1、December 2009DEUTSCHE NORM English price group 8No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 07.100.30!$ZK“1559640w
2、ww.din.deDDIN ISO 16649-2Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of -glucuronidase-positivePart 2: Colony-count technique at 44 C using5-bromo-4-chloro-3-indolyl -D-glucuronide (ISO 16649-2:2001)English version of DIN ISO 16649-2:2009-12Mikrobiologie von
3、Lebensmitteln und Futtermitteln Horizontales Verfahren fr die Zhlung von -Glucuronidase-positiven Teil 2: Koloniezhlverfahren bei 44 C mit 5-Brom-4-Chlor-3-Indol- -D-Glucuronid(ISO 16649-2:2001)Englische Fassung DIN ISO 16649-2:2009-12www.beuth.deDocument comprises pagesEscherichia coli Escherichia
4、coli 12DIN ISO 16649-2:2009-12 Contents 2National foreword .3 Introduction .4 1 Scope 5 2 Normative references 5 3 Terms and definitions .5 4 Principle6 5 Diluent and culture media.6 6 Apparatus and glassware .7 7 Sampling.7 8 Preparation of test sample7 9 Procedure .7 9.1 Test portion, initial susp
5、ension and dilutions 7 9.2 Inoculation and incubation.7 9.3 Counting the colony-forming units8 10 Expression of results 8 10.1 General8 10.2 Calculation8 10.3 Estimation of low numbers.9 10.4 Method of calculation: Special cases10 10.5 Confidence limits.10 Test report 11 Page 11 National Annex NA (i
6、nformative) Bibliography.3 5.1 Diluent5.2 Culture medium: Tryptone-bile-glucuronic medium (TBX).6 .6 Bibliography 12 DIN ISO 16649-2:2009-12 National foreword This standard has been prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France). The responsible German body in
7、volved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-01-06 AA Mikrobiologische Lebensmitteluntersuchung einschlielich Schnellverfahren. The text of ISO 16649-2:2001 has been ad
8、opted in this standard without any modification. DIN ISO 16649 consists of the following parts, under the general title Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of -glucuronidase-positive Escherichia coli: Part 1: Colony-count technique at 44 C using membr
9、anes and 5-bromo-4-chloro-3-indolyl-D-glucuronide Part 2: Colony-count technique at 44 C using 5-bromo-4-chloro-3-indolyl-D-glucuronide Part 3: Most probable number technique using 5-bromo-4-chloro-3-indolyl-D-glucuronide (ISO/TS; Technical Specification) ISO 7218 referred to in clause 2 has not bee
10、n adopted as national standard. The revised version ISO 7218:2007 has been published as DIN EN ISO 7218:2007-11. The DIN Standard corresponding to the International Standard referred to in this document is as follows: ISO 6887-1 DIN EN ISO 6887-1 National Annex NA (informative) Bibliography DIN EN I
11、SO 6887-1, Microbiology of food and animal feeding stuffs Preparation of the test samples, of initial suspension and of decimal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions 3DIN ISO 16649-2:2009-12 Microbiology of
12、 food and animal feeding stuffs Horizontal method for the enumeration of -glucuronidase-positive Escherichia coli Part 2: Colony-count technique at 44 C using 5-bromo-4-chloro-3-indolyl -D-glucuronide IntroductionBecause of the large variety of food and feed products, this horizontal method may not
13、be appropriate in everydetail for certain products. In this case, different methods which are specific to these products may be used ifabsolutely necessary for justified technical reasons. Nevertheless, every attempt should be made to apply thishorizontal method as far as possible.When this part of
14、ISO 16649 is next reviewed, account will be taken of all information then available regarding theextent to which this horizontal method has been followed and the reasons for deviations from this method in thecase of particular products.The harmonization of test methods cannot be immediate and, for c
15、ertain groups of products, InternationalStandards and/or national standards may already exist that do not comply with this horizontal method. It is hopedthat when such standards are reviewed they will be changed to comply with this part of ISO 16649 so thateventually the only remaining departures fr
16、om this horizontal method will be those necessary for well-establishedtechnical reasons.This International Standard describes two horizontal methods (ISO 16649-1 and ISO 16649-2) for the enumerationof G62-glucuronidase-positive Escherichia coli.The user may choose either ISO 16649-1 or ISO 16649-2.
17、Either part is for general application. However,ISO 16649-1 should be used for foodstuffs which may contain severely stressed cells.41 ScopeThis part of ISO 16649 specifies a horizontal method for the enumeration of G62-glucuronidase-positive Escherichiacoli in products intended for human consumptio
18、n or for the feeding of animals. It uses a colony-count techniqueat 44 C on a solid medium containing a chromogenic ingredient for detection of the enzyme G62-glucuronidase.WARNING Strains of Escherichia coli which do not grow at 44 C and, in particular, those that areG62-glucuronidase negative, suc
19、h as Escherichia coli O157, will not be detected.2 Normative referencesThe following normative documents contain provisions which, through reference in this text, constitute provisions ofthis part of ISO 16649. For dated references, subsequent amendments to, or revisions of, any of these publication
20、sdo not apply. However, parties to agreements based on this part of ISO 16649 are encouraged to investigate thepossibility of applying the most recent editions of the normative documents indicated below. For undatedreferences, the latest edition of the normative document referred to applies. Members
21、 of ISO and IEC maintainregisters of currently valid International Standards.ISO 6887-1, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension anddecimal dilutions for microbiological examination Part 1: General rules for the preparation of the initialsuspens
22、ion and decimal dilutions.ISO 7218, Microbiology of food and animal feeding stuffs General rules for microbiological examinations.3 Terms and definitionsFor the purposes of this part of ISO 16649, the following terms and definitions apply.3.1G62-glucuronidase-positive Escherichia colibacteria which
23、at 44 C form typical blue colony on tryptone-bile-glucuronide medium (TBX) under the conditionsspecified in this part of ISO 166493.2enumeration of G62-glucuronidase-positive Escherichia colidetermination of the number of colony-forming units (CFU) of G62-glucuronidase-positive Escherichia coli, per
24、 millilitreor per gram of sample, when test and calculations are carried out in accordance with this part of ISO 16649DIN ISO 16649-2:2009-12 54Principle4.1 Duplicate plates of tryptone-bile-glucuronic medium (TBX) are inoculated with the specified quantity of thetest sample or the initial suspensio
25、n.Under the same conditions, using decimal dilutions of the test sample or of the initial suspension, two plates perdilution are inoculated.The dishes are incubated for 18 h to 24 h at 44 C Gb1 1 C then examined to detect the presence of colonies which,from their characteristics, are considered to b
26、e G62-glucuronidase-positive Escherichia coli.4.2 The number of colony-forming units (CFU) of G62-glucuronidase-positive Escherichia coli per gram or permillilitre of sample is calculated (see clause 10).5 Diluent and culture mediaFor current laboratory practice, see ISO 7218.5.1 DiluentSee ISO 6887
27、-1 or the specific International Standard dealing with the product to be examined.5.2 Culture medium: Tryptone-bile-glucuronic medium (TBX)5.2.1 CompositionEnzymatic digest of casein 20,0 gBile salts No. 3 1,5 g5-Bromo-4-chloro-3-indolyl G62-D-glucuronic acid (BCIG) 144 G6dmolaDimethyl sulfoxide (DM
28、SO)b3mlAgar9gto18gcWater 1 000 mlae.g. 0,075 g of cyclohexylammonium salt.bDimethyl sulfoxide is harmful by inhalation and contact. The use of a fumecupboard when handling is advised. Because of this toxicity, a diluentrecommended by the manufacturer may be used.cDepending on the gel strength of the
29、 agar.5.2.2 PreparationDissolve the BCIG in the dimethyl sulfoxide or in the diluent recommended by the manufacturer. Dissolve allcomponents in the water and heat to boiling.Adjust the pH, if necessary, so that after sterilization, it is 7,2 Gb1 0,2 at 25 C.Sterilize the medium in the autoclave set
30、at 121 C for 15 min. Immediately cool the medium in the water bath (6.3)at 44 Cto47C.DIN ISO 16649-2:2009-12 66 Apparatus and glasswareUsual microbiological equipment (see ISO 7218) and, in particular, the following.6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave).6.2 Incu
31、bators, capable of operating at 44 C Gb1 1 C.6.3 Water-bath, capable of being maintained at 44Cto47C.6.4 Test tubes, flasks or bottles of suitable capacity.6.5 Pipettes or micropipettes, total delivery (blow out), having wide openings and having a nominal capacityof 1 ml and 10 ml, graduated respect
32、ively in 0,1 ml and 0,5 ml divisions.6.6 Petri dishes, of approximately 90 mm diameter.6.7 pH-meter, capable of measuring to an accuracy of Gb1 0,1 pH unit.Its minimum measuring threshold shall be 0,01 pH unit. The pH-meter shall be equipped with either manual orautomatic temperature equalization.7
33、SamplingIt is important that the laboratory receive a sample which is truly representative and has not been damaged orchanged during transport or storage.Sampling is not part of the method specified in this part of ISO 16649. If there is no specific International Standard,it is recommended that the
34、parties concerned come to an agreement on this subject.8 Preparation of test samplePrepare the test sample in accordance with the specific International Standard appropriate to the productconcerned. If there is no specific International Standard available, it is recommended that the parties concerne
35、dcome to an agreement on this subject.9 Procedure9.1 Test portion, initial suspension and dilutionsSee ISO 6887-1 and any specific International Standard appropriate to the product.9.2 Inoculation and incubation9.2.1 Using a sterile pipette or a micropipette (6.5), transfer to a sterile Petri dish (
36、6.6) 1 ml of the test sample (ifliquid), or 1 ml of the initial dilution (101) in the case of other products.Inoculate two plates per dilution.Repeat the procedure with the further decimal dilutions, if necessary, using a new sterile pipette for each dilution.DIN ISO 16649-2:2009-12 79.2.2 Pour into
37、 each Petri dish approximately 15 ml of the TBX medium (5.2), previously cooled at 44Cto47Cin the water bath (6.3).Carefully mix the inoculum with the medium and allow the mixture to solidify, with the Petri dishes standing on acool horizontal surface.The time which elapses between the distribution
38、of the inoculum in a dish and pouring of the medium shall notexceed 15 min.9.2.3 Invert the inoculated dishes (9.2.2) so that the bottom is uppermost and place them in an incubator (6.2)setat44C for 18 h to 24 h. The total incubation time shall not be longer than 24 h.WARNING If the presence of stre
39、ssed cells is suspected, incubate for an initial period of 4 h at 37 C,and then raise the incubation temperature to 44 C for 18 h to 24 h. The incubation temperature shall notexceed 45 C.9.3 Counting the colony-forming unitsAfter the specified period of incubation (9.2.3) count the typical CFU of G6
40、2-glucuronidase-positive Escherichia coli ineach dish containing less than 150 typical CFU and less than 300 total (typical and non-typical) CFU.If they form part of the retained dishes, the dishes containing 0 typical CFU should be taken into consideration inthe different calculation methods define
41、d in clause 10.10 Expression of results10.1 GeneralThe calculation in 10.2 takes into account those cases most frequently encountered when conducting tests inaccordance with good laboratory practice. Some special, fairly improbable, cases can arise (e.g. very different CFUnumbers between the two dis
42、hes from the same dilution, or very different proportions from that of the dilution factorbetween the dishes from two successive dilutions). It is then necessary that the counting results be examined,interpreted and possibly rejected by a competent microbiologist.10.2 CalculationFor a result to be v
43、alid, in general it is considered that it is necessary to count the CFU on at least one dishcontaining as a minimum 15 blue CFU.Calculate N, the number of CFU of G62-glucuronidase-positive Escherichia coli present in the test sample per millilitreor per gram, as the weighted mean from two successive
44、 dilutions using the following equation:12(0,1)aNVn n dG3dG2bGe5(1)whereaGe5is the sum of the CFU counted on all the dishes retained from two successive dilutions, at least one ofwhich contains a minimum 15 blue CFU;n1is the number of dishes retained at the first dilution;V is the volume of inoculum
45、, in millilitres, applied to each dish;DIN ISO 16649-2:2009-12 8n2is the number of dishes retained at the second dilution;d is the dilution factor corresponding to the first dilution retained d = 1 in the case (liquid products)where the directly inoculated test sample is retained.Round off the resul
46、ts to two significant figures (see ISO 7218).Take as the result the number of G62-glucuronidase-positive Escherichia coli per millilitre (liquid products) or per gram(other products) expressed as a whole number to two significant figures (if below 100) or as a number between 1,0and 9,9 multiplied by
47、 the appropriate power of 10.10.3 Estimation of low numbers10.3.1 If the two dishes at the level of the test sample (liquid products) or of the initial suspension (other products)or of the first inoculated or retained dilution contain less than 15 blue CFU, calculate NE, the number of CFU ofG62-gluc
48、uronidase-positive Escherichia coli present in the test sample, as the arithmetical mean from two parallelplates using the following equation:EcNVndG3dGd7Gd7Ge5(2)wherecGe5is the sum of the blue CFU counted on the two dishes;V is the volume of the inoculum, in millilitres, applied to each dish;n is
49、the number of dishes retained (n = 2 in this case);d is the dilution factor to the initial suspension or the first inoculated or retained dilution d =1inthecase(liquid products) where the directly inoculated test sample is retained.Round off the results to two significant figures (see ISO 7218).Express the result as follows:Gbe estimated number of G62-glucuronidase-positive Escherichia coli per millilitre (liquid products) or per gram (otherproducts): NE= Y.10.3.2 If the two dishes at the level of the test samp
