5.1: Swimming bacteria

5.1: Swimming bacteria kdorfman Fri, 11/04/2011 - 14:05

Observing Swimming Bacteria

E. coli for 5.1

E. coli for 5.1 kdorfman Fri, 11/04/2011 - 14:08

Strain RP437 - wildtype for chemotaxis

At least 2 days before lab:

Streak all chemotaxis strains on LB agar.

Night before lab

Pick a single colony from the RP437 plate, inoculate into ~5 mL minimal medium + HMLTT; shake at 30C

Morning of lab

Inoculate ~200┬ÁL into ~45 mL minimal medium + HMLTT

At class time, OD should be ~0.02

Don't let them reach stationary phase!

Making motility movies

Making motility movies kdorfman Fri, 11/04/2011 - 14:42

To make movies with microscopes in 360:

Frames: 50
Interval: 0.1 sec
exposure: 16.38 msec
pixel type: 8 bit
binning: 4

use x4 list (mark 1) stage position list

shutter open

Materials for 5.1

Materials for 5.1 kdorfman Fri, 11/04/2011 - 14:40

Slides

coverslips

perfusion chambers (Coverwell 622503 from Grace Bio-Labs) - clean with distilled water

Microscopes

lens paper

NOTE: The optics are bad with the plastic coverslip on the perfusion chamber. If you peel the silicone gasket off the plastic coverslip and replace it with a glass coverslip, you can see much better! (See if there are thinner gaskets.)

Double-sided tape! Put 4 strips of double sided tape on a slide, lower a coverslip over them, and voila! chambers for bacteria! Make sure the tape strips are longer than the coverslip is wide, so you can introduce cells or media without mixing them.

Try double-sided tape with a liner so it is easier to cut and handle, e.g. MMM4010 at WBMason. Scotch Double-Sided Mounting Tape, Industrial Strength, 1" x 60", Clear/Red Liner

For phase to work, the condenser must be at maximum height!

Phase rings must be aligned! Telescope in microscope bulb drawer can be used to align rings. Loosen the chrome screws, adjust the ring with the little black wheels, then tighten. Repeat for each ring/objective combination.

Darkfield may work, too, but condenser must be at maximum height as well.

Reagents for 5.1

Reagents for 5.1 kdorfman Fri, 11/04/2011 - 14:49

Minimal Medium + HMLTT

Check stocks in refrigerator before making any solutions!

Plus, for extra-mile experiments:

  • 0.01 M glutamic acid

  • pH 5.06 (adjust with acetic acid)

  • 0.01 M galactose

  • 1mM CoCl2