Hints for Microscopy

From FreeBio

Finding those little buggers on the scope can be tricksy. Here are some things to make life a bit easier after hours of peering into that little green field...

1. Concentrate the cells before looking at them. My current method is to take 0.5 ml of a solution at around 0.1 - 0.3 OD600, spin them down, aspirate, and resuspend in 15 ul. Then dot 1 ul of this on a slide.

2. In the 10X, turn the coarse focus knob forward, bringing the stage up as far as it can go (it won't touch the objective). Now turn the knob back towards you, bringing the stage done. In the field of view, the lens will focus first on random specks, go blurry, focus on more random specks, then the bacteria. They should look like lots and lots of little dots.

3. In the 100X, add a drop of oil to the slide. Raise the stage until the lens touches the oil, breaking the surface tension. Now turn the fine focus forwards. Bacteria should appear in about three full turns of the knob!

4. Picture taking - I like to use Biosketch Phase (100 msec) for phase pictures, BioWire GFP (250 msec) for GFP and YFP, and BioWire CFP (250 msec) for CFP.