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.

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	160-180°C 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 1000°C	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 77°C) 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 80-100°C 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 100°C 10-30 mins	minimal equipment required	cumbersome: not practical for everyday lab use not reliably sporicidal	small instruments and equipment
autoclaving	steam under pressure 121°C/15 psi for 15-90 mins (gravity displacement autoclave) 132°C/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:

Clean the bulb at least every 2 weeks; turn off power and wipe with an alcohol-moistened cloth.
Blue light output is not an indication of the lamp's effectiveness; measure radiation output at least twice yearly with a UV meter or replace the bulb when emission declines to 70% of its rated output.
Post warning signs to discourage personnel from entering areas where there is potential exposure to UV light.
Wear UV protective goggles, caps, gowns and gloves in rooms with UV installations.

6.2.2.2 Miscellaneous Physical Methods

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

Infrared radiation: used for heat treatment of small metal and glass items.
Microwaves: used for treatment of liquids, nonmetallic objects, and biohazardous waste.
Gamma irradiation: disrupts DNA and RNA in living organisms, and is used by hospital and laboratory suppliers for materials that do not tolerate heat and pressure (i.e., autoclaving) or chemical treatments.
Membrane filtration: physically removes particulates (e.g., microorganisms) from heat-sensitive pharmaceutical and biological fluids. The size of the particles removed is determined by the pore size of the filter membrane.

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:

number and nature of microbes to be destroyed (e.g., spores vs vegetative cells, bacteria vs viruses),
type and configuration of item to be disinfected (fissures, crevices and enclosures may shield organisms),
purpose of treatment (e.g., disinfection vs sterilization),
interaction with other active chemicals,
whether the item is covered with soil which might inactivate the disinfectant,
contact time required for disinfection,
toxicity to individuals, culture systems, environment, residual toxicity on items,
pH, temperature, hardness of available dilution water,
cost.

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

**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:

² "high tested" provides 70-72% available chlorine; chlorinated lime or bleaching powder provides approximately 35% available chlorine

³ appproximately 60% available chlorine

⁴ approximately 25% available chlorine

⁵ To avoid shipping of this extremely reactive product, reagents ("base" and "activator") from commercially available kits are mixed with water to generate chlorine dioxide immediately prior to use

⁶ 10% povidone-iodine provides 1% available iodine

⁷ An iodophor stock solution may actually be a less effective germicide than its dilution. For example, a full-strength (10%) solution of povidone-iodine provides approximately 10 times less free available iodine than a 1/100 dilution. Iodophors must be used at the manufacturer's recommended concentrations.

**TABLE 4A** Summary of recommended concentrations, contact times, advantages and disadvantages of non-halogen chemical germicides. The wide ranges of effective concentrations and contact times cited reflect the interdependence of time and concentration as well as factors such as resistance of the particular class or strain of target microorganism(s) and desired effect. Also, some germicides are available in combinations (e.g., glutaraldehyde/phenol or peracetic acid/alcohol mixtures) which are synergistic whereby the components in combination produce a greater antimicrobial effect than the sum of their individual effects.
	Effective Concentrations and Contact Times	Advantages	Disadvantages	Examples of Laboratory Uses
Alcohols	70-80% ethanol 60-95% isopropanol 10-30 minutes	low toxicity rapid action low residue non-corrosive	rapid evaporation limits contact time flammable, eye irratant may damage rubber, plastic, shellac ineffective against bacterial spores	skin disinfectant (antiseptic) surface decontamination benchtop, cabinet wipedown
Phenolic Compounds	400-50,000 ppm (.05-1.5%) 10-30 minutes	tolerant of organic load, "hard" dilution water leaves an active residue (may be desirable on some surfaces) biodegradable	pungent odour, corrosive, some forms toxic not sporicidal; limited activity against viruses leaves a residual film (undesirable in culture systems) may support growth of bacteria¹	instruments and equipment disinfection disinfection of floors and other surfaces antiseptic soaps and lotions
Quaternary Ammonium Compounds	500-15,000 ppm (.05-1.5%) 10-30 minutes	combined detergent and germicidal activity stable working dilutions have low toxicity	non sporicidal, limited activity against viruses, mycobacteria most formulations not readily biodegradable may support growth of bacteria²	surface decontamination equipment wipedown antiseptic formulations available floors and walls
Hydrogen Peroxide	3-30% aqueous solution 10-60 minutes 6% for 30 minutes may kill spores	rapid action no residue low toxicity environmentally safe	limited sporicidal activity corrosive to some metals potentially explosive at high concentrations stock solutions irritating to skin and eyes	surface decontamination instruments and equipment
Peracetic Acid (PAA)	.001-.3% aqueous solution gas phase: 2-4% 5-120 minutes	broad spectrum sporicidal at low temperatures can tolerate organic load rapid action nontoxic decomposition products leaves no residue	pungent odour corrosive to some metals shelf life of dilutions is less than 1 week stock solutions irritating to skin and eyes stock must be protected from heat, light gas phase: respiratory irritant, fire hazard above 55°C	instruments and equipment gas phase sterilization of chambers for germ-free animals

**Table 4B** - Summary of recommended concentrations, contact times, advantages and disadvantages of non-halogen chemical germicides. The wide ranges of effective concentrations and contact times cited reflect the interdependence of time and concentration as well as factors such as resistance of the particular class or strain of target microorganism(s) and desired effect. Also, some germicides are available in combinations (e.g., glutaraldehyde/phenol or peracetic acid/alcohol mixtures) which are synergistic whereby the components in combination produce a greater antimicrobial effect than the sum of their individual effects.
	Effective Concentrations and Contact Times	Advantages	Disadvantages	Examples of Laboratory Uses
Aldehydes:
Glutaraldehyde	0.5-2.5% alkalinized aqueous solution 2-30 mins; up to 12 hours to kill all spores	broad spectrum does not corrode metal can tolerate organic load	expensive pH, temperature dependent pungent odour toxic: skin, eye, respiratory tract irritant activated solutions have less than 2-week shelf life	cold sterilant and fixative surface decontamination instruments, equipment, glassware
Formalin (37% aqueous formaldehyde)	3-27% formalin (1-10% formaldehyde) in 70-90% alcohol 10-30 minutes	broad spectrum inexpensive does not corrode metal can tolerate organic load	pungent odour skin, eye and respiratory tract irritant potential carcinogen (animal studies) may require 24 hrs or more to kill all spores	cold sterilant and fixative surface decontamination instruments and equipment
Formaldehyde (gas)	1-3 hours	as for formalin effective penetration	as for formulin flammable porr penetration of covered surfaces	on site decontamination of biological safety cabinet HEPA filters enclosed areas
Ethylene Oxide Gas	50-1200 mg/L 1-12 hours	broad spectrum no heat or moisture evolved penetrates packaging materials	flammable, reactive toxic: potential carcinogen and mutagen some sterilized items may need more than 24 hours for outgassing	heat or moisture sensitive supplies, instruments and equipment

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