BS-808-1986.pdf

BRITISH STANDARD BS 808:1986 Method for Assessing the efficacy of disinfectants by the modified Chick-Martin test UDC 615.28 + 648.63:615.076.7 Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 00:58:12 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 808:1986 This British Standard, having been prepared under the direction of the Disinfectants Standards Committee, was published under the authority of the Board of BSI and comes into effect on 31 July 1986 © BSI 09-1999 First published September 1938 First revision July 1986 The following BSI references relate to the work on this standard: Committee reference DIC/12 Draft for comment 85/52447 DC ISBN 0 580 15267 7 Committees responsible for this British Standard The preparation of this British Standard was entrusted by the Disinfectant Standards Committee (DIC/-) to Technical Committee DIC/12, upon which the following bodies were represented: Association of Public Analysts British Association for Chemical Specialities British Food Manufacturing Industries Research Association British Medical Association Campden Food Preservation Research Association Department of Trade and Industry (Laboratory of the Government Chemist) Hospital Infection Society Institute of Trading Standards Administration Institution of Environmental Health Officers Medical Research Council Public Health Laboratory Service Society for Applied Bacteriology Society of Chemical Industry Amendments issued since publication Amd. No.Date of issueComments Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 00:58:12 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 808:1986 © BSI 09-1999i Contents Page Committees responsibleInside front cover Forewordii 1Scope1 2Definitions1 3Principle1 4Sterilization1 5Reagents and materials1 6Apparatus3 7Preparation of test culture3 8Sampling and stock disinfectant solutions3 9Preliminary tests4 10Procedure4 11Calculation of phenol coefficient4 12Test report5 Appendix A Preparation of disinfectant dilutions6 Table 1 Phenol working solutions: concentrations2 Table 2 Disinfectant test dilutions: concentrations6 Publications referred toInside back cover Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 00:58:12 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 808:1986 ii © BSI 09-1999 Foreword This British Standard has been prepared under the direction of the Disinfectants Standards Committee. It supersedes BS 808:1938, which is withdrawn. The Chick-Martin test was devised at the Lister Institute and first described in 1908 (J Hygiene, 1908, VIII, 655). It was designed primarily to estimate the value of disinfectants to be used outside the body. An essential feature of the test is the inclusion of organic matter. As disinfectants have frequently to act in the presence of large amounts of organic matter, this feature of the test is of considerable practical importance. However it is alleged that the particular form of organic matter (dried faeces) used in the test was variable in character and unpleasant to handle. When BSI was asked to prepare a British Standard based on the Chick-Martin test it constituted a committee to examine the possibility of substituting yeast for faeces in the test. The committee found that yeast was free from the objections alleged against faeces; it also found that further modifications of the original technique produced more consistent results. This work was published as BS 808:1938. This revision of the test introduces the following main changes: a) replacement of the broth by Rideal-Walker broth, single-strength; b) replacement of the organism by Salmonella typhi (NCTC 786); c) deletion of the appendix giving the method for determination of the crystallizing point of the phenol, on the grounds that specification of analytical reagent grade is sufficient; d) modifications to the procedure for obtaining the yeast suspension, in order to obtain a more consistent material. A British Standard does not 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 front cover, pages i and ii, pages 1 to 6, 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 the inside front cover. Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 00:58:12 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 808:1986 © BSI 09-19991 1 Scope This British Standard describes a modification of the original Chick-Martin test for assessing the efficacy of disinfectants used externally to the human body. The modification is to substitute yeast for dried faeces, the essential feature of the test being the inclusion of organic matter. NOTEThe titles of the publications referred to in this British Standard are listed on the inside back cover. 2 Definitions For the.purposes of this British Standard the following definition applies. phenol coefficient the quotient of the mean of the highest concentration of phenol permitting growth in a challenge medium and the lowest concentration not permitting such growth to the corresponding mean concentration of the disinfectant under test 3 Principle A challenge medium containing Salmonella typhi and organic matter (yeast) is added to a range of dilutions of the disinfectant under test and of phenol and the mixtures incubated for 48 h at 37 °C. The mixtures are then examined for growth of the organism, and the phenol coefficient (see clause 2) calculated. 4 Sterilization Wherever sterile reagents, media, materials or apparatus are referred to, or an instruction to sterilize them appears, the sterility shall be that achieved either by being kept at: a) 170 °C to 175 °C for not less than 1 h in an oven (dry sterilization); or b) 121 ± 1 °C for not less than 15 min in an autoclave (wet sterilization). Cotton wool shall not, however, be subjected to more than 2 h dry sterilization. Manipulation of sterile material and of bacterial cultures shall be carried out aseptically. 5 Reagents and materials 5.1 General All reagents shall be of recognized biological or analytical grade, and distilled water or water otherwise produced shall comply with BS 3978. All solutions and media shall be freshly prepared. 5.2 Rideal-Walker broth Dissolve 25 g of Oxoid nutrient broth No. 21) (Code CM 67) in each litre of distilled water. Adjust the pH value, if necessary, to a value that will result in a pH value between 7.3 and 7.5 at 25 °C after the broth is sterilized (normally adjustment before sterilization to pH 7.6 is suitable). Dispense the broth in 5 mL and in 10 mL quantities into the test tubes (6.2.6). Plug or cap the tubes and sterilize as described in clause 4. Do not sterilize the medium in bulk and do not sterilize the medium a second time. 5.3 Test organism Salmonella typhi (NCTC 786)1). This organism can be obtained from the National Collection of Type Cultures (NCTC), subject to its “Conditions of Supply: Hazardous Pathogens” (available from NCTC), i.e. requests for supply must be signed or countersigned by a person whose signature is on file in the NCTC. NOTESalmonella typhi has been categorized by the Advisory Committee on Dangerous Pathogens as a hazard group 3 pathogen and work with viable material should be conducted according to their recommendations for containment level 3. However in the case of Salmonella typhi (NCTC 786), following a hazard/risk assessment initiated by the British Association for Chemical Specialities (BACS), and accepted by the Health and Safety Executive, work with Salmonella typhi (NCTC 786) need not be confined to a microbiological safety cabinet provided that the BACS Code of Practice2) is followed. A fresh culture shall be obtained every 3 months and a fresh initial culture (7.1) prepared. 5.4 Phenol dilutions 5.4.1 Stock solution. Prepare a 50 g/L solution of phenol in sterile water (see clause 4) dissolving 5.0 g of phenol in sterile water and diluting to the mark in a 100 mL one-mark volumetric flask (6.2.10). Use this stock solution to prepare the working solutions. 1) For information on the availability of these reagents and materials, apply to the Enquiry Section, BSI, Linford Wood, Milton Keynes MK14 6LE, enclosing a stamped addressed envelope for reply. 2) Obtainable from BACS, John Marshalls House, 246 High Street, Sutton, Surrey SM1 1PA. Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 00:58:12 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 808:1986 2 © BSI 09-1999 5.4.2 Working solutions. Select four appropriate consecutive dilutions from Table 1 for the determination, based on previous experience or, if necessary, after undertaking a ranging test as described in 9.2. Table 1 Phenol working solutions: concentrations Prepare 100 mL of the first dilution required by adding, by means of a burette the appropriate amount of stock solution (5.4.1) to a 100 mL measuring cylinder and making up to 100 mL with water also measured by means of a burette. After mixing, remove 10 mL of this first dilution by means of a delivery pipette (6.2.8) and place 2.5 mL of this in the first test-tube (6.2.6), discard the remaining 7.5 mL. Add 10 mL of water to the measuring cylinder, by means of a pipette (6.2.3). After mixing by repeated inversion of the cylinder, remove a further 10 mL, add 2.5 mL of this to the second test-tube of the series, discarding the remaining 7.5 mL, and add a further 10 mL of water to the measuring cylinder. Continue the dilution process until the required range of dilutions has been obtained. 5.5 Yeast suspension 5.5.1 Yeast, 250 g moist bakers yeast. NOTEIt is best to obtain the yeast locally so that it is as fresh as possible. 5.5.2 Crumble the yeast (5.5.1) by hand into a 1 L beaker and slowly add 500 mL of water, creaming and stirring with a heavy glass rod until all the lumps are mixed in. Weigh three suitable dishes with a reasonably large surface area (the base of a 90 mm glass Petri dish is ideal) and record their masses. Thoroughly stir the yeast suspension and add 10 ± 0.1 g, weighed to an accuracy of 1 mg, of yeast suspension from the beaker to each of the dishes. Place the rest of the yeast suspension in a refrigerator at 0 °C to 4 °C. Place each of the dishes in a drying oven (6.2.11) at 105 °C and dry. Allow to cool in a desiccator and reweigh. Dry the dishes and contents in the oven, cool and reweigh and continue to repeat these procedures until two consecutive weighings are within 1 mg. Calculate the mean dry mass of yeast as a percentage of the moist material. NOTEAn initial period of 4 h in the drying oven followed by one or two 1 h periods have been found to cover most cases. Measure the volume of the yeast suspension by transferring from the beaker to a 2 L measuring cylinder (6.2.7) swirling it to mix as it is poured out and making sure that no lumps remain. Record the volume. Calculate the volume of diluent to be added to obtain a 5 % (m/m) suspension, calculated on the dry mass of the yeast. Add that amount of diluent to the cylinder using some of this diluent to wash out the beaker. Transfer 100 mL aliquot portions into screw-capped bottles. Sterilize (see clause 4). NOTEBy marking the level in the bottle before autoclaving, those which show any excessive loss in the autoclaving process can be discarded. Allow the bottles to cool slowly and then store them in a refrigerator at 0 °C to 4 °C for at least 6 weeks before use. Take one bottle of the prepared yeast suspension, empty the contents into a sterile 150 mL beaker, and by means of a pH meter (6.2.12) determine the pH of the suspension. Add, with stirring, 0.1 mol/L sodium hydroxide solution from a burette until a pH of 6.9 to 7.1 is obtained. Measure the amount of sodium hydroxide solution required. To each of the other bottles in the batch add aseptically the calculated amount of sterile 1 mol/L sodium hydroxide solution. 5.6 Yeast and Salmonella typhi suspension For the determination, prepare a mixture in the proportion of 48 mL of yeast suspension (5.5), at room temperature, and 2 mL of well mixed Salmonella typhi culture (5.3), in a small flask and allow to stand in a water bath at 20 °C. Make the suspension not more than 1 h before it is added to the dilutions of phenol or test disinfectant. Shake well before use. 5.7 Solid culture medium Dissolve agar in the required volume of freshly prepared broth (5.2) to give an agar content of 15 g/L to 20 g/L. Adjust the pH value, if necessary, to a value that will result in a pH value of 7.4 ± 0.1 at 25 °C after sterilization. Dispense the medium into the tubes (6.2.6), sterilize them as described in clause 4 and allow to set in the form of slopes. % 2.47 2.22 2.0 1.8 1.62 1.458 1.312 1.18 1.062 Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 00:58:12 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 808:1986 © BSI 09-19993 6 Apparatus 6.1 General The apparatus shall be sterile (see clause 4) and scrupulously clean immediately before use. 6.2 Ordinary microbiological laboratory apparatus, especially as given in 6.2.1 to 6.2.12. 6.2.1 Inoculating loop.A loop, 4 mm internal diameter, formed at one end of a piece of 0.376 mm diameter platinum or platinum-iridium alloy wire. A holder, formed by a thin metal rod, or tube, shall be fastened at the other end of the wire and the distance from the loop to the holder shall be 38 ± 1 mm. The loop shall be at an angle to the length of wire so that, when the loop is lifted vertically from the surface of a liquid, the plane of the loop is approximately horizontal. 6.2.2 Incubator(s), capable of being controlled at 37 ± 1 °C and at 22 ± 1 °C. 6.2.3 Pipettes, 5 mL or 10 mL capacity complying with class B of BS 1583, or an automatic pipette or dispenser, capable of dispensing 5 mL or 10 mL to the same degree of accuracy. 6.2.4 Dropping pipettes, delivering 2.5 mL or 10 mL or, preferably, an automatic pipette, with sterile tip, capable of dispensing 2.5 mL or 10 mL accurately. 6.2.5 Graduated pipette delivering 1 mL, complying with class B of BS 700-1. 6.2.6 Test tubes, complying with BS 3218, nominal size 16 mm × 150 mm. 6.2.7 Measuring cylinders, stoppered, 100 mL and 200 mL, graduated in 10 mL increments, and 2 L. 6.2.8 Delivery pipette, delivering up to 10 mL, graduated to the tip. 6.2.9 Water bath, capable of being maintained at 20 ± 0.5 °C. 6.2.10 One-mark volumetric flask, 100 mL, complying with BS 1792. 6.2.11 Drying oven, capable of being controlled at 105 ± 2 °C. 6.2.12 pH meter 7 Preparation of test culture 7.1 Initial culture The test organism (5.3) is distributed in fre