An activity that decreases the microbial load to a level deemed proper to avoid contamination or infection is decontamination. The suitability of a decontamination procedure depends on occasions. For example, surgical instruments must be sterile. However, this level of microbial disinfection is pointless for environmental areas, such as walls and floors.

The application of a chemical to living tissue to prevent infection refers as antisepsis. Example substances are iodine compounds and hand washing antimicrobial soaps.

Sterilization

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

Autoclaves

The most efficient and reliable method of sterilization in laboratory is offered by autoclaves. Critical process factors are exposure, temperature, time. Also ensuring that materials are packaged to allow the steam to penetrate throughout the load is another crucial parameter. Size of the load and the packing density of the chamber affects the sterilization time. Usual laboratory autoclaves function at 121 °C and 15 psi. All users must review the operating manual periodically. Instructions should be prominently posted. When removing processed material, heat resistant gloves and face protection must be used. Slowly crack the door and wait a few minutes before fully opening it.

For dry loads, addition of 250-500 mL of water to the load pan in order to aid steam generation is necessary. Do not tightly cap bottles and test tubes. Autoclave bags must be closed loosely to allow steam to penetrate.

Autoclave tape is not a fail-safe indicator of sterilization; it blackens after only brief exposure to a temperature of 121 °C.

Some autoclave tapes contain lead which makes it necessary to dispose of these tapes as Hazardous Waste. For properly eliminating this hazardous waste stream, laboratories must use lead-free autoclave tape.

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).

Chemical Sterilization

Chemical sterilization is mainly used for heat-sensitive patient-care instruments which enter body cavities or normally sterile areas. This process necessitates prolonged contact times with relatively high concentrated solutions. As a result, prior to dilution, these toxic products must be treated as hazardous chemicals. Cautiously follow manufacturers’ directions regarding dilution, contact time and personal protective equipment. Some sterilants require specific ventilation systems in order to remove hazardous gases and vapors.

Disinfection

Elimination of virtually all pathogenic microorganisms on inanimate objects with the exception of large numbers of bacterial endospores is called disinfection.

Disinfection should be established if hazardous organisms are destroyed. Microorganisms can be grouped according to 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).

Table 7.2. offers a framework for the selection of the proper disinfectant.

When using any disinfectant:

  • Label instructions must be followed for dilution and time needed for desired level of disinfection must be contacted.
  • Disinfectants that require pre-use dilution must be treated as hazardous chemicals while mixing.
  • Wear a proper lab coat, the correct type of chemical-resistant glove, and fit goggles (not glasses).
  • Clean contaminated surfaces that may have become contaminated at the end of the task.
  • Choose the disinfectant with the lowest possible toxicity.

Considerations for selecting and using disinfectants:

  • Rough surfaces require a longer contact time than smooth ones.
  • Surface compatibility-bleach will corrode many metals, after use rinse it with water. Based on their composition, instruments vary in their ability to withstand disinfectants.
  • Organic compounds will inactivate some disinfectants; a second treatment may be necessary once visible contamination (and hence, most organic debris) has been removed. The removal of visible ‘soil’ is the 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 labelled "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. Table 7.2 summarizes the disinfectant activities:

Table 7.2 Summary of disinfectant activities

Disinfectant

Disinfection Level

1*

2*

3*

4*

5*

Comments

Alcohols

(ethyl and isopropyl)
60-85%

intermediate

+

+

-

+/-

+

Not sporicidal; evaporates quickly so that adequate contact time may not be achieved, high concentrations of organic matter diminish effectiveness; flammable.

Phenolics

(0.4%-5%)

intermediate

+

+

+/-

+

+

Not sporicidal; phenol penetrates latex gloves; eye/skin irritant; remains active upon contact with organic soil; may leave residue.

Glutaraldehyde
(2-5%)

High

+

+

+

+

+

Used to sterilize surgical instruments that cannot be autoclaved; strong odor; sensitizer; use with adequate ventilation. Not for use on environmental surfaces.

Quaternary Ammonium
(0.5-1.5%)

Low

+

+

-

-

+/-

May be ineffective against Pseudomonas and other gram – bacteria; recommendation limited to environmental sanitation (floors, walls). Low odor, irritation.

Iodophors
(30-1,000 ppm iodine)

intermediate

+

+

+

+/-

+/-

Inactivated by organic matter.

Chlorine

(100-1,000 ppm)

intermediate

+

+

+

+/-

+

Not sporicidal; inactivated by organic matter; fresh solutions of hypochlorite should be prepared daily; corrosive; irritating to eyes and skin.


* 1. Bacteria; 2. Lipophilic Viruses; 3. Hydrophilic Viruses; 4. Mycobacterium Tuberculosis; 5. Fungi

Adopted from Columbia University, Medical Center.

Using Bleach as a Disinfectant

As a strong oxidizing agent, the sodium hypochlorite in household bleach is an effective disinfectant for the known and potential infectious materials. However, as sodium hypochlorite breaks down into salt and water, it is recommended that the solution is made fresh daily. 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.

Bleach stock must be stored in an opaque plastic bottle at room temperature. Initial hypochlorite concentration is affected by the rate of degradation, the volume remaining and the ambient temperature. 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 offers an useful monitoring means.

All bleach brands are not manufactured to the same potency. Depending on manufacturer, the potency of commercial bleach is between 3.25 and 6.15% hypochlorite.

Proper gloves must be worn while handling bleach, because it can be corrosive on some surfaces, including steel. Bleach residue on non-porous surfaces must be wiped off with 70% ethanol or water. As ammonia and bleach can react to produce a highly toxic product, bleach should not be used in conjunction with other household cleaning products that contain ammonia. Pre-filled spray bottles that contain a 1:10 mixture are appropriate for using in the lab. Aspiration of tissue culture media into a collection flask, under vacuum, is one of the most commonly performed laboratory procedures. SU Policy requires that such media may be decontaminated prior to disposal in the municipal sewer system (Waste Management). Following these instructions guaranties effective decontamination.

Bleach must be added before aspiration, and undiluted bleach must be added to fill 10% of the final volume of the collection flask. Bleach is an active decontaminant. Also, its strong oxidizing properties will turn the phenol red indicator in tissue culture media from pink to yellow/clear. Aspiration flasks containing pink liquid is a sign of insufficient bleach concentration. So they should be topped off with fresh bleach until a yellow/clear colour is obtained. The collection flasks must be emptied when they are 3/4 full.

References and sources for information from the relevant websites and documentation of different universities, NGOs and government agencies used in the preparation of this website are provided at references.

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