Sterilization of DMSO-Nile Blue A methods - (Aug/31/2007 )
I've read through some posts on DMSO and how it "melts" the membrane filter. I had experienced the same thing before whilst filtering DMSO-Nile Blue.
I'm isolating microorganisms with lipid inclusion and I need Nile Blue A (or Nile Red) as an indicator. First, I prepared Nile Blue in DMSO and then sterilize the my using the syringe filter. A volume is added to molten agar together with glucose before pouring.
Problem is, I have contamination in my plates. I do not know if it comes from the DMSO-Nile (solution gushed out instead of a slower trickle) or the (filter-sterilized) glucose.
Q: Can I sterilize the DMSO-Nile mixture in an autoclave? Will it adversely affect the function of the dye?
Really need to get my plates ready before I can start isolating my bacteria.
Thanks in advance.
Firstly do not autoclave DMSO, flashing point of DMSO is about 87°C. At 3% volume there is an explosion/fire risk.
Pure DMSO on its own is sterile. Cell membranes can not form under such conditions.
I think you could add the DMSO/dye into a volume of medium and then filter sterile the medium/DMSO solution. Since the DMSO is diluted, it won't damage the filter membrane.
Alternatively you may filter sterilisation the DMSO, using nylon membrane filters (0.2 um). I am told they are DMSO safe.
hmm... I find it hard to believe that the glucose solution is contaminated. It was filter steriled wasn't it? (0.2um)
Make sure your containers are sterile. No point filter sterilising a solution only to place it in contaminated containers.
Is your working environment clean? If you are working in a laminar flow cabinate make sure it has been decontaminated and that the filters are still good. If you are working on a bench top with a bensen burner, make sure you are not working near an open window, near doors or under an air duct. Also get the lab coat clean... and no talking while pouring plates...aerosols.