General Cell Culture

This page covers general things you need to know to get started in cell culture work.

Sterile Technique

You cannot be a slob in tissue culture work. Bacteria and yeasts will outcompete your cells for growth and kill them in short time, leaving you farther and farther from your degree. Be totally paranoid! Here are some tips:

• Buy a small spray bottle at a grocery store and keep 70% ethanol in it. Use this liberally on everything. When you sit down to the hood, spray it. When you go to use a pipettor, nuke it. Spray your gloved hands regularly, and certainly everytime they touch non-sterile objects or are removed from the hood. Spray and rub down every bottle or flask that you bring into the hood from outside.
• Never leave spilled media or serum anywhere in the hood. Clean it up, for any microbes present will gladly use it as their headquarters during their raids on your cultures.
• Work efficiently and smoothly in the hood. Jerky movements with the automatic pipet fling media everywhere and disrupt the laminar flow of air in the hood that keeps it sterile.

Growth of Your Cell Culture

Growth Vessels

Mammalian cells are grown in plates with various formats, such as 96-well and 24-well, or in flasks, such as T25 and T75. Plates are placed in the incubator with a lid, and in general the media should not fill the wells more than 1/2-way (i.e. 200 µL for a 96-well plate and 1 mL for a 24-well plate. If you can, avoid using the outside wells of the plates for growing cells, and just pipette media into these to combat evaporation in these wells.

Flasks are placed in the inubator on their largest side, with the neck of the flask bent upward and the cap on loosely to facilitate exchange of gas (some flasks have filtered tops...these can be screwed tight). The number associated with the flask, like 75 for a T75 flask, is the surface area of the largest side in square cm. General culture volumes are 25 mL for a T75 and 5 mL for a T25.

The Incubator

The incubator must be kept clean and sterile, have a full tank of CO2, and have a tray full of sterile water in it to keep the environment humid. If it is not sterile, bacteria and/or fungus will grow in your cultures. To prevent this or in case of a contamination problem, take the inside metal panels and racks out of the incubator (yes, take it apart), autoclave them unwrapped for 30 min. Then put them back in the incubator still hot, spraying them with ethanol just before you put them in. As a general maintenance, every month or so move your cultures to another incubator and spray ethanol over the entire inside surface of the incubator. Let the ethanol evaporate before you put your cultures back in, for the ethanol fumes are not good for them.

Keep your eye on the CO2 tank! If it runs out over the weekend, you are in for trouble. The CO2 regulates the pH of the growth media your cells are in. If the CO2 runs out, your media will turn a reddish-purple from the indicator in the media, meaning your cultures are now very alkaline, and probably dead. Most cells require a 5% CO2 environment.

Keep your incubator humid! The tray of water in the incubator will do this nicely. Add a few drops of the antimicrobial agent they sell for water baths to the tray of water to keep yeast and bacteria from growing in it. If the incubator runs dry, the water in your cultures will evaporate. This is especially true in the outside wells of the culture plates.

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