SIST ISO 15553:2010

INTERNATIONAL ISO
STANDARD 15553
First edition
2006-11-15
Water quality — Isolation and
identification of Cryptosporidium oocysts
and Giardia cysts from water
Qualité de l'eau — Isolement et identification des oocystes de
Cryptosporidium et des kystes de Giardia

Reference number
©
ISO 2006
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ii © ISO 2006 – All rights reserved

Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Terms 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 water. 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 about Cryptosporidium and Giardia. 31
Annex H (informative) Details of manufacturers. 32
Bibliography . 36

Foreword
ISO (the International Organization 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 has the right to be represented on that committee. International organizations, governmental and
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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 committees 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 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.
ISO 15553 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
iv © ISO 2006 – All rights reserved

Introduction
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 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/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.
INTERNATIONAL STANDARD ISO 15553:2006(E)

Water 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 examination 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 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 quality 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 is 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 with 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
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 parasite 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 used
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 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.
Table 1 — Membrane filters/filtration systems used for the concentration of parasites
from water samples
Membrane filter/filtration system Application
Concentration of 10-litre to 200-litre (or more) samples of
TM a
Pall Envirochek STD
water
TM
Pall Envirochek HV Concentration of 10-litre to 1 000-litre samples of water ®
IDEXX Filta-Max Concentration of 10-litre to 1 000-litre samples of water
a
It 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 particulate 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 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 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
TM 1)
4.1 Reagents required for eluting Pall Envirochek STD capsule filters
4.1.1 Deionized water, 0,2 µm 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).
TM 1)
4.2 Reagents required for eluting Pall Envirochek HV capsule filters
4.2.1 Deionized water, 0,2 µm 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 users of this International Standard and does not constitute an endorsement by ISO of these products.
2 © ISO 2006 – All rights reserved

4.2.4 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).
® 1)
4.3 Reagents required for eluting IDEXX Filta-Max filters
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, for 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 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 suitable suppliers.
4.4.5 4′,6′-Diamidino-2-phenylindole dihydrochloride dihydrate (DAPI) freeze dried reagent.
Store according to the manufacturer's 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 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.
Store at (5 ± 3) °C, never allow the suspension to freeze and check quality regularly. Ideally, suspensions of
oocysts and cysts should be no more than 3 months ol
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