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1、BRITISH STANDARD BS 6068-4.3: 1989 Water quality Part 4: Microbiological methods Section 4.3 Detection and enumeration (most probable number) of faecal streptococci by the multiple tube technique UDC 556.11:628.1.03:628.191:614.777:579.68.083.1:579.862.1.087.23:543.39.062 Licensed Copy: sheffieldun
2、sheffieldun, na, Tue Dec 05 01:26:50 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 6068-4.3:1989 This British Standard, having been prepared under the direction of the Environment and Pollution Standards Policy Committee, was published under the authority of the Board of BSI and comes into effect on
3、 29 September 1989 BSI 07-1999 The following BSI references relate to the work on this standard: Committee reference EPC/44 Draft for comment 83/54980 DC ISBN 0 580 17295 3 Foreword This Section of BS 6068, which has been prepared under the direction of the Environment and Pollution Standards Policy
4、 Committee, is related to ISO 7899-1:1984 “Water quality Detection and enumeration of faecal streptococci Part 1: Method by enrichment in a liquid medium”. ISO 7899-1 was prepared by subcommittee 4, Microbiological methods, of Technical Committee 147, Water quality, of the International Organization
5、 for Standardization (ISO) as a result of discussion in which the UK participated. This Section of BS 6068 differs from ISO 7899-1:1984 in that the catalase test has been removed, the incubation conditions in the isolation stage have been changed, and the scope has been expanded. This British Standa
6、rd is being published in a series of Parts subdivided into Sections that will generally correspond to particular international standards. Sections are being, or will be, published in Parts 1 to 7 which, together with Part 0, are listed below. Part 0: Introduction; Part 1: Glossary; Part 2: Physical,
7、 chemical and biochemical methods; Part 3: Radiological methods; Part 4: Microbiological methods; Part 5: Biological methods; Part 6: Sampling; Part 7: Precision and accuracy. NOTEThe tests described in this Section of BS 6068 should only be carried out in laboratories with suitable facilities and b
8、y suitably qualified persons with an appropriate level of microbiological expertise. Standard microbiological procedures should be followed throughout. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their corr
9、ect application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, pages 1 to 4, an inside back cover and a back cover. This standard has been updated (see copyr
10、ight date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover. Amendments issued since publication Amd. No.Date of issueComments Licensed Copy: sheffieldun sheffieldun, na, Tue Dec 05 01:26:50 GMT+00:00 2006, Uncontrolled Copy, (c) BSI B
11、S 6068-4.3:1989 BSI 07-1999i Contents Page ForewordInside front cover 0Introduction1 1Scope1 2Definitions1 3Principle1 4Diluents, culture media and reagents1 5Apparatus2 6Sampling2 7Procedure2 8Expression of results3 9Test report3 Appendix A Further microbiological information relevant to water exam
12、ination for faecal streptococci4 Publications referred toInside back cover Licensed Copy: sheffieldun sheffieldun, na, Tue Dec 05 01:26:50 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ii blank Licensed Copy: sheffieldun sheffieldun, na, Tue Dec 05 01:26:50 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS
13、 6068-4.3:1989 BSI 07-19991 0 Introduction The presence and extent of faecal pollution is an important factor in assessing the quality of a body of water. Examination of samples for the presence of faecal streptococci1), which normally inhabit the intestines of man and other warm-blooded animals, ca
14、n provide an indication of such pollution. Opinions vary in different countries as to which streptococcal species should be included in the term “faecal streptococci”, however in this Section of BS 6068 they are restricted to those possessing Lancefields Group D antigen. Although serological confirm
15、ation is not included in the method, the selectivity of the media used is such as to give a reasonable estimate of the numbers of Group D streptococci present. 1 Scope This Section of BS 6068 describes a method for the detection and enumeration of faecal streptococci in water samples by culture in a
16、 liquid medium in multiple tubes and the calculation of their most probable numbers in the sample. The method can be applied to all types of water, including turbid water. It is not suitable for the analysis of large volumes of water containing few faecal streptococci (see BS 6068-4.4). NOTEThe titl
17、es of the publications referred to in this standard are listed on the inside back cover. 2 Definitions For the purposes of this Section of BS 6068, the following definitions apply. 2.1 presumptive faecal streptococci organisms capable of aerobic growth at 37 0.5 C in a liquid culture medium containi
18、ng glucose and sodium azide with the production of acid within 48 h 2.2 faecal streptococci presumptive faecal streptococci as defined in 2.1 which can also grow at 44 0.25 C in the presence of bile salts and sodium azide, and hydrolyse aesculin 3 Principle Test portions of the sample, and/or diluti
19、ons of it, are inoculated into a series of containers of a selective liquid culture medium containing glucose and sodium azide. The containers are incubated for 24 h and 48 h at 37 C and examined. Confirmatory tests are then carried out on each culture showing acid production. The cultures are subcu
20、ltured to a confirmation medium and incubated at 44 C for up to 6 h to demonstrate growth in the presence of bile salts and sodium azide, and the hydrolysis of aesculin. Statistical tables are used to calculate the most probable number of presumptive faecal streptococci and confirmed faecal streptoc
21、occi likely to be present in 100 mL of the sample from the numbers of containers giving positive confirmatory results. 4 Diluents, culture media and reagents WARNING. All selective media described in this Section of BS 6068 contain sodium azide. As this substance is highly toxic and mutagenic, preca
22、utions should be taken to avoid contact with it, especially by the inhalation of fine dust during the preparation of commercially available dehydrated complete media. Azide-containing media should not be mixed with strong inorganic acids, as toxic hydrogen azide (HN3) may be produced. Solutions cont
23、aining azide can also form explosive compounds when in contact with metal pipework and sinks. Azides can be decomposed safely by an excess of a nitrite solution. 4.1 Basic material For the preparation of culture media and diluents use chemicals of a recognized analytical grade and ingredients of uni
24、form quality. For the preparation of culture media use glass distilled or deionized water, free from substances which may inhibit bacterial growth, or water of equivalent purity that complies with grade 3 of BS 3978. Alternatively use commercially available dehydrated culture media. Prepare such med
25、ia strictly in accordance with the manufacturers instructions. 4.2 Diluents For making sample dilutions use one of the diluents given in BS 6068-4.2. 1) See Appendix A for further microbiological information relevant to water examination for faecal streptococci. Licensed Copy: sheffieldun sheffieldu
26、n, na, Tue Dec 05 01:26:50 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 6068-4.3:1989 2 BSI 07-1999 4.3 Isolation medium: glucose azide broth The composition of the isolation medium shall be as follows: Dissolve the ingredients in the water by boiling. Adjust the pH, if necessary, with sodium hydro
27、xide solution (40 g/L) or hydrochloric acid (36.5 g/L) so that after sterilization the pH is 7.2 0.1 at 25 C. Distribute single strength medium in 5 mL volumes and double strength medium in 10 mL and 50 mL volumes into suitably-sized containers (see 7.1) and sterilize for 15 min at 121 C. 4.4 Confir
28、mation medium: bile-aesculin-azide agar The composition of the confirmation medium shall be as follows: Dissolve the ingredients in the water by boiling. Adjust the pH if necessary, with sodium hydroxide solution (40 g/L) or hydrochloric acid (36.5 g/L) so that after sterilization the pH is 7.2 0.1
29、at 25 C. Distribute the solution into screw-capped containers and sterilize for 15 min at 121 C. Cool to 50 C using the water bath (5.4) and pour into Petri dishes to a depth of at least 3 mm. Allow the medium to set on a cool, horizontal surface. Store at 4 C in sealed conditions to prevent drying.
30、 5 Apparatus 5.1 Ordinary microbiological laboratory equipment, including Petri dishes, test tubes and culture bottles or flasks. Apart from apparatus, which is supplied sterile, glassware and other equipment shall be sterilized in accordance with BS 6068-4.2. 5.2 Incubator, capable of being maintai
31、ned at 37 0.5 C. 5.3 Incubator, capable of being maintained at 44 0.25 C. 5.4 Water bath, capable of being maintained at 50 1 C. 6 Sampling Take the samples and deliver them to the laboratory in accordance with BS 6068-4.2, BS 6068-6.1, BS 6068-6.2, and BS 6068-6.3. 7 Procedure 7.1 General Follow th
32、e instructions for the preparation of the sample (see clause 6). Make dilutions, prepare test portions, inoculate the isolation medium with the test portions and carry out the multiple tube technique as a whole in accordance with BS 6068-4.2. For test portion volumes of 1 mL or less use single stren
33、gth medium (4.3) and for test portion volumes of 5 mL or more use equal volumes of double strength medium (4.3). 7.2 Isolation Inoculate test portions of the sample into a series of containers of isolation medium (4.3) and mix thoroughly. Incubate the containers at 37 0.5 C using the incubator (5.2)
34、. Examine the containers after incubation for 22 2 h and consider all cultures showing a yellow colour, indicating acid production, either throughout the whole container or only in the lower part, as giving a positive reaction. Incubate for a further 22 2 h any containers not showing a colour change
35、. After this additional incubation period re-examine these containers and consider even a faint colour change to reddish purple indicative of acid production. NOTEComparison of inoculated and incubated containers with an uninoculated and incubated container of medium will assist the colour-change as
36、sessment. Consider all containers showing positive reactions as containing presumptive faecal streptococci and carry out the confirmatory test described in (7.3). Single strength Double strength beef extract4.5 g9.0 g tryptone15.0 g30.0 g glucose7.5 g15.0 g sodium chloride (NaCl)7.5 g15.0 g sodium a
37、zide (NAN3)0.2 g0.4 g bromocresol purple (ethanol solution 15 g/L)1 ml2 ml water1 000 ml1 000 ml. tryptone17.0 g peptone3.0 g yeast extract5.0 g ox-bile, dehydrated10.0 g sodium chloride (NaCl)5.0 g aesculin1.0 g ammonium iron (III) citrate0.5 g sodium azide (NAN3)0.15 g agar12 g to 20 ga water1 000
38、 mL. a In accordance with the manufacturers instructions. Licensed Copy: sheffieldun sheffieldun, na, Tue Dec 05 01:26:50 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 6068-4.3:1989 BSI 07-19993 7.3 Confirmation After mixing to resuspend the contents of the containers showing positive reactions (see
39、 7.2), streak a loopful from each of these cultures on to the confirmation medium (4.4) and incubate at 44 0.25 C using the incubator (5.3). Development of a black or brown colour in or around the inoculum within 6 h confirms the presence of faecal streptococci. NOTEThe use of a “heavy” or thick ino
40、culum will increase the speed at which results are obtained, if the inocula are well spaced out many isolates can be tested on one plate of medium. 8 Expression of results Calculate the results in accordance with BS 6068-4.2. Express the results as the most probable number of presumptive faecal stre
41、ptococci (7.2) and confirmed faecal streptococci (7.3) per 100 mL of sample. 9 Test report The test report shall give the following information: a) a reference to this Section of BS 6068; b) all details necessary for complete identification of the sample; c) the results obtained expressed in the ter
42、ms given in clause 8; d) any other information relevant to the method. Licensed Copy: sheffieldun sheffieldun, na, Tue Dec 05 01:26:50 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 6068-4.3:1989 4 BSI 07-1999 Appendix A Further microbiological information relevant to water examination for faecal str
43、eptococci For routine water examination purposes, faecal streptococci may be described generally in microbiological, though not taxonomic terms, as Gram-positive cocci forming pairs and chains which are non-sporing, oxidase and catalase negative, possess Lancefields Group D antigen and can hydrolyze
44、 aesculin. They can grow both aerobically and anaerobically in the presence of bile salts and in concentrations of sodium azide which are inhibitory to coliform organisms and most Gram-negative bacteria. Streptococcus faecalis and some related species can reduce 2,3,5-triphenyltetrazolium chloride (
45、TTC) to the insoluble red dye formazan; other species reduce TTC weakly or not at all. Licensed Copy: sheffieldun sheffieldun, na, Tue Dec 05 01:26:50 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 6068-4.3:1989 BSI 07-1999 Publications referred to BS 3978, Specification for water for laboratory use.
46、 BS 6068, Water quality. BS 6068-4.2, General guide to the enumeration of micro-organisms by culture. BS 6068-4.4, Detection and enumeration of faecal streptococci by the membrane filtration technique. BS 6068-6.1, Guidance on the design of sampling programmes. BS 6068-6.2, Guidance on sampling tech
47、niques. BS 6068-6.3, Guidance on preservation and handling of samples. Licensed Copy: sheffieldun sheffieldun, na, Tue Dec 05 01:26:50 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 6068-4.3: 1989 BSI 389 Chiswick High Road London W4 4AL BSI British Standards Institution BSI is the independent nation
48、al body responsible for preparing British Standards. It presents the UK view on standards in Europe and at the international level. It is incorporated by Royal Charter. Revisions British Standards are updated by amendment or revision. Users of British Standards should make sure that they possess the
49、 latest amendments or editions. It is the constant aim of BSI to improve the quality of our products and services. We would be grateful if anyone finding an inaccuracy or ambiguity while using this British Standard would inform the Secretary of the technical committee responsible, the identity of which can be found on the inside front cover. Tel: 020 8996 9000. Fax: 020 8996 7400. BSI offers members an individual updating service called PLUS which ensures that subscribers automatically receive the latest editions