Department of Biological Sciences,

1000 Hilltop Circle

Baltimore, MD 21250

General Laboratory Procedures, Equipment Use, and Safety Considerations 

 VI. Sterile Technique

1. All media, including plates, liquid media and top agar must be autoclaved immediately after it is prepared. It is best to prepare media in several small bottles, only opening one at a time. Check the bottle for contamination before you use it by gently swirling it and looking for cloudy material in the center. Always grow up a small amount of broth alone when growing cells overnight. A small amount of contamination is not always evident until the media is incubated at 37 deg C.
2. Use a flame on inoculating loops and on the lips of media bottles before and after pipetting from them. Never leave a media or agar bottle open on the bench and don's take an individually-wrapped pipet out of its protective wrapper until you are ready to use it (i.e., don't walk across the room with an unwrapped pipet). Always use a fresh, sterile pipet or pipet tip when pipetting culture media, and never go back into a media bottle or cell culture with a used pipet.
3. To prevent wide-scale, untraceable contamination, each person should have his own stock of liquid culture media, top agar, plates, 100% glycerol, glycerol stocks of cells, etc. and don't share.
4. Overnight cultures should be grown only from a single colony on a fresh plate or from a previously-tested glycerol stock that was grown from a single colony. To prepare an overnight culture from a glycerol stock, take an individually-wrapped 1-ml pipet and a culture tube of media to the -80 deg C freezer. Quickly remove the cap from the freezer vial containing the glycerol stock, scrap a small amount of ice from the surface of the culture, replace the cap on the freezer vial, and place the pipet into the culture tube. Sufficient numbers of bacteria are present in the ice in order for the culture to grow to saturation in 16 hours. Never let the glycerol stock thaw.
5. Think about what you are doing. The best defense is common sense.

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Last updated on 3/2/2010