1、BRITISH STANDARD BS4285-3.9.2: 1992 Microbiological examination for dairy purposes Part3: Methods for detection and/or enumeration of specific groups of microorganisms Section3.9 Detection of Salmonella Subsection3.9.2 Screening method using electrical conductance IMPORTANT NOTE.It is essential that
2、 Parts0 and1 and Section2.1, which are published separately, be read in conjunction with this Subsection.BS4285-3.9.2:1992 This British Standard, having been prepared under the direction of the Agriculture and Food Standards Policy Committee, was published under the authority of the Standards Board
3、and comes into effect on 15 June 1992 BSI 06-1999 The following BSI references relate to the work on this standard: Committee reference AFC/14 Draft for comment91/52990DC ISBN 0 580 20916 4 Committees responsible for this British Standard The preparation of this British Standard was entrusted by the
4、 Agriculture and Food Standards Policy Committee (AFC/-) to Technical Committee AFC/14, upon which the following bodies were represented: AFRC Institute of Food Research Association of British Preserved Milk Manufacturers Association of Public Analysts British Food Manufacturing Industries Research
5、Association British Laboratory Ware Association Campden Food and Drink Research Association Dairy Trade Federation Department of Agriculture Northern Ireland Department of Trade and Industry (Laboratory of the Government Chemist) Milk Marketing Board Milk Marketing Board for Northern Ireland Ministr
6、y of Agriculture, Fisheries and Food Public Health Laboratory Service Scottish Milk Marketing Board Society for Applied Bacteriology United Kingdom Dairy Association Amendments issued since publication Amd. No. Date CommentsBS4285-3.9.2:1992 BSI 06-1999 i Contents Page Committees responsible Inside
7、front cover Foreword ii 1 Scope 1 2 References 1 3 Definitions 1 4 Principle 1 5 Safety precautions 1 6 Culture media, reagents and sera 1 7 Apparatus 5 8 Sampling 5 9 Preparation of the test sample 5 10 Procedure 5 11 Control cultures 9 12 Expression of results 9 13 Test report 9 Figure 1 Procedure
8、 2 Figure 2 Typical conductance response curves 7 Table 1 Reactions of Salmonella to biochemical tests 9 Table 2 Interpretation of confirmatory tests 9 List of references Inside back coverBS4285-3.9.2:1992 ii BSI 06-1999 Foreword This Subsection of BS4285 has been prepared under the direction of the
9、 Agriculture and Food Standards Policy Committee. BS4285-3.9:1987 describes a classical method for the detection of Salmonella. It has been renumbered as Subsection3.9.1 to allow the following screening test using electrical conductance to be issued as Subsection3.9.2. This Subsection incorporates t
10、he isolation and confirmation procedures described in ISO6785:1985 Milk and milk products Detection of Salmonella, but it is technically different from that standard principally because it allows a choice of at least two of the selective solid media from the four described. A British Standard does n
11、ot purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct 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
12、front cover, pages i and ii, pages1 to10, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on theinside front cover.BS4285-3.9.2:1992 BSI 06-1999 1 1 Scope This Subsectio
13、n of BS4285 describes a screening method for the detection of Salmonella in milk products and other dairy products using electrical conductance. WARNING. It is essential to observe the safety precautions in clause5. 2 References 2.1 Normative references This Part of BS4285 incorporates, by reference
14、, provisions from specific editions of other publications. These normative references are cited at the appropriate points in the text and the publications are listed on the inside back page. Subsequent amendments to, or revisions of, any of these publications apply to this Subsection of BS4285 only
15、when incorporated in it by updating or revision. 2.2 Informative references This Subsection of BS4285 refers to other publications that provide information or guidance. Editions of these publications current at the time of issue of this standard are listed on the inside back page, but reference shou
16、ld be made to the latest editions. 3 Definitions For the purposes of this British Standard, the following definitions apply. 3.1 salmonella microorganisms which change the conductance of the selective media in a typical manner, form typical colonies on solid selective media and which display the bio
17、chemical and serological characteristics described when the tests are carried out in accordance with this standard 3.2 detection of Salmonella determination of the presence or absence of these microorganisms, in a particular mass or volume of product, when the tests are carried out in accordance wit
18、h this standard 4 Principle 4.1 General The detection of Salmonella by electrical techniques necessitates four successive stages as described in4.2 to4.5 (see Figure 1). 4.2 Pre-enrichment in liquid medium The non-selective pre-enrichment broth is inoculated with the test portion and incubated at37
19、C for16h to20h. 4.3 Enrichment and detection in selective liquid media One or two selective media in conductance cells are inoculated with the culture obtained from4.2 and incubated at37 C in an electrical impedance detection unit. This allows the selective growth of Salmonella during which specific
20、 substrates are metabolized causing a change in the electrical conductivity of the medium that is indicative of the presence of the organism. The diagnostic reactions are dependent on the fermentation of mannitol or dulcitol with the simultaneous reduction of trimethylamine-N-oxide, and/or the decar
21、boxylation of lysine. The incubation period shall be not less than24h at37 C. 4.4 Plating out and recognition From the cultures obtained (see4.3), two selective solid media are inoculated. The two selective solid media, are incubated at37 C and examined after20h to24h and, if necessary, after40h to4
22、8h to check for the presence of colonies which, from their characteristics, are considered to be presumptive Salmonella. 4.5 Confirmation Colonies of presumptive Salmonella (see4.4) are sub-cultured and confirmed by means of appropriate biochemical and serological tests. 5 Safety precautions 5.1 The
23、 procedure specified in this Subsection of BS4285 shall be carried out only in laboratories with suitable facilities and under the control of a qualified microbiologist. 5.2 These procedures shall not be performed in quality control laboratories, or in food manufacturing or processing premises, wher
24、e there is a risk of contamination of the environment. 5.3 Full bacteriological precautions shall be taken at all times whilst carrying out the procedure specified in this Subsection of BS4285. Particular attention shall be given to the sterilization of used equipment and media after testing suspect
25、 samples and prior to disposal or re-use. 6 Culture media, reagents and sera 6.1 Basic materials To improve the reproducibility of the results, it is recommended that dehydrated basic components or complete dehydrated media should be used for the preparation of culture media. The manufacturers instr
26、uctions shall be rigorously followed when preparing the culture media.BS4285-3.9.2:1992 2 BSI 06-1999 Figure 1 ProcedureBS4285-3.9.2:1992 BSI 06-1999 3 The chemicals used for preparing the culture media and the reagents shall be of recognized analytical grade. The water used shall be distilled or de
27、ionized water, free from substances that might inhibit growth of microorganisms under the test conditions. The pH measurements shall be carried out by means of the pH meter(7.7). If the prepared culture media and reagents are not used immediately, they shall, unless otherwise specified, be stored in
28、 the dark at between0 C and5 C, but for no longer than1month, and in conditions that prevent any change in their composition. 6.2 Culture media and reagents 6.2.1 Pre-enrichment medium: modified buffered peptone water Preparation Dissolve all ingredients in the water by steaming them. Adjust the pH,
29、 if necessary, so that after sterilization it is7.2+0.1 at25 C. Distribute the medium in quantities of225ml into flasks of suitable capacity. Sterilize for15 min at(121 1) C. 6.2.2 Single selective enrichment medium:trimethylamine-N-oxide broth I (TMAO broth I) WARNING. Extreme care should be taken
30、with the laboratory use of sodium hydrogen selenite and selenite solutions because of their potentially toxic effect. Do not pipette by mouth under any circumstances, and avoid inhalation of dust. 6.2.2.1 Base Preparation Dissolve the components of the medium in the water, adjust the pH to7.2 (if ne
31、cessary), and steam for10 min. NOTECare should be taken to avoid overheating of the medium which results in the production of an orange-red precipitate. If this occurs, discard the medium. 6.2.2.2 L-cystine solution Preparation Dissolve the L-cystine in the sodium hydroxide solution and dilute to100
32、ml with distilled water in a one-mark volumetric flask. Sterilize by filtration. Do not autoclave. 6.2.2.3 Complete medium Preparation Cool the base solution and add the L-cystine solution aseptically. Adjust the pH, if necessary, so that it is7.2 0.1 at25 C. Use the medium on the day of preparation
33、. Mix the solution thoroughly and dispense appropriate quantities (usually2ml,5ml or10ml) into sterile conductance measuring cells, in accordance with the manufacturers instructions. Composition peptone 10.0g sodium chloride 5.0g disodium hydrogen orthophosphate dodecahydrate (Na 2 HPO 4 .12H 2 O) 9
34、.0g potassium dihydrogen orthophosphate (KH 2 PO 4 ) 1.5g glucose 5.0g L-lysine monohydrochloride 5.0g water 1000ml Composition peptone 5.0g mannitol 5.0g trimethylamine-N-oxide dihydrate (CH 3 ) 3 NO.2H 2 O 5.6g disodium hydrogen orthophosphate dihydrate (Na 2 HPO 4 .2H 2 O) 10.0g sodium hydrogen s
35、elenite 4.0g water 1000ml Composition L-cystine 0.1g sodium hydroxide solution,40g/l 15ml water to a final volume of 100ml Composition Base (see6.2.2.1) 1 000ml L-cystine solution (see6.2.2.2) 10mlBS4285-3.9.2:1992 4 BSI 06-1999 6.2.3 Medium A for two tube selective enrichment procedure: trimethylam
36、ine-N-oxide broth II (TMAO broth II) WARNING. Extreme care should be taken with the laboratory use of sodium hydrogen selenite and selenite solutions because of their potentially toxic effect. Do not pipette by mouth under any circumstances and avoid inhalation of dust. 6.2.3.1 Base Preparation Diss
37、olve the components of the medium in the water, adjust the pH to7.2 (if necessary), and steam for10 min. NOTECare should be taken to avoid overheating of the medium which results in the production of an orange-red precipitate. If this occurs, discard the medium. 6.2.3.2 L-cystine solution See 6.2.2.
38、2. 6.2.3.3 Complete medium Preparation Cool the base solution and add the L-cystine solution aseptically. Adjust the pH, if necessary, so that it is7.2 0.1 at25 C. Use the medium on the day of preparation. Mix the solution thoroughly and dispense appropriate quantities (usually2ml,5ml or10ml) into s
39、terile conductance measuring cells, in accordance with the manufacturers instructions. 6.2.4 Medium B for two tube selective enrichment procedure: modified lysine decarboxylase medium (MLM) Preparation, Dissolve the components of the medium in the water, adjust the pH, if necessary, so that after st
40、eaming it is7.0 0.1 at25 C. Steam for10min. NOTECare should be taken to avoid overheating the medium which results in the production of an orange-red precipitate. If this occurs, discard the medium. Dispense appropriate quantities (usually2ml,5ml or10ml) in sterile conductance measuring cells, in ac
41、cordance with the manufacturers instructions. 6.2.5 Selective solid media 6.2.5.1 General The selective solid media shall be at least two of those specified in6.2.5.2 to6.2.5.5, chosen either by the person carrying out the test or by the person commissioning the test. 6.2.5.2 Bismuth sulfite agar. C
42、omplying with6.2.4 of BS4285-3.9.1:1987. 6.2.5.3 Brilliant green/phenol red agar. Complying with5.2.5.2 of BS4285-3.9.1:1987. 6.2.5.4 Hektoen enteric agar. Complying with5.2.5.4 of BS4285-3.9.1:1987. 6.2.5.5 XLD agar. Complying with5.2.5.3 of BS4285-3.9.1:1987. 6.2.6 Nutrient agar. Complying with5.2
43、.6 of BS4285-3.9.1:1987. 6.2.7 Triple sugar/iron agar (TSI agar). Complying with5.2.7 of BS4285-3.9.1:1987. 6.2.8 Urea agar. Complying with5.2.8 of BS4285-3.9.1:1987. 6.2.9 Lysine decarboxylation medium. Complying with5.2.9 of BS4285-3.9.1:1987. Composition peptone 5.0g dulcitol 5.0g trimethylamine-
44、N-oxide dihydrate (CH 3 ) 3 N0.2H 2 O 5.6g disodium hydrogen orthophosphate dihydrate (Na 2 HPO 4 .2H 2 O) 10.0g sodium hydrogen selenite 4.0g water 1 000ml Composition Base (see6.2.3.1) 1000ml L-cystine solution (see6.2.2.2) 10ml Composition lactalbumin hydrolysate 5.0g L-lysine monohydrochloride 1
45、0.0g glucose 2.0g sodium chloride 5.0g sodium hydrogen selenite 2.0g water 1000mlBS4285-3.9.2:1992 BSI 06-1999 5 6.2.10 Reagent for detection of -galactosidase.Complying with5.2.10 of BS4285-3.9.1:1987. 6.2.11 Reagents for Voges-Proskauer (VP) reaction. Complying with5.2.11 of BS4285-3.9.1:1987. 6.2
46、.11.1 VP medium. Complying with5.2.11.1 of BS4285-3.9.1:1987. 6.2.11.2 Creatine solution (N-amidinosarcosine). Complying with5.2.11.2 of BS4285-3.9.1:1987. 6.2.11.3 1-naphthol, ethanolic solution. Complying with5.2.11.3 of BS4285-3.9.1:1987. 6.2.11.4 Potassium hydroxide solution. Complying with5.2.1
47、1.4 of BS4285-3.9.1:1987. 6.2.12 Reagents for indole reaction. Complying with5.2.12 of BS4285-3.9.1:1987. 6.2.12.1 Tryptone/tryptophan medium. Complying with5.2.12.1 of BS4285-3.9.1:1987. 6.2.12.2 Kovacs reagent. Complying with5.2.12.2 of BS4285-3.9.1:1987. 6.2.13 Semi-solid nutrient agar. Complying
48、 with5.2.13 of BS4285-3.9.1:1987. 6.2.14 Saline solution. Complying with5.2.14 of BS4285-3.9.1:1987. 6.2.15 Toluene 6.3 Sera Sera complying with5.3 of BS4285-3.9.1:1987. 7 Apparatus NOTEFor details of apparatus including its preparation and sterilization, see BS4285-1.2. 7.1 Ordinary microbiological
49、 laboratory apparatus. 7.2 Oven or incubator, capable of being controlled at (50 5) C, for drying the surface of agar plates. 7.3 Incubator, capable of being controlled at (37 1) C. 7.4 Instrument for the measurement of electrical conductance. Instruments are required which are capable of measuring and recording changes in the electrical conductance of the selective enrichment broths, and subsequently to display these changes with respect to time over the duration of the incubation period. Commercial instruments specifically desig
