INTERNATIONAL STANDARD ISO 6887-1 Second edition 2017-03 Microbiology of the food chain — Preparation of test samples, initial suspension and decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial suspension and decimal dilutions Microbiologie de la chne alimentaire — Préparation des échantillons, de la suspension mère et des dilutions décimales en vue de l’examen microbiologique — Partie 1: Règles générales pour la préparation de la suspension mère et des dilutions décimales Reference number ISO 6887-1:2017(E) © ISO 2017 ISO 6887-1:2017(E) COPYRIGHT PROTECTED DOCUMENT © ISO 2017, Published in Switzerland All rights reserved Unless otherwise specified, no part o f this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission Permission can be requested from either ISO at the address below or ISO’s member body in the country o f the requester ISO copyright o ffice Ch de Blandonnet • CP 401 CH-1214 Vernier, Geneva, Switzerland Tel +41 22 749 01 11 Fax +41 22 749 09 47 copyright@iso.org www.iso.org ii © ISO 2017 – All rights reserved ISO 6887-1:2017(E) Contents Page Foreword iv Introduction v Scope Normative references Terms and definitions Principle Diluents 5.1 Basic materials 5.2 Diluents for general use 5.2.1 Peptone salt solution 5.2.2 Buffered peptone water 5.2.3 Double-strength buffered peptone water 5.3 Diluents for special purposes 5.4 Distribution and sterilization of the diluent 5.5 Performance testing for diluents Apparatus Sampling Preparation of samples 8.1 General 8.2 Frozen products 8.2.1 General 8.2.2 Small samples defrosted before testing 8.2.3 Large pieces or blocks sampled while frozen 8.5 Liquid and non-viscous products 8.6 Acidic products 8.7 High-fat (over 20 %) foods 8.8 Multi-component products 8.9 Packaged products 8.10 Surface samples (swabs and other devices) 10 Specific procedures 10 10 9.2 Duration of the procedure 11 9.3 Pooling and compositing procedures for qualitative tests 11 10 Further dilutions 11 10.1 Decimal dilution series 11 10.2 Other dilution series 12 Annex A (informative) Illustrations of pooling and compositing procedures 13 Annex B (informative) Method for sampling frozen test pieces or blocks 18 Annex C (informative) Data showing reliability of test results according to size of test portions 20 Annex D (informative) Verification protocol for pooling samples for qualitative tests 23 Bibliography 26 8.3 H ard and dry p ro ducts 8.4 D ehydrated and o ther low- mo is ture p ro ducts 9.1 Tes t p o rtio n and initial s us p ens io n (p rimary dilutio n) © ISO 2017 – All rights reserved iii ISO 6887-1:2017(E) Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies) The work o f preparing International Standards is normally carried out through ISO technical committees Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters o f electrotechnical standardization The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part In particular the different approval criteria needed for the di fferent types o f ISO documents should be noted This document was dra fted in accordance with the editorial rules of the ISO/IEC Directives, Part (see www.iso org/directives) Attention is drawn to the possibility that some o f the elements o f this document may be the subject o f patent rights ISO shall not be held responsible for identi fying any or all such patent rights Details o f any patent rights identified during the development o f the document will be in the Introduction and/or on the ISO list of patent declarations received (see www.iso org/patents) Any trade name used in this document is in formation given for the convenience o f users and does not constitute an endorsement For an explanation on the meaning o f ISO specific terms and expressions related to formity assessment, as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology This second edition cancels and replaces the first edition (ISO 6887-1:1999), which has been technically revised A list of parts in the ISO 6887 series can be found on the ISO website iv © ISO 2017 – All rights reserved ISO 6887-1 : 01 7(E) Introduction Because o f the large variety o f food and animal feed products, this horizontal method might not be appropriate in every detail for certain products In this case, di fferent methods which are specific to these products can be used i f absolutely necessary for justified technical reasons When this document is next reviewed, account will be taken of all information then available regarding the extent to which this horizontal method has been followed and the reasons for deviations from this method in the case of particular products The harmonization of test methods cannot be immediate and for certain groups of products, International Standards and/or national standards may already exist that not comply with this horizontal method It is hoped that when such standards are reviewed, they will be changed to comply with this document so that eventually, the only remaining departures from this horizontal method will be those necessary for well-established technical reasons This document defines the general rules for the preparation o f samples, initial suspensions and subsequent dilutions for microbiological examination The remaining parts o f ISO 6887 give specific rules for the preparation o f samples and initial suspensions, each covering the variety o f food and feed products and environmental samples to which ISO 6887 applies For a number o f products, it is necessary to take special precautions, especially when preparing the initial suspension, because o f the physical state o f the product (such as dry products, highly viscous products) or the presence o f inhibitory substances (such as spices, high salt content) or the acidity, etc These are covered in general terms in this document Any special diluents or practices required for particular products or microorganisms in specific standard methods take priority over the general rules listed in the ISO 6887 series These can include the following: — specific rehydration procedures for foods o f low water activity to minimize osmotic shock; — the use o f adequate temperatures to aid suspension o f cocoa, gelatine, milk powder, etc.; — resuscitation procedures for the improved recovery o f stressed microorganisms resulting from food processing and storage; — homogenization procedures and duration specific to certain products (e.g cereals) and/or to certain determinations (e.g yeasts and moulds) © ISO 2017 – All rights reserved v INTERNATIONAL STANDARD ISO 6887-1:2017(E) Microbiology of the food chain — Preparation of test samples, initial suspension and decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial suspension and decimal dilutions WARNING — The use of this document may involve hazardous materials, operations and equipment It is the responsibility o f the user o f this document to establish appropriate sa fety and health practices and to determine the applicability o f regulatory limitations be fore use Scope T h i s c u ment defi ne s genera l r u le s for the aerobic prep a ration o f the i n iti a l s u s p en s ion and o f d i lution s for microbiological examinations of products intended for human or animal consumption T h i s c u ment i s appl ic able to the genera l c a s e a nd o ther p ar ts apply to s p e ci fic group s o f pro duc ts as mentioned in the foreword Some aspects might also be applicable to molecular methods where matrice s c a n b e a s s o ci ate d with i n h ibition o f the P C R s tep s and s e quently a ffe c t the te s t re s u lt This document excludes preparation of samples for both enumeration and detection test methods where prep aration i n s truc tion s are de ta i le d i n s p e c i fic I nternationa l Sta nda rd s Normative references T he fol lowi ng c u ments are re ferre d to i n the tex t i n s uch a way th at s ome or a l l o f thei r content s titute s re qu i rements o f th i s c u ment For date d re ference s , on ly the e d ition cite d appl ie s For u ndate d re ference s , the late s t e d ition o f the re ference d c ument (i nclud i ng a ny amend ments) appl ie s ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for microbiological examinations ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and performance testing of culture media Terms and definitions For the pu r p o s e s o f th i s c u ment, the fol lowi ng term s and defi n ition s apply ISO and IEC maintain terminological databases for use in standardization at the following addresses: — IEC Electropedia: available at http://www.electropedia org/ — ISO Online browsing platform: available at http://www.iso org/obp 3.1 laboratory sample s ample prep are d for s end i ng to the lab orator y a nd i ntende d for i n s p e c tion or te s ti ng [SOURCE: ISO 7002:1986, A.19] © ISO 2017 – All rights reserved ISO 6887-1 : 01 7(E) 3.2 composite sample mixed sample of a number of the same items of food, animal feed, animals or environment, from which a te s t p or tion i s ta ken for exam i nation i n the lab orator y N o te to entr y: S e e i l lu s tratio n o f a co mp o s ite s a mp le i n Annex A 3.3 pooled sample mixed sample of a number of the same items of food, animal feed, animals or environment, where the comple te m i xtu re i s the te s t p or tion a nd i s ta ken a s a whole N o te to entr y: S e e i l lu s tratio n o f a p o ole d s a mp le i n for e xam i nation i n the lab orator y Annex A 3.4 tes t sample sample prepared from the test and from which (3.1 (3.5) are taken laboratory sample test portion s ) accord i ng to the pro ce du re s p e c i fie d i n the me tho d o f N o te to entr y: P rep a ratio n o f the l ab orator y s a mp le b e fore the te s t p o r tion i s ta ken i s i n fre quentl y u s e d i n microbiological examinations [SOURCE: ISO 7002:1986, A.47] 3.5 tes t portion measured (volume or mass) representative sample taken from the preparation of the (3.6) laboratory sample (3.1) for use in the in itial suspen sion N o te to entr y: S ome ti me s prep a ration o f the laboratory sam ple but th i s i s i n fre quentl y u s e d i n m ic rob iolo gic a l e xa m i n ation s (3.1) is required before the test portion is taken, 3.6 initial suspension pri mar y d i lution s u s p en s ion, s olution or emu l s ion ob tai ne d a fter a weighe d or me a s u re d qua ntity o f the pro duc t u nder exam i nation (or o f a te s t s ample prep a re d from the pro duc t) h as b e en m i xe d with, norma l ly, a n i ne - fold qua ntity o f d i luent, a l lowi ng l arge p ar ticle s , i f pre s ent, to s e ttle N o te to entr y: Ni ne - fo ld qu a ntitie s o f d i luent a re no r m a l l y u s e d to pro duce a de c i m a l d i lutio n s er ie s , b ut o ther ratio s m ay b e re qu i re d for s p e c i fic p u r p o s e s , s uch a s to enu merate low nu mb ers 3.7 further dilution s u s p en s ion or s olution ob tai ne d by m i xi ng a me as u re d volu me o f the initial suspen sion x- fold volu me o f d i luent and by rep e ati ng th i s op eration with suitable for the inoculation of culture media, is obtained fur ther (3.6) with an d i lution s unti l a d i lution s erie s , N o te to entr y: Ten- fold d i lution s a re no rm a l l y u s e d to p ro duce a de c i m a l d i lution s er ie s , b ut o ther ratio s m ay b e re qu i re d for s p e c i fic p u r p o s e s 3.8 pooled test portions mixture of test portions from a number of the same items of food, animal feed, animals or environment, where the complete mixture is the test portion examined N o te to entr y: S e e i l lu s tratio n o f p o o le d te s t p or tion s i n Annex A © ISO 2017 – All rights reserved ISO 6887-1 : 01 7(E) 3.9 pooled (pre-) enriched tes t portions individually (pre-)enriched test portions from a number o f the same items o f food, animal feed, animals or environment, from which specified volumes are combined for further examination Note to entry: See illustration o f pooled (pre-)enriched test portions in Annex A 3.10 specific standard International Standard or guidance document describing the examination o f a specific product (or group o f products) for the detection or enumeration o f a specific microorganism (or group o f microorganisms) Principle Preparation of the initial suspension (3.6) in such a way as to obtain as uni form a distribution as possible of the microorganisms contained in the test portion (3.5) Preparation, i f necessary, o f further dilutions (3.7) in order to reduce the number of microorganisms per unit volume to allow, after incubation, observation of their growth or not (in the case of tubes or bottles) or colony counting (in the case o f plates), as stated in each specific standard NOTE In order to restrict the range of enumeration to a given optimum interval, or if high numbers of microorganisms are foreseen, it is possible to inoculate only the necessary (decimal) dilutions needed to achieve the enumeration according to the calculations described in ISO 7218 Diluents 5.1 Basic materials To improve the reproducibility o f test results, it is recommended that either ready-made diluents or dehydrated basic components or a dehydrated complete preparation should be used In all cases, the manu facturer’s instructions shall be followed rigorously Chemical products shall be o f recognized analytical quality and suitable for microbiological examinations The water used shall be distilled water or o f equivalent quality (see ISO 7218 or ISO 11133) For more detailed rules on preparation and performance testing of culture media, see ISO 11133 5.2 Diluents for general use 5.2.1 5.2.1.1 Peptone salt solution Composition Enzymatic digest o f casein Sodium chloride Water 5.2.1.2 1,0 g 8,5 g 000 ml Preparation Dissolve the components in the water in flasks, bottles or test tubes (6.4) by heating, i f necessary Adjust the pH i f necessary so that, a fter sterilization, it is 7,0 ± 0,2 at 25 °C © ISO 2017 – All rights reserved ISO 6887-1 : 01 7(E) 5.2.2 5.2.2.1 Buffered peptone water Composition Peptone a Sodium chloride Disodium hydrogen phosphate dodecahydrate (Na2 HPO ·12H O) b ‡ Potassium dihydrogen phosphate (KH PO 4) ‡ Water 10,0 g 5,0 g 9,0 g 1,5 g 000 ml a For example, enzymatic digest o f casein b I f disodium hydrogen phosphate with a di fferent water content is used, amend the mass o f the ingredient accordingly For example, in case of anhydrous disodium hydrogen phosphate (Na2 HPO 4), use 3,57 g ‡ Buffer ingredients, see 5.2.3 5.2.2.2 Preparation Dissolve the components in the water in flasks, bottles or test tubes (6.4), by heating i f necessary Adjust the pH, i f necessary, so that a fter sterilization, it is 7,0 ± 0,2 at 25 °C 5.2.3 Double-strength buffered peptone water This diluent may be necessary for high acid samples (see 8.6 ) and is prepared by dissolving double the quantities o f a complete dehydrated medium in 000 ml o f water and processing in the same manner I f the diluent is prepared from individual ingredients, only double the quantities o f the two bu ffer ingredients (marked ‡) are required 5.3 Diluents for special purposes See the specific standard or part o f ISO 6887 appropriate to the product concerned 5.4 Distribution and sterilization o f the diluent Dispense the diluent in volumes as necessary for the preparation o f the initial suspensions into vessels (6.4) of appropriate capacity Dispense further diluent in volumes as necessary for the preparation o f the (decimal or other ratio) dilutions into vessels (6.4) of appropriate capacity The tolerance allowable on final diluent volumes, a fter sterilization, shall not exceed ±2 % In order to enumerate several groups o f microorganisms using di fferent culture media, it may be necessary to distribute all the diluents (or some o f them) in quantities greater than 9,0 ml into vessels (6.4) of appropriate size Stopper the vessels loosely to allow for expansion on heating Sterilize in the autoclave at 121 °C ± °C for 15 (see ISO 7218) After autoclaving, check that the volumes from a proportion of the batch of diluent prepared are within the permitted tolerance o f ±2 % This may be achieved either destructively by emptying the contents © ISO 2017 – All rights reserved ISO 6887-1 : 01 7(E) A C o m p o s i t e d s a m p l e s A number of items are composited into one sample and mixed before the test portion is taken as shown in Figure A.1 F 14 i g u r e A — C o m p o s i t i n g s a m p l e s © ISO 2017 – All rights reserved ISO 6887-1 : 01 7(E) A.3 Pooled samples A number o f items o f the same type are pooled into one sample and the whole mixture is used as the test portion (see Figure A.2) Figure A.2 — Pooling samples © ISO 2017 – All rights reserved 15 ISO 6887-1 : 01 7(E) A.4 Pooled test portions T he te s t p or tion s from a nu mb er o f item s o f the s ame typ e a re m i xe d and the whole m i xtu re i s u s e d a s the test portion for subsequent examination (see Figure A.3) Figure A.3 — Pooling test portions 16 © ISO 2017 – All rights reserved ISO 6887-1 : 01 7(E) A P o o l e d ( p r e - ) e n r i c h e d t e s t p o r t i o n s T he te s t p or tion s o f a nu mb er o f item s o f the s a me typ e are ( pre -) en riche d a nd then a s p e c i fie d volu me from each culture is combined for subsequent examination (see Figure A.4) F i © ISO 2017 – All rights reserved g u r e A — P o o l i n g ( p r e - ) e n r i c h e d t e s t p o r t i o n s 17 ISO 6887-1:2017(E) Annex B (informative) Method for sampling frozen test pieces or blocks B N o n - h o m o g e n e o u s b l o c k s For non-homogenous blocks (compressed, conglomerated, frozen or deep-frozen pieces) of mass 25 kg to 30 kg, the perforation points shall be as shown in Figure B.1 F 18 i g u r e B — P r o c e d u r e f o r n o n - h o m o g e n e o u s b l o c k s © ISO 2017 – All rights reserved ISO 6887-1:2017(E) B H o m o g e n e o u s t e s t p i e c e s For homogeneous pieces, the perforation points and depth limits shall be as shown in Figure B.2 Dimensions in millimetres Key cauterized area tray F i © ISO 2017 – All rights reserved g u r e B — P r o c e d u r e f o r h o m o g e n e o u s s a m p l e s 19 ISO 6887-1 : 01 7(E) Annex C (informative) Data showing reliability of test results according to size of test portions C G e n e r a l Data are available to show that the larger the test portion used, the less variance occurs between replicate test results o f the same sample types [6] The data presented in this annex are based on an experimental protocol developed and used in The Netherlands and France C D u t c h s t u d y c o m p a r i n g d i ff e r e n t s a m p l e s i z e s a n d e ff e c t s o f s a m p l e homogenization Samples o f 600 g from three sample types (pre-cut vegetables, Chinese rice dish and milk shake or so ft ice cream) were used to compare di fferences in the homogeneity o f the individual sample types, together with four different sample preparation procedures The test portions for two samples were taken without prior homogenization, while two test samples were homogenized before taking the test portions (see Table C.1) T a b l e C — S t u d y d e s i g n S ample preparation method T1 T2 T3 T4 t o s h o w e ff e c t s o f s a Homogenization of s ample No Yes (100 g) Yes (100 g, to with diluent) No m p l e p r e p a r a t i o Tes t portion 10 g 10 g 20 g 35 g n m e t h o d a n d t e s t p o r t i o n s i z e Tes t portion dilution in 10 in 10 in in 10 For samples prepared without homogenization, two test portion sizes were used: the minimum of 10 g specified for enumeration tests in many specific standards (T1) and a larger test portion o f 35 g (T4) For the two samples prepared with homogenization, test samples of 100 g were taken and homogenized: one (T2) was homogenized directly, while the other (T3) was diluted to with diluent be fore homogenization All sample preparations were tested for aerobic colony count, using a final dilution factor o f in 10 The results o f the study were tested for variance using the F-test and these are shown in Table C.2 20 © ISO 2017 – All rights reserved ISO 6887-1 : 01 7(E) Table C.2 — E ffect o f four sample preparation techniques on the variance o f test results from three sample types Matrix (no o f Results s amples) Pre-cut vegetables (18) Chinese rice dish (22) Milk shake or soft ice cream (8) TO TALS S ample S ample S ample S ample preparation preparation preparation preparation Mean Standard deviation Variance Mean Standard deviation Variance Mean Standard deviation Variance Mean Standard deviation Variance T1 T2 T3 T4 (10 g) (10 g) (10 g) (3 g) 0,150 0,128 0,016 0,285 0,261 0,068 0,094 0,142 0,020 0,201 0,212 0,045 0,164 0,179 0,032 0,218 0,239 0,057 0,064 0,035 0,001 0,171 0,200 0,040 0,111 0,071 0,005 0,104 0,072 0,005 0,069 0,042 0,002 0,101 0,068 0,005 0,172 0,183 0,003 0,216 0,237 0,056 0,115 0,092 0,008 0,182 0,199 0,040 These data are in agreement with other published work[6] showing that the least variance for all three sample types o f di ffering homogeneity was obtained when the largest samples (100 g) were homogenized, with or without prior to dilution C.3 French study comparing seven di fferent sample preparation techniques Samples o f three di fferent sample types (pâté, cheese and mixed salad) were prepared using seven different techniques (T1 to T7) involving variations of sample size and homogenization as shown in Table C.3 Table C.3 — Study design to show e ffects o f sample preparation method and test portion size S ample preparation Homogenization of method s ample T1 T2 T3 T4 T5 T6 T7 No No Yes (whole sample) Yes (whole sample, to with diluent) No No Yes (100 g, to with diluent) Tes t portion Tes t portion dilution 10 g from area 10 g taken from areas 10 g 20 g 35 g from area 35 g taken from areas 20 g in 10 in 10 in 10 in in 10 in 10 in The study was similar to that described in C.2, with a further variation of taking the test portions from a single area of the sample (T1 and T5) or from five di fferent areas (T2 and T6) across the sample Technique T7 was similar to technique T4 except that a sample of 100 g was used rather than the whole sample All sample preparations were tested for aerobic colony count, using a final dilution factor o f in 10 The results o f the study were tested for variance and these are shown in Table C.4 © ISO 2017 – All rights reserved 21 ISO 6887-1 : 01 7(E) T a b l e C — E ff e c t o f s e v e n s a m p l e p r e p a r a t i o n t e c h n i q u e s o n t h e v a r i a n c e o f t e s t r e s u l t s f r o m three sample types T e c h n i q u e ( t e s t p o r t i o n s i z S ample size Homogenized ( Y/N ) N o o f s a m p l e s S ample types RE S ULTS (55 s amples in duplicate) T1 T2 T3 T4 T5 T6 T7 (10 g) (10 g) (10 g) (2 g) (3 g) (3 g) (2 g) Whole Whole Whole ≈10 g Whole Whole N N 100 g Y (1 to with diluent) 16 e ) sd Variance N N Y 129 124 130 P âté , P âté , cheese 0,81 0,66 0,59 0,35 0,36 0,13 cheese cheese T he s e data a l s o i l lu s trate that the le as t varia nce P âté , for Whole or Y (1 to with diluent) 135 P âté , cheese 0,17 0,03 10 P âté , P âté 0,33 0,11 mixed salad 0,30 0,09 P âté , mixed salad 0,1 0,02 a l l th re e s a mple typ e s o f d i fferi ng homo geneity was obtained when the largest samples (100 g) were homogenized, with or without prior to dilution 22 © ISO 2017 – All rights reserved ISO 6887-1:2017(E) Annex D (informative) Verification protocol for pooling samples for qualitative tests D.1 Pooling tests D.1.1 General Two procedures for pooling test portions at different stages of qualitative testing are detailed in the main text (see 9.3 ) This annex describes a protocol suitable for veri fying that the pooling procedure has no effect on the number of false-negative results obtained after examination of different matrices by qualitative methods Only test portions from the same type o f product or environmental sample are to be pooled and the chosen pooling procedure is to be verified be fore subsequent use on routine samples Only samples from the same origin/source (e g same batch, lot) are to be pooled and then only at the request o f the client Samples from different origin/sources, such as from different clients, are not to be pooled D.1.2 Inocula Use a standard suspension o f an appropriately stressed strain (see ISO 16140-2) o f the test microorganism appropriate to the method being investigated Inoculate test portions of the matrix at a level o f approximately c fu per 25 g (or ml), o f which not more than o f 300 replicate tests should contain no detectable organisms per 25 g NOTE The size o f the test portion is normally 25 g (or 25 ml) for qualitative tests, but alternative quantities can be used provided this is taken into account The stress conditions applied should mimic the type o f stress encountered by the target microorganism when present in a naturally contaminated sample o f the product or environmental sample Certified or other re ference materials with a known range o f the appropriate microorganism may be used for preparation of the inocula D.1.3 Sample preparation Add or mix an appropriate quantity o f the suspension o f microorganisms to a known quantity o f uncontaminated product or environmental sample to provide su fficient material for the series o f tests to be undertaken I f samples are inoculated in bulk, ensure that this is su fficiently homogeneous Alternatively, inoculate replicate pre-weighed 25 g (or ml) quantities o f matrix to give su fficient test portions containing the defined level o f microorganisms Retain a suitable quantity o f the uninoculated matrix to use as sterile controls and to mix with the inoculated samples i f preliminary tests show that the inoculum level is too high D.1.4 Pooling test portions D.1.4.1 Preliminary re ference test Add the inoculum (D.1.2 ) containing approximately cfu of the test microorganism to the initial suspension of the 25 g (or ml) test portion in 225 ml (pre-)enrichment broth, incubate and complete the examination according to the method being investigated © ISO 2017 – All rights reserved 23 ISO 6887-1 : 01 7(E) A “detected” result confirms that the inoculum was recovered from the inoculated test portion o f 25 g (or ml) D.1.4.2 Verification protocol The example described is pooling o f test portions from 10 samples o f the same type for the detection of Salmonella (by ISO 6579-1) but the protocol may be used for di fferent numbers of samples, other microorganisms and other procedures Mix 10 test portions of 25 g (or ml) from each sample (total 250 g or ml) with 250 ml of pre-warmed pre-enrichment medium Add the inoculum (D.1.2 ) containing approximately c fu Salmonella and incubate the entire culture in accordance with the standard procedure, ensuring that the incubation temperature is achieved within a suitable time After incubation, subculture and complete the examination according to the method being investigated If Salmonella is detected, pooling of 10 × 25 g (or ml) test portions o f the sample type is verified for the selected test strain and the stress conditions applied If S almonella is not detected, repeat the pooling using fewer test portions until a positive result is obtained D.1.5 Pooling (pre-)enrichment portions D.1.5.1 Preliminary re ference test Carry out the re ference test according to D.1.4.1 D.1.5.2 Verification protocol The example described is pooling o f pre-enrichment portions from 10 samples o f the same type for the detection of Salmonella (by ISO 6579-1) but the protocol may be used for di fferent numbers of samples, other microorganisms and other procedures Add the inoculum (D.1.2 ) containing approximately c fu Salmonella to one of ten initial suspensions of 25 g (or ml) of the sample in 225 ml of pre-enrichment medium and incubate all ten suspensions in accordance with the standard procedure Inoculate a defined volume o f the inoculated pre-enrichment culture and equal volumes o f the other uninoculated pre-enrichment cultures into an appropriate volume of the selective medium, ensuring that the specified ratio for this sub-culturing step is maintained Incubate the selective medium and complete the examination according to the method being investigated D.1.5.3 Interpretation o f results If Salmonella is detected, pooling of 10 pre-enrichment cultures from 25 g (or ml) test portions of the sample type is verified for the selected test strain and the stress conditions applied If S almonella is not detected, repeat the pooling using fewer pre-enrichment cultures until a positive result is obtained in all replicate tests D.2 Confirmation o f findings Evidence to suggest that one or other o f the pooling procedures described provides a satis factory means o f testing multiple samples requires confirmation Little reliance should be placed on the results of a single trial and the chosen protocol should be repeated at least five times, and ideally eight to ten times, using di fferent samples o f the same matrix 24 © ISO 2017 – All rights reserved ISO 6887-1 : 01 7(E) typ e/targe t m icro orga ni s m combi nation to en s u re, with re a s onab le pre ci s ion, that the te s t i s c ap able of detecting the target organism at the lower effective concentration © ISO 2017 – All rights reserved 25 ISO 6887-1 : 01 7(E) Bibliography [1 ] [2 ] [3 ] [4] [5 ] [6] 26 ISO 7002, Agricultural food products — Layout for a standard method of sampling from a lot ISO/TS 17728, Microbiology of the food chain — Sampling techniques for microbiological analysis of food and feed samples ISO 18593, Microbiology of food and animal feeding stuffs — Horizontal methods for sampling techniques from surfaces using contact plates and swabs ISO 16140-2, Microbiology of the food chain — Method validation — Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method ISO 6579-1, Microbiology of the food chain — Horizontal method for the detection, enumeration and serotyping of Salmonella — Part 1: Detection of Salmonella spp CORRY J.E.L.C., JARVIS B., HEDGES A.J Minimising the between-sample variance in colony counts on foods Food Microbiol 2010, 27 pp 598–603 © ISO 2017 – All rights reserved ISO 6887-1 : 01 7(E) ICS 07.100.30 Price based on 26 pages © ISO 2017 – All rights reserved