1、BRITISH STANDARD BS 6734:2004 Antimicrobial efficacy of disinfectants for veterinary and agricultural use Method ICS 11.080.20; 71.100.35 BS 6734:2004 This British Standard, having been prepared under the direction of the Standards Policy and Strategy Committee, was published on 13 October 2004 BSI
2、13 October 2004 The following BSI references relate to the work on this British Standard: Committee reference CH/216 Draft for comment DC 04/30113130 ISBN 0 580 44526 7 Committees responsible for this British Standard The preparation of this British Standard was entrusted to Technical Committee, CH/
3、216, Chemical disinfectants and antiseptics, upon which the following bodies were represented: Association of British Healthcare Industries British Association for Chemical Specialities British Medical Association British Society of Soil Science Campden and Chorleywood Food Research Camping and Cara
4、vanning Club Ltd Caravan Club Chemical Industries Association Department for Environment, Food and Rural Affairs Health Protection Agency Health and Safety Executive Hospital Infection Society Laboratory of the Government Chemist Ltd Local Government Association Society for Applied Microbiology Soci
5、ety of Chemical Industry Trading Standards Institute UK Cleaning Products Industry Association Veterinary Medicines Directorate Amendments issued since publication Amd. No. Date CommentsBS 6734:2004 BSI 13 October 2004 i Contents Committees responsible Inside front cover Foreword ii 1S c o p e 1 2T
6、e r m s a n d d e f i n i t i o n s 1 3 Principle 1 4 Sterilization 1 5 Reagents and materials 1 6A p p a r a t u s 3 7 Preparation of test culture 4 8 Preparation of challenge medium 5 9 Preparation of dilutions 5 10 Test procedure 7 11 Test result 8 12 Test report 8 Bibliography 9BS 6734:2004 ii B
7、SI 13 October 2004 Foreword This British Standard has been prepared by Technical Committee CH/216. It supersedes BS 6734:1986, which was withdrawn in April 2004 due to lack of confidence. The concerns about BS 6734:2004 expressed by committee members have been addressed in drafting this standard. Th
8、is publication does not purport to include all the necessary provisions of a contract. Users are responsible for its 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 fr
9、ont cover, pages i and ii, pages 1 to 9 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued.BS 6734:2004 BSI 13 October 2004 1 1 Scope This British Standard describes a method for determining the antibacterial efficacy of disinfectants in
10、tended for use in livestock farming applications. The method described in this standard is applicable to disinfectants intended for use against tuberculosis. 2 Terms and definitions For the purposes of this British Standard, the following terms and definitions apply. 2.1 antibacterial efficacy lowes
11、t concentration (greatest dilution) of a disinfectant that will reduce the bacterial population in a challenge medium by at least a factor of 10 4 3 Principle A challenge medium containing Mycobacterium fortuitum is added to a series of dilutions of a product in hard water. After a contact time of 6
12、0 min at 4 C, a portion of each mixture is deactivated and then subcultured into five replicate tubes of recovery broth, which are incubated and examined for growth. NOTE 1 The test organism has been placed in Category 2 by the Advisory Committee on Dangerous Pathogens. The procedure is repeated, as
13、 necessary, until two dilutions differing in concentration by a ratio 9:10 are obtained, one of which produces growth in more than three of the five replicates and the other of which shows no growth in two or more of the five replicates. NOTE 2 Absence of growth in two or more of the five replica cu
14、ltures is equivalent to a reduction of at least 99.99 % in the initial colony count. 4 Sterilization Wherever sterile reagents, media, materials or apparatus are referred to, or an instruction to sterilize them is given, sterility shall be that achieved by being kept at either: a) 170 C to 175 C for
15、 not less than one hour in an oven (dry sterilization 1) ); or b) C for not less than 15 min in an autoclave (wet sterilization). Carry out manipulation of sterile material and bacterial cultures aseptically. 5 Reagents and materials 5.1 General All reagents shall be of recognized biological or anal
16、ytical grade. Water shall be free from substances that are toxic or inhibiting to the bacteria. It shall be freshly glass distilled or, if distilled water of adequate quality is not available, water for injectable preparations (EDQM) and not demineralized water. All solutions and media other than ye
17、ast suspension shall be freshly prepared so as to be fit for purpose, i.e. support bacterial growth as appropriate and not possess inhibitory properties. 5.2 Diluent. Dissolve 0.305 g of anhydrous calcium chloride (CaCl 2 ) and 0.139 g of magnesium chloride hexahydrate (MgCl 2 .6H 2 O) in water and
18、dilute to one litre. NOTE This is standard hard water of 342 mg/kg hardness in accordance with Emulsion Stability Test WHO/M/13 in Specifications for pesticides used in public health 1. 1) This method is suitable for dry glassware only. 121 +3 0BS 6734:2004 2 BSI 13 October 2004 5.3 Test organism My
19、cobacterium fortuitum, NCTC 8573, NCIMB 10384 2) , 3) . 5.4 Solid culture medium. Blood Agar Base 2) , 4) Composition: 5.5 Liquid culture media. 5.5.1 Sterile recovery broth. Nutrient Broth No 2 2) , 4) . Composition: 5.5.2 Challenge culture broth. Add 0.05 % volume by volume polysorbate 80 2) ,5)to
20、 the liquid medium (5.5.1). Sterilize (see Clause 4). 5.6 lnactivator. Add, aseptically, 0.5 ml of sterile horse serum to 9.5 ml portions of liquid culture medium (5.5.1) when cool. 5.7 Yeast, 250 g moist bakers yeast. NOTE It is recommended that the yeast is obtained locally, so that it is as fresh
21、 as possible. 5.8 Yeast suspension. Place three suitable dishes with a reasonably large surface area (the base of a 90 mm glass Petri dish is ideal) in a drying oven at a temperature not greater than (105 2) C (6.2g) and dry. Allow to cool in a desiccator. Weigh to the nearest 1 mg and record their
22、masses. Crumble the yeast (5.7) by hand into a one-litre beaker and slowly add 500 ml of the diluent (5.2), creaming and stirring with a heavy glass rod until all the lumps are mixed in. Thoroughly stir the yeast suspension and transfer (10 0.1) g of it, weighed to an accuracy of 1 mg, from the beak
23、er to each of the dried, weighed dishes. Place each of the dishes in a drying oven at a temperature of (105 2) C (6.2g) and dry, then allow to cool in a desiccator and reweigh. Repeat these procedures until two consecutive weighings are within 1 mg. Calculate the mean dry mass of yeast as a percenta
24、ge of the moist material. NOTE Four hours in the drying oven, followed by one or two further one hour periods in the oven have been found to be satisfactory in most cases. 2) For information on the availability of these reagents and materials, contact BSI Customer Services, British Standards House,
25、389 Chiswick High Road, London W4 4AL. 3) NCTC 8573 is the designation given by the National Collection of Type Cultures and NCIMB 10384 is the designation provided by the National Collection of Marine and Industrial Bacteria. This information is given for the convenience of users of this standard a
26、nd does not constitute an endorsement by BSI of the product named. Corresponding strains supplied by other culture collections may be used if they can be shown to lead to the same results. 4) To improve the reproducibility, it is recommended that commercially available dehydrated material is used fo
27、r the preparation of culture media. The manufacturers instructions for the preparation of these products should be rigorously followed. a) meat extract powder 10.0 g/l; b) peptone 10.0 g/l; c) sodium chloride 5.0 g/l; d) agar 15.0 g/l. Sterilize (see Clause 4). a) meat extract powder 10.0 g/l; b) pe
28、ptone 10.0 g/l; c) sodium chloride 5.0 g/l; Sterilize (see Clause 4). 5) Tween 80 is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and does not constitute an endorsement by BSI of the product named.BS 6734:2004 BSI 13
29、 October 2004 3 During the operation to calculate the mean dry mass, place the remainder of the yeast suspension in a refrigerator at 2 C to 8 C. Measure the volume of the remaining yeast suspension by transferring from the beaker to a two-litre measuring cylinder, swirling it to mix as it is poured
30、 out and making sure that no lumps remain. Record the volume (2 ml). Calculate the volume of diluent to be added to obtain a 5 % mass by mass suspension calculated on the dry mass of the yeast. Add that amount of diluent to the cylinder using some of the diluent to wash out the beaker. Transfer 100
31、ml portions into screw-capped bottles. Sterilize (see Clause 4). Mark the level in the bottles before autoclaving and discard those which show any excessive loss in the autoclaving process. Allow the bottles to cool slowly and then store them in a refrigerator at 2 C to 8 C for at least 8 weeks and
32、not more than 14 weeks before use. Take one bottle of the prepared yeast suspension, empty the contents into a 150 ml beaker and, by means of a pH meter, determine the pH of the suspension. Add, with stirring, sodium hydroxide solution, at a concentration of 0.2 mol/l, from a burette until a pH of 6
33、.9 to 7.1 is obtained. Calculate the amount of sodium hydroxide solution required (0.1 ml). To each of the other bottles in the batch add aseptically the calculated amount of sterile 0.2 mol/l sodium hydroxide solution determined. 5.9 Ringers solution, quarter-strength. Make a quarter-strength Ringe
34、rs solution in accordance with either a) or b). a) Dissolve 9.00 g of sodium chloride, 0.42 g of potassium chloride, 0.48 g of calcium chloride hexahydrate and 0.20 g of sodium hydrogen carbonate in water and dilute to 1 000 ml. Add one volume of the solution to three volumes of water to give a quar
35、ter-strength solution. b) Dissolve one quarter-strength Ringers solution tablet in the appropriate volume of water, to obtain a quarter-strength solution. Then, in common for both solutions, sterilize (see Clause 4). Prior to use, dispense (9 0.1) ml volumes into suitable sterile containers (e.g. te
36、st tubes with closures, Universal containers). 6 Apparatus 6.1 Cleanliness The standard of cleanliness normally associated with bacteriological procedures is required but, when disinfectants based on quaternary ammonium compounds have been used, special care in the cleaning of glassware is necessary
37、. The following cleaning procedure is satisfactory and shall be used in cases of dispute, but otherwise, it is permissible for any method that can be shown to give equivalent results and is known to be satisfactory to be used. Wash glassware in cold, 62 g/l nitric acid solution and allow to stand ov
38、ernight in the acid. Rinse successively with 4 g/l sodium hydroxide solution, tap water, 6 g/l nitric acid solution and tap water until the acid is removed. Finally, rinse with distilled water.BS 6734:2004 4 BSI 13 October 2004 6.2 Ordinary microbiological laboratory apparatus, is required especiall
39、y: a) pipettes, 1.0 ml graduated at 0.01 ml intervals or automatic pipettes with sterile barrier tips, capable of dispensing 0.1 ml and 1.0 ml accurately; b) pipettes, 10 ml graduated at 0.1 ml intervals or an automatic pipette with a sterile tip, capable of dispensing 2.5 ml and 10 ml accurately; c
40、) universal container culture bottles, 25 ml to 35 ml; d) test tubes, 150 mm 19 mm diameter with suitable plastic caps; e) water bath, capable of being controlled at (4 0.5) C and between 45 C and 50 C; f) incubator, capable of being controlled at (37 1) C; g) drying oven, capable of being controlle
41、d at (105 2) C; h) electromechanical agitator, e.g. Vortex mixer 6) ; i) balance, capable of reading to 1 mg; j) timer, capable of reading to 1 s; k) graduated stoppered measuring cylinders; l) refrigerator, capable of maintaining a temperature of 2 C to 8 C; m) pH meter, capable of reading to 0.01
42、pH units; n) bacteriological loop. Measuring equipment shall be calibrated as appropriate. NOTE Universal container culture bottles c) may be used instead of test tubes d). 7 Preparation of test culture 7.1 Initial culture The test organism (5.3), distributed in freeze-dried form, shall be opened in
43、 accordance with the suppliers instructions. Using a sterilized pipette, add approximately 0.5 ml of the sterilized challenge culture broth (5.5.2) to the contents of the tube and incubate for seven days in the incubator (6.2f) controlled at (37 1) C. From this initial culture, prepare the stock cul
44、ture (7.2) and, from that, the broth cultures (7.3). 7.2 Stock culture Spread a loopful of the initial culture (7.1) over the surface of a slope of the sterilized solid culture medium (5.4) in a universal bottle (6.2c). Incubate for seven days in the incubator (6.2f) controlled at (37 1) C, and stor
45、e in a refrigerator between 2 C and 8 C until required. Use stock cultures so stored within one month, although subcultures can be taken before the expiry of that date and used within another month. However, do not take more than six serial subcultures before resorting to a new freeze-dried culture
46、(5.3). 6) Vortex is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and does not constitute an endorsement by BSI of the product named.BS 6734:2004 BSI 13 October 2004 5 7.3 Broth culture Inoculate a 10 ml volume of the
47、 challenge culture broth (5.5.2) in a test tube or universal container (6.2c) or d) from the stock culture (7.2) and incubate in the incubator (6.2f) at (37 1) C. Progressively subculture into fresh challenge culture broth every seven days, incubating the inoculum at (37 1) C for 7 days 4 h. Use sub
48、cultures 1 to 4 for the preparation of the challenge medium (Clause 8). After four broth-to-broth subcultures, restart the process using a fresh stock culture (7.2). 7.4 Colony count Immediately before preparing the challenge medium (Clause 8) make serial decimal dilutions of the broth culture (7.3)
49、 in the quarter-strength Ringers solution (5.9), mixing before taking samples. Prepare duplicate pour plates from 1 ml portions of the sixth, seventh and eighth dilutions and 15 ml portions of the sterilized solid culture medium (5.4) after the latter has been melted and equilibrated at 45 to 50 C. Allow to set. Invert the plates and incubate in the incubator (6.2f), controlled at (37 1) C, in the inverted position for 7 days and the
