1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58Cryptosporidium oocysts and Giardia cysts from waterICS 07.100.20Water quality Isolation and identi
2、fication of BRITISH STANDARDBS ISO 15553:2006BS ISO 15553:2006This British Standard was published under the authority of the Standards Policy and Strategy Committee on 29 December 2006 BSI 2006ISBN 0 580 49843 3Amendments issued since publicationAmd. No. Date Commentscontract. Users are responsible
3、for its correct application.Compliance with a British Standard cannot confer immunity from legal obligations.National forewordThis British Standard was published by BSI. It is the UK implementation of ISO 15553:2006. The UK participation in its preparation was entrusted by Technical Committee EH/3,
4、Water quality, to Subcommittee EH/3/4, Microbiological methods.A list of organizations represented on EH/3/4 can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a Reference numberISO 15553:2006(E)INTERNATIONAL STANDARD ISO15553Firs
5、t edition2006-11-15Water quality Isolation and identification of Cryptosporidium oocysts and Giardia cysts from water Qualit de leau Isolement et identification des oocystes de Cryptosporidium et des kystes de Giardia BS ISO 15553:2006ii iiiContents Page Foreword iv Introduction v 1 Scope . 1 2 Term
6、s and definitions. 1 3 Principle. 1 4 Reagents 2 5 Apparatus 4 6 Sampling and transport 6 7 Procedure 7 8 Quality control procedures 14 9 Reporting of results 14 Annex A (normative) Preparation of reagents. 16 Annex B (informative) Concentration of oocysts and cysts from small (10 l) volumes of wate
7、r. 19 Annex C (informative) Calibration of eyepiece graticule 24 Annex D (informative) Preparation of positive controls and recovery tests 25 Annex E (informative) Examples of indicative performance data . 28 Annex F (informative) Alternative methods. 30 Annex G (informative) Further information abo
8、ut Cryptosporidium and Giardia 31 Annex H (informative) Details of manufacturers. 32 Bibliography . 36 BS ISO 15553:2006iv Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International
9、 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, a
10、lso take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical commit
11、tees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the pos
12、sibility 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. ISO 15553 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4, Microbiological methods. BS ISO 15553:
13、2006vIntroduction Cryptosporidium and Giardia are protozoan parasites that can cause enteric illness in humans. Both organisms are characterized by an ability to survive in the aquatic environment. Cryptosporidium in particular is resistant to chlorine at the concentrations used in the treatment of
14、drinking and swimming pool waters. Consequently the absence of vegetative bacteria as indicators of faecal contamination does not necessarily indicate the absence of Cryptosporidium oocysts or Giardia cysts. The methods described in this document may be used to determine whether Cryptosporidium and/
15、or Giardia are present in water supplies. The techniques have been selected on the basis of method development and peer review publication of the data thus obtained. They are further selected to give comparable recoveries of the methods or reagents used in the isolation of the organisms. BS ISO 1555
16、3:2006blank1Water quality Isolation and identification of Cryptosporidium oocysts and Giardia cysts from water 1 Scope This International Standard specifies a method that is applicable for the detection and enumeration of Cryptosporidium oocysts and Giardia cysts in water. It is applicable for the e
17、xamination of surface and ground waters, treated waters, mineral waters, swimming pool and recreational waters. This method does not allow identification to species level, the host species of origin or the determination of viability or infectivity of any Cryptosporidium oocyst or Giardia cyst which
18、may be present. These procedures are for use by experienced analysts who have successfully completed competency tests prior to commencing analysis. In addition, such analysts should continue to demonstrate competency by examining seeded samples at regular intervals and taking part in external qualit
19、y assurance schemes. NOTE Bodies resembling Cryptosporidium or Giardia in morphology can be present and these may be mistaken for oocysts or cysts. Results should be interpreted with care. Where there is doubt about the identity of oocysts or cysts or where an unusually high result is obtained, it i
20、s advisable to have the slides examined by experts from other laboratories to confirm or refute the findings. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 Cryptosporidium protozoan parasite, concentrated and selected from water samples wit
21、h the methods described, which reacts with specific anti-Cryptosporidium antibodies and exhibits the typical morphological characteristics described in 7.4 of this International Standard NOTE A more complete definition of the parasite and the different genotypes and species is given in Annex G. 2.2
22、Giardia protozoan parasite, concentrated and selected from water samples with the methods described, which reacts with specific anti-Giardia antibodies and exhibits the typical morphological characteristics described in 7.4 of this International Standard NOTE A more complete definition of the parasi
23、te and the different species is given in Annex G. 3 Principle 3.1 Concentration from water The isolation of Cryptosporidium and Giardia from water requires the use of a procedure which allows the volume of the sample to be reduced whilst retaining any oocysts and cysts. The concentration procedure u
24、sed however, is dependent upon the water type which is to be analysed, the volume of sample and the amount of particulate material in the sample. This document describes the use of two concentration techniques for varying volumes of water using cartridge filtration and elution followed by low speed
25、centrifugation (7.1). Additional methods for the recovery of oocysts and cysts from small volumes of water or very turbid waters are given in Annex B. Some examples of recovery data for these techniques are given in Annex E. BS ISO 15553:20062 Table 1 Membrane filters/filtration systems used for the
26、 concentration of parasites from water samples Membrane filter/filtration system Application Pall EnvirochekTMSTD aConcentration of 10-litre to 200-litre (or more) samples of water Pall EnvirochekTMHV Concentration of 10-litre to 1 000-litre samples of water IDEXX Filta-Max Concentration of 10-litre
27、 to 1 000-litre samples of water aIt has been shown by some laboratories that this technique may be used successfully for larger volumes of water although the manufacturers instructions may only include volumes up to 200 litres. 3.2 Purification and further concentration After concentration of parti
28、culate material from filter eluates, oocysts and cysts are isolated using immunomagnetic separation (IMS) (7.2). Oocysts and cysts are attached to para-magnetic beads coated with specific antibody, the beads are separated from the unwanted particulate material using a magnet and then the oocysts and
29、 cysts are dissociated from the beads using acid and neutralized using alkali before immunostaining. 3.3 Detection of Cryptosporidium and Giardia After IMS, organisms are labelled with monoclonal antibody (mAb) conjugated to a fluorochrome, usually fluoroscein isothiocyanate (FITC). In addition, any
30、 nuclear material is labelled with a nucleic acid stain to aid identification (7.3). Each sample is then examined for the presence of labelled Cryptosporidium oocysts and Giardia cysts using epifluorescence and differential interference contrast (DIC) microscopy (7.4). 4 Reagents 4.1 Reagents requir
31、ed for eluting Pall EnvirochekTMSTD capsule filters 1)4.1.1 Deionized water, 0,2 m filtered at the point of use. 4.1.2 Laureth 12 detergent. 4.1.3 Tris buffer, pH 7,4 (A.1.1). 4.1.4 EDTA solution, 0,5 mol/l, pH 8,0 (A.1.2). 4.1.5 Antifoam A. 4.1.6 Elution buffer (A.1.3). 4.2 Reagents required for el
32、uting Pall EnvirochekTMHV capsule filters 1)4.2.1 Deionized water, 0,2 m filtered at point of use. 4.2.2 Pre-treatment buffer (A.1.4). 4.2.3 Laureth 12 detergent 1) All products and reagents are examples of suitable products available commercially. This information is given for the convenience of us
33、ers of this International Standard and does not constitute an endorsement by ISO of these products. BS ISO 15553:200634.2.4 Tris buffer, pH 7,4 (A.1.1). 4.2.5 EDTA solution, 0,5 mol/l, pH 8,0 (A.1.2). 4.2.6 Antifoam A. 4.2.7 Elution buffer (A.1.3). 4.3 Reagents required for eluting IDEXX Filta-Maxfi
34、lters 1)4.3.1 Phosphate buffered saline (PBS) (A.2.1). 4.3.2 Polyoxyethylene(20)sorbitan monolaurate (Tween 20). Store at room temperature (20 5) C. Expiry date one year. 4.3.3 Elution buffer (A.2.2). 4.4 Concentration and detection reagents 4.4.1 Methanol, analytical grade. 4.4.2 Magnetic beads, fo
35、r the detection of Cryptosporidium and Giardia. Expiry date printed by the manufacturer. NOTE See Annex H for a list of suitable suppliers. 4.4.3 Fluorescently labelled monoclonal antibodies (mAbs) against Cryptosporidium and Giardia. Store at (5 3) C. Expiry date as stated by the manufacturer. When
36、 stains are prepared from concentrated material using a diluent supplied by the manufacturer, the prepared solution is stored at (5 3) C for no longer than 6 months. NOTE See Annex H for a list of suitable suppliers. 4.4.4 Immunofluorescence mounting medium (A.3.1). NOTE See Annex H for a list of su
37、itable suppliers. 4.4.5 4,6-Diamidino-2-phenylindole dihydrochloride dihydrate (DAPI) freeze dried reagent. Store according to the manufacturers instructions. Expiry date printed by the manufacturer on each vial. 4.4.6 DAPI stock solution (A.3.2). 4.4.7 DAPI working solution (A.3.3). 4.4.8 Phosphate
38、 buffered saline (PBS) (A.2.1). 4.4.9 Non-fluorescing immersion oil. Store at room temperature (20 5) C. 4.4.10 Stock suspensions of Cryptosporidium parvum oocysts and Giardia lamblia cysts. BS ISO 15553:20064 Store at (5 3) C, never allow the suspension to freeze and check quality regularly. Ideall
39、y, suspensions of oocysts and cysts should be no more than 3 months old. Stock suspensions should be checked microscopically to confirm that they are monodispersed and discarded if clumps or aggregates are detected. In addition, if mAb and DAPI staining become weak and oocysts become deformed, they
40、should also be discarded. 4.4.11 Parasite storage medium (A.3.4). 5 Apparatus Use usual laboratory equipment and, in particular, the following. 5.1 Scientific apparatus, required for concentration using Pall EnvirochekTMSTD or HV. 2)5.1.1 Sampling capsule, EnvirochekTMSTD or HV (Pall). 5.1.2 Perista
41、ltic pump, capable of a flow rate of 2 l/min. 5.1.3 Silicon tubing, for use with the peristaltic pump. 5.1.4 Seeding container, 10 l, if seeding filters is required. 5.1.5 Wrist-action shaker, with arms for the agitation of the EnvirochekTMSTD or HV sample capsules. 5.1.6 Centrifuge, capable of a mi
42、nimum of 1 100 g. 5.1.7 Centrifuge tubes, conical, plastic, screwtop, 250 ml capacity. 5.1.8 Centrifuge tubes, conical, plastic, screwtop, 50 ml capacity. NOTE A flow meter and flow restrictor are required for taking water samples with the filter. 5.2 Specific apparatus, required for concentration u
43、sing IDEXX Filta-Max. 2)5.2.1 Sampling housing, Idexx Filta-Max. 5.2.2 Sampling module, Idexx Filta-Max. 5.2.3 Filter membranes, Idexx Filta-Max. 5.2.4 Laboratory pump, capable of supplying 500 kPa (5 bar) pressure. 5.2.5 Peristaltic pump, capable of flow rate of 4 l/min. 5.2.6 Silicon tubing, for u
44、se with peristaltic pump. 5.2.7 Seeding container, 10 l, if seeding filters is required. 5.2.8 Wash station, automatic or manual, and wash station clamp set, Idexx Filta-Max. 5.2.9 Vacuum set, includes plastic hand pump, waste bottle, tubing and magnetic stirring bar. Idexx Filta-Max. 2) All apparat
45、us are examples of suitable products available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of these products. BS ISO 15553:200655.2.10 Tubing set, includes elution tube, and middle section, concentr
46、ator tube and base, with line tap and steel rod Idexx Filta-Max. 5.2.11 Membrane, for tubing set. 5.2.12 Plastic bag, for washing membrane. 5.2.13 Centrifuge, capable of 1 100 g. 5.2.14 Centrifuge tubes, conical, plastic, 50 ml capacity. 5.2.15 Forceps. NOTE A flow meter and flow restrictor are requ
47、ired for taking water samples with the filter. 5.3 General apparatus 2). 5.3.1 Incubator, at (36 2) C. 5.3.2 Refrigerator, at (5 3) C. 5.3.3 Magnetic stirrer, and magnetic stirring bars. 5.3.4 Vortex mixer. 5.3.5 Wash bottles, polypropylene, 250 ml. 5.3.6 Calibrated micropipettes, adjustable: 1 l to
48、 10 l with 1 l to 10 l tips; 20 l to 200 l with 10 l to 200 l tips; 200 l to 1 000 l with 100 l to 1 000 l tips. 5.3.7 pH meter. 5.3.8 Magnetic particle concentrators, with suitable tubes. 5.3.9 Well microscope slides, with special hydrophobic coating and coverslips. 5.3.10 Epifluorescence microscop
49、e, with a UV filter (350 nm excitation, 450 nm emission), FITC filter (480 nm excitation, 520 nm emission) filters,TMdifferential interference contrast (DIC) optics and an eye piece graticule. Total magnification 1 000 . 5.3.11 Microscope stage micrometer, 1 mm, ruled in 100 units. 5.3.12 Eyepiece graticule, ruled in 100 units. 5.3.13 Humidity chamber, e.g. consisting of a tightly sealed plastic container containing damp paper towels on which the slides are placed. 5.3.14 10 l containers, gradu
