I am using specific probes for detecting and quantify bacteria with FACS, however in spite, it is supposed to work (Previous studies and In silico analysis of the probes), I only get high fluorescence but unspecific hybridization.

I have tried different variables to increase the specificity:

-Formamide concentration in the Hyb buffer (20mM Tris 7.0, 0.1% SDS, 0.9M NaCl)

-Increase wash steps and change pH (SSC20x)

-Change hybridization temperatures (From 48° to 70°) 4 hrs with agitation

-Reduce the concentration of the probes

Probes:

Universal GCT GCC TCC CGT AGG AG- 6FAM

Bifidobacteria GAT AGG ACG CGA CCC CAT-CY5

Lactobacillus ACA TGG AGT TCC ACT-CY3

In silico analysis: Mathfish

All the bacteria are pure cultures washed in PBS, fixed in Alcohol 50% or formaldehyde 3.7% and the permeability of the bacteria have been tested with PI

However, none of these changes had a significant change. Is there anything else that I could try?

Thank you in advance