Correct storage of cells protocol help.

[Shane.vontelin.van.breda]

Hi everyone,

I am having the correct storage of my cells. I am growing M. tuberculosis to McFarland 1 then I store them as aliquots in eppies at -80DegC. I store them in the brith they are grown in, 7H9 with a small amount of glycerol and OADC.

When I do my assay, I thaw the cells at room temperature then I dilute them accordingly and do my assay.

When I do the assay the growth takes abnormally long. So I think that my cells are being killed off by storing them this way or the cold temperature is having some type of effect on them and the cells just grow slower?

I would like to know what is the correct way to store cells at such a low temperature? Do you guys think they are being killed off in the freezer or the cold temperature is effecting their growth in broth?

Thanks