🦠 Cryptosporidium and Giardia in water — capsule filtration, immunomagnetic separation and immunofluorescence microscopy

Microbiology ISO 15553

Overview

Cryptosporidium and Giardia are parasitic protozoa causing intestinal diseases in humans. Both resting forms — oocysts and cysts — survive for a long time in the aquatic environment, and the oocysts of Cryptosporidium are resistant to chlorine at the concentrations used in treating drinking water and swimming-pool water. For that reason, as the introduction to the standard states, the absence of vegetative indicator bacteria of faecal contamination is not proof of the absence of these protozoa — the result of a test for E. coli or enterococci says nothing about them.

ISO 15553:2006 describes the detection and enumeration of Cryptosporidium oocysts and Giardia cysts in surface water and groundwater, in treated water, in mineral waters and in swimming-pool and recreational water. The method does not permit identification to species, nor establishing the host from which they come, nor assessing the viability or infectivity of the forms found; the standard also stipulates that the examination may be carried out only by experienced analysts who have passed competence tests and who are regularly checked on fortified samples and in proficiency testing. Objects morphologically similar to oocysts and cysts may appear in the preparation — in case of doubt or of an unusually high result the standard recommends passing the preparations for assessment to experts from another laboratory.

The standard has not been adopted into the collection of Polish Standards — the PKN catalogue contains no national adoption of it, and Polish methodological work (NIZP–PZH) refers expressly to ISO 15553:2006. The Regulation of the Minister of Health of 22 May 2026 on the quality of water intended for human consumption now in force (Journal of Laws 2026 item 748) does not name Cryptosporidium or Giardia among the parameters of water or among the microbiological parameters for which reference methods have been indicated; the examination is therefore carried out outside the obligatory scope, for the purposes of risk assessment and epidemiological investigations. The earlier legal state is described in a 2012 paper of NIZP–PZH: the regulation in force at that time indicated the need for testing for Cryptosporidium sp. in water from surface or mixed intakes when Clostridium perfringens had been detected in the treated water, whereby — as the authors note — such tests were not carried out in routine control in Poland. The first edition of 2006 remains current: ISO confirmed it in a systematic review on 22 March 2021, and a further review, begun on 15 January 2026, was closed on 5 June 2026 — its outcome has not yet been recorded in the life-cycle card. No superseding standard has been indicated. The life-cycle card at the same time places the standard at STAGE 90.92, that is "to be revised" — just as with ISO 16200-1, which has its own entry in this knowledge base: the standard is still the current version, but ISO has referred it for revision, so when citing it in a procedure it is worth checking whether a new edition has appeared. In the NIZP–PZH studies carried out according to the assumptions of this standard on water from surface intakes (the Vistula, the Zegrze Reservoir) Giardia cysts were detected in all samples (1.0–38.9 per 10 l), and Cryptosporidium oocysts in nearly half of the samples (0.5–2.5 per 10 l).

THE POLISH TITLE IN THIS ENTRY IS OUR WORKING TRANSLATION, not an official title: the standard has not been adopted into the collection of Polish Standards.

Method principle

Isolating the protozoa from water requires reducing the volume of the sample while retaining the oocysts and cysts. The standard describes two concentration steps, carried out one after the other: capsule filtration with elution, and then low-speed centrifugation. From the concentrated suspension of solid particles the oocysts and cysts are isolated by immunomagnetic separation (IMS): they bind to paramagnetic beads coated with a specific antibody, the beads are separated with a magnet from the unwanted matter, after which the oocysts and cysts are detached from the beads with acid and neutralised with alkali. The isolated organisms are labelled with a monoclonal antibody conjugated with a fluorochrome (usually fluorescein isothiocyanate, FITC), and the nuclear material with a nucleic acid stain; the preparation is examined under an epifluorescence microscope with differential interference contrast (DIC).

Applications

• Control of water intended for consumption from surface and mixed intakes (an examination outside the obligatory scope of Journal of Laws 2026 item 748)
• Assessment of the efficiency of treatment — barriers against resting forms resistant to chlorine
• Examination of surface water and groundwater abstracted by waterworks
• Swimming-pool and recreational water (the standard names them expressly in the scope)
• Mineral waters and treated water distributed in the network
• Epidemiological investigations where an outbreak of a water-borne disease is suspected

Key parameters

Standard

Standard number

ISO 15553:2006

Title (PL)

Jakość wody — Izolowanie i identyfikacja oocyst Cryptosporidium i cyst Giardia z wody

Title (EN)

Water quality — Isolation and identification of Cryptosporidium oocysts and Giardia cysts from water

Step-by-step procedure

1. Taking the sample

   Choose the volume to suit the kind of water and the sensitivity required: 10–100 l as a spot sample carried to the laboratory, or up to 1000 l filtered at the point of sampling. At large volumes connect the capsule in line in the direction of flow indicated by the manufacturer, with the flow meter read before and after sampling; do not exceed the recommended flow rates. Filtering 1000 l may take up to 24 h.

2. Transport and storage

   Carry spot samples and filters in the dark, at ambient temperature. In the laboratory store at (5 ± 3) °C if the examination does not begin at once. Bring a chilled filter to room temperature before beginning the elution.

   🌡 Temperature: (5 ± 3) °C

3. Elution of the filter

   Stand the Envirochek STD capsule upright with the vent valve upwards, take off both caps and drain off the water remaining in the sample through the filter. Fit the lower cap, fill the capsule with elution buffer so that it covers the filter by about 1 cm, close it and shake horizontally at 600 ± 25 cycles/min. After the first shaking pour the washings into a 250 ml conical tube, add a further portion of buffer and shake again, changing the position of the vent valve.

   ⏱ Time: 2 × (5 min ± 30 s)

4. Centrifugation and measurement of the pellet volume

   Centrifuge the combined washings at 1100 × g for 15 min without braking in the deceleration phase. Read the pellet volume immediately after centrifugation. If it exceeds the value recommended by the manufacturer of the IMS kit, divide the sample into sub-samples.

   ⏱ Time: 15 min

5. Aspirating the supernatant and equalising the volume

   Aspirate the supernatant with a pipette at a vacuum of less than 20 kPa, leaving 2–5 ml above the pellet; where the pellet is not visible take particular care. Make up with deionised water to 9 ml and vortex for 10–15 s. The suspension may be stored at (5 ± 3) °C until the IMS.

   ⏱ Time: 10–15 s of vortexing

6. Immunomagnetic separation (IMS)

   Bind the oocysts and cysts to paramagnetic beads coated with a specific antibody, separate the beads from the remaining matter with a magnet, and then detach the organisms from the beads with acid and neutralise with alkali before the immunological staining.

7. Staining the preparation

   Label the isolated organisms with a monoclonal antibody conjugated with a fluorochrome (usually FITC), and the nuclear material with a nucleic acid stain (DAPI). Incubate the preparations in a humid chamber and mount them with an immunofluorescence medium.

8. Microscopic examination

   Examine the preparation under an epifluorescence microscope at a total magnification of 1000×, with the FITC filter (excitation 480 nm, emission 520 nm) and the UV filter (350/450 nm) and in DIC contrast. Structures morphologically similar to oocysts and cysts may give false positive results — in case of doubt or of an unusually high result pass the preparation for assessment to experts from another laboratory.

9. Quality control and recovery

   The standard provides separate procedures for quality control (clause 8) and for the preparation of positive controls and recovery tests (Annex D). In the practice of NIZP–PZH samples of tap water of 20 l volume were fortified with a weighed reference suspension (99 ± 1.4 oocysts and 99 ± 1.6 cysts) and the recovery was calculated from that.

10. Giving the result

    The result is given as the number of Cryptosporidium oocysts and Giardia cysts in the volume of water examined (in Polish work usually per 10 l). The test report should note the volume of water filtered and the filtration system used, since the sensitivity of the determination depends on them.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Cryptosporidium and Giardia are pathogenic to humans (intestinal diseases) — treat samples, concentrates and reference suspensions as infectious material
• Cryptosporidium oocysts are resistant to chlorine at the concentrations used in water treatment — disinfection with hypochlorite is not a reliable way of decontaminating equipment after work with this material
• Store reference suspensions at (5 ± 3) °C and never freeze them; do not use suspensions which have clumped or whose staining is weakening
• The examination is carried out only by analysts who have passed competence tests and who are regularly checked on fortified samples and in external proficiency testing programmes
• Methanol and the solvents used for fixing the preparations — work in a fume cupboard, away from sources of ignition, in gloves and protective goggles

Frequently asked questions