1、May 2016 English price group 12No part of this translation 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 07.100.99; 85.040; 85.060!%UV“250519
2、1www.din.deDIN ISO 8784-1Pulp, paper and board Microbiological examination Part 1: Enumeration of bacteria and bacterial spores based on disintegration (ISO 8784-1:2014),English translation of DIN ISO 8784-1:2016-05Faserstoff, Papier und Pappe Mikrobiologische Untersuchung Teil 1: Zhlung von Bakteri
3、en und bakteriellen Sporen nach Desintegration (ISO 8784-1:2014),Englische bersetzung von DIN ISO 8784-1:2016-05Ptes, papiers et cartons Analyse microbienne Partie 1: Dnombrement des bactries et des spores bactriennes bas sur la dsintgration (ISO 8784-1:2014),Traduction anglaise de DIN ISO 8784-1:20
4、16-05www.beuth.deDocument comprises 19 pagesDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritative.05.16 A comma is used as the decimal marker. Contents PageNational foreword 3Introduction 51 Scope . 62 Normative references 63 Terms and de
5、finitions . 64 Principle 75 Culture media and diluents . 75.1 General . 75.2 Water . 75.3 Culture media for total bacteria count and spore count . 75.4 Diluents . 76 Apparatus and equipment 86.1 General . 86.2 List of equipment 87 Sampling 88 Preparation of the test material . 98.1 General . 98.2 De
6、termination of dry-matter content . 98.3 Weighing . 98.4 Disintegration 99 Determination of the total bacterial count and spore count . 109.1 General 109.2 Plating for total bacterial count . 109.3 Plating for spore count 119.4 Incubation 1110 Enumeration of the colonies . 1111 Calculation and repor
7、t . 1111.1 Calculation . 1111.2 Interpretation . 1211.3 Report 1312 Test report 13Annex A (informative) Dilution fluid . 14Annex B (informative) Precision 15Bibliography .19DIN ISO 8784-1:2016-05 2Foreword .4National foreword This document (ISO 8784-1:2014) has been prepared by Technical Committee I
8、SO/TC 6 “Paper, board and pulps” (Secretariat: SCC, Canada). The responsible German body involved in its preparation was DIN-Normenausschuss Papier, Pappe und Faserstoff (DIN Standards Committee Paper, Board and Pulps), Working Committee NA 074-02-01 AA Chemisch-technische Prfverfahren fr Papier, Pa
9、ppe, Faserstoff und Chemiezellstoff. ISO 8784-1:2014 has been adopted without modifications. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 186:2002 DIN EN ISO 186: 2002-08 ISO 638:2008 DIN EN ISO 638:2009-01 ISO 4833:2003 DIN EN ISO 4
10、833:2003-06 ISO 7213:1981 DIN EN 27213:1993-11 ISO 7218 DIN EN ISO 7218 ISO 11133 DIN EN ISO 11133 ISO/TS 19036 DIN ISO/TS 19036 (DIN SPEC 10125) National Annex NA (informative) Bibliography DIN EN ISO 186:2002-08, Paper and board Sampling to determine average quality DIN EN ISO 638:2009-01, Paper,
11、board and pulps Determination of dry matter content Oven-drying method DIN EN ISO 4833:2003-06, Microbiology of the food chain Horizontal method for the enumeration of microorganisms Part 1: Colony-count at 30 degrees C by the pour plate techniqueN1)DIN EN 27213:1993-11, Pulps; sampling for testing
12、DIN EN ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations DIN EN ISO 11133, Microbiology of food, animal feed and water Preparation, production, storage and performance testing of culture media DIN ISO/TS 19036 (DIN SPEC 10125)
13、, Microbiology of food and animal feeding stuffs Guidelines for the estimation of measurement uncertainty for quantitative determinations N1) National footnote: Now replaced by DIN EN ISO 4833-1:2013-12 and DIN EN ISO 4833-2:2013-12. DIN ISO 8784-1:2016-05 3 ForewordISO (the International Organizati
14、on for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of 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 ha
15、s 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 of electrotechnical standardization.The p
16、rocedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the edit
17、orial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent righ
18、ts identified during the development of 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 information given for the convenience of users and does not constitute an endorsement.For an expla
19、nation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee responsible for this docum
20、ent is ISO/TC 6, Paper, board and pulps, Subcommittee SC 2, Test methods and quality specifications for paper and board.This third edition cancels and replaces the second edition (ISO 8784-1:2005), which has been technically revised.The second edition was applicable to yeast and mould, as well as ba
21、cteria. The following main changes have been made with respect to the previous edition: This third edition is only applicable to bacteria and bacterial spores, and no longer applicable to yeast and mould; incubation temperature changed from (37 C 1 C) to (32 C 2 C) (9.4); 2 parallel determinations a
22、re to be made (Clause 8 and Clause 9); the result can be reported “as received” in addition to reporting on a dry-mass basis (8.2, 11.1, and Clause 12).ISO 8784 consists of the following parts, under the general title Pulp, paper and board Microbiological examination: Part 1: Enumeration of bacteria
23、 and bacterial spores based on disintegrationDIN ISO 8784-1:2016-05 4 IntroductionThis part of ISO 8784, which deals with the microbiological examination of dry market pulp, paper, and paperboard, is broadly based on ISO 48331although the conditions are not identical. However, it provides specific a
24、mplification where necessary. It is intended for the estimation of colony-forming units, CFU, aerobic bacteria, and bacterial spores.Because of the exacting techniques required in aseptic procedures, reproducible good quality results can only be ensured by skilled microbiological technicians.DIN ISO
25、 8784-1:2016-05 5 Pulp, paper and board Microbiological examination Part 1: Enumeration of bacteria and bacterial spores based on disintegration1 ScopeThis part of ISO 8784 specifies a method for determining the total number of colony-forming units of bacteria and bacterial spores in dry market pulp
26、, paper, and paperboard after disintegration. The enumeration relates to specific media.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For
27、 undated references, the latest edition of the referenced document (including any amendments) applies.ISO 186:2002, Paper and board Sampling to determine average qualityISO 7213:1981, Pulps Sampling for testingISO 638:2008, Paper, board and pulps Determination of dry matter content Oven-drying metho
28、d3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1bacteriamicroscopic, single-celled organisms that possess a prokaryotic type of cell structure, which reproduce by fission and are able to grow under the test conditions specified in this part of
29、ISO 87843.2bacterial sporeshighly resistant, dormant structuresEXAMPLE Endospores from certain genera of bacteria.3.3total bacterial countnumber of colony-forming units (CFU) of bacteria and bacterial spores formed after incubation in a standard culture medium, under the test conditions specified in
30、 this part of ISO 87843.4spore countnumber of colony-forming units (CFU) of bacterial spores formed after incubation in a standard culture medium, under the test conditions specified in this part of ISO 8784DIN ISO 8784-1:2016-05 6 4 PrincipleThis poured plate method involves enumeration of colonies
31、 in a standard culture medium. A fibre suspension, prepared from paper, paperboard, or pulp samples, is plated in agar. Two parallel determinations are made. For enumeration of bacterial spores, the fibre suspension is heated for 10 min at 80 C prior to plating. The plates are incubated at 32 C for
32、48 h. The total numbers of bacteria or bacterial spores are enumerated by counting the colonies formed in the agar.The mean value of 2 parallel determinations is calculated and the results are expressed as the number of CFU per gram of sample.5 Culture media and diluents5.1 GeneralAll substrates and
33、 diluents shall be appropriately sterilized. When preparing the culture medium, make sure that the ingredients are completely dissolved by mixing while heating prior to dispensing into suitable containers for sterilization. See ISO 111332for quality assurance and guidelines on preparation and produc
34、tion of culture media.5.2 WaterWhen water is mentioned in a formula, use distilled water or purified water, see ISO 111332.5.3 Culture media for total bacteria count and spore countCulture medium shall be prepared as follows, or from commercially available dehydrated culture media according to the m
35、anufacturers instructions. Ready-to-use medium may be used when its composition is comparable to that given in this part of ISO 8784. To test the performance of the medium, see ISO 111332.Plate count agar (PCA) composition per litre:Tryptone 5,0 gYeast Extract 2,5 gDextrose 1,0 gAgar 15,0 gWater 1 0
36、00 mlFinal pH 7,0 0,2If PCA is not available, Tryptone glucose extract (TGE) agar may be used (see A.3). The use of TGE as an alternative culture medium is acceptable if it gives comparable results as the standard culture medium. The culture medium used shall be stated in the test report (see Clause
37、 12).5.4 DiluentsRingers solution (see A.1) is preferred, although other isotonic solutions may be used. Ringers tablets are commercially available.To facilitate the release of cells from the fibres, it is recommended to add 20 l of Tween 80 (see A.2) per litre to the Ringers solution prior to steri
38、lization by autoclaving.The diluent used and if Tween 80 has been added, shall be stated in the test report (see Clause 12).DIN ISO 8784-1:2016-05 7 6 Apparatus and equipment6.1 GeneralAll laboratory equipment and parts of the equipment in direct contact with the sample and the diluent or the cultur
39、e medium shall be sterilized.NOTE For advice on standard microbiological equipment, see ISO 72184.6.2 List of equipment6.2.1 Use ordinary microbiological laboratory equipment, and the following.6.2.2 Suitable wrapping material, e.g. aluminium foil (non-coated and inert), ready-to-use envelopes of di
40、fferent sizes or self-closing plastic bags, all of which are commercially available.6.2.3 Disintegrator, high speed electrical blender with metal (preferably stainless steel) or glass cup that can be sterilized.NOTE Other homogenizing system with equivalent efficiency may be used.6.2.4 Incubator, ca
41、pable of maintaining a constant temperature of 32 C 2 C.6.2.5 Petri dishes, having a diameter of 90 mm (standard) or 140 mm to 150 mm (alternative).6.2.6 Pipettes, of wide-mouth type suitable volume.The width of the mouth must be large enough so that a 1 % fibre suspension can easily be drawn into t
42、he pipette tip.NOTE A suitable volume is 10 ml or 50 ml.6.2.7 Water bath, capable of maintaining a temperature of 80 C 2 C.6.2.8 Colony-counting equipment or magnifying device, with a magnification between 1,5 and 2,5 shall be used.NOTE The use of an additional lens might be necessary to increase th
43、e magnification, up to 10 , to facilitate the counting of pin-point bacterial colony-forming units and also to ensure that no other particles except bacterial colonies are counted (see Clause 10).6.2.9 Balance, with an accuracy of 0,01 g.6.2.10 Sterilizing unit, an autoclave capable of sterilization
44、 at 121 C.7 SamplingMake sure that the sampling procedure is performed using aseptic techniques.If the sample is to represent a lot of paper or paperboard, the sampling shall be in accordance with ISO 186:2002. From each unit of paper or paperboard to be sampled, cut several top layers and discard t
45、hem to eliminate surface contamination. Use a sterile knife to cut through several layers of the paper or board sample, producing a stack of sheets. Discard the top sheet.DIN ISO 8784-1:2016-05 8 If the sample is to represent a lot of pulp, the sampling shall be in accordance with ISO 7213:1981. Fro
46、m each unit of dry market pulp to be sampled, discard several top sheets from each bale to eliminate surface contamination.In other cases, sample a sufficient number of units so that the test material is representative of the paper, the paperboard, or the dry market pulp to be tested. In all samplin
47、g and examination procedures, make sure that the test material taken is representative of the sample received.Ideally, a sample should contain at least four sheets, each of them having a minimum size of 200 mm 250 mm of dry market pulp, paper, or paperboard (at least 2 sheets for testing and 2 protective sheets).NOTE For paperboard or thicker material, it might be sufficient to use only 1 sheet for each parallel determination. For thinner paper, more than 2 sheets can be used for each parallel determination.After sampling, wrap the unexpose
