Basically my experiment looks like this: (1) Culture on LJ's (2 weeks @ 37 degrees) -> (2) transfer colony (aseptically) to 10 - 20ml 7H9 media with glycerol (incubate @ 37 degrees) -> (3) grow until McFarland 3 -> (4) store in aliquots at -80 degrees -> (5) thaw frozen samples and grow in 7H9 until McFarland 1 or 3 (when needed for experiments) -> (6) perform experiment MABA (microtiter alamar blue assay) (McFarland 1).
(1) Does my experiment look acceptable up to MABA?
(2) What is the correct method for storing at -80? Can I just freeze my cells in broth and store in eppies?
(3) What is the correct method for using my cells from the -80 fridge? Is thawing at room temp ok? Or should I incubate them in eppies for 24H before growing in 7H9?
(4) Instead of freezing them should I maybe use a continuous culturing method? (I prefer freezing because I am not continually doing experiments).
(5) For the MABA (microtiter alamar blue assay) I need 10^5 CFU/ml (MIC determinations and determination of synergy with different antibiotics). Can I determine the CFU/ml in duplicate for cells stored at -80 and then grown to McFarland 1, and assume the CFU/ml will be round about the same for the others stored at -80? The assay takes place in a 96 well plate and literature says a positive result can take up to 5 - 7 days but I only get one in about two weeks. Any idea why? Is it just strain differences.
(6) Could this long incubation period have an effect on my MICs?