LIVE VIRUS WORKER WEB TRAINING

Decontamination Procedures

Decontamination is a process or treatment that renders an instrument or environmental surface safe to handle. A decontamination procedure can be as simple as clean-up with detergent and water or as thorough as sterilization. Sterilization and disinfection are two ways to address microbial contamination.
 

Sterilization is the use of physical or chemical processes to destroy all microbial life, including highly resistant forms, such as bacterial spores.

Disinfection is the elimination of essentially all pathogenic non-spore forming microorganisms but not necessarily all microbial forms from work surfaces and equipment. Effectiveness is influenced by a number of factors, including: types and numbers organisms; amount of organic matter; the object being disinfected; the disinfectant being used; exposure time, temperature and concentration.

When to Decontaminate

All material and equipment contaminated with or containing potentially infectious agents should be decontaminated:

• upon completion of procedures involving the use of biologically-active materials
• in the event of spills of such materials
• at least daily
• before being washed, stored, or discarded

In most Princeton University laboratories, decontamination is accomplished by steam heat sterilization in an autoclave, or by surface application of or placement in a chemical disinfectant solution, such as 1:10 bleach solution or its equivalent.

Sterilization by Steam Autoclave

Sterilization by steam autoclave (saturated steam under pressure of approximately 15 psi to achieve a chamber temperature of at least 250^oF for a designated time) is the preferred and most convenient method to rapidly destroy all forms of microbial life. Steam autoclaves are used to sterilize glassware, instruments, gloves, liquids in bottles, biological waste, dressings, and other materials needing such treatment.  However, to do this, material to be sterilized must come into contact with live steam. Bags or containers should be left open during autoclaving or water (~200ml) should be added to sealed bags to generate steam.  Heat indicator tape should be used with each autoclave load to indicate that sterilization has been completed. Autoclave sterility monitoring should be conducted on a regular basis using biological indicators (such as B. stearothermophilus spore strips) placed among treated materials and at locations throughout the autoclave. The spores, which are more resistant to heat than most microbials, provide validation of general microbial destruction when they are effectively inactivated (250oF for 13 minutes) by autoclave operation. (See section that follows regarding safe use of the autoclave.)
 

Safety Considerations in Use of the Steam Autoclave
Autoclaves sterilize by steam under pressure, typically at a pressure of a little under 2 atm at temperatures of up to 275^oF.  Since they are heated pressure vessels, they should be checked periodically to ensure that the seals to the closures are in good condition. They should also be equipped with safety devices to prevent excessive temperatures and pressures.  Prior to using a steam autoclave, you should be thoroughly trained in safe techniques and acceptable practices.

Fortunately, most autoclaves are designed so that they cannot be opened while the chamber is under pressure.  However, the materials inside will still be very warm, and removing them too hastily or forgetting to wear insulating gloves could very likely cause burns to your hands and the item being handled to be dropped.

Liquids placed in sealed bottles may explode, and liquids in ordinary glass bottles instead of Pyrex containers designed for temperatures and pressures may rupture.  If the unit is set to exhaust rapidly, as might be done for instrument sterilization, boiling may take place in bottles of liquids, with a subsequent loss of liquid into the autoclave.  Flammable liquids or chemicals which could become unstable at the temperatures reached in the autoclave should not be run through the sterilizing cycle.

The following guidelines should always be followed when using an autoclave:

• Never attempt to open the door while the machine is operating.
• Always check the jacket pressure gauge to make sure that it is reading 0 PSI before opening the door.
• Do not overfill containers when autoclaving liquids.
• Use a catch basin to capture overflow when autoclaving any materials.
• Periodically check chamber trap and clean as necessary.
• When preparing materials for disposal, do not autoclave more than can easily be placed in an appropriate disposal container.

If the machine is not operating properly, notify the laboratory supervisor. Do not attempt to make repairs. This should be done only by a trained technician.

Chemical Disinfectant Use

The most practical use of chemical disinfectants is for surface decontamination and, when used in sufficient concentration, as a decontaminant for liquid wastes prior to final disposal down the drain.

Liquid Decontamination
 

• add liquid chlorine bleach to provide a final 1:10 dilution of bleach to culture solution
• let stand at least 20 minutes
• discard down the drain

Surface Decontamination

• wipe with 1:10 dilution of chlorine bleach to water, or
• wipe with iodophor disinfectant (per label concentration), or
• wipe with 70% or 95% alcohol

• Leave cabinet fan on for any spill inside the biosafety cabinet
• Cover spill with paper toweling
• Apply disinfectant proceeding from the outer edge of the spill to its center
• Leave disinfectant in place for 20 minutes
• Discard spill material in biohazardous waste

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Biosafety Cabinets

Biosafety cabinets (BSCs) are used as the primary means of containment for working safely with infectious microorganisms.  Biosafety cabinets are only one part of an overall biosafety program, which requires consistent use of good microbiological practices.

BSCs are designed to prevent biological exposure to personnel and the environment and may also protect experimental material from being contaminated when appropriate practices and procedures are followed. Three kinds of biosafety cabinets, designated as Class I, II and III have been developed to meet varying research and clinical needs.
 

Biosafety Cabinet Classifications

Type of Cabinet	Agent Classification	Average Inlet Velocity	Protection
Partial Containment
Class I	Low & Moderate Risk (BSL 2, 3)	75	User only
Class II, type A	Low & Moderate Risk (BSL 2, 3)	75 (work opening fixed)	User & material
Class II, type B	Low & Moderate Risk (BSL 2, 3)	100 (work opening at 8”)	User & material
Absolute Containment
Class III	High Risk (BSL 3, 4)		User & material

Biological safety cabinets use high efficiency particulate air (HEPA) filters in their exhaust and/or supply systems.  These filtered cabinets are primarily designed to protect against exposure to particulates, including biological agents used in the cabinet.  You should be aware that exposure protection from radionuclides and toxic chemicals is only provided with use of certain BSCs.  BSCs Class II, Type B are capable of providing such protection.

The Class I BSC

The Class I BSC provides personnel and environmental protection, but no product protection. It is similar in air movement to a chemical fume hood, but has a HEPA filter in the exhaust system to protect the environment (See Figure).
 
 

	A. Front Opening B. Sash C. Exhaust HEPA D. Exhaust plenum

The Class II BSC

The Class II BSCs provide personnel, environmental and experimental material protection. The Class II BSC is the type commonly used in biological laboratories at Princeton. Air flow is drawn from the room around the operator into the front grille of the cabinet, which provides personnel protection. In addition, the downward laminar flow of HEPA-filtered air provides protection for experimental material inside the cabinet. Because cabinet air has passed through the exhaust HEPA filter, it is contaminant-free (environmental protection), and may be recirculated back into the laboratory (Type A) or ducted out of the building (Type B).
 

A. Front Opening B. Sash C. Exhaust HEPA filter D. Supply HEPA filter E. Rear plenum F. Blower

The Class III BSC

The Class III biological safety cabinet is most suitable for work with biohazardous agents requiring BSL 3 or 4 containment.  Because this level of work is not conducted at Princeton University, no Class III cabinets are found in laboratories here.  The Class III cabinet (See Figure) is a totally enclosed, HEPA filter-ventilated cabinet fitted with glove ports and decontamination capablities for entry and exit of material.  It offers the highest degree of personnel and environmental protection from infectious aerosols.
 
 
 

A. Glove ports with O-ring for attaching arm-length gloves to cabinet B. Sash C. Exhaust HEPA D. Supply HEPA E. Double-ended autoclave or pass-through box

Procedure for Using Biosafety Cabinets

1. Plan the procedure carefully; determine all materials needed for work in the cabinet
2. Turn on lights
3. Check air intake and exhaust to ensure no blockage
4. Turn on fan
5. Use 70-95% ethanol or commercial disinfectants to decontaminate work surface
6. Keep storage in the cabinet to a minimum
7. Arrange necessary items in the cabinet to limit the need for excessive movement that disrupts cabinet air flow
8. Recognize that movement into and out of the cabinet creates small eddy currents at the face of the cabinet, which can disrupt the protective air barrier.

Other Considerations for Biosafety Cabinets
Remember that the use of a biosafety cabinet is not a substitute for good laboratory practice.  Aerosols can escape from the cabinet and contaminate hands, arms and front of user.

Biosafety cabinets must be certified by an outside firm annually if they are being used to protect the user.  If they are only being used for product protection, then the researcher must decide how often the cabinet gets certified.

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Laboratory Waste Streams

Each lab should have the following Biological Waste Streams Chart posted:

BIOLOGICAL LABORATORY WASTE STREAMS*

CATEGORY	DESCRIPTION	CONTAINER	LOCATION	HANDLING	ROUTING
Non-recyclable waste glass	Plate glass, pyrex, light bulbs, broken glass chemical containers, Non-contaminated** plastic serological pipettes	Tall cardboard container with plastic liner	Corridor and/or Lab	Removed by custodial staff when full	Solid waste     (not recyclable)
Empty chemical containers	Intact, clean triple-rinsed glass and plastic (#1 and #2) containers;                  recyclable without caps	Special plastic bucket with half lid	Corridor	Removed by custodial staff when full	Recyclable
Lab trash	Non-contaminated** gloves, bench paper, packaging materials, foil, plastic bags, paper towels, weighing boats, bottle caps, fly media, fly embryo plates, culture plates (with or without media); culture and centrifuge tubes (with or without media), filter flasks	Standard waste can with 4 mil liner	Lab	Removed by custodial staff when full	Solid waste
Regulated medical waste
All sharps	All Pasteur and other glass pipettes, needles, syringes, scalpel blades, razor blades, slides, coverslips	Labeled sharps container	Lab	Placed in medical waste boxes by lab occupants	Medical waste
Other medical waste	Experimentally cultured stocks, plates or other materials meeting New Jersey definition (See *** below) Ethidium bomide gels	Standard medical waste box with red plastic liner	Lab	Sealed and placed in corridor by lab occupants	Medical waste

* Does include not radioactive or hazardous chemical waste.

** Non-contaminated applies to any material not having prior contact with infectious agents.  The New Jersey Regulated Medical Waste regulations define infectious agents as a virus or a bacteria capable of being communicated by invasion and multiplication in body tissues and capable of causing adverse health impacts in humans.

*** Other Regulated Medical Waste.  Any solid waste generated in the diagnosis, treatment, or immunization of human beings or animals in research pertaining thereto, or in the production of biologicals in the following categories:  cultures and stocks of infectious agents and associated pathological wastes, human blood and blood products, sharps (used or unused), animal waste (contaminated animal carcasses and animal bedding exposed to agents infectious in humans).
 

All sharps are considered regulated medical waste.  This category includes all Pasteur and other glass pipettes, needles, syringes, scalpel blades, razor blades, slides, and coverslips.  You must place these sharps in a labeled sharps container located in the lab.  You then place these containers, when full, into the medical waste boxes (with red bag liners) and these boxes are disposed of as regulated medical waste.

Other medical waste includes experimentally cultured stocks, plates or other materials meeting the New Jersey definition¹.  Also in this category are Ethidium Bromide gels.  These materials should be placed directly into the medical waste box.  These boxes, when full, should be sealed with red biohazard tape and placed in the corridor for pickup.

These boxes, when full, be sealed, generator label applies to the box and the box placed in the corridor for pickup.

It is important that the exterior of the medical waste box and the outside of the sharps container being placed into the medical waste box be labeled to indentify the generating lab. Adhesive-backed lables have been provided tot he generating departments by Building Services and should be used for this purpose.

The next category of waste is non-recyclable waste glass.  This category includes plate glass, Pyrex©, light bulbs, broken glass chemical containers, and non-contaminated  plastic serological pipettes.  These materials should be placed in the tall cardboard containers with heavy plastic liners (picture) in the corridors or labs.  These cardboard containers are removed by the custodial staff when full.

Empty, intact chemical containers are recyclable and should be triple-rinsed and placed, without caps, in special rubbermaid collection cans with side of the lid cut open. These cans are located in the corridors and are emptied by the custodial staff when full.

Aside from radioactive or chemical waste, everything else in the lab should be considered regular lab trash.  Examples include non-contaminated² gloves, bench paper, packaging materials, foil, plastic bags, paper towels, weighing boats, bottle caps, fly media, fly embryo plates, culture plates (with or without media), culture and centrifuge tubes (with or without media), and filter flasks.
 

Lab trash should be placed in the standard waste can as long as it is lined with a standard 4 mil plastic liner.  These cans are emptied by the custodial staff when full.
 
 
 

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<sub>1.  Any solid waste generated in the diagnosis, treatment, or immunization of human beings or animals, in research pertaining thereto, or in the production or testing of biologicals, in the following categories:  cultures and stocks (of infectious agents and associated pathological wastes, human blood and blood products, sharps (used or unused), animal waste (contaminated animal carcasses and animal bedding exposed to agents infectious in humans).
2. Non-contaminated applies to any material not having been in contact with an infectious agent.  The New Jersey Regulated Medical Waste regulations define infectious agent as “any organism (such as a virus or bacteria) capable of being communicated by invasion and multiplication in body tissues and capable of causing disease or adverse health impacts in humans”.</sub>

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The following links are provided as additional Biosafety references and information.

NIH

CDC/NIH

Health Information Sheet

Guidelines for BSL1 and BSL2

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