Plating with Glass Beads

MATERIALS:

Bacterial Cells

Diluting broth

30 6mm borosilicate beads (autoclaved in glass tubes and/or glass beakers)

Small autoclaved plastic funnel

2% X-Gal in DMF

20mM IPTG

Sterile 15ml screw cap test tube

Single well rectangular media plates

METHOD:

  1. Gather materials under laminar flow hood.

  2. Titer bacterial stock via serial dilutions or if the titer has already been determined dilute bacterial stock with broth to a population of ~ 500 colonies/plate. (For titering, smaller quantities of dilutions can be made, for mass plating prepare several mls.)

  3. Beginning with the first plate, remove lid and set to side, dispense 35ul IPTG, 70ul X-Gal and 100ul bacterial cells. Plate most dilute cells first and work up. Be sure to vortex cells thoroughly and immediately dispense 100ul onto media. Dispense solutions in different spots (close to each other, X-Gal will precipitate out of solution if mixed with the other 2 components, will be fine if dispensed aside from each other, but close to each other and than mixed via the beads.)

  4. Pour sterile glass beads onto plate, cover and shake vigorously in a circular motion, clockwise and counter clockwise for approximately 1.5 minutes, dependent on how dry the media is, approximately 132 rpm. Agitate vigorously making sure the beads are well distributed over the entire plate (may have to rotate 180° a few times) until balls no longer move freely and quiet down (balls are than dry and can be returned via funnel to tube).

  5. Cover with lid and invert plate. Go on to the next plate.

  1. After all the plates are inoculated, place them in an incubator as per protocol temperature and time. When plating is complete, rinse funnel tubes or beakers and beads with 2% micro, pour into strainer and rinse thoroughly with dwater and finally rinse with ddwater. Beads can be rinsed with 70% EtOH to ensure drying.

  2. When beads are dry, count 30/tube and autoclave.