Short communicationResponse surface methodology to optimize peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) in Saccharomyces cerevisiae

- S. cerevisiae in situ hybridization was optimized by response surface methodology.
- PNA hybridization temperature, time and formamide concentration were modelled.
- This work impacts the study of yeast's dynamics in fermentations.

Saccharomyces cerevisiae (S. cerevisiae) is a crucial fermenting microorganism for the beer, wine and bread industry. As such, an accurate and rapid method for its identification and monitoring is required. In here, a peptide nucleic acid (PNA) probe was first designed to target S. cerevisiae by fluorescence in situ hybridization (FISH). This PNA-FISH method was then systematically optimized, employing response surface methodology (RSM). The interaction between the critical hybridization temperature, time and formamide concentration and their effect on the FISH efficiency were modelled. The model predicted optimal fluorescence intensity upon hybridization at 53.9 °C, during 57.8 min and using 43.8% (v/v) formamide in the hybridization buffer, which was experimentally confirmed. RSM showed to be a valuable tool to optimize and better understand the dynamics of yeast FISH, which can impact the performance evaluation of related fermentation processes.