Improved quantification of Dehalococcoides species by fluorescence in situ hybridization and catalyzed reporter deposition.
Chlorinated ethenes contamination of soil and groundwater is a widespread problem in most industrialized countries. To date, there is a general consensus in the literature that members of the genus Dehalococcoides are required for complete dechlorination of these compounds. The availability of specific identification tools to track their distribution in the field is therefore a topic of particular relevance in environmental studies. These microorganisms have been successfully visualized by fluorescence in situ hybridization (FISH) in highly active dechlorinating cultures. However, FISH detection of Dehalococcoides under low activity conditions can be strongly hampered by their small cell size and low ribosome content. In this study, catalyzed reporter deposition (CARD)-FISH was employed as an alternative detection method. In a trichloroethene (TCE) dechlorinating enrichment culture, CARD-FISH, using proteinase K as a permeabilization pre-treatment, was found to be significantly superior to conventional FISH in terms of both microscopic visualization and quantification efficiency (about 30%). An application of this method on contaminated aquifer samples is also presented and discussed.
Fazi
Stefano
S
Water Research Institute, IRSA-CNR, Via Reno 1, 00198 Rome, Italy.
Aulenta
Federico
F
Majone
Mauro
M
Rossetti
Simona
S
D041862:Chloroflexi / Q000235:genetics* / Q000302:isolation & purification*; D015169:Colony Count, Microbial / Q000379:methods*; D019286:Endopeptidase K / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008853:Microscopy; D010539:Permeability; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D014752:Vinyl Chloride / Q000378:metabolism
D006735:Horseradish Peroxidase; D019286:Endopeptidase K; D014752:Vinyl Chloride