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Health & Safety Manual - Biological Safety

2.7 Decontamination

2.7.1 Sterilization
2.7.1.1 Autoclaves
2.7.1.2 Dry Heat
2.7.1.3 Chemical Sterilization
2.7.2 Disinfection
2.7.3 Using bleach as a disinfectant


Decontamination refers to any activity that reduces the microbial load to a level deemed suitable to prevent contamination or infection.  The appropriateness of a decontamination procedure is situation-dependent.  For example, surgical instruments must be sterile but this level of microbial killing is unnecessary for environmental surfaces such as floors and walls.

Antisepsis refers to the application of a chemical to living tissue to prevent infection.  Examples include iodine compounds and antimicrobial soaps for hand washing.

2.7.1 Sterilization
Sterilization refers to the destruction of all microbial life, including bacterial endospores.

2.7.1.1 Autoclaves - Autoclaves provide the most efficient and reliable method of sterilization for most laboratory applications.  The critical process factors are temperature, exposure time, and ensuring that materials are packaged to allow the steam to penetrate throughout the load.  Sterilization time will vary in relation to the size of the load and the packing density of the chamber.  Typical laboratory autoclaves operate at 121°C and 15 psi.  All users must review the operating manual periodically.  Instructions should be prominently posted.

Use heat resistant gloves and face protection, particularly when removing processed material; crack the door slowly and wait a few minutes before fully opening it.

For dry loads, add 250-500 ml. of water to the load pan to aid in steam generation.  Autoclave bags should be closed loosely to allow steam to penetrate; do not tightly cap bottles and test tubes.

Autoclave tape is not a fail-safe indicator of sterilization; it blackens after only brief exposure to a temperature of 121°C.  When used for sterilizing infectious waste, autoclave performance must be periodically validated by using B. stearothermophilus spore vials.  Place a vial in a hard-to-reach area of a mock challenge load and attach a string to facilitate removal after autoclaving.  Incubate as directed; a lack of turbidity indicates that the autoclave is achieving sterilizing conditions.  

Some autoclave tapes contain lead which makes it necessary to dispose of these tapes as Hazardous Waste.  Laboratories must use lead-free autoclave tape to eliminate this hazardous waste stream Information concerning lead-free autoclave tape can be found at:
http://www.ehs.columbia.edu/GetTheLeadOut2.pdf

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2.7.1.2 Dry Heat - Dry heat is used for materials (some glassware, instruments, and anhydrous materials) that are sensitive to moisture or the corrosion it may cause.  Consult the manufacturers of such items for recommendations for appropriate sterilization procedures.  Dry heat requires higher temperatures and a longer exposure times than autoclaving. Dry heat for 2-4 hours at 160°C is needed to sterilize a load requiring 30 minutes at 121°C in an autoclave.  This method may also be validated by using spore vials; see autoclave section (above).

2.7.1.3 Chemical Sterilization - Chemical sterilization is chiefly used for heat-sensitive patient-care instruments that enter body cavities or normally sterile areas.  This process requires prolonged contact times with relatively highly concentrated solutions.  As a result, these products, especially prior to dilution, may be toxic and should be treated as hazardous chemicals.  Carefully follow manufacturers’ directions regarding dilution, contact time, personal protective equipment.  Some sterilants require that specific ventilation systems be in place to remove hazardous gases and vapors. 

Manufacturers of “Cidex” (active ingredient: glutaraldehyde) have developed “Cidex-OPA”.  Its active ingredient, ortho-phthalaldehyde, is less toxic and irritating than glutaraldehyde and is the preferred product at the University for aldehyde-based sterilization.

2.7.2 Disinfection
Disinfection refers to the elimination of virtually all pathogenic microorganisms on inanimate objects with the exception of large numbers of bacterial endospores.

Disinfection encompasses a continuum of outcomes in terms of the types of organisms destroyed.  Microorganisms can be grouped in terms of decreasing resistance to disinfectants as follows: bacterial endospores (B. subtilis, clostridium spp); Mycobacteria; nonlipid or small viruses (poliovirus, rhinovirus); fungi; vegetative bacteria; and lipid or medium sized virus (herpes simplex, HIV, HBV).

The table at the end of this section provides a framework for the selection of the appropriate disinfectant. The label on a commercial product will note its type of ‘cidal’ action (‘tuberculocidal’, ‘sterilant’). These claims may not appear unless the manufacturer has submitted data to the EPA supporting such claims. The lists of EPA registered disinfectants can be obtained from your campus EH&S office or found at http://www.epa.gov/oppad001/chemregindex.htm

The EPA does not independently audit such results and research indicates that in real life situations some products do not perform as claimed. This result from manufacturers testing their products in best-case situations, e.g., on a smooth surface, at an optimal pH, in a buffer solution instead of a solution containing organic material which partially inactivates some disinfectants. For high risk pathogens, investigators may devise their own test to confirm a product’s claim or consult EH&S.

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When using any disinfectant:

  • Follow label instructions for dilution and contact time needed for desired level of disinfection.
  • Disinfectants that require pre-use dilution should be treated as hazardous chemicals during mixing. Wear a lab coat, the correct type of chemical-resistant glove, and goggles, not glasses.
  • Clean contaminated surfaces as soon as possible and any surface that may have become contaminated at the end of the task..
  • Select the disinfectant with the lowest toxicity possible

Considerations for selecting and using disinfectants:

  • Nature of surface-rough surfaces require a longer contact time than smooth ones.
  • Surface compatibility-bleach will corrode many metals, rinse with water after use; instruments vary in their ability to withstand disinfectants based on their composition.
  • Organic matter will inactivate some disinfectants; a second application may be necessary once visible contamination (and hence, most organic debris) has been removed. The removal of visible ‘soil’ may be the single most critical factor in assuring effective decontamination.
  • Resistance of microorganisms, e.g. bacterial endospore vs. vegetative bacteria.
  • Number of microorganisms present, overnight culture vs. a recently inoculated one.

The Bloodborne Pathogens Standard requires that products labeled "tuberculocidal hospital disinfectant" be used on surfaces and equipment when the Standard is in force. Household bleach, usually at a 1/10 dilution, also satisfies this requirement and may be used in these cases. Bleach solutions lose potency over time and should be prepared fresh daily.

Summary of Disinfectant Activities

2.7.3 Using bleach as a disinfectant

The sodium hypochlorite in household bleach is a strong oxidizing agent that is an effective disinfectant for the known, and potential, infectious materials at Columbia University. However, over time the sodium hypochlorite breaks down to salt and water.
When bleach and water are mixed together, 1:10, to create a cleaning or disinfecting solution, the solution rapidly begins to lose needed disinfecting properties. Therefore, it is recommended that the solution is made fresh daily.

Stock bleach should be stored in an opaque plastic bottle at room temperature. The rate of degradation depends on the initial hypochlorite concentration, the ambient temperature and the volume remaining. Manufacturers are not required to put an expiration date on the bottle. A good practice is to mark the bottle with the receive date, and replace bleach that was received more than 6 months prior. Colorimetric test strips for hypochlorite concentration provide an easy and useful monitoring tool.

Not all bleach is created equal; the potency of commercial bleach is between 3.25 and 6.15% hypochlorite, depending on manufacturer. Ultra regular CLOROX liquid bleach contains a higher concentration. As an additional measure of surety, it is possible to determine the production date of this product from the last four digits of the serial number on the bottle. The first of these digits identifies the year of production (3=2013, 4=2014) and the other three indicate the day of the year of production (002= Jan 2nd, 364=Dec 30th). For example, E614293 = production date 10/20/2014 (October 20th is the 293rd day of 2014). Note that this scheme is only applicable to Clorox bleach.

Clorox

Gloves should be worn while handling bleach. Bleach can be corrosive on some surfaces, including steel. Bleach residue on non-porous surfaces should be wiped off with water or 70% ethanol. Bleach should not be used in conjunction with other household cleaning products that contain ammonia; the two can react to produce a highly toxic product. Pre-filled spray bottles that mix at the nozzle are a convenient way of generating a 1:10 mixture for use in the lab.

Aspiration of tissue culture media into a collection flask, under vacuum, is one of the most commonly performed laboratory procedures. University Policy requires that such media be decontaminated prior to disposal in the municipal sewer system.

Effective decontamination is simple, following these instructions. Before aspiration, add undiluted bleach to fill 10% of the final volume of the collection flask. Bleach is an effective decontaminant with the added advantage that its strong oxidizing properties will turn the phenol red indicator in tissue culture media from pink to yellow/clear. Aspiration flasks containing pink liquid indicate insufficient bleach concentration, and should be topped off with fresh bleach until a yellow/clear color is achieved prior to additional aspiration or disposal. Empty the collection flasks when they are 3/4 full, or at least weekly.

An adhesive magnet (shown below) is available to post on biosafety cabinets that summarizes effective aspiration flask and bleach management. Please email biosafety@columbia.edu for a complimentary copy.

BSC magnet

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