Standard Aseptic Laboratory Procedures^[1]

When working with bacteria and other microorganisms it is essential to practice aseptic technique. This ensures that the cultures being studied are not contaminated by organisms from the environment or that the organisms are not released into the environment, which would contaminate both the laboratory and laboratory personnel. Although most of the microorganisms we will use in the lab are not pathogenic (disease producing), it is a good idea to treat all cultures as potential health hazards. If you master the following standard aseptic laboratory techniques and make sure that you automatically follow them for every lab situation, you won’t have any problems when you do encounter work involving pathogens. Bottom line: Aseptic technique generally relies on common sense and good organization.

1. Wash your hands with soap and water before and after working with biological cultures.

2. Wipe the lab bench or work area with disinfectant before and after the lab activities.

3. Tie back long hair, roll up your sleeves, and wear a lab coat. Do not wear a brimmed hat or cap.

4. Keep hands and pens/pencils away from face and mouth.

5. Arrange all necessary equipment and glassware to ensure clear working area (i.e., no reaching over materials).

6. Sterilize instruments used to manipulate microorganisms before and after use. Once you put your loop or instrument down on the bench (even for a second) it is no longer sterile and must be re-flamed. Sterile swabs are only to be used one time and are then to be discarded in a waste container. You need to use a sterile instrument when subsampling as well.

7. Report all spills and accidents, no matter how small, to the lab instructor.

8. Cultures and media will remain sterile if they are not opened, but you cannot study microorganisms if you never open the culture lids. If available, use sterile culturing hoods (designed with special air flow and containment features that reduce contamination). If not available, you can reduce contamination by practicing the following:

a. When opening bottles containing media or cultures, keep these in a tilted position, so that air borne microorganisms are less likely to land inside.

b. To avoid condensation and reduce contamination in agar petri plate cultures, be sure to keep the plates inverted. When you open the culture, lift the bottom (containing the agar) out of the lid, leaving the lid on the bench surface. Quickly turn the bottom over, do your manipulations on the agar surface, place the bottom back into the lid. When opening petri plates containing liquid media or cultures that cannot be inverted, do not remove the lid completely. Hold the lid at a 45° angle, opening it just enough to do your manipulations.

c. Do not place your head directly over or breathe directly on the microorganism cultures, sterile solutions, or culture medium.

9. All biologically contaminated materials must be disposed of in a safe manner. Wastes and used cultures must be autoclaved or disinfected before they can be discarded into the regular garbage. Follow the procedures below:

a. All materials (glass, contaminated and uncontaminated) must be discarded into the appropriate containers or placed in the areas designated. For example, all biologically contaminated material (e.g., toothpicks, swabs, latex gloves, etc.) are to be placed in biohazard waste; all used slides and pipettes go into the glass waste.

b. Never remove cultures or materials from the laboratory.

c. When a container is full, obtain an empty container for your discard materials or notify the technician of the situation. Forcing materials into full containers creates hazards that may expose you and others to unnecessary contamination.