How to sterilize disposable electroporation cuvettes - For Pichia Pastoris transformation (Jun/28/2005 )
Recently I was trying to use an electroporation system to transform Pichia Pastoris, Since some of my colleagues here use 75% alcohol to sterilize the disposable electroporation cell. I am wondering whether this is reliable ,since I find Pichia pastoris can produce ascospores which is very tough,ie it is very resistant to average sterilization agents.Although autoclave is a very effective method,it can oxidize the electrode and alter it's conductivity.Further more, plastic and metal(here refer to aluminum,the material used for the electrod in the disposalbe electroporation cell) will enlarge at a different extent when heated, eventually,after several times of autoclaving, the platic body of the cell will be crevassed and lose it's insulation(this is quite common,I have found a lot of cases!).
So,I am wondering is there any better cost-efficient method available?
i think that this topic may help as it discuss on reusing cuvettes...
For the wash of oour cuvettes, we now wash them in 0,1M NaOH three times (5' each). Good rinse by H2O, and three times by ddH2O, finally alcool two or three times. treatment is ok.
Thanks for your advise,Fred!
75% alcohol is ok for ordinary bacteria who do not produce a spore.
But other organizm who produce a spore is highly resistance to 75% alcohol.Pichia Pastoris produce ascospore, thus it poses a threat to my ordinary rescuing plan.I just make a small experiment to test whether 75% alcohol is efficient killing Pichia Pastoris!
spore are not resistant to 0,1M NaOH 3x5', are they???
Try UV (or if you can, gamma radiation). Ultrasonication is pretty effective too.
I did an experiment the last few days, and I found 75％is almost very effective in killing Pichia Pastoris.I picked one colony of Pichia each and resuspended them in 1.5 ml of 75% alcohol and sterilized water as a control.
Mix them well,as keep them over night.The next day,centrifuge at 1500 to collect the pichia cell body abandon the supernatant,then resuspend them in 150 microliter sterilized water and spread them onto a YPD plate each.Incubate at 30 degree.Now 3 days passed,and no colony appear on the plate which have been spread the alcohol treated Pichia,but the controled one have thrived over the entire plate! Amazing!
To Fred: Anyway, I find although sodium hydroxide is very effective against spores(5%,I find it over the web),but since the electrod of the electroporation cuvette is made of aluminum,it will react with NaOH and give out hydrogen which meas aluminum will disolve in the NaOH solotion.So,I feel this might be harmful to the electroporation cuvette.
From what I recall, the P.pastoris strain used in expression does not form spore. Sorry, I cannot remember the exact reference. But from Pichia protocol, it stays mostly in happloid stage.
So there should be no problem with your re-use cuvette.