title: Improved DNA-FISH for cytometric detection of Candida spp.
abstract: OBJECTIVE
  We developed improved methods for DNA-based fluorescence in situ hybridization (FISH) for rapid detection of Candida spp. and Candida albicans via flow cytometry.
  
  
  RESULTS
  Two previously reported C. albicans-targeted DNA probes were evaluated against whole cells of C. albicans and related Candida species using a rapid, high-temperature hybridization protocol. One probe (CalB2208) was shown for the first time to be suitable as a FISH probe. Although cell labelling for both probes was relatively bright, we were able to substantially improve our results by altering fixation and hybridization conditions. For fixation, a 60 : 40 mixture of 10% buffered formalin and ethanol was most effective. Probe intensity was improved as much as ten-fold through the use of unlabelled helper probes, and buffer containing 0·9 mol l−1 NaCl plus 10% formamide yielded the best hybridizations for both probe/helper cocktails. Although optimal labelling occurred with longer hybridizations, we found that C. albicans could be completely differentiated from the nontarget yeast Rhodotorula glutinis after only 15 min using the brightest probe (Calb-1249) and that a formal washing step was not required. Specificities of probe/helper cocktails under optimal conditions were determined using a panel of target and nontarget cell types, including four strains of Candida dubliniensis. Calb-1249 cross-reacted slightly with Candida parapsilosis and strongly with both Candida tropicalis and C. dubliniensis. In contrast, we found that CalB2208 was exclusive for C. albicans. The molecular basis of this specificity was confirmed by DNA sequencing.
  
  
  CONCLUSIONS
  We describe DNA probe-based approaches for rapid and bright labelling of Candida spp. and for specific labelling of C. albicans without cross-reaction with C. dubliniensis. Our work improves upon previously described methods.
authors.affiliation: Department of Food Science & Human Nutrition, Iowa State University, Ames, IA 50011-1061, USA.
authors.forename: B
authors.initials: B
authors.lastname: Bisha
authors.forename: H J
authors.initials: HJ
authors.lastname: Kim
authors.forename: B F
authors.initials: BF
authors.lastname: Brehm-Stecher
mesh_terms: D002175:Candida / Q000235:genetics / Q000302:isolation & purification*; D002176:Candida albicans / Q000235:genetics / Q000302:isolation & purification; D041022:Candida tropicalis / Q000235:genetics / Q000302:isolation & purification; D015342:DNA Probes; D005434:Flow Cytometry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009693:Nucleic Acid Hybridization; D017422:Sequence Analysis, DNA
chemical_list: D015342:DNA Probes