Insti-Spore Si3.2 test slides for fluidized endospores
Fluidized endospores are impregnated with and embedded within silica. This makes the endospore impossible to stain using either the MAA or Insti-Spore methods. We have developed an improved test, Insti-Spore Si3.2, that mitigates this problem. The test consists of a non-toxic Treatment that removes silica from the endospore wall. The v3.2 permeability-treated Test Slide consists of two wells: SYTO/PI and DAPI/PI. The treatment procedure allows fluorescent probes to enter the cell and target the DNA. Endospores subsequently are visible within the remaining silica matrix. When visualized in fluorescence mode using eiter the DAPI LP filter set for the the DAPI/PI well or the SYTO LP filter set for the SYTO/PI well reveals endospores as red/pink ovals embedded in a dim gray or green fluorescent silica matrix. Since the endospore itself is stained (not the endospore cell wall) the resulting fluorescent ovals are smaller than typical non-fluidized endospores. Think of them as the "Donut Hole" vs "Ghost Football".
Kit components: Treatment solution plus IS-Si3.2 probe slides.
We no longer use HF. The yellow tube contains the non-toxic solubilization components plus 12 Insti-Spore Si3.2 test slides.
Procedure
Set your pipetter to 400µl.
Use molecular grade water
Add 400µl water to the Treatment Solution vial (yellow)
Mix the resultant solution
The solution is well mixed when you see bubbles
Acquire a very small amount of sample
A toothpick-end amount
Add sample to Test Solution vial
Vortex 15s
This is a very important step. Simple shaking will not work.
Incubate the sample 30 minutes at 42C temperature.
The preferred method is to use a 42C waterbath.
You may also place the vial on a 42C warming tray.
In the meantime set the 40mm coverslip...
... in a clean, safe place
During sample incubation set your pipetter to 10µl
And remove the seal top layer
Withdraw 10µl treated sample
You will do this twice. Once for each well.
Add 10µl Treated sample to each well of the IS-Si3.2 slide.
Dry the sample slide at 42C for about 5 minutes
In the meantime set your pipetter to 7µl and find your immersion oil
Make certain you use non-fluorescent immersion oil.
After drying add 7µl immersion oil to each well
Adding 7µl immersion oil
7µl immersion oil in both wells
Apply the 40mm coverslip carefully
Remember: you get only one chance.
Remove and discard the coverslip tab
The slide is now ready to be imaged
The SYTO/PI well (closest to the slide label) should be excited with your SYTO (LP) filter set (WB). The DAPI/PI well (furthest from label) should be excited using the UV (LP) filter set (WU).
