CHAPTER 9

BIOLOGICAL SAFETY

7.0 DISINFECTION AND STERILIZATION

 

7.1  Definitions

7.2  Waste Disposal

7.3  General Procedures

7.4  Choosing a Method

7.5  Selecting a Disinfectant

7.6  Sterilization Methods

7.7  Autoclaving Procedure of Biological Waste


7.1       Definitions

Decontamination the application of microbiocidal steam, gas, solid (granular) or liquid chemical agents in situations in which microbes may be protected from contact by extraneous matter. Decontamination implies the destruction of or removal of microorganisms to some lower level, but not necessarily total destruction.  Sterilization, disinfection and antisepsis are forms of decontamination 

Sterilization the total destruction of all living organisms (including spores) by processing in steam sterilizers (autoclaves), with ethylene oxide autoclaves or by chemical (high level) sterilization. 

Disinfection to destroy all nonspore forming organisms that could pose a potential hazard to humans or compromise the integrity of the equipment.  Disinfection implies the use of antimicrobial agents on inanimate objects (floors, bench tops, equipment).

Antisepsis the application of a liquid antimicrobial chemical to living tissue either human or animal. The objective is to prevent sepsis by either destroying potentially infectious organism or inhibiting their growth and multiplication.

 

7.2       Waste Disposal

Laboratory waste needs to be segregated according to it nature and means of disposal. It is important to follow the proper disposal means for safety reasons but also in a concern for the cost of disposal and the threat to the environment. Please try to keep the waste generated to a minimal amount. 

         Normal non hazardous waste (clear bags)

This type of waste is handled by housekeeping and should contain non hazardous material such as paper, clean cell culture disposable plastic wear, non infectious nucleic acid and molecular biology disposable plastic wear and biological waste that has been chemically disinfected and autoclaved (pans from biological hoods containing bleach). 

         Biohazardous waste to be autoclaved  (red bags)

This type of waste is generated by BSL2 laboratories and should be in red bags placed in a secondary container. The bags are picked up by housekeeping and are then taken to a centralized area on campus for autoclaving and disposal.  

         Hazardous waste autoclaved prior to leaving the lab and then autoclaved in centralized place on campus before disposal.

This type of waste is generated by high and maximum containment laboratories, for which all waste has to be autoclaved prior to leaving the facility. These are bags that come in a different color, but make sure they are autoclave approved or they will melt in the autoclave, causing extensive damage to the unit.

Once this waste is out of the facility it is placed in a secondary container and is picked up by housekeeping and taken to a centralized area on campus for another autoclave cycle followed by disposal. 

         Hazardous material to be incinerated (Yellow bags).

This type of waste is to be placed in yellow bags and is very limited to certain research laboratories and chemotherapy areas. Approval for such means of disposal must be obtained by EHS. 

 

7.3       General Procedures

Disinfect frequently all floors, cabinet tops, and equipment where biohazardous materials are used. 

         Decontaminate all infectious materials and contaminated equipment prior to being washed, stored, or discarded.

         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.

         Decontaminate, properly store, or dispose of all biohazardous materials at the end of each day.

         Be aware that agar and other materials may interfere with the germicidal actions of chemical disinfectants, thus requiring pre-cleaning, higher concentrations, or longer contact time.

         Ensure sterilization by using suitable indicators with each autoclave load.

         Use clearly marked holding containers such as "NON-INFECTIOUS" or "BIOHAZARDOUS TO BE  AUTOCLAVED".

 

7.4       Choosing a Method

The method of choice for sterilization or disinfection will depend on two factors:

         the target organism (the biological agent)  and

         the characteristic(s) of the materials or areas to be decontaminated.

 

7.5       Selecting a Disinfectant

Use the following table to aid in the selection of an appropriate disinfectant.      

DESCRIPTIONS OF COMMONLY USED DISINFECTANTS

Substance

Description

Alcohols

Ethyl or isopropyl alcohol at 70-80% concentration is a good general purpose disinfectant, not effective against bacterial spores.  At BSL2/3/4, primarily used for control of environmental contamination.  Not recommended for use with blood borne pathogens.

Phenols

Effective against vegetative bacteria, fungi and lipid-containing viruses; unpleasant odor; toxic by skin contact.

Quaternary

Ammonium

Compounds

Cationic detergents, which are strongly, surface active; extremely effective against lipoviruses; not effective against bacterial spores; may be neutralized by anionic detergents (soaps).

Chlorine

Bleach

Low concentrations (10%) active against vegetative bacteria and most viruses; higher concentration (50%) 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).

Sodium Hydroxide

 

Sodium hydroxide at 1-2N for 1-2h can be used to disinfect material contaminated with Prions (BMBL)

 

 

7.6       Sterilization Methods

Wet Heat (Steam)  

This method requires approximately 15psi pressure with a chamber temperature of at least 250mF (121mC).  The cycle time begins when the materials being sterilized reach the predetermined temperature.  Then the length of time is dependent upon the volume size of the load (usually 30-60 minutes).  Monitor steam sterilization effectiveness with a biological indicator, Geobacillus stearothermophilus. 

Dry Heat                                  

This is less effective than steam, and requires more time (two to four hours) and a higher temperature (320-338mF or 160-170mC).  Monitor dry heat sterilization with a Bacillus subtilis biological indicator. 

Biological Test Packs

 EHS monitors autoclaves used for waste decontamination.  This process utilizes spores of Geobacillus stearothermophilus

Ethylene Oxide Gas (EO)

Ethylene oxide sterilization in no longer available at UTMB.  Contact EHS Biological and Chemical Safety for additional information if needed.

 

7.7       Autoclaving Procedure of Biological Waste

All biological waste that requires autoclaving should always be placed in a secondary container when being transported to and from the autoclave for decontamination. 

Waste bag that require autoclaving shall be double bagged and placed in a container for autoclaving. This will prevent spills in the autoclave. 

Bottles with liquids will also be placed in a secondary container prior to being autoclaved. Autoclave tape should be placed on the bottle to verify proper sterilization.

Waste contained in trays will be placed in a secondary container labeled and autoclaved. DO NOT over fill trays with waste. The tray must be closed and all material should be immersed in disinfecting liquid. 

Use caution when removing items from autoclave. Steam and hot liquids may seriously burn you.  Autoclave gloves, face shields, and rubber aprons are available for added protection.  

Before disposing of the autoclave waste read the printout of the run to assure proper sterilization time, temperature, pressure. If an error is noted the waste must be re-autoclaved and facility maintenance should be notified.