title: Application of laser scanning cytometry followed by epifluorescent and differential interference contrast microscopy for the detection and enumeration of Cryptosporidium and Giardia in raw and potable waters.
abstract: OBJECTIVE
  The main goal of this study was to validate a new laser scanning cytometry method (ChemScanRDI) that couples immunofluorescence detection with differential interference contrast (DIC) confirmation, against manual microscopic enumeration of Giardia and Cryptosporidium (oo)cysts. This study also assessed the basic performance of the new Association Française de Normalisation (AFNOR) NF T 90-455 method for Giardia and Cryptosporidium (oo)cyst enumeration with respect to (oo)cyst yield, linearity, repeatability, influence of turbidity and detection limit in raw and potable waters.
  
  
  RESULTS
  The new standard method relies on cartridge (Envirocheck) filtration, immunomagnetic separation purification, immunofluorescence staining and detection followed by DIC confirmation. The recovery was 30-50% for both parasites at seeding levels from 30 to 230 (oo)cysts. The method is linear from 0 to around 400 seeded (oo)cysts and the yield does not significantly vary for turbidity levels from 10 to 40 Formazin Nephelometric Units (FNU). The results were obtained using manual microscopic enumeration of the (oo)cysts. The ChemScanRDI yielded counts that were at least equivalent to those obtained using manual microscopy for both parasites in raw and potable water concentrates, for seeding levels of 10-300 or 10-100, respectively. The purification and labelling method proposed by the supplier of theChemScanRDI (Chemunex) reached very similar recoveries to the AFNOR protocol (70-86% in both cases).
  
  
  CONCLUSIONS
  Laser scanning cytometry can be used as a more standardized alternative to manual enumeration as part of the new AFNOR standard method.
  
  
  CONCLUSIONS
  By using laser scanning cytometry instead of manual microscopy, laboratories could circumvent the limitations of manual microscopy, namely: low sample throughput, operator subjectivity and operator fatigue. The study further supports the drive to incorporate laser scanning cytometry in the standard methods for Giardia and Cryptosporidium enumeration.
authors.affiliation: Vivendi Water Anjou Recherche, Saint Maurice, France. marie-renee.de-roubin@generale-des-eaux.net
authors.forename: M-R
authors.initials: MR
authors.lastname: de Roubin
authors.forename: J-S
authors.initials: JS
authors.lastname: Pharamond
authors.forename: F
authors.initials: F
authors.lastname: Zanelli
authors.forename: F
authors.initials: F
authors.lastname: Poty
authors.forename: S
authors.initials: S
authors.lastname: Houdart
authors.forename: F
authors.initials: F
authors.lastname: Laurent
authors.forename: J-L
authors.initials: JL
authors.lastname: Drocourt
authors.forename: S
authors.initials: S
authors.lastname: Van Poucke
mesh_terms: D000818:Animals; D003458:Cryptosporidium / Q000254:growth & development / Q000302:isolation & purification*; D005618:Fresh Water / Q000469:parasitology*; D005872:Giardia / Q000254:growth & development / Q000302:isolation & purification*; D019044:Image Cytometry / Q000379:methods*; D018189:Immunomagnetic Separation; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008857:Microscopy, Interference / Q000379:methods*; D033661:Oocysts / Q000302:isolation & purification; D010274:Parasitology / Q000295:instrumentation / Q000379:methods; D014881:Water Supply