title: Optimization of a two-step permeabilization fluorescence in situ hybridization (FISH) assay for the detection of Staphylococcus aureus.
abstract: BACKGROUND
  Aspects of the fluorescence in situ hybridization (FISH) method for the detection of clinically important bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli, were investigated for optimization.
  
  
  METHODS
  Various approaches to optimizing the FISH procedure were taken and different methods were compared. To save time, hybridization and washing buffers were prepared beforehand and stored at -20 °C and mixed to their final formamide and NaCl concentrations just before use. The use of 50-ml tubes for hybridization incubation reduced drying out, reagent wastage, and reaction times.
  
  
  RESULTS
  A two-step permeabilization FISH assay was developed that used phosphate-buffered saline as a buffer for lysostaphin. It could detect bacteria with DNA probes conjugated to fluorophores with a higher signal intensity and the less expensive biotinylated DNA probes with minimal cell lysis in 1 hr.
  
  
  CONCLUSIONS
  The two-step assay might be used when the FISH signal is weak, bacterial numbers are low or if there is a need to use other reporter molecules.
authors.affiliation: Macquarie University, Faculty of Science, Sydney, New South Wales, Australia. tomxlawson@gmail.com
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Lawson
authors.forename: Russell E
authors.initials: RE
authors.lastname: Connally
authors.forename: Subramanyam
authors.initials: S
authors.lastname: Vemulpad
authors.forename: James A
authors.initials: JA
authors.lastname: Piper
mesh_terms: D000704:Analysis of Variance; D004926:Escherichia coli / Q000302:isolation & purification; D005559:Formamides; D016591:Histocytological Preparation Techniques / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008248:Lysostaphin; D025202:Molecular Diagnostic Techniques / Q000379:methods; D009113:Muramidase; D012965:Sodium Chloride; D013211:Staphylococcus aureus / Q000302:isolation & purification*; D013212:Staphylococcus epidermidis / Q000302:isolation & purification; D019809:Streptavidin
chemical_list: D005559:Formamides; D012965:Sodium Chloride; C031066:formamide; D019809:Streptavidin; D009113:Muramidase; D008248:Lysostaphin