1、BRITISH STANDARD BS 4285-5.3: 1987 Microbiological examination for dairy purposes Part 5: Ancillary methods Section 5.3 Test for inhibitory substances UDC 637.1.055.07:57.042.2BS4285-5.3:1987 This British Standard, having been prepared under the directionof the Dairying Standards Committee, was publ
2、ished under the authority ofthe Board of BSI and comes intoeffect on 30June1987 BSI 08-1999 BS4285 first published March1968 Supplement No. 1 to BS4285:1968 first published April1970 First revision Section5.3 June1987 The Committees responsible forthis British Standard are shown in Part0 The followi
3、ng BSI references relate to the work on this standard: Committee reference DAC/4 Draft for comment 85/55315 DC ISBN 0 580 15844 6 Foreword This Section of BS4285 has been prepared under the direction of the Dairying Standards Committee. It supersedes B.5 of BS4285:1968, which is deleted by amendment
4、. 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 comprise
5、s a front cover, an inside front cover, pagesi andii, pages1 to4, 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. Amendments issued since publ
6、ication Amd. No. Date of issue CommentsBS4285-5.3:1987 BSI 08-1999 i Contents Page Foreword Inside front cover 1 Scope 1 2 Definitions 1 3 Principle 1 4 Test organism and culture media 1 5 Apparatus 2 6 Sampling 2 7 Preparation of test sample 2 8 Test culture, test plates and controls 2 9 Procedure
7、3 10 Expression of results 3 11 Interpretation of results 3 12 Confirmation of results for raw milks 4 13 Test report 4 Publications referred to Inside back coverii blankBS4285-5.3:1987 BSI 08-1999 1 NOTEIt is essential that Parts0 and 1, which are published separately, be read in conjunction with t
8、his Section. 1 Scope This Section of BS4285 describes a method for testing for penicillin and other inhibitory substances in milk and milk powders. NOTEThe titles of the publications referred to in this standard are listed on the inside back cover. 2 Definitions For the purposes of this Section of B
9、S4285 the following definitions apply. 2.1 inhibitory substances materials that inhibit the growth of a test organism 2.2 penicillin an antibiotic containing a beta-lactam ring in the molecule and which is inactivated by the enzyme penicillinase NOTESome synthetic penicillin-type antibiotics may not
10、 be inactivated by penicillinase under the conditions of the test and will therefore be classified with inhibitors other than penicillin. 3 Principle A disc of absorbent paper impregnated with sample is placed on the surface of an agar medium inoculated with Bacillus stearothermophilus. After incuba
11、tion, a clear zone around the disc in the otherwise cloudy medium indicates an inhibitory substance. 4 Test organism and culture media 4.1 Test organism The test organism shall be Bacillus stearothermophilus var. calidolactis: NCDO 1780 or NCIB 11780 1) . 4.2 Medium for agar slopes 4.2.1 Composition
12、. The composition of the medium for the agar slopes shall be as follows: 4.2.2 Preparation. Dissolve the components in the water with heat. Adjust the pH so that after sterilization it is7.4 0.1 at 25 C. Dispense8mL to10mL volumes into screw-capped test-tubes. Plug with cotton wool. Sterilize at121
13、1 C for15min and allow to set in sloped position. NOTEThe quantity of agar should be varied according to the manufacturers instructions to give a firm gel. 4.3 Culture broth 4.3.1 Composition. The composition of the culture broth shall be as follows: 4.3.2 Preparation. Dissolve the components in the
14、 water. Adjust the pH so that after sterilization it is8.0 0.1 at25 C. Sterilize at121 1 C for15min. 4.4 Plate count agar 4.4.1 Composition. The composition of the plate count agar shall be as follows: 4.4.2 Preparation. Dissolve the components in the water with heat. Adjust the pH so that after ste
15、rilization it is8.0 0.1at25 C. Sterilize at121 1 C for15min. NOTEThe quantity of agar should be varied according to the manufacturers instructions to give a firm gel. 4.5 Standard penicillin solution Prepare a standard solution of penicillin of100i.u./mL by dissolving sodium or potassium benzyl peni
16、cillin in water in a stoppered bottle (see5.2). Use on the day of preparation and maintain at2 C to5 C. 4.6 Penicillinase solution Prepare a penicillinase solution of1000i.u./mL by dissolving penicillinase in water. Store at2 C to 5 C for not more than2weeks. 1) Obtainable from the National Collecti
17、on of Food Bacteria, AFRC Food Research Institute, Shinfield, Reading RG2 9AT or the National Collection of Industrial and Marine Bacteria, Torry Research Station, PO Box 31, Aberdeen AB9 8DG respectively. yeast extract 2 g peptone 5 g meat extract 1 g sodium chloride 5 g agar 15 g water 1000 m L ye
18、ast extract 10 g tryptone 20 g glucose 0.5 g water 1000 mL yeast extract 2.5 g tryptone 5 g glucose 1 g agar 15 g water 1000 mLBS4285-5.3:1987 2 BSI 08-1999 4.7 Milk free from inhibitory substances Prepare a 100g/L solution of inhibitor-free milk by the reconstitution in water of skimmed milk powder
19、 previously tested and found to be free from inhibitory substances. Alternatively, dispense in bottles of sufficient capacity a quantity of fresh bulk milk tested and found to be free from inhibitory substances. In either case heat for1h at 100 C. Store at2 C to5 C for not more than1week. Carry out
20、a test on nine discs from each batch of paper discs using water to verify that the discs do not give a clear zone when controlled at55 1 C for2.5h to5h on the test plates (see7.3.4) in the incubator (5.5). 5 Apparatus NOTEFor details of apparatus including its preparation and sterilization seeBS4285
21、-1.2. 5.1 Ordinary microbiological laboratory apparatus 5.2 Sample bottles, with closures which do not contain inhibitors. NOTERubber stoppers are not suitable. 5.3 Petri dishes. NOTE 1To ensure an even depth of agar medium, which is essential for accuracy, care should be taken to select a good qual
22、ity flat-bottomed brand. NOTE 2Sterile disposable plastics Petri dishes complying with BS611-2 are recommended. 5.4 Forceps, clean, dry, with fine points. 5.5 Incubator, capable of being controlled at55 1 C. 5.6 Waterbaths, capable of being controlled at37 1 C and80 1 C. 5.7 Antibiotic assay paper d
23、iscs, 12mm to13mm in diameter. 6 Sampling Take the laboratory sample in accordance with BS4285-1.1. 7 Preparation of test sample 7.1 Prepare the test sample from the laboratory sample in accordance with BS4285-1.1. Keep the test sample in a sample bottle(5.2). 7.2 Test raw and heat processed milks u
24、ndiluted. Either test them on the day the sample is taken, or store them at 20 C for as short a time as possible. 7.3 Reconstitute milk powder by adding to sterile distilled water to give a100g/L solution. 8 Test culture, test plates and controls 8.1 Maintenance of test culture Keep the test organis
25、m (4.1) on agar slopes (4.2) in tubes with screw caps and cotton wool plugs. Inoculate a tube in streaks, using a loop of the test culture and incubate for 48h in the incubator (5.5) controlled at55 1 C. After incubation, flame the cotton wool plug of the tube, then force it a little way into the tu
26、be and close with a screw cap. Keep the culture at2 C to5 C for not more than4months. 8.2 Propagation of the test culture 8.2.1 Aseptically transfer10mL of the culture broth(4.3) to a150mL sterile conical flask. 8.2.2 Inoculate the culture broth in the flask (8.2.1) with a loop of the culture from t
27、he agar slope (8.1). Alternatively, use0.1mL of an existing liquid culture (8.2.2) provided that this is no more than36h old and has been kept at2 C to5 C. Incubate for16h to18h in the incubator (5.5) controlled at55 1 C. 8.2.3 When using a culture from an agar slope (8.1), or a liquid culture (8.2.
28、2) which is more than36h old, carry out the subculturing procedure described in 8.2.1 and 8.2.3 at least twice with no more than an interval of36h between subculturing. Keep the culture at2 C to5 C during any intervening period. It is essential that the culture be uniformly turbid. If it contains fl
29、ocks or sediment discard it and prepare a new culture from the agar slopes(8.1). NOTEThe liquid culture (8.2.2) should have a viable colony count between50million/mL and100million/mL when incubated at 55 1 C for48h on a plate count agar medium(4.4). 8.3 Preparation of test plates 8.3.1 Melt the plat
30、e count agar (4.4) and cool to55 C, for example, by placing the medium for30min in the incubator (5.5) controlled at55 1 C. 8.3.2 Add1part of freshly prepared liquid culture(8.2.2) to5parts of the plate count agar(8.3.1) at55 1 C in a test tube or bottle, and mix thoroughly. 8.3.3 For reference purp
31、oses, mark off and number squares of sides25mm on the bottom of the test plates using a ruler and wax pencil or other suitable means. 8.3.4 Transfer a quantity of inoculated agar medium(8.3.2) calculated to give a layer0.8mm to1.0mm thick to a sterile Petri dish (5.3) previously heated in the incuba
32、tor (5.5) to55 1 C. Prepare further test plates as required.BS4285-5.3:1987 BSI 08-1999 3 Allow the medium to solidify on a cold horizontal surface, previously checked with a spirit level. Replace the lids on the dishes and then invert the lidded dishes to minimize condensation on the surface of the
33、 agar medium. 8.3.5 The test plates thus prepared should be used preferably on the same day, but they may be kept several days provided that they are cooled immediately after preparation and kept in a sealed polyethylene bag at2 C to5 C. 8.4 Penicillin controls NOTEThe penicillin solutions (8.4.1 to
34、 8.4.3) should be prepared on the day the test is carried out. 8.4.1 Prepare a working solution of penicillin by making up1.25mL of the standard penicillin solution (4.5) to 1000mL with water. This working solution contains 0.125i.u./mL. 8.4.2 Prepare a solution containing0.005i.u./mL penicillin by
35、adding48mL of the inhibitor-free milk(4.7) to2mL of the working solution (8.4.1) and mixing. 8.4.3 Prepare a solution containing0.01i.u./mL penicillin by adding46mL of the inhibitor-free milk(4.7) to4mL of the working solution (8.4.1) and mixing. 8.5 Penicillinase control 8.5.1 Mix the test sample (
36、see clause7) thoroughly and transfer10mL to a suitable sterile wide-mouthed bottle. 8.5.2 Add about0.4mL of the penicillinase solution(4.6) and mix. Incubate for at least30min in the waterbath (5.6) controlled at37 1 C. 9 Procedure 9.1 Dip a paper disc (5.7) into the well-mixed test sample (see clau
37、se7) by means of the forceps (5.4). Remove any excess by touching the disc against the side of the sample bottle. Place the disc flat on the surface of a test plate (8.3.4) at the centre of a marked square and press down gently with the forceps. Randomize the distribution of this disc and the seven
38、described subsequently (9.3 to 9.5) over the test plates. 9.2 Repeat the procedures described in9.1 using another disc. 9.3 Repeat the procedures described in 9.1 and 9.2 using the penicillin control milk (8.4.2) containing0.005i.u./mL penicillin, instead of the sample. 9.4 Repeat the procedures des
39、cribed in9.1 and 9.2 using the penicillin control milk (8.4.3) containing0.01i.u./mL penicillin, instead of the sample. 9.5 Repeat the procedures described in9.1 and 9.2 using the penicillinase control (8.5), instead of the sample. 9.6 When all eight discs have been placed on the inoculated agar med
40、ium, incubate the plate with the cover downwards for 2“ h to5h in the incubator (5.5) controlled at 55 1 C. 9.7 After incubation examine the plates in front of a suitable light source for clear zones of inhibition around the paper discs. 10 Expression of results Determine the average diameters of th
41、e clear zones of inhibition for the sample, the penicillin milk controls (8.4.2) and (8.4.3), and the penicillinase control (8.5) and record these diameters. 11 Interpretation of results 11.1 Clear zones around the discs (9.3) containing the penicillin milk control corresponding to0.005i.u./mL penic
42、illin should be perceptible; there should be larger zones around the discs (9.4) containing the penicillin control corresponding to0.01i.u./mL penicillin. NOTEThe complete absence of clear zones of inhibition could be due to errors in technique or due to insensitivity of the culture. In these circum
43、stances repetition of the procedure should be commenced using a new culture. As the test is qualitative, however, useful information may be obtained in the meantime by repeating the test method replacing one of the pencillin control milk samples with a penicillin milk control sample of higher streng
44、th, e.g.0.02i.u./mL. 11.2 The presence of clear zones around the discs(8.4) containing the sample of milk indicates the presence in the milk of substances inhibitory to the test organism. 11.3 If there are no clear zones around the discs containing the penicillinase control (9.5), but there are clea
45、r zones around the discs containing the sample (9.1), the inhibitory substance in the sample is penicillin. The level of penicillin in the sample is judged by comparing the zone diameters produced by the test sample and the penicillin control milks containing0.005i.u./mL and0.01i.u./mL (9.3 and9.4).
46、 11.4 If the average diameter of the clear zones around the discs (9.5) containing the penicillinase control is equal to the average diameter of the clear zones around the discs (9.1) containing the sample, the inhibitory substance is not penicillin.BS4285-5.3:1987 4 BSI 08-1999 11.5 If there are cl
47、ear zones around the discs (9.5) containing the penicillinase control which are smaller in average diameter than that of the clear zones around the discs (9.1) containing the sample, this contains penicillin together with other inhibitory substances. Clear zones of any size around the penicillinase
48、discs (9.5) are attributable to inhibitory substances other than penicillin, while those around the sample discs (9.1) are attributable to penicillin and other inhibitory substances. NOTETesting of samples with and without the addition of penicillinase provides an indication of whether the inhibitor
49、y substance is penicillin, however, a small reduction of inhibition associated with penicillinase may be no more than a reflection of the intrinsic properties of the impure enzyme preparations commonly available. 12 Confirmation of results for raw milks Transfer about2mL of the test sample to a16mm 160mm sterile tube taking care not to wet the sides of the tube. Place the tube for10min in a waterbath (5.6) controlled at80 1 C with the top level of the milk at least20mm below the water level. Cool to
