Team:TU Darmstadt/Protocols/Bacterial cell culture

Bacterial cell culture

Materials

Equipment

• Bunsen burner
• Pipettes with pleus ball
• Micropipettes with sterile tips
• Flow bench

Chemicals & consumables

• Culture tubes with metal caps
• Growth medium like LB, DYT or SOC
• [http://en.wikipedia.org/wiki/Parafilm Parafilm]
• Autoclaved glass pipette tubes

Procedure

Starting culture: under sterile conditions add about 5mL (two fingers high) of medium to a culture tube and insert the stock or picked colony.

Step by step:

1. Cultivate the stock on agar plate e.g. until colonies grow (incubation usually at 37°C)
2. 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
3. Remove the tube cap, flame the top of the culture tube, pipette in 5 ml, flame the top of the tube, and cap it
4. Pick a single colony (to asure the cells are from the same single clonal population) and transfer it to the medium
   • by tapping a small (0.1 μl) pipette tip (held on a pipette) on the surface of the plate. Uncap a tube, flame the top, tip the tube so as to transfer cells from the pipette tip to the surface of the media without touching the inside of the tube with the non-sterile portion of the pipetter, flame, cap.
   • by picking up the colony with a sterile toothpick
   • by using a sterile metal loop (sterile by flaming) to place the colony in the tube
5. 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.
6. Vortex each tube for 1-2 seconds to mix well.
7. Take the tubes to incubat (usually at 37°C) in an incubator or warm room.
8. Wait overnight (e.g. for miniprep) or until your cells have reached the desired concentration.

Notes

References

• http://openwetware.org/wiki/Bacterial_cell_culture