarm to room tem- perature. Inoculate 0,5 ml of working culture. Incu- bate at 37 “C + 1 “C while shaking at 100
44、 min- + IO min-. - Measure turbidity every 30 min. At a turbidity corresponding to a cell density of approxi- mately IO* cfu/ml (based on data obtained in 9.2), take the inoculum culture from the incubator and quickly cool on melting ice. Use within 2 h. NOTE 7 It is essential that the culture is qu
45、ickly cooled to prevent loss of F-pili by the cells, which will negatively influence recovery. Melt bottles of ssTYGA (A.3) cool to 44 “C to 50 “C, aseptically add calcium-glucose solution (A. 1) (0,5 ml/50 ml) and distribute 2,5 ml aliquots into cul- ture tubes with caps, placed in a water bath at
46、45 “C + 1 “C. To each tube, add 1 ml of sample (or dilution or concentrate). Examine each volume or di- lution step at least in duplicate. Add 1 ml of inoculum culture, mix carefully and pour the contents over the surface of a 9 cm TYGA plate (A.2). Distribute evenly, allow to solidify on a perfectl
47、y horizontal, cool surface and incubate the plates upside-down at 37 “C + 1 “C for 18 h + 2 h. - NOTES 8 Do not stack more than 6 (preferably 4) plates. 9 The addition of ice-cold sample and host culture to the top-agar may lead to a sharp drop in temperature and solidification of the medium. Assure
48、 a sufficient time inter- val between these two steps to allow reheating. However, make sure that inoculated tubes remain in the water bath for not more than 10 min. Count the number of plaques appearing on each plate within 4 h, using indirect oblique light. In parallel with the series of plates de
49、scribed under 10.1, prepare a similar series with RNase-solution (A.5) added to the tubes of ssTYGA until a final con- centration of 40 pg/ml is obtained (i.e. 100 1 of RNase solution to 2,5 ml of ssTYGA in a tube). NOTES 11 Confirmatory tests should at least be carried out a) when examining new sampling points; b) regularly at fixed sampling points when c) always at fixed sampling points when IVRNase/N is usually 10 %; d) if large, (probably seen. circular, clear plaques with smooth edges somatic Salmonella phages) are regularly 12 In rare cases, RNA-phages may not be inhibited
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