BS-4285-3.10.3-1989.pdf

british standard bs 4285-3.10.3: 1989 staphylococcus aureus: most probable number enumeration microbiological examination for dairy purposes part 3: methods for detection and/or enumeration of specific groups of microorganisms section 3.10 staphylococcus aureus subsection 3.10.3 enumeration using the most probable number technique noteit is essential that parts 0 and 1, which are published separately, are read in conjunction with this subsection. udc 637.1.055.07:579.8:620.1 licensed copy: sheffieldun sheffieldun, na, thu nov 30 08:12:18 gmt+00:00 2006, uncontrolled copy, (c) bsi bs 4285-3.10.3:1989 this british standard, having been prepared under the direction of the dairying standards committee, was published under the authority of the board of bsi and comes into effect on 31 august 1989 bsi 04-1999 the committees responsible for this standard are shown in part 0. the following bsi references relate to the work on this standard: committee reference dac/4 draft for comment 87/53481 dc isbn 0 580 17091 8 foreword this subsection of bs 4285 has been prepared under the direction of the dairying standards committee. the method was given neither in bs 4285:1968 nor in supplement no. 1 (1970). the method described in this subsection is for use for enumeration of coagulase-positive staphylococcus aureus at levels lower than about 1 c.f.u.1)/ml of test sample or primary dilution. a limitation of the applicability of the most probable number technique is the large error inherent in the method (see bs 4285-2.6). for enumeration of coagulase-positive staphylococcus aureus at levels higher than 1 c.f.u./ml of test sample or primary dilution, the colony count technique described in subsection 3.10.1 should be used. the method described in subsection 3.10.2 should be used when enumeration is not needed and it is only required to know whether coagulase-positive staphylococcus aureus is present or absent in a known quantity of test sample. 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 4, 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. 1) colony forming unit. amendments issued since publication amd. no.date of issuecomments licensed copy: sheffieldun sheffieldun, na, thu nov 30 08:12:18 gmt+00:00 2006, uncontrolled copy, (c) bsi bs 4285-3.10.3:1989 bsi 04-1999i contents page forewordinside front cover 1scope1 2definition1 3principle1 4diluents and culture media1 5apparatus2 6sampling3 7preparation of the test sample3 8procedure3 9results4 10test report4 figure 1 interpretation of coagulase test results4 publications referred toinside back cover licensed copy: sheffieldun sheffieldun, na, thu nov 30 08:12:18 gmt+00:00 2006, uncontrolled copy, (c) bsi ii blank licensed copy: sheffieldun sheffieldun, na, thu nov 30 08:12:18 gmt+00:00 2006, uncontrolled copy, (c) bsi bs 4285-3.10.3:1989 bsi 04-19991 1 scope this subsection of bs 4285 describes a method for the enumeration of coagulase-positive staphylococcus aureus in milk and milk products. note 1see the foreword regarding the applicability of subsections 3.10.1, 3.10.2 and 3.10.3. note 2the titles of the publications referred to in this standard are listed on the inside back cover. 2 definition for the purposes of this subsection of bs 4285, the following definition applies. staphylococcus aureus gram-positive cocci which grow in the enrichment broth specified, forming typical and/or atypical colonies on the surface of the defined selective culture medium and which show a strongly positive coagulase reaction notefor the purposes of this subsection the confirmation of staphylococcus aureus is based on strongly positive coagulase reactions, but it is recognized that some strains of staphylococcus aureus give weakly positive coagulase reactions. these latter strains may be confused with other bacteria but they may be distinguished from such other bacteria by the use of additional tests such as the production of thermonuclease. 3 principle inoculation of a series of decimal dilutions of the test portion into an enrichment medium is followed by incubation. subculturing on baird-parker agar is followed by incubation. identification and confirmation of any staphylococcus aureus colonies found then follows. the final step is the calculation from tables of the most probable number of bacteria likely to give the positive reactions observed in the dilution series. 4 diluents and culture media notein order to improve the reproducibility of the results it is recommended that, for the preparation of the culture media, dehydrated basic components or complete dehydrated media and, for the egg-yolk emulsion (see 4.4.2.3), a commercially available preparation be used, prepared in accordance with the manufacturers instructions. if the culture media and the diluent are not used immediately, they should be kept in the dark at 2 c to 5 c, in conditions that prevent any change in their composition. 4.1 diluents use diluents described in bs 4285-1.2. 4.2 enrichment broth (giolitti and cantoni) with tween 80 4.2.1 base medium. the composition of the base medium shall be as follows: prepare the base medium as follows. dissolve the ingredients in water, heating and shaking to obtain complete solution. cool to room temperature and adjust the ph so that after sterilization it will be 6.9 0.1 at 25 c. dispense in amounts of 19 ml in 20 mm 200 mm tubes. sterilize the medium for 15 min at 121 1 c. notethe solution may be stored for up to 1 month at 2 c to 5 c. 4.2.2 double strength medium. use the same ingredients as for the base medium (4.2.1) but use half the quantity of water and distribute in 10 ml quantities in 20 mm 200 mm test tubes. 4.2.3 potassium tellurite solution. the composition of the potassium tellurite solution shall be as follows: prepare the potassium tellurite solution as follows. dissolve the potassium tellurite in the distilled water with minimal heating. sterilize by filtration. note 1the solution may be stored for up to 3 months at 2 c to 5 c. note 2it is essential to select the product carefully as to source and reliability and to undertake preliminary bacteriological tests to ensure that the potassium tellurite available is suitable for the purpose. 4.2.4 directions for use. immediately before use, heat the tubes containing either the basal medium (4.2.1) or the double strength medium (4.2.2), in boiling water or flowing steam for 20 min at 100 c to expel the air. cool to 45 c and add 0.1 ml of the potassium tellurite solution (4.2.3). 4.3 water agar prepare a 20 g/l agar solution in water. sterilize for 15 min at 121 1 c. before use heat in boiling water or flowing steam at 100 c to expel the air and cool to 45 c. notethe solution may be stored for up to 1 month at 2 c to 5 c. 4.4 agar medium (baird-parker) 4.4.1 base medium. the composition of the base medium shall be as follows: tryptone10.0 g meat extract5.0 g yeast extract5.0 g lithium chloride5.0 g mannitol20.0 g sodium chloride5.0 g glycine1.2 g sodium pyruvate3.0 g tween 801.0 g distilled water1 000 ml potassium tellurite solution (dipotassium trioxotellurate)1.0 g distilled water100 ml tryptone10.0 g yeast extract1.0 g licensed copy: sheffieldun sheffieldun, na, thu nov 30 08:12:18 gmt+00:00 2006, uncontrolled copy, (c) bsi bs 4285-3.10.3:1989 2 bsi 04-1999 prepare the base medium as follows. dissolve the components or the complete agar base in the water by boiling. adjust the ph so that after sterilization it will be 7.2 0.1 at 25 c. transfer the medium in quantities of 90 ml to flasks or bottles of capacity not more than 300 300 ml. sterilize for 15 min at 121 1 c. notethe medium may be stored for up to 1 month at 2 c to 5 c. 4.4.2 solutions 4.4.2.1 potassium tellurite solution, complying with 4.2.3. 4.4.2.2 sodium pyruvate solution. the composition of the sodium pyruvate solution shall be as follows: prepare the sodium pyruvate solution as follows. dissolve the sodium pyruvate in part of the water. make up to the final volume. sterilize by filtration. notethe solution may be stored for up to 1 month at 2 c to 5 c. 4.4.2.3 egg-yolk emulsion concentration approximately 20 %(v/v). if a commercial preparation is not available, prepare the egg-yolk emulsion as follows. wash the shells of fresh hens eggs in a liquid detergent, rinse in running water and dip into 70 % (v/v) ethanol and drain. using aseptic procedures, break each egg into a petri dish and separate the white from the yolk by tilting the dish with the lid slightly open. add the yolks to a sterile measuring cylinder with four volumes of sterile water and mix thoroughly. heat the mixture in a water bath controlled at 45 1 c for 2 h and leave for 18 h to 24 h at 2 c to 5 c to allow a precipitate to form. collect the supernatant emulsion aseptically. notethe emulsion may be stored for up to 72 h at 2 c to 5 c. 4.4.3 complete medium. the composition of the complete medium shall be as follows: prepare the complete medium as follows. melt the base medium and cool it to approximately 45 c by means of water bath (5.3). add the other liquids, mixing well after each addition. 4.4.4 preparation of agar plates. place 15 1 ml of the complete medium (4.4.3), cooled to approximately 45 c, in sterile petri dishes (5.4) and allow to solidify. notethe plates may be stored, prior to drying, at 2 c to 5 c for up to 24 h. dry the plates, with the lids off and the agar surface downwards, in the oven or incubator (5.6), controlled at 50 1 c, for 30 min. 4.5 brain-heart infusion broth the composition of the brain-heart infusion broth shall be as follows: prepare the brain-heart infusion broth as follows. dissolve the components or the complete medium in the water by boiling. adjust the ph so that after sterilization it will be 7.4 0.1 at 25 c. transfer the culture medium to tubes or bottles in quantities of 10 ml. sterilize the medium for 15 min at 121 1 c. notethe medium may be stored for up to 6 months at 2 c to 5 c. 4.6 rabbit plasma use commercially available dehydrated rabbit plasma and rehydrate it according to the manufacturers instructions. add ethylenediaminetetra-acetic acid (edta) solution, if necessary, to give an edta concentration of 1 g/l in the rehydrated plasma solution. 5 apparatus notefor details of apparatus, including its preparation and sterilization, see bs 4285-1.2. 5.1 ordinary microbiological laboratory apparatus. 5.2 water bath or steamer, capable of being maintained at 100 c (for melting agar media and heating giolitti and cantonis enrichment broth). 5.3 water bath, capable of being maintained at 45 1 c. 5.4 petri dishes, of diameter 90 mm. meat extract5.0 g glycine12.0 g lithium chloride5.0 g agar12 g to 20 g (according to the manufacturers instructions) water1 000 ml sodium pyruvate20.0 g water100 ml base medium (4.4.1)90 ml potassium tellurite solution (4.4.2.1)1.0 ml sodium pyruvate solution (4.4.2.2)5.0 ml egg-yolk emulsion (4.4.2.3)5.0 ml peptone10 g dehydrated calf brain infusion12.5 g dehydrated beef heart infusion5.0 g glucose2.0 g sodium chloride5.0 g disodium hydrogenorthophosphate (na2hpo4)2.5 g water1 000 ml licensed copy: sheffieldun sheffieldun, na, thu nov 30 08:12:18 gmt+00:00 2006, uncontrolled copy, (c) bsi bs 4285-3.10.3:1989 bsi 04-19993 5.5 total delivery pipettes, of 1 ml nominal capacity. 5.6 drying cabinet, oven or incubator, ventilated (for drying the surface of agar plates) capable of being maintained at 50 1 c. 5.7 incubator, capable of being maintained at 37 1 c. 5.8 loop, of platinum, nichrome or plastics. 5.9 test tubes and bottles of suitable capacity. 5.10 vortex mixer. 6 sampling take the laboratory sample in accordance with bs 4285-1.1. 7 preparation of the test sample prepare the test sample from the laboratory sample in accordance with bs 4285-1.1. 8 procedure 8.1 enrichment in giolitti and cantonis enrichment broth following the procedure described in bs 4285-2.6, take three tubes of double-strength broth (4.2.2) and transfer to each of these tubes, using a pipette, 10 ml of the liquid test sample or 10 ml of the primary dilution. take three tubes of the single-strength broth (4.2.1) and transfer to each of these tubes, using a pipette, 1 ml of the liquid test sample, or 1 ml of the primary dilution. for each of the following dilutions (from 1 in 10 or 1 in 100, according to the circumstances) take three tubes of single-strength broth (4.2.1). transfer 1 ml of the respective dilutions into each of these tubes. change the pipette for each dilution. carefully mix the inoculum with the medium but avoid any introduction of air. pour a plug of melted agar (4.3) to form a layer on top of the medium and allow it to solidify. incubate for 24 h in the incubator (5.7) controlled at 37 1 c. subculture any tube of the giolitti and cantoni enrichment broth showing any blackening or black precipitate, as described in 8.2. otherwise incubate the tubes for a further 24 h at 37 1 c and subculture as described in 8.2. 8.2 subculturing on baird-parker agar remove the plugs of agar from the incubated tubes (8.1) using the following technique. with a spatula, cut the agar plug along two perpendicular diameters and, if necessary, insert the spatula all around the plug. agitate the tube, if necessary using the vortex mixer (5.10), so that the pieces of the plug fall to the bottom of the tube and, at the same time, the bacteria are placed in suspension. using the loop (5.8) spread about 0.01 ml of the agitated broth on a plate of agar medium (4.4) so as to give isolated colonies after incubation. allow the plates to dry with their lids on for about 15 min at room temperature. invert the plates and incubate them for 24 h and for a further 24 h in the incubator (5.7) controlled at 37 1 c. 8.3 selection of plates and interpretation after incubation for 24 h, mark on the bottom of the plates the positions of any typical colonies present (see note). re-incubate the plates for a further 24 h and mark any newly developed typical colonies. also mark any atypical colonies present (see note). select for confirmation (see 8.4) five typical and/or five atypical colonies, as the case may be, from each plate (presumptive staphylococcus aureus). notetypical colonies are black, shining and convex, 1 mm to 1.5 mm in diameter after incubation for 24 h and 1.5 mm to 2.5 mm in diameter after incubation for 48 h, although colonies of staphylococcus aureus of less than 1 mm may be obtained after 48 h. typical colonies are surrounded by a clear zone which may be partially opaque. after incubation for 24 h an opalescent ring, immediately in contact with the colonies, may appear in this clear zone. atypical colonies may have one of the following morphologies: a) black, shiny narrow white margin with the clear zone absent or scarcely visible and egg-yolk negative or weakly positive; b) less black than a) with a rough, dry texture. atypical colonies are formed frequently by strains of staphylococcus aureus contaminating milk products. 8.4 confirmation (coagulase test) from the surface of each selected colony (see 8.3), remove an inoculum with a sterile wire and transfer it to a tube or bottle of brain-heart infusion broth (4.5). incubate for 20 to 24 h in the incubator (5.7) controlled at 37 1 c. add 0.1 ml of each culture aseptically to 0.3 ml of the rabbit plasma (4.6) (unless other amounts are specified by the manufacturer) in sterile tubes of dimensions 10 mm 75 mm or bottles, and incubate for 4 h to 6 h in the incubator controlled at 37 1 c. examine for clotting of the plasma. consider the coagulase test to be positive if the volume of the clot occupies more than three-quarters of the original volume of the liquid (3+ or 4+ in figure 1). licensed copy: sheffieldun sheffieldun, na, thu nov 30 08:12:18 gmt+00:00 2006, uncontrolled copy, (c) bsi bs 4285-3.10.3:1989 4 bsi 04-1999 as a control, add 0.1 ml of sterile brain-heart infusion broth (4.5) to the recommended quantity of rabbit plasma (4.6) and incubate without inoculation. if the control plasma shows no signs of clotting, consider the test to be valid. 9 results conclude that coagulase-positive staphylococci are present in the corresponding tube of enrichment broth if at least three colonies which clot the rabbit plasma are obtained in accordance with 8.5. calculate the results and express them as described in bs 4285-2.6. 10 test report the test report shall give the number and date of this standard, i.e. bs 4285-3.10.3:1989 and state the results obtained. it shall also state any operating conditions not specified in this standard or regarded as optional, as well as any circumstances that may have influenced the result. the report shall include all details required for complete identification of the sample. figure 1 interpretation of coagulase test results licensed copy: sheffieldun sheffieldun, na, thu nov 30 08:12:18 gmt+00:00 2006, uncontrolled copy, (c) bsi bs 4285-3.10.3:1989 bsi 04-1999 publications referred to bs 4285, microbiological examination for dairy purposes. bs 4285-0, general introduction. bs 4285-1, guide to general procedures. bs 4285-1.1, sampling and preparation of sample. bs 4285-1.2, diluents, media and apparatus and their preparation and sterilization. bs 4285-1.3, procedures for obtaining incubation conditions. bs 4285-2, methods of general application for enumeration of microorganisms. bs 4285-2.6, enumeration of microorganisms by most probable number techni