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McGill Laboratory Biosafety Manual - Second edition, 1997
 


6 Sterilization and Disinfection in the Laboratory

It is important to distinguish between sterilization and disinfection. Whereas sterilization results in destruction of all forms of microbial life, disinfection results in destruction of specific pathogenic microorganisms. A more detailed description of disinfection levels can be found in the Glossary at the back of this manual (Section 13).

6.1 Microbial Resistance to Physical and Chemical Agents

Microorganisms vary in their resistance to destruction by physical or chemical means. A disinfectant that destroys bacteria may be ineffective against viruses or fungi. There are differences in susceptibility between gram-negative and gram-positive bacteria, and sometimes even between strains of the same species. Bacterial spores are more resistant than vegetative forms, and non-enveloped, non-lipid-containing viruses respond differently than do viruses which have a lipid coating. Information on the susceptibilty of a particular microorganism to disinfectants and physical inactivation procedures can be found in the material safety data sheet (MSDS) for that agent. MSDSs provide additional details such as health hazards associated with the microorganism, mode of transmission, containment requirements and spill response procedures. The Environmental Safety Office has available, and can provide to individuals, MSDSs on a number of infectious microorganisms.

6.2 Physical Sterilants and Disinfectants

6.2.1 Heat Sterilization and Decontamination

Generally, sterilization is best achieved by physical methods such as steam or dry heat, which are less time-consuming and more reliable than chemical germicides. A summary of physical agents which employ heat for control of microorganisms can be found in Table 2. Of these physical procedures, steam autoclaving is the most practical option for the majority of laboratories for both sterilization and decontamination purposes.

Details on the use of an autoclave are given in Section 8.8.
 
TABLE 2 - Outline of the properties of heat decontamination methods. For everyday laboratory purposes, autoclaving is the preferred method, unless the item cannot withstand the heat and/or moisture of autoclaving.
Principle/Conditions Advantages Disadvantages Uses
Dry Heat Thermal inactivation: destroys by oxidation Non-corrosive
Simple design and principle
Less effective than moist heat; requires longer times and/or higher temperatures Materials that are damaged by, or are impenetrable to, moist heat
Hot Air Oven
  • 160-180C for 2-4 hours
  • penetrates water-insoluble materials (e.g., grease and oil)
  • less corrosive to metals and sharp instruments than steam
  • slow diffusion, penetration
  • loading, packing critical to performance
  • not suitable for reusable plastics
  • anhydrous materials, such as oils, greases and powders
  • laboratory glassware, instruments
  • closed containers
  • Red-heat Flame
  • oxidation to ashes (burning)
  • rapid
  • initial contact with flame can produce a viable aerosol
  • possibility of accidental fire
  • inoculating loops, needles
  • Incineration
  • oxidation to ashes (burning)
  • 1-60 minutes: temperatures may exceed 1000C
  • reduces volume of waste by up to 95%
  • improper use may lead to emission of pathogens in smoke
  • requires transport of infectious waste
  • excess plastic (>20%) content reduces combustibility
  • for decontamination of waste items prior to disposal in landfill
  • Moist Heat Irreversible coagulation of (microbial) proteins More rapid and more effective than dry heat
    Pasteurization
  • heating to below boiling point (generally 77C) for up to 30 minutes
  • can be used on heat sensitive liquids and medical devices
  • low cost
  • not reliably sporicidal
  • milk and dairy products
  • some heat-sensitive medical equipment
  • Tyndallization (Fractional Sterilization)
  • heating to 80-100C for 30 mins on successive days, with incubation periods in between
  • resistant spores germinate and are killed on the second and third days
  • time consuming
  • not reliably sporicidal
  • heat sensitive materials such as bacteriologic media, solutions of chemicals, biological materials
  • Boiling
  • maximum temperature obtainable is approximately 100C 10-30 mins
  • minimal equipment required
  • cumbersome: not practical for everyday lab use
  • not reliably sporicidal
  • small instruments and equipment
  • autoclaving
  • steam under pressure
  • 121C/15 psi for 15-90 mins (gravity displacement autoclave)
  • 132C/27 psi for 4-20 minutes (pre-vacuum autoclave)
  • minimal time required
  • most dependable sterilant for lab use
  • loading and packing critical to performance
  • shielding dirt must first be removed
  • maintenance and quality control essential
  • damages heat-sensitive itmes
  • penetration of sterile glassware, media and instruments
  • decontamination of reusable supplies and equipment
  • decontamination of infectious waste

  • 6.2.2 Other Physical Agents of Sterilization and Disinfection

    6.2.2.1 Ultraviolet Light (Germicidal Lamps)

    The light (approximately 260 nm wavelength) emitted by UV lamps is germicidal, and can be used to reduce the number of pathogenic microorganisms on exposed surfaces and in air. However, UV light has poor penetrating power; accumulations of dust, dirt, grease or clumps of microorganisms may shield microorganisms from the direct exposure required for destruction. UV light presents skin and eye burn hazard, and factors such as lamp age and poor maintenance can reduce performance. For safe and reliable use of germicidal lamps:

    6.2.2.2 Miscellaneous Physical Methods

    The procedures listed below are included for the reader's interest:

    6.3 Chemical Sterilants and Disinfectants

    Instruments or materials which cannot withstand sterilization in a steam autoclave or dry-air oven can be sterilized with a gas such as ethylene oxide or a broad spectrum liquid chemical germicide. Chemical decontamination of surfaces may also be necessary for very large or fixed items. Since liquid chemical germicides generally require high concentrations and several hours of exposure time for sterilization purposes, they are usually used for disinfection rather than for sterilization purposes. The majority of chemical disinfectants have toxic properties: follow the manufacurer's directions for use and wear the appropriate personal protective equipment (e.g., gloves, eye protection, apron), especially when handling stock solutions.

    Choice of a chemical germicide for use on contaminated equipment, supplies, laboratory surfaces or biohazardous waste depends upon a number of factors, including:

    Direct contact between germicide and microorganism is essential for disinfection. Microorganisms can be shielded within air bubbles or under dirt, grease, oil, rust or clumps of microorganisms. Agar or proteinaceous nutrients and other cellular material can, either directly (through inactivation of the germicide) or indirectly (via physical shielding of microorganisms) reduce the efficacity of some liquid germicides.

    No one chemical germicide is effective for all disinfection or sterilization purposes. A summary of chemical germicides, their use, effective concentrations, advantages and disadvantages can be found in Tables 3, 4A and 4B.
     
    TABLE 3 - Summary of concentrations used, contact times, advantages and disadvantages and uses of some of the halogen-releasing chemical germicides. The wide ranges of effective concentrations and contact times cited are due to a number of factors, including the interdependence of time and concentration, the variability in resistance of different microorganisms, the amount of organic material present and the desired effect (e.g., low-level vs high-level disinfection)
    Effective Concentrations, Contact Times Advantages Disadvantages Examples of Uses
    Chlorine Compounds: Sodium hypochlorite solution 1 (liquid bleach)
  • 100-10,000 ppm (.01-1%) free chlorine
  • 10-60 minutes (>= 3,000 ppm for broad spectrum)
  • broad spectrum
  • inexpensive
  • widely available
  • bactericidal at low temperature
  • toxic, corrosive to skin and metals
  • unstable at optimum effective pH of 6
  • inactivated by organic matter
  • deteriorates under light and heat: shelf life of dilutions is less than 1 week
  • general disinfectant
  • waste liquids
  • surface decontamination
  • emergency spill clean up
  • instrument disinfection
  • Calcium hypochlorite2 granules, powder, tablets
  • as for liquid bleach
  • as for liquid bleach but more stable
  • as for liquid bleach above, except shelf life is longer
  • as for liquid bleach
  • NaDCC3 (Sodium dichloroisocyanurate) powder, granules, tablets
  • as for liquid bleach
  • more stable than hypochlorites
  • stable at pH 6.0
  • toxic, corrosive
  • inactivated by organic matter
  • as for liquid bleach
  • Chloramine-T4 (Sodium tosylchloramide) powder or tablets
  • as for liquid bleach
  • more stable, less affected by organic matter than hypochlorites
  • longer activity than hypochlorites
  • deteriorates under humidity, light and heat
  • as for liquid bleach
  • Chlorine dioxide5
  • demand-release of chlorine dioxide in situ
  • longer activity than other chlorine compounds
  • less corrosive, less toxic than other chlorine compounds
  • effective at pH 6-10
  • aqueous solutions decompose under light
  • instrument disinfection
  • gas sterilization of germ-free animal chambers
  • Iodine Preparations: Iodophors6
  • 30-1,000 ppm (.003-.1%) free iodine
  • 10-30 minutes
  • broad spectrum
  • germicidal over a wide pH range
  • generally nonstaining, less toxic and less irritating than aqueous or alcoholic iodine solutions
  • not consistently sporicidal
  • efficacy reduced by organic matter
  • some iodophor solutions support growth of Pseudomonas7
  • germicidal soaps and antiseptics
  • surface decontamination
  • work surface wipedown
  • instrument disinfection
  • Notes: