The Laboratory Biosafety Guidelines: 3rd Edition 2004

Chapter 8
Decontamination

8.1 Introduction

It is a basic biosafety principle that all contaminated materials be decontaminated prior to disposal. Decontamination includes both sterilization (the complete destruction of all microorganisms, including bacterial spores) and disinfection (the destruction and removal of specific types of micro-organisms). A list of various decontaminants, their effectiveness against different microbial groups, their important characteristics and their most appropriate application in research and clinical laboratories have been amply summarized by others^(1-4). It is the responsibility of all laboratory workers to ensure the effective use of products for decontamination of materials, equipment, and samples from containment zones; of surfaces and rooms; and of spills of infectious materials.

These procedures represent a critical containment barrier whereby failure in the decontamination procedure can result in occupational exposure to infectious agents and/or the unintentional release of agents from a containment facility. Employee infection with M. tuberculosis as a result of exposure to contaminated waste has been documented⁽⁵⁾. Reports of infections with Coxiella burnetii in commercial laundry workers, presumably due to improper decontamination of laboratory coats and gowns prior to laundering, also exist in the literature^(6,7). Employees should be responsible for leaving their laboratory clothing for laundering in a designated spot or area.

Most Canadian jurisdictions have prepared or are preparing guidelines or regulations concerning the management of biomedical waste. The treatment procedures used at each laboratory are subject to the standards in place for that province or territory. The Canadian Council of Ministers of the Environment (CCME) has also developed minimum national guidelines for defining, handling, treating and disposing of biomedical wastes⁽⁸⁾. The intent of the guidelines is to promote uniform practices and set minimum national standards for managing biomedical waste in Canada. Provincial, territorial and municipal regulatory authorities should also be consulted as they may specify more stringent requirements.

As mentioned in the operational requirements elsewhere in this document, all contaminated materials must be decontaminated before disposal or cleaning for reuse. The choice of method is determined by the nature of the material to be treated. This may include, but is not limited to, laboratory cultures, stocks and clinical specimens; laboratory equipment, sharps and protective clothing; and other items that have come into contact with infectious materials. Laboratory bench tops and surfaces are to be decontaminated after any spill of potentially infectious materials and at the end of the working day. Laboratory rooms and large pieces of equipment may also require decontamination (i.e., prior to servicing, maintenance, transfer to other settings or reassignment). Specific written protocols must be developed and followed for each process. Employees must be trained in all decontamination procedures specific to their activities and should know the factors influencing the effectiveness of the treatment procedure, as discussed briefly below.

8.2 Autoclaves

Infectious laboratory wastes (petri dishes, pipettes, culture tubes, glassware, etc.) can be effectively decontaminated in either a gravity displacement or prevacuum autoclave. Prevacuum autoclaves remove air from the chamber by pulling a vacuum (except for liquid cycles) before the saturated steam enters the autoclave chamber, resolving problems with air entrapment during removal of air by gravity displacement. The effectiveness of decontamination by steam autoclaving depends upon various loading factors that influence the temperature to which the material is subjected and the contact time. Particular attention must be given to packaging, including the size of containers and their distribution in the autoclave. Containers of waste must allow steam penetration and must be arranged in the autoclave in a manner that permits free circulation of steam. Tight-fitting containers do not permit steam penetration. Piling containers above one another and overloading can result in decontamination failure.

Effective operating parameters for autoclaves should be established by developing standard loads and their processing times through the use of thermocouples and biological indicators placed at the centre of the load (i.e., the most difficult areas of the load to decontaminate). Biological indicators are also used for routine monitoring (e.g., weekly, based on the frequency of use) of the sterilization process. A biological indicator is a standardized population of bacterial spores intended to demonstrate favourable sterilization conditions in the load. Attention must be paid to appropriate selection of the indicator, as the design and construction vary depending on its intended use (e.g., liquid versus dry load, self-contained system, enzyme-based rapid method). Chemical indicators are meant to be used in conjunction with biological indicators and physical monitors (i.e., pressure and temperature readings). They provide instant results for day-to-day monitoring that the load has been processed; however, they must not be used as the sole indicator of sterility. Favero presents a comprehensive overview of the types of biological and chemical/physical indicators and their recommended use⁽⁹⁾. The results of biological indicator testing should be kept on file.

8.3 Chemical Disinfection

Chemical disinfectants are used for the decontamination of surfaces and equipment that cannot be autoclaved, such as specimen containers and other items removed from containment, and for clean up of spills of infectious materials, rooms and animal cubicles, and a variety of other items for which heat treatment is not feasible. The initial choice of a chemical disinfectant depends upon the resistance of the microorganisms of concern. The most susceptible are vegetative bacteria, fungi and enveloped viruses^(2,3). Mycobacteria and nonenveloped viruses are less susceptible; bacterial spores and protozoan cysts are generally the most resistant^(2-4). Consideration should also be given to practicability, stability, compatibility with materials and health hazards⁽¹⁾.

There are usually striking differences between the activity of disinfectants when employed under actual laboratory conditions as opposed to the controlled, standardized testing methods used to generate efficacy data for product registration. A review of the official protocols used to assess disinfectant activity is under way⁽¹⁰⁾. The effectiveness of the disinfectants can be influenced by a number of factors: presence of organic material (e.g., blood, serum, sputum) that decreases the effect of hypochlorites⁽⁴⁾ ; temperature; relative humidity; concentration; and contact time^(2,4). In some cases, it may be beneficial for laboratories to conduct in-use disinfectant efficacy testing to evaluate a product's performance in the field, under conditions of use. A basic method to evaluate surface disinfectants involves the artificial contamination of a surface and immersion in the appropriate dilution of the disinfectant; thereafter the disinfectant is neutralized by dilution and checked to determine whether all microorganisms have been killed⁽¹¹⁾. A similar protocol can be used to verify the effectiveness of disinfectants used in discard containers: an inoculum is added to the disinfectant, which after a predetermined contact time is neutralized by dilution, and an aliquot is examined for growth⁽¹¹⁾.

Selection of an appropriate disinfectant can become a dismaying task given the numerous products on the market. There are increasing numbers of disinfectants being developed and new disinfectant options being explored⁽¹²⁾. However, the active components of disinfectants belong to relatively few classes of chemicals, and understanding the capabilities and limitations of each class of chemicals (e.g., hypochlorites, quaternary ammonium compounds, phenolics, iodines, alcohols) will allow choice of a product based on relative effectiveness.

8.4 Gaseous Decontamination of Rooms

Gaseous decontamination of rooms is generally only necessary at containment levels 3 and 4 under particular circumstances (e.g., after a spill or accidental release of infectious materials, for removal of large equipment items from containment, before maintenance work on contaminated systems, before retesting of HVAC control systems). Because of the potential for exposure to the hazardous chemicals (formaldehyde) used, gaseous decontamination of rooms should be done only by highly trained personnel. The two-person rule should always apply to this operation, and both individuals should be trained and fitted in the use of appropriate respiratory protection. The recommended protocol involves the depolymerization of paraformaldehyde into a well-sealed room to provide a concentration in air of 10.6 g/m ³ (0.3 g/ft³ )⁽¹⁾. After a contact time of at least 6 hours, the formaldehyde is neutralized with ammonium carbonate (using 1.1 times the weight of formaldehyde) before venting and aeration⁽¹⁾. The room and surrounding space should be monitored for airborne levels of formaldehyde, and only when levels are below the exposure limits can the area be considered safe for re-entry without protective clothing. Successful gaseous decontamination requires an ambient temperature of at least 21^o C and a relative humidity of 70%⁽¹⁾. Biological indicators should be used to monitor the effectiveness of the gaseous decontamination procedure⁽¹³⁾.

Vaporized hydrogen peroxide has been proposed as a safer alternative to gaseous decontamination with formaldehyde. In the sterilization process, 30% liquid hydrogen peroxide is vaporized to yield approximately 1200 ppm. The vapour breaks down into nontoxic oxygen and water. Vaporized hydrogen peroxide has been successfully used as a nondestructive sterilant for the decontamination and removal of laboratory equipment and materials (e.g., telephone, camera, computer, pipette, electric drill) from a containment laboratory⁽¹⁴⁾. Future commercial applications may address today's constraints associated with the cumbersome size of the vaporized hydrogen peroxide generator and limitations in the space capable of being decontaminated (e.g., pass-through boxes, small rooms).

8.5 Liquid Effluent Treatment Systems

Liquid effluent treatment systems are used in containment level 4 laboratories (and containment level 3 laboratories handling nonindigenous animal pathogens) for decontaminating liquid waste streams from sinks, showers, autoclave chambers and other drains. These systems represent a secondary treatment system, as no infectious micro-organisms are disposed of directly into the drain without prior treatment (i.e., the addition of chemical disinfectants). The decontamination parameters (i.e., time and temperature for heat-based systems) must be defined and must be effective against the microorganisms of concern. The internal temperature and pressure of the effluent tanks and the decontamination time should be logged throughout the cycle. Chemical-based decontamination systems may be practical on a small scale where smaller volumes of liquid effluent require treatment. Decontaminated liquids released from the treatment system must meet all applicable regulations (e.g., municipal bylaws for temperature, chemical/metal content, suspended solids, oil/grease and biochemical oxygen demand).

8.6 Irradiation

Gamma irradiation (e.g., 60 Co) can be used for the decontamination of heat-sensitive materials and is an effective means of decontaminating chemicals and solvents removed from a containment facility. The efficacy of the treatment technology depends on the penetration of the treated items by gamma irradiation and, therefore, on the density of the treated substance as well as the strength of the irradiation source⁽²⁾.

Microwave irradiation is not widely used for decontamination in containment facilities. As in steam autoclaving, heat is the critical factor for eliminating viable microorganisms. The factors that affect microwave treatment include the frequency and wavelength of the irradiation, the duration of exposure and the moisture content of the material to be decontaminated^(15,16).

Ultraviolet irradiation (UV) should not be relied upon as the sole method of decontamination for materials removed from containment facilities. UV has limited penetrating power and is primarily effective against unprotected microbes on exposed surfaces or in the air⁽¹⁾. It can be effective in reducing airborne and surface contamination provided that the lamps are properly cleaned, maintained and checked to ensure that the appropriate intensity is being emitted.

8.7 Incineration

Incineration has traditionally been the chosen method for processing anatomical biomedical waste and animal carcasses. In most cases, wastes to be incinerated have to be packaged and transported off-site in accordance with provincial or territorial legislation. Materials removed from containment laboratories for off-site incineration should initially be treated at the containment barrier, preferably by autoclaving. Effective incineration depends on proper equipment design; on provision for the time, temperature, turbulence and air required for complete oxidation; and on careful feeding of the unit. Modern incinerators have two chambers with an ideal temperature in the primary chamber of at least 800^o C and in the secondary chamber of at least 1000^o C^(2,15). Loads with high moisture content may lower the processing temperature. There are no microbial standards for stack discharge, but there are for emission of particulate matter and selected chemical contaminants⁽⁸⁾. Provincial or territorial regulatory authorities should be consulted for additional requirements regarding incinerator operations and emissions.

8.8 New Technologies

Growing concern with air pollution has caused many regulatory agencies to introduce more stringent standards for incinerators with a resultant explosion in alternative waste treatment systems. Most of these systems use one or more of the following methods: heating by means of microwaves, radio waves, hot oil, hot water, steam or superheated gases; exposure to chemicals such as hypochlorite, chlorine dioxide or sodium hydroxide; subjecting the waste to heated chemicals; and exposing the medical waste to irradiation sources^(15,16). A description of many of these technologies and their advantages and disadvantages have been previously summarized^(15,16). A modified rendering process has been shown to be an effective alternative and has been successfully used to decontaminate infected animal carcasses⁽¹⁷⁾.New technologies are subject to the approval of provincial or territorial regulatory authorities, and laboratories should consult these authorities before purchasing products or implementing new approaches to decontamination.

References

1. Vesley,D.,Lauer,J.L.,andHawley,R.J. Decontamination, sterilization, disinfection and antisepsis. In:Fleming,D.O.,and Hunt, D.L. Biological safety principles and practices. Washington, DC: ASM Press, 2000;383-402.

2. Dychdala, G.R., Gottardi, W., Block, S.S., Wickramanayake, G.B., Larson, E.L., Morton, H.E., O'Connor, D.O., Rubino, J.R., Merianos, J.J., Lopes, J.A., Denton, G.W., Rossmoore, H.W., May,  
   O.W., Opperman, R.A., Gitlitz, M.H., Beiter, C.B., and Yeager, C.C. Part III: disinfectants and antiseptics (section A: by chemical type). In: Block, S.S. Disinfection, sterilization and preservation. Malvern, PA: Lea & Febiger, 1991;131-364.

3. Hugo, W.B., and Russell, A.D. Types of antimicrobial agents. In: Russell, A.D., Hugo, W.B., and Ayliffe, GA.J. Disinfection, preservation and sterilization. Oxford, UK: Blackwell Science Ltd, 1999;5-94.

4. Russell, A.D. Factors influencing the efficacy of antimicrobial agents. In: Russell, A.D., Hugo, W.B., and Ayliffe, GA. J. Disinfection, preservation and sterilization. Oxford, UK: Blackwell Science Ltd., 1999;95-123.

5. Weber, A.M., Boudreau, U.V., and Mortimer, V.D. A tuberculosis outbreak among medical waste workers. J Am Biol Safety Assoc 2000; 2:570-88.

6. Collins, C.H., and Kennedy, D.A. Laboratory-acquired infections. In: Laboratory-acquired infections: history, incidence, causes and preventions. Oxford, UK Butterworth-Heinemann, 1999;1-37.

7. Richmond, J.Y., and McKinney, R.W. Biosafety in microbiological and biomedical laboratories. Washington, DC: U.S. Government Printing Office, 1999.

8. Canadian Council of Ministers of the Environment. Guidelines for the management of biomedical waste in Canada. Toronto, ON: Canadian Standards Association. CCME EPC-WM-42E, 1992.

9. Favero, M.S. Developing indicators for monitoring sterilization. In: Rutala, W.A. Disinfection, sterilization and antisepsis in health care. Washington, DC: Association for Professionals in Infection Control and Epidemiology Inc., 1998;119-32.

10. Sattar, S.A. Microbicidal testing of germicides: an update. In: Rutala, W.A. Disinfection, sterilization and antisepsis in health care. Washington, D.C.: Association for Professionals in Infection Control and Epidemiology Inc., 1998;225-240.

11. Best, M., Sattar, S.A., Springthorpe, V.S., and Kennedy, M.E. Efficacies of selected disinfectants against Mycobacterium tuberculosis. J Clin Microbiol 1990;28:2234-39.

12. Springthorpe, M.S. New chemical germicides. In: Rutala, W.A. Disinfection, sterilization and antisepsis in health care. Washington, DC: Association for Professionals in Infection Control and Epidemiology Inc., 1998;273-80.

13. Abraham, G., Leblanc, P.M., Smith, P.M., and Nguyen, S. The effectiveness of gaseous formaldehyde decontamination assessed by biological monitors. J Am Biol Safety Assoc 1997;2:30-38.

14. Best, M. Efficacy of vaporized hydrogen peroxide against exotic animal viruses. Appl Environ Microbiol 1997;63:3916-18.

15. Salkin, I.F., Krisiunas, E., and Thumber, W.L. Medical and infectious waste management. In: Richmond, J.Y. Anthology of biosafety II: facility design considerations. Mundelein, IL: American Biological Safety Association, 2000;140-60.

16. Turnber, W.L. Biohazardous waste: risk assessment, policy and management. New York, NY: John Wiley & Sons Inc., 1996.

17. Thompson, L., Best, M., and Langevin, P. Biological efficacy testing of liquid effluent and tissue/carcass sterilization systems. Mundelein, IL: American Biological Safety Association, 1998.

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