PRINCIPLES OF STERILIZATION AND DISINFECTION: SIGNIFICANCE IN MICROBIOLOGY

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Killing of Prions and Thermophilic Archaea

The standard sterilization methods used in medical applications are capable of causing irreversible damage to medically relevant microorganisms such as bacteria, protozoans, fungi, and helminths including worm eggs. Much more extreme processes are required to inactivate prions, such as autoclaving at 121oC for 4.5 hours or at 134oC for 30 minutes. Hyperthermophilic archaea forms have also been discovered in recent years that proliferate at temperatures of 100oC and higher and can tolerate autoclaving at 121oC for one hour. These extreme life forms, along with prions, are not covered by the standard definitions of sterilization and sterility. Disinfection is a specifically targeted antimicrobial treatment with the objective of preventing transmission of certain microorganisms. The purpose of the disinfection procedure is to render an object incapable of spreading infection. Preservation is a general term for measures taken to prevent microbe-caused spoilage of susceptible products (e.g., pharmaceuticals, foods). Decontamination is the removal or count reduction of microorganisms contaminating an object. The objective of aseptic measures and techniques is to prevent microbial contamination of materials or wounds. In antiseptic measures, chemical agents are used to fight pathogens in or on living tissue, for example in a wound.

The Kinetics of Pathogen Killing
Killing microorganisms with chemical agents or by physical means involves a first-order reaction. This implies that no pathogen-killing method kills off all the microorganisms in the target population all at once and instantaneously. Plotting the killing rate against exposure time in a semilog coordinate system results in a straight-line curve. Sigmoid and asymptotic killing curves are exceptions to the rule of exponential killing rates. The steepness of the killing curves depends on the sensitivity of the microorganisms to the agent as well as on the latter’s effectiveness. The survivor/exposure curve drops at a steeper angle when heat is applied, and at a flatter angle with ionizing radiation or chemical disinfectants. Another contributing factor is the number of microorganisms contaminating a product (i.e., its bioburden): when applied to higher organism concentrations, an antimicrobial agent will require a longer exposure time to achieve the same killing effect.

Standard sterilization methods extend beyond killing all microorganisms on the target objects to project a theoretical reduction of risk, i.e., the number of organisms per sterilized unit should be equal to or less than 10–6. The D value (decimal reduction time), which expresses the time required to reduce the organism count by 90%, is a handy index for killing effectiveness. The concentration (c) of chemical agents plays a significant role in pathogen-killing kinetics. The relation between exposure time (t) and c is called the dilution coefficient (n): t . Cn = constant. Each agent has a characteristic coefficient n, for instance five for phenol, which means when c is halved the exposure time must be increased by a factor of 32 to achieve the same effect. The temperature coefficient describes the influence of temperature on the effectiveness of chemical agents. The higher the temperature, the stronger the effect, i.e., the exposure time required to achieve the same effect is reduced. The coefficient of temperature must be determined experimentally for each combination of antimicrobial agent and pathogen species.

Mechanisms of Action of sterilants and disinfectants used for sterilization and disinfection processes
When microorganisms are killed by heat, their proteins (enzymes) are irreversibly denatured. Ionizing radiation results in the formation of reactive groups that contribute to chemical reactions affecting DNA and proteins. Exposure to UV light results in structural changes in DNA (thymine dimers) that prevent it from replicating. This damage can be repaired to a certain extent by light (photoreactivation). Most chemical agents (alcohols, phenols, aldehydes, heavy metals, oxidants) denature proteins irreversibly. Surfactant compounds (amphoteric and cationic) attack the cytoplasmic membrane. Acridine derivatives bind to DNA to prevent its replication and function (transcription)

Physical Methods of Sterilization and Disinfection

HEAT
The application of heat is a simple, cheap and effective method of killing pathogens. Methods of heat application vary according to the specific application.

  • Pasteurization. This is the antimicrobial treatment used for foods in liquid form (milk):
    — Low-temperature pasteurization: 61.5oC, 30 minutes; 71oC, 15 seconds.
    — High-temperature pasteurization: brief (seconds) of exposure to 80–85oC in continuous operation.
    — Uperization: heating to 150oC for 2.5 seconds in a pressurized container using steam injection.

  • Disinfection. Application of temperatures below what would be required for sterilization. Important: boiling medical instruments, needles, syringes, etc. does not constitute sterilization! Many bacterial spores are not killed by this method.
  • Dry heat sterilization. The guideline values for hot-air sterilizers are as follows: 180oC for 30 minutes, 160oC for 120 minutes, whereby the objects to be sterilized must themselves reach these temperatures for the entire prescribed period.
  • Moist heat sterilization. Autoclaves charged with saturated, pressurized steam are used for this purpose:
    — 121oC, 15 minutes, one atmosphere of pressure (total: 202 kPa).
    — 134oC, three minutes, two atmospheres of pressure (total: 303 kPa).
    In practical operation, the heating and equalibriating heatup and equalizing times must be added to these, i.e., the time required for the temperature in the most inaccessible part of the item(s) to be sterilized to reach sterilization level. When sterilizing liquids, a cooling time is also required to avoid boiling point retardation.

The significant heat energy content of steam, which is transferred to the cooler sterilization items when the steam condenses on them, explains why it is such an effective pathogen killer. In addition, the proteins of microorganisms are much more readily denatured in a moist environment than under dry conditions.

RADIATION

  • Nonionizing radiation. Ultra-violet (UV) rays (280–200 nm) are a type of nonionizing radiation that is rapidly absorbed by a variety of materials. UV rays are therefore used only to reduce airborne pathogen counts (surgical theaters, filling equipment) and for disinfection of smooth surfaces.
  • Ionizing radiation. Two types are used:
    — Gamma radiation consists of electromagnetic waves produced by nuclear
    disintegration (e.g., of radioisotope 60Co).
    — Corpuscular radiation consists of electrons produced in generators and accelerated to raise their energy level.
    Radiosterilization equipment is expensive. On a large scale, such systems are used only to sterilize bandages, suture material, plastic medical items, and heat-sensitive pharmaceuticals. The required dose depends on the level of product contamination (bioburden) and on how sensitive the contaminating microbes are to the radiation. As a rule, a dose of 2.5 x 104 Gy (Gray) is considered sufficient. One Gy is defined as absorption of the energy quantum one joule (J) per kg.

FILTRATION

Liquids and gases can also be sterilized by filtration. Most of the available filters catch only bacteria and fungi, but with ultrafine filters, viruses and even large molecules can be filtered out as well. With membrane filters, retention takes place through small pores. The best-known type is the membrane filter made of organic colloids (e.g., cellulose ester). These materials can be processed to produce thin filter layers with gauged and calibrated pore sizes. In conventional depth filters, liquids are put through a layer of fibrous material (e.g., asbestos). The effectiveness of this type of filter is due largely to the principle of adsorption. Because of possible toxic side effects, they are now practically obsolete.

Chemical Methods of Sterilization and Disinfection

Ethylene oxide (C2H4O). This highly reactive gas (C2H4O) is flammable, toxic, and a strong mucosal irritant. Ethylene oxide can be used for sterilization at low temperatures (20–60oC). The gas has a high penetration capacity and can even get through some plastic foils. One drawback is that this gas cannot kill dried microorganisms and requires a relative humidity level of 40–90% in the sterilizing chamber. Ethylene oxide goes into solution in plastics, rubber, and similar materials, therefore sterilized items must be allowed to stand for a longer period to ensure complete desorption.

Aldehydes. Formaldehyde (HCHO) is the most important aldehyde. It can be used in a special apparatus for gas sterilization. Its main use, however, is in disinfection. Formaldehyde is a water-soluble gas. Formalin is a 35% solution of this gas in water. Formaldehyde irritates mucosa; skin contact may result in
inflammations or allergic eczemas. Formaldehyde is a broad-spectrum germicide for bacteria, fungi, and viruses. At higher concentrations, spores are killed as well. This substance is used to disinfect surfaces and objects in 0.5–5% solutions. In the past, it was commonly used in gaseous form to disinfect the air inside rooms (5 g/m3). The mechanism of action of formaldehyde is based on protein denaturation. Another aldehyde used for disinfection purposes is glutaraldehyde.

Alcohols. The types of alcohol used in disinfection are ethanol (80%), propanol (60%), and isopropanol (70%). Alcohols are quite effective against bacteria and fungi, less so against viruses. They do not kill bacterial spores. Due to their rapid action and good skin penetration, the main areas of application of alcohols are surgical and hygienic disinfection of the skin and hands. One disadvantage is that their effect is not long-lasting (no depot effect). Alcohols denature proteins.

Phenols. Lister was the first to use phenol (carbolic acid) in medical applications. Today, phenol derivatives substituted with organic groups and/or halogens (alkylated, arylated, and halogenated phenols), are widely used. One common feature of phenolic substances is their weak performance against spores and viruses. Phenols denature proteins. They bind to organic materials to a moderate degree only, making them suitable for disinfection of excreted materials.

Halogens. Chlorine, iodine, and derivatives of these halogens are suitable for use as disinfectants. Chlorine and iodine show a generalized microbicidal effect and also kill spores. Chlorine denatures proteins by binding to free amino groups; hypochlorous acid (HOCl), on the other hand, is produced in aqueous solutions, then disintegrates into HCl and ½O2 and thus acts as a powerful oxidant. Chlorine is used to disinfect drinking water and swimming-pool water (up to 0.5 mg/l). Calcium hypochlorite (chlorinated lime) can be used in nonspecific disinfection of excretions. Chloramines are organic chlorine compounds that split off chlorine in aqueous solutions. They are used in cleaning and washing products and to disinfect excretions. Iodine has qualities similar to those of chlorine. The most important iodine preparations are the solutions of iodine and potassium iodide in alcohol (tincture of iodine) used to disinfect skin and small wounds. Iodophores are complexes of iodine and surfactants (e.g., polyvinyl pyrrolidone). While iodophores are less irritant to the skin than pure iodine, they are also less effective as germicides.

Oxidants. This group includes ozone, hydrogen peroxide, potassium permanganate, and peracetic acid. Their relevant chemical activity is based on the splitting off of oxygen. Most are used as mild antiseptics to disinfect mucosa, skin, or wounds.

Surfactants. These substances (also known as surface-active agents, tensides, or detergents) include anionic, cationic, amphoteric, and nonionic detergent compounds, of which the cationic and amphoteric types are the most effective. The bactericidal effect of these substances is only moderate. They have no effect at all on tuberculosis bacteria (with the exception of amphotensides), spores, or nonencapsulated viruses. Their efficacy is good against Gram-positive bacteria, but less so against Gram-negative rods. Their advantages include low toxicity levels, lack of odor, good skin tolerance, and a cleaning effect.

Practical Disinfection
The objective of surgical hand disinfection is to render a surgeon’s hands as free of microorganisms as possible. The procedure is applied after washing the hands thoroughly. Alcoholic preparations are best suited for this purpose, although they are not sporicidal and have only a brief duration of action. Alcohols are therefore often combined with other disinfectants (e.g., quaternary ammonium compounds, QACs). Iodophores are also used for this purpose. The purpose of hygienic hand disinfection is to disinfect hands contaminated with pathogenic organisms. Here also, alcohols are the agent of choice. Alcohols and/or iodine compounds are suitable for disinfecting patient’s skin in preparation for surgery and injections. Strong-smelling agents are the logical choice for disinfection of excretions (feces, sputum, urine, etc.). It is not necessary to kill spores in such applications. Phenolic preparations are therefore frequently used.

Contaminated hospital sewage can also be thermally disinfected (80–100oC) if necessary. Surface disinfection is an important part of hospital hygiene. A combination of cleaning and disinfection is very effective. Suitable agents include aldehydes and phenol derivatives combined with surfactants. Instrument disinfection is used only for instruments that do not cause injuries to skin or mucosa (e.g., dental instruments for work on hard tooth substance). The preparations used should also have a cleaning effect. Laundry disinfection can be done by chemical means or in combination with heat treatment. The substances used include derivatives of phenols, aldehydes and chlorine as well as surfactant compounds. Disinfection should preferably take place during washing. Chlorine is the agent of choice for disinfection of drinking water and swimming-pool water. It is easily dosed, acts quickly, and has a broad disinfectant range.

The recommended concentration level for drinking water is 0.1–0.3 mg/l and for swimming-pool water 0.5 mg/l. Final room disinfection is the procedure carried out after hospital care of an infection patient is completed and is applied to a room and all of its furnishings. Evaporation or atomization of formaldehyde (5 g/m3), which used to be the preferred method, requires an exposure period of six hours. This procedure is now being superseded by methods involving surface and spray disinfection with products containing formaldehyde. Hospital disinfection is an important tool in the prevention of cross-infections among hospital patients. The procedure must be set out in written form for each specific case so that everyone, including patients and hospital staff plays their individual roles to ensure the proper containment of the spread of pathogens within the hospital environment.

Further reading

Kayser F.H., Bienz K.A., Eckert J. and Zinkernagel R.M (2005). Kayser Medical Microbiology. Thieme Verlag, Ru¨digerstraße 14, 70469 Stuttgart, Germany  

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