4.1: Sterile technique

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Sterile technique is ESSENTIAL when working with microorganisms! It is important to protect strains from contamination with other strains and from the many undefined microbes in the environment. Large numbers of diverse microorganisms are all around us - in the air, on laboratory surfaces, on your skin and on your clothing. True to their name, microorganisms are too small to be detected by the eye, but they grow rapidly in laboratory culture media. Correct transfer techniques and the use of sterile reagents are usually enough to prevent contamination.

Some simple precautions will reduce the possibility of contamination:

Wipe down a small working area on the lab bench with 70% ethanol.
Light a Bunsen burner in your work area while working with strains. The burner produces an updraft that prevents airborne microorganisms from falling into cultures.
Use sterile reagents, micropipette tips, and test tubes. Tips and microcentrifuge tubes should be kept in covered containers when not in use.
Minimize contamination from clothing and body surfaces. Pull back and secure long hair. Avoid touching or breathing on sterile surfaces that will contact microorganisms.
Avoid talking when you are transferring strains.
Work quickly! Minimize the time that tops are removed from vessels containing microorganisms or media.
Keep caps right-side up to prevent contamination from airborne microbes.

The culture media and reagents that we will use have been sterilized by either autoclaving or filtration. An autoclave is a chamber that uses pressurized steam to kill cells on surfaces or in solutions, using temperatures near 121 ̊C and pressures from 30-40 psi. (For comparison, atmospheric pressure is ~15 psi.) Ultrafiltration is used in the place of autoclaving when solutions contain temperature-sensitive compounds. The pores in the filters are typically 0.2 or 0.45 μm in diameter. These pores are sufficiently small to prevent the passage of bacteria, but not viruses or macromolecules.