Insti-Fluor L/D for viability staining
The accepted method for determining bacteria viability is to double-stain with SYTO and PI. Insti-Fluor L/D test slides come pre-loaded with SYTO plus PI for bacterial viability staining. Here's how they are used:
Dilute sample (arrow) into water and vortex
Samples from plates or other concentrated sources need to be diluted with water. Here we have a small amount of plated bacteria sample (Erwinia herbicola) that we are putting into 0.5ml water.
Vortex the sample 15s
This makes a uniform bacterial suspension
Remove the protective top layer
Here we are using an Insti-Fluor L/D test slide, rather than making SYTO and PI solutions.
Set your pipetter to 10µl
Add 10µl sample suspension to the center of the well
Use the pipette tip to spreasd the sample throughout the well
Apply the coverslip
Since you want to test viability, you should NOT dry the sample.
Press gently around the permiter of the coverglass
Observe using Blue Excitation and a LP Em filter (WB)
Living cells stain only with SYTO, so they fluoresce bright green. Dead cells also absorb PI, which quenches SYTO fluorescence. Thus Dead cells fluoresce red.
