with 5%, 7%, and 10% sucrose
Incubate briefly in LPGM in a 2 mL tube in the Ferris wheel, at a slow spin speed, parallel to the direction of rotation so the tube goes upside down and the liquid moves from top to bottom of the tube.
Can image at 4x on a poly-lysine coated slide . Make a vaseline circle, put some pollen suspension inside it, cover with coverslip.
Set up time-lapse imaging: 20 images every 5 minutes. Find a field of view with several pollen grains in focus at once, all starting to germinate.
In a multiwell plate The surface of one well of a 12 well plate can be covered by as little as 400uL water.
Need to cover with poly-lysine. Already have ~50 mL of 0.1% working solution.
So can coat ~125 wells
Well diameter = ~22 mm
In a cover-slip bottom 60 mm dish
500 uL coats the well.
Coverslip diameter = 30 mm
How many unique fields of view can we get in one dish?
Pollen tubes grow ~0.2 um/sec = 12um/min = 0.012 mm/min
|magnification||field diam (mm)||field diam (um)|
Reasonably good optics at 10x; camera adds magnification.
Coat all wells on 3 12-well plates, one for each concentration of sucrose.