1、December 2009DEUTSCHE NORM English price group 9No 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!$X“1562553ww
2、w.din.deDDIN ISO 16649-1Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of -glucuronidase-positivePart 1: Colony-count technique at 44 C using membranes and5-bromo-4-chloro-3-indolyl -D-glucuronide (ISO 16649-1:2001)English version of DIN ISO 16649-1:2009-12Mikro
3、biologie von Lebensmitteln und Futtermitteln Horizontales Verfahren fr die Zhlung von -Glucuronidase-positiven Teil 1: Koloniezhlverfahren bei 44 C mit Membranen und5-Brom-4-Chlor-3-Indol- -D-Glucuronid (ISO 16649-1:2001)Englische Fassung DIN ISO 16649-1:2009-12SupersedesDIN 10110:1994-08www.beuth.d
4、eDocument comprises pagesEscherichia coli Escherichia coli 14DIN ISO 16649-1:2009-12 Contents Introduction .2Page National foreword .3 5 1 Scope 6 2 Normative references 6 3 Terms and definitions .6 4 Principle7 5 Diluent and culture media.7 5.1 Diluent.7 5.2 Culture media.7 6 Apparatus and glasswar
5、e .9 7 Sampling.9 8 Preparation of test sample9 9 Procedure .10 9.1 Test portion, initial suspension and dilutions 10 9.2 Resuscitation .10 9.3 Transfer to selective medium and incubation 10 9.4 Counting the colony-forming units (CFU).10 10 Expression of results 10 10.1 General10 10.2 Calculation10
6、10.3 Estimation of low numbers.11 10.4 Method of calculation: Special cases12 10.5 Confidence limits.13 11 Test report 13 Bibliography 14 National Annex NA (informative) Bibliography.4 DIN ISO 16649-1:2009-12 National foreword This standard has been prepared by Technical Committee ISO/TC 34 “Food pr
7、oducts” (Secretariat: AFNOR, France). The responsible German body involved 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 eins
8、chlielich Schnellverfahren. The text of ISO 16649-1:2001 has been adopted 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
9、Escherichia coli: Part 1: Colony-count technique at 44 C using membranes 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; T
10、echnical Specification) ISO 7218 referred to in clause 2 has not been 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
11、 EN ISO 6887-1 Amendments This standard differs from DIN 10110:1994-08 as follows: a) For isolation, the membranes from the resuscitation stage are transferred to tryptone-bileglucuronide agar (TBX), containing a chromogenic ingredient (BCIG) for detection of the enzyme glucuronidase. b) The indole
12、test (8.2) has been deleted. Previous editions DIN 10110: 1992-11, 1994-08 3DIN ISO 16649-1:2009-12 National Annex NA (informative) Bibliography DIN EN ISO 6887-1, Microbiology of food and animal feeding stuffs Preparation of the test samples, of initial suspension and of decimal dilutions for micro
13、biological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions 4DIN ISO 16649-1:2009-12 Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of -glucuronidase-positive Escherichia coli Part 1: Colony-count technique at
14、 44 C using membranes and 5-bromo-4-chloro-3-indolyl -D-glucuronide IntroductionBecause of the large variety of food and feed products, this horizontal method may not be appropriate in everydetail for certain products. In this case, different methods which are specific to these products may be used
15、ifabsolutely necessary for justified technical reasons. Nevertheless, every attempt should be made to apply thishorizontal method as far as possible.When this part of ISO 16649 is next reviewed, account will be taken of all information then available regarding theextent to which this horizontal meth
16、od 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 certain groups of products, InternationalStandards and/or national standards may already exist that do not comply with this horizontal
17、 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 from this horizontal method will be those necessary for well-establishedtechnical reasons.This International Standard describes two hor
18、izontal 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. Either part is for general application. However,ISO 16649-1 should be used for foodstuffs which may contain severely stressed cells.5
19、1 ScopeThis part of ISO 16649 specifies a horizontal method for the enumeration of G62-glucuronidase-positive Escherichiacoli in products intended for human consumption or for the feeding of animals. It uses a colony-count techniqueafter resuscitation using membranes and incubation at 44 C on a soli
20、d medium containing a chromogenicingredient 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, such as Escherichia coli O157, will not be detected.2 Normative referencesThe followin
21、g 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 publicationsdo not apply. However, parties to agreements based on this part of ISO 16649 are e
22、ncouraged 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 of ISO and IEC maintainregisters of currently valid International Standards.ISO 68
23、87-1, 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 initialsuspension and decimal dilutions.ISO 7218, Microbiology of food and animal feeding stuffs
24、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 at 44 C form typical blue colonies on tryptone-bile-glucuronide medium (TBX) under
25、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 millilitreor per gram of sample, when test and calculations are carried out in a
26、ccordance with this part of ISO 16649DIN ISO 16649-1:2009-12 64Principle4.1 A specified quantity of the test sample or initial suspension is inoculated onto cellulose membranes overlaidon mineral-modified glutamate agar (MMGA), then incubated at 37 Cfor4h.Under the same conditions, using decimal dil
27、utions of the test sample or of the initial suspension, two plates perdilution are inoculated.4.2 For isolation, the membranes from the resuscitation stage on the MMGA are transferred to tryptone-bile-glucuronide agar (TBX), then incubated at 44 C for 18 h to 24 h.4.3 The number of colony-forming un
28、its (CFU) of G62-glucuronidase-positive Escherichia coli per gram or permillilitre of sample is calculated from the number of typical blue CFU (see clause 10).5 Diluent and culture mediaFor current laboratory practice, see ISO 7218.5.1 DiluentSee ISO 6887-1 or the specific International Standard dea
29、ling with the product to be examined.5.2 Culture media5.2.1 Resuscitation medium: Mineral-modified glutamate agar (MMGA)5.2.1.1 CompositionSodium glutamate 6,35 gLactose 10,0 gSodium formate 0,25 gL-()-Cystine 0,02 gL-()-Aspartic acid 0,02 gL-(+)-Arginine 0,024 gThiamine 0,001 gNicotinic acid 0,001
30、gPantothenic acid 0,001 gMagnesium sulfate heptahydrate (MgSO4Gd77H2O) 0,100 gAmmonium iron(III) citratea0,010 gCalcium chloride dihydrate (CaCl2Gd72H2O) 0,010 gDipotassium hydrogen phosphate (K2HPO4)0,9Ammonium chloride 2,5 gAgar 9gto18gbWater 1 000 mlaIron content at least 15 % (by mass).bDependin
31、g on the gel strength of the agar.DIN ISO 16649-1:2009-12 75.2.1.2 PreparationDissolve the ammonium chloride in the water. Add the other components and heat to boiling.Adjust the pH, if necessary, so that after sterilization it is 6,7 Gb1 0,2 at 25 C.Transfer aliquots of up to 500 ml to suitable con
32、tainers (6.10).Sterilize in the autoclave (6.1) set at 115 C for 10 min.5.2.1.3 Preparation of agar platesPour 12 ml to 15 ml of the medium into sterile Petri dishes (6.11) and allow to solidify.Dry the plates (see ISO 7218). The plates may be stored at 3 C Gb1 2 C for up to 5 days.The agar should b
33、e dry enough to allow excess moisture to disappear within 15 min of spreading the inoculum(1 ml).5.2.2 Selective medium: Tryptone-bile-glucuronic medium (TBX)5.2.2.1 CompositionEnzymatic digest of casein 20,0 gBile salts No. 3 1,5 g5-Bromo-4-chloro-3-indolyl-G62-D-glucuronic acid (BCIG) 144 G6dmolaD
34、imethyl sulfoxide (DMSO)b3mlAgar9gto18gcWater 1 000 mlaFor example, 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.cDependi
35、ng on the gel strength of the agar.5.2.2.2 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 th
36、e medium in the autoclave (6.1) set at 121 C for 15 min.5.2.2.3 Preparation of agar platesProceed as described in 5.2.1.3.DIN ISO 16649-1:2009-12 86 Apparatus and glasswareUsual microbiological equipment (see ISO 7218) and, in particular, the following.6.1 Apparatus for dry sterilization (oven) or w
37、et sterilization (autoclave).6.2 Incubators, capable of being maintained at 37 C Gb1 1 C and at 44 C Gb1 1 C.6.3 Drying cabinet or ventilated oven, capable of being maintained between 25 C Gb1 1 Cand50C Gb1 1 C, ora laminar airflow cabinet.6.4 Refrigerator (for storage of prepared media), capable of
38、 operating at 3 C Gb1 2 C.6.5 Sterile and non-inhibitive membranes, made of cellulose acetate or mixed esters of cellulose, with0,45 m to 1,2 m pore size and 85 mm diameter.6.6 Blunt-ended forceps, sterile, of approximately 12 cm length.6.7 pH-meter, capable of measuring to an accuracy of Gb1 0,1 pH
39、 unit.Its minimum measuring threshold shall be 0,01 pH unit. The pH-meter shall be equipped with either manual orautomatic temperature equalization.6.8 Pipettes, total delivery (blow out), having wide openings and having a nominal capacity of 1 ml, graduatedin 0,1 ml divisions.6.9 Measuring cylinder
40、s, of appropriate capacity for preparation of the media.6.10 Test tubes, bottles or flasks, of suitable capacity for sterilization and storage of culture media.6.11 Petri dishes, of approximately 90 mm diameter.6.12 Spreaders, made of glass or plastic, for example hockey sticks made from a glass rod
41、 of approximately3,5 mm diameter and 20 cm length, bent at right angles about 3 cm from one end and with the cut ends madesmooth by heating.7 SamplingIt is important that the laboratory receive a sample which is truly representative and has not been damaged orchanged during transport or storage.Samp
42、ling 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 parties concerned come to an agreement on this subject.8 Preparation of test samplePrepare the test sample in accordance with the specific International Stand
43、ard appropriate to the productconcerned. If there is no specific International Standard available, it is recommended that the parties concernedcome to an agreement on this subject.DIN ISO 16649-1:2009-12 99 Procedure9.1 Test portion, initial suspension and dilutionsSee ISO 6887-1 and any specific In
44、ternational Standard appropriate to the product.9.2 Resuscitation9.2.1 Using sterile forceps (6.6), aseptically place a membrane (6.5) onto the dried surface of each of two platesof the MMGA (5.2.1.3), taking care to avoid trapping air bubbles beneath the membranes. Gently flatten themembranes with
45、a sterile spreader (6.12), if necessary.Using a sterile pipette (6.8), add 1 ml of the test sample or the initial suspension to the centre of each membrane.Using a sterile spreader, spread the inoculum evenly over the whole membrane surface, avoiding any spillage fromthe membrane.9.2.2 Repeat the pr
46、ocedure as specified in 9.2.1 with the further decimal dilutions, if necessary, using anothersterile pipette and another sterile spreader for each dilution.9.2.3 Leave the inoculated plates in a horizontal position at room temperature for approximately 15 min until theinoculum has soaked through the
47、 membrane into the agar. Incubate the plates for 4 h Gb1 1 h in the incubator (6.2)setat37C, with the membrane/agar surface uppermost.9.3 Transfer to selective medium and incubation9.3.1 After resuscitation, using sterile forceps (6.6), transfer membranes from MMGA (resuscitation medium) toplates of
48、 TBX medium (5.2.2.3).WARNING The moist membrane will adhere to the agar surface. Avoid trapping air bubbles. Do not use aspreader.9.3.2 Incubate the plates for 18 h to 24 h in the incubator (6.2) set at 44 C, and not more than 45C, with themembrane/agar surface uppermost. Do not stack dishes more t
49、han three high.9.4 Counting the colony-forming units (CFU)After the specified period of incubation (9.3.2), count the typical CFU of G62-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 defined in clause 10.10 Expression of results10.1 GeneralThe calculation in 10.2 take
