Disinfection and Sterilization
Biological safety depends on proper cleanup and removal of potentially harmful agents. Disinfection and sterilization are two ways to help ensure biological safety in the laboratory.
- Disinfection: Reduction of the number of pathogenic organisms by the direct application of physical or chemical agents.
- Sterlization: Total destruction of all living organisms. The following sections discuss guidelines and procedures for biological disinfection and sterilization. General Guidelines Choosing the best method for disinfection and sterilization is very important. The proper method depends on the following:
- Target organisms to be removed
- Characteristics of the area to be cleaned Once you have chosen the proper method for disinfection or sterilization, follow these guidelines to ensure laboratory safety:
- Frequently disinfect all floors, cabinet tops, and equipment where biohazardous materials are used.
- Use autoclavable or disposable materials whenever possible. Keep reusable and disposable items separate.
- Minimize the amount of materials and equipment present when working with infectious agents.
- Sterilize or properly store all biohazardous materials at the end of each day.
- Remember that some materials may interfere with chemical disinfectants -- use higher concentrations or longer contact time.
- Use indicators with autoclave loads to ensure sterilization.
- Clearly mark all containers for biological materials (e.g., BIOHAZARDOUS - TO BE AUTOCLAVED.).
8.1 Types of DisinfectantUse the following table to aid in the selection of disinfectants:
|Alcohols||Ethyl or isopropyl alcohol at 70-80% concentration is a good general purpose disinfectant; not effective against bacterial spores.|
|Phenols||Effective against vegetative bacteria, fungi, and viruses containing lipids; unpleasant odor.|
|Formaldehyde||Concentration of 5-8% formalin is a good disinfectant against vegetative bacteria, spores, and viruses; known carcinogen; irritating odor.|
|Quaternary Ammonium Compounds||Cationic detergents are strongly surface active; extremely effective against lipoviruses; ineffective against bacterial spores; may be neutralized by anionic detergents (i.e., soaps).|
|Chlorine||Low concentrations (50-500 ppm) are active against vegetative bacteria and most viruses; higher concentrations (2,500 ppm) are required for bacterial spores; corrosive to metal surfaces; must be prepared fresh; laundry bleach (5.25% chlorine) may be diluted and used as a disinfectant.|
|Iodine||Recommended for general use; effective against vegetative bacteria and viruses; less effective against bacterial spores; Wescodyne diluted 1 to 10 is a popular disinfectant for washing hands.|
NOTE: See the Radiation Safety chapter for information pertaining to the use of ultraviolet lights as a method of disinfection.
8.2. Sterilization Methods
There are three common methods for sterilizing laboratory materials: wet heat, dry heat, and ethylene oxide gas.
When used properly, the damp steam heat from an autoclave effectively sterilizes biohazardous waste. Sterilization occurs when contaminated materials reach 15 psi pressure at 250°F or 121°C for at least 30 minutes.
IMPORTANT: For the autoclave process to be effective, sufficient temperature, time, and direct steam contact are essential.
Every TAMUS-HSC components that autoclaves biohazardous waste should have written documentation to ensure the waste is sterile. Parameters for sterilization and standard operating procedures should include requirements for verifying sterilization.
Potential problems with wet heat sterilization and autoclaves include the following:
- Heavy or dense loads require higher temperature for sterilization.
- Poor heat conductors (e.g., plastic) take longer to sterilize.
- Containers may prevent steam from reaching the materials to be sterilized.
- Incomplete air removal from the chamber can prevent contact between the steam and the load.
- Deep trays can interfere with air removal.
- Tightly stacked loads can impede steam circulation and air removal.
- Double-bagging will impede steam penetration.
- Carcasses do not allow steam penetration.
- Some bags and containers rated as autoclavable have thermal stability but they do not allow steam penetration.
To ensure that all materials are sterile, always test autoclave loads. Remember, however, that some sterilization indicators are incomplete. Autoclave tape, for example, verifies sufficient external temperature exposure, but it does not indicate internal equipment temperature, exposure time, or steam penetration. Thermocouples or other instrumentation can also indicate temperature, but they do not verify sterility. A biological indicator is the most effective monitor to ensure sterility. Commercially available strips or vials of Bacillus species endospores, for example, are suitable biological indicators.
Dry heat is less effective than wet heat for sterilizing biohazardous materials. Dry heat requires more time (two to four hours) and a higher temperature (320-338°F or 60-170°C) to achieve sterilization. A Bacillus species biological indicator can verify dry heat sterilization.
Ethylene Oxide Gas
Ethylene oxide gas is lethal to all microorganisms. Because it is also a known carcinogen and potentially explosive (freon and carbon dioxide mixtures are stable), minimize your exposure and use extreme care when working with this gas. Ethylene oxide sterilizers and aerators must be properly vented. Ethylene oxide gas is most effective with heat-resistant organisms and heat sensitive equipment. The effectiveness of ethylene oxide gas may be affected by the following:
- The antimicrobial activity of ethylene oxide increases with increased temperature. Normal sterilization temperature is 120-140°F or 49- 60°C.
- Ethylene Oxide Concentration:
- Sterilization time decreases with increased gas concentration. Normal concentration is 500-1000 mg/L.
- Relative humidity of 30-60% is necessary.
- Exposure Time:
- Follow the manufacturer's recommendations.