Bacterial cell culture

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(new protocol for beginners to in vivo work, please supplement)
m (typo)
Line 12: Line 12:
#Streak an [[Pour plates|agar plate]] from glyerol stock.  Incubate plates overnight in the 37 °C room.
#Streak an [[Pour plates|agar plate]] from glyerol stock.  Incubate plates overnight in the 37 °C room.
-
#Take your plates from the warm room.  Take an aliquot of the [Endy:Preparing Antibiotic Stocks|antibiotic](s) needed from the freezer, and set it on the bench to thaw.
+
#Take your plates from the warm room.  Take an aliquot of the [[Endy:Preparing Antibiotic Stocks|antibiotic]](s) needed from the freezer, and set it on the bench to thaw.
#Flame a glass pipette, open the bottle of medium and flame the mouth, measure out the amount you need to fill your tubes, flame the cap and recap the bottle as quickly as possible.
#Flame a glass pipette, open the bottle of medium and flame the mouth, measure out the amount you need to fill your tubes, flame the cap and recap the bottle as quickly as possible.
#Remove the tube cap, flame the top of the culture tube, pipette in 5 ml, flame the top of the tube, and cap it.   
#Remove the tube cap, flame the top of the culture tube, pipette in 5 ml, flame the top of the tube, and cap it.   

Revision as of 16:52, 18 July 2005

Materials

  • Glass culture tubes with metal caps and labels
  • Medium from media room or customized
  • Fireboy or Bunsen burner
  • Glass pipette tubes
  • Motorized pipette
  • Micropipettes and sterile tips

Procedure

For a typical liquid culture, use 5 ml of appropriate medium. The amount in each tube does not have to be exact if you are just trying to culture cells for their precious DNA.

  1. Streak an agar plate from glyerol stock. Incubate plates overnight in the 37 °C room.
  2. Take your plates from the warm room. Take an aliquot of the antibiotic(s) needed from the freezer, and set it on the bench to thaw.
  3. Flame a glass pipette, open the bottle of medium and flame the mouth, measure out the amount you need to fill your tubes, flame the cap and recap the bottle as quickly as possible.
  4. Remove the tube cap, flame the top of the culture tube, pipette in 5 ml, flame the top of the tube, and cap it.
  5. Pick up one colony by tapping a small 0.1 microliter pipette tip (on a pipette) on the surface of the plate. Uncap a tube, flame the top, eject the tip into the tube, flame, cap. You can also use a sterile toothpick for transfer, but this method gives a clear indication of which tubes have been inoculated.
  6. Pipette the desired amount of antibiotic into each tube along the wall. Do not put the non-sterile part of the pipette inside the tube and use a new tip for each tube.
  7. Vortex each tube for 1-2 seconds to mix well.
  8. Take the tubes to the 37 °C room. Turn the rotating rack off using the dial to decrease the speed. Do not use the switch because the motion is too abrupt. Add your tubes in a balanced layout. If you have an odd number, there are extra tubes for balance in the box on the floor next to the machine. Dial the machine back up to 7.
  9. Wait overnight or until your cells have reached the desired concentration.
Personal tools