11. Decon Draft
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Risk Management Serv ices
www.riskmanagement.ubc.ca
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Decontamination and SterilizationMethods
Laboratory Biological Safety
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Definitions
Sterilization:any process, physical or chemical, which resultsin the absence of all life on or in an object. This term appliesespecially to the destruction of microorganisms, including
bacteria, fungi, and their spores, and the inactivation of viruses
Decontamination:to destroy, remove, or neutralize livingorganisms, toxic agents or chemical carcinogens on a surface or
object (this does not imply either total destruction or totalremoval); to make an object safe for unprotected individuals
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Disinfection:to use a chemical agent to kill or inactivate mostvegetative bacteria, fungi, and viruses but not necessarilyspores. This term applies to a chemical used on inanimate
surfaces not to living tissues
Germicide: a substance used to destroy a specificmicroorganism
Algicidean agent that kills algae
Bacteriocide- an agent that kills vegetative bacteria andpossibly some less resistant spores (commercial term)
Fungicidean agent that kills fungi
Sporicidean agent that kills spores
Virucidean agent that inactivates, destroys or kills viruses
Tuberculocidean agent that kills mycobacteria
Definitions
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General Precautions
All areas where infectious materials have been handled need tobe decontaminated a minimum of daily. Ideally an area will bedecontaminated before and after work is performed
All infectious materials and/or contaminated equipment orapparatus should be decontaminated before being washed,removed, stored, or discarded
All biohazardous materials should be placed in an appropriately
marked refrigerator, waste or incubator, sterilized or otherwiseconfined at the end of each work day
Do not store materials in the autoclave!
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General Precautions
Dry hypochlorites, or any otherstrong oxidizing material, MUSTNOTbe autoclaved with organicmaterials such as paper, cloth, oroils
If organic material in the autoclavecatches fire, the oxidizer will
stimulate the fire and may cause anexplosion because of the highpressure in the autoclave
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Lab Benches
GOOD
B D
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Examples of Biological Waste
Waste Type Examples
Microbiology laboratory
(Biohazardous)
laboratory cultures, stocks of specimens of micro-
organisms, live or attenuated vaccines, human or
animal cell cultures used in research, and laboratory
material that has come into contact with any of the
above
Animal animal tissues, organs, body parts, carcasses,
bedding, animal blood and blood products
Human anatomical human tissue, organs, body parts
Human blood and blood
products
human fluid blood/blood products, items saturated or
dripping with blood, body fluids contaminated with
human blood, human body fluids removed for
diagnosis or during surgery, treatment or autopsy
Clinical & laboratory
waste sharps
needles, syringes, blades or laboratory glass capable
of causing punctures/cuts
defined as per BC Hazardous Waste Regulation
http://www.bclaws.ca/EPLibraries/bclaws_new/document/LOC/freeside/--%20E%20--/Environmental%20Management%20Act%20SBC%202003%20c.%2053/05_Regulations/27_63_88%20Hazardous%20Waste%20Regulation/63_88_01.xml#section1http://www.bclaws.ca/EPLibraries/bclaws_new/document/LOC/freeside/--%20E%20--/Environmental%20Management%20Act%20SBC%202003%20c.%2053/05_Regulations/27_63_88%20Hazardous%20Waste%20Regulation/63_88_01.xml#section1 -
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Biological Waste Minimization Measures
SEGREGATE uncontaminatedsolid waste from biomedical andbiohazardous waste (all risk groups)
Use products with less environmental impact:
Petri dishes with 35% less plastic
Glassware that can be decontaminated and reused
Refillable pipette racks Other reusable or recyclable products
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Methods of Decontamination
Heat or autoclaving
Autoclaving is the most dependable procedure for ensuring thecomplete destruction of microorganisms. It generally involvesheating in a chamber employing saturated steam under a relativepressure of 103 kPa (15 psi) to achieve a chamber temperature ofat least 121OC for a minimum of 60 minutes
Chemical decontaminants
Chemical decontaminants are toxic chemicals that can kill certaintypes of microorganisms
Radiation
This method is not used in academic laboratories
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Sterilization by Heat
Moist Heat Dry Heat
Temperature Time Temperature Time
100 oC 20 hours 120 oC 8 hours
110 oC 2.5 hours 140 oC 2.5 hours
115 oC 50 minutes 160 oC 1 hour
121 oC 15 minutes 170 oC 40 minutes
125 oC 6.5 minutes 180 oC 20 minutes
The following table demonstrates the heat equivalency betweenMoist and Dry Heat
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Autoclave
When autoclaving, the materials beingsterilized must come into contact with steamand heat for actual sterilization to result. It isfor this reason that the use of some form of
efficacy indicator must be done with each cycle
Sterilization Indicators: Routine Testing
Autoclave Tape
Autoclave Charts
Chemical
Biological
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This protocol describes the autoclavetreatment conditions and requirements for
laboratory solid waste contaminated with RiskGroup 1 and 2 Biohazardous Materials(Category B Infectious Substances), inLaboratories in UBC Point Grey Campus.
This treatment will allow the safemanagement of the resulting waste as non-hazardous solid waste in accordance with thewaste delisting approved by the Ministry ofEnvironment.
The new procedure can be found herealongwith the training videos.
New Disposal Procedure: Solid Waste
Contaminated with Microbiological Risk Group 1and Risk Group 2 Agents
http://riskmanagement.ubc.ca/environment/hazardous-waste-management/new-disposal-procedures-risk-groups-1-2http://riskmanagement.ubc.ca/environment/hazardous-waste-management/new-disposal-procedures-risk-groups-1-2 -
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Resistance to Chemical Decontaminants
Bacterial Spores
Bacillus subtilis, Clostridium sporogenes
MycobacteriumMycobacterium tuberculosis
Nonlipid or Small VirusesPoliovirus, Coxsackievirus, Rhinovirus
FungiTrichophyton sp., Cryptococcus sp., Candida sp.
Microorganisms can be more or less resistant to decontaminationand the general order is (most to least resistant):
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Resistance to Chemical Decontaminants
FungiTrichophyton sp., Cryptococcus sp., Candida sp.
Vegetative BacteriaPseudomonas aeruginosa, Staphylococcus aureus, Salmonella
choleraesuis
Lipid or Medium-Size VirusesHerpes simplex virus, Cytomegalovirus, Respiratory syncytial virus,
Hepatitis B virus, Human Immunodeficiency virus
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Microorganisms are affected by chemical decontaminantsthrough:
Cell lysis
Protein coagulation or denaturation Enzyme denaturation or inactivation
Destruction of enzyme substrates
Chemical Decontaminants
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Decontaminant Effectiveness
The effectiveness of a decontaminant is
influenced by a number of factors:
Organic Load Surface Topography
Method of Application
Concentration
Contact Time
Temperature Relative Humidity
pH and Stability
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The ideal decontaminant would be a broad spectrum agent able toact effectively against all biohazards. It would be fast acting, noteasily inactivated, non-toxic to the user, non-corrosive,economical, easy to use, easy to dispose of and have a long "uselife". Unfortunately, there is no one chemical agent that canfulfill all of these requirements
There are several categories of liquid decontaminants, each of
which has different characteristics and different ranges ofusefulness. Some agents are not compatible, so always follow themanufacturers instructions regarding mixing agents, dilution,storage and use of the decontaminant
Types of Chemical Decontaminants
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Liquid Decontaminants
Alcohols
Ethyl or isopropyl alcohol in a concentration of 70 - 85%by volume is often used
Pros:easy to use and non corrosive Cons:extremely flammable, evaporates easily, inactivated
by organic matter, and no cleansing properties
Effective:lipid-containing viruses, enveloped viruses, andvegetative bacteria. Longer contact times are needed for
fungi and some mycobacteria Ineffective:non-enveloped viruses and bacterial spores
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Chlorine Compounds
This halogen is a universal decontaminant active againstall microorganisms including bacterial spores. Chlorinecombines with protein and rapidly decreases inconcentration in its presence. It is a strong oxidizingagent
Pros:inexpensive, readily available, and broad spectrum
Cons:corrosive, short use life, and inactivated by
organic material Effective:everything
Ineffective:14C (produces radioactive CO2) should notbe used with any radioisotope where the reaction wouldcreate a volatile compound
Examples:Bleach, Javex, Presept, Alcide
Liquid Decontaminants
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Formaldehydes
Formaldehyde for use as a decontaminant is usuallymarketed at about 37% concentration of the gas in watersolution (referred to as formalin), or as a polymerizedcompound called paraformaldehyde. Formaldehyde, in aconcentration of 5% active ingredient (18.5 g/lformaldehyde), is an effective liquid decontaminant
Pros:broad spectrum and less susceptible to inactivationby organic material
Cons:long contact times (30+ minutes), loss of activityat cooler temperatures, pungent odour, expensive, andshort use life
Effective:everything
Examples:Cidex 7, Sporocidin, and Sonacide
Liquid Decontaminants
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Gas Decontaminants
Formaldehydes
The chemical of choice for space decontamination
Used at a concentration of 10,000 ppm (TLV 0.3 ppm with
a ceiling of 0.5 ppm) It has limited penetrating abilities
It is a suspected carcinogen
It is a sensitizer
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Radiation Decontaminants
Ionizing Radiation Ionizing radiation will destroy all microorganisms Gamma radiation - either Co-60 or Cs-137
None of these methods are presently used at UBC. The risk fromthe radiation outweighs the decontamination benefits
Nonionizing Radiation Ultraviolet radiation the short wavelengths of light beyond the
violet end of the visible spectrum
Produces thymine dimers in cells which leads to the death of themicroorganism
A narrow range of the UV spectrum has antimicrobial activity butis limited by poor penetrating power
May not lead to total decontamination of the area UV lights need to be changed every 3-6 months to be effective
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Most chemicals are whitepowders, so if you dont cleanup after yourself no one willknow what the white powder
left behind is
Clean-up
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Clean-up
All spaces where biological work isperformed need to bedecontaminated at least once daily
Not only is this space messy, thereis also a very large possibility ofcontamination and makesreproducible data difficult to obtain
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Decontamination and Sterilization are notequivalent.
There are several methods of decontamination, each with
advantages and dis-advantages: Heat/autoclaving,
Chemical decontaminants
Radiation
It is the responsibility of the supervisor to ensure that workersare appropriately trained on how to clean up a biological spillfor their specific projects.
The next module will discuss Lab Emergency Procedures
Summary
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