17 September 2016

Lecture #4: STERILIZATION AND DISINFECTION





Microbial populations should be effectively controlled for the health condition of the general public. One can remove, inhibit or kill microorganism by physical agents, physical processes or chemical agents. There are various techniques and agents available, they act in many different ways and each has its own limitations in practical applications.

There are terms used to describe the physical processes and chemical agents and these terms are important in labeling drugs and chemicals used against microorganisms.

Sterilization – the process of destroying all forms of microbial life. A sterile object, therefore, is free of living microorganisms.

Bactericidal – having the property of killing the bacteria. The action is irreversible, i.e., the killed organisms can no longer reproduce even after being removed from contact with the agent.

Bacteriostatic – having the property of inhibiting bacterial multiplication. When the agent is removed, the bacteria resume multiplication.

Antiseptic – a substance that prevents the growth of microorganisms either by destroying cells or inhibiting their growth and activity. It is usually associated with substances applied to the body.

Disinfectant – usually a chemical agent that kills the growing forms, but not the resistant spore forms. This term is applied to inanimate objects. These chemicals may be toxic to tissues.

Septic – is characterized by the presence of pathogenic microbes while aseptic is the absence of pathogenic microbes.

Sanitizer – an agent that reduces the bacterial population to a safe level. This is commonly applied to inanimate objects and generally employed in the daily care of equipment and utensil in food and plants, restaurants and other eating places.

Antimicrobial agents – a chemical or biological agent that destroys or inhibits growth of microorganisms.


Conditions influencing antimicrobial action

There are many factors that must be considered in the application of any physical or chemical agent. Among these are:

1.      Temperature – an increase in temperature in the presence of a chemical agent, hastens the destruction of microorganisms.

2.      Kind of microorganisms – species of microorganisms have different susceptibility pattern to physical and chemical agents. In spore–forming bacteria, the growing, vegetative forms are more susceptible than the spore forms.

3.      Physiologic state of the cells – young, actively metabolizing cells are more easily destroyed than old. Dormant cells in the case of an agent which causes damage through interference with metabolism.

4.      Environment – the consistency of the material (aqueous or viscous) in which the microorganism is grown markedly influence the penetration of the agent.


Mode of action antimicrobial agents

1.      Damage to the cell wall – e.g. enzymes lysozyme, penicillin

2.      Alteration of cell permeability – e.g. phenolic compounds, synthetic detergents, soaps and quaternary ammonium compounds.

3.      Alteration of protein and nucleic acid molecules – e.g. high temperatures and high concentration of chemicals.

4.      Inhibition of enzyme action – e.g. cyanide inhibits cytochrome oxidase, fluoride inhibits glycolysis, trivalent arsenic compounds block TCA cycle and strong oxidizing agents such as halogens and hydrogen peroxide.

5.      Inhibition of nucleic acid synthesis – e.g. certain synthetic chemicals and some naturally occurring substances.

Antimetabolite or metabolic analogue – is a substance that is structurally related to but slightly different from, the natural metabolite. Metabolite is a chemical substance participating in metabolism.


Physical agents

A.    High Temperature – when combined with high humidity, it is considered to be the most effective method of killing microorganisms.

These terms are used to indicate the degree of heat resistance of bacteria:

1.      Thermal death point (TDP) – refers to the lowest temperature at which suspension of bacteria is killed in 10 minutes.

2.      Thermal death time (TDT) – refers to the shortest period of time required to kill a suspension of bacteria at a prescribed temperature and under specific conditions.

Spore–formers have 2 TDP and TDT:

a.      Vegetative state – 100oC; 10 – 15 minutes
b.      Spore state – 121oC, 20 – 30 minutes

3.      Decimal reduction time – the time in minutes to reduce the population by 90%

Moist heat – kills microorganisms by coagulating their proteins and is much more rapid and effective than dry heat.

1.      Steam under pressure – the most practical and dependable agent for sterilization. It has the advantage of rapid heating, penetration and moisture in abundance which facilitate coagulation of proteins.

The autoclave is the apparatus used and the temperature and pressure is under control. The most important in operating this apparatus is that the temperature is maintained and the air should be completely be replaced with saturated steam. It is the high temperature that kills the microorganisms and not the high pressure. The material to be sterilized should be at 121oC (115 lbs/sq.in.) for 15 to 20 minutes.

2.      Fractional or Intermittent sterilization (Tyndallization) – Arnold sterilizer is used employing free flowing steam at 100oC for 30 minutes on 3 successive days with incubation period in between sterilization to allow the spore forming bacteria to germinate and be killed or destroyed the next day.

3.      Boiling – exposure of the materials at 100oC for 2 to 3 minutes will kill all vegetative forms but not the spore forms even for many hours.

4.      Pasteurization – a controlled heat treatment which kills certain types of bacteria but will not kill all microorganisms.

Two methods:

a.      LTH (Low Temperature Holding) or Batch method – exposed at 62oC for 30 minutes

b.      HTST (High Temperature, Short time) or Flush process – exposed at 72oC for 15 minutes.

5.      Inspissation – thickening through evaporization. This is used in the sterilization of high protein media that cannot withstand high temperatures of the autoclave. Usual application is at 75 – 80oC for 2 hours or successive days.

Dry heat – kills microorganism by oxidizing their chemical constituents

1.      Hot–air sterilization – recommended to sterilize laboratory glasswares using a special electric or gas oven at temperature of 160oC for 2 hours. Oven is the apparatus used.

2.      Incineration – actual burning of materials at 300–400oC. It is used for the destruction of carcasses, infected laboratory animals and other infected material to be disposed of.

B.     Low temperature – considered to be a microbiostatic because it reduces the rate of metabolism. Low temperatures are useful for preservation of cultures and cannot be depended upon for disinfection or sterilization.

C.     Dessication and lyophilization – the mechanism of microbial inhibition is dehydration. The effect is chiefly bacteriostatic. They are recommended for the preservation of foods, bacterial cultures and viruses.

D.    Osmotic pressure – the mechanism of action is plasmolysis; the cells are dehydrated therefore, they are unable to metabolize and grow. Most of the microorganisms are inhibited by high concentrations of salts and sugars. This fact forms the basis of preserving foods by “salting” or by concentrated sugar solution.

E.     Sonic titration and vibration – there is a breaking of the cell wall of the bacteria. These are used in research work of cellular constituents.

F.      Radiation – when it passes through the cells, it creates free hydrogen and hydroxyl radicals and some peroxides which in turn cause different kinds of intracellular damage. There have been many developments in the application of ionizing radiation to sterilize biological materials and this is called cold sterilization which is being developed in the food and pharmaceutical industries.

1.      X–rays – are lethal to microorganisms and they have considerable energy and penetration ability but they are impractical for purposes of controlling microbial population because they are very expensive in large amount and they are difficult to utilize efficiently since they are given off in all directions from their point of origin.

2.      Ultraviolet light – has very little ability to penetrate matter, only those microorganisms that are directly exposed to ultraviolet light are susceptible to destruction. They are widely used in hospital operating rooms and aseptic filling rooms in pharmaceutical industry and for treatment of contaminated surfaces in food and dairy industry.

3.      Gamma rays – are practically used in sterilization of materials of considerable thickness or volume because of great penetrating power and their microbicidal effect.

The “target” theory of action was made based on the effect of ionizing radiations on the cells and this means that the radiant–energy particle makes a “direct hit” on some substances within the cell, causing ionization which results in bacterial death.

·         If ultraviolet radiation is not too extensive, the cell can repair the damage by excision of dimers which is accomplished by:

a.      Photoreactivation – light induced mechanism
b.      Excision repair – dark reaction

G.    Titration – is used to remove microorganism in some biological fluid like animal serum or enzymes and some vitamins or antibiotics that are thermolabile, i.e., destroyed by heat.

A variety of bacteriological filters have been available and they are made up different material like diatomaceous earth like in Berkefield filter, asbestos pad in Seitz filter, unglazed porcelain in Chamberland–Pasteur filter, sintered glass in Morton filter and cellulose ester in membrane or molecular filter. Most filters are available in several grades, based on the average size of the pores. It is not porosity alone as the only factor preventing the passage of organisms. Other factors contributing the efficiency of filtration include electric charge of the filters and carried by the organisms and the nature of the fluid being filtered.

The development of high–efficiency particulate air (HEPA) filters has made it possible to deliver clean air to an enclosure such as cubicle or room and together with system of laminar flow, it is now used extensively to provide dust and bacteria free air.

Chemical agents

Chemical agents do, however, have value in disinfection, that is, reducing the numbers of and eliminating certain dangerous microbes. Among the disinfectant used are:

1.      Acids

a.      Acidic disinfectants function by destroying the bonds of nucleic acids and precipitating proteins. Acids also change the pH of the environment making it detrimental to many microorganisms. Concentrated solutions of acids can be caustic, cause chemical burns, and can be toxic at high concentrations in the air. These characteristics limit their use. The antimicrobial activity of acids is highly pH dependent. Acids have defined but limited use as disinfectants.

b.      Acetic acid is usually sold as glacial acetic acid (95% acetic acid) which is then diluted with water to make a working solution concentration of 5%. The concentrated form is corrosive to the skin and lungs, but the typical dilution (5%) is considered non–toxic and non–irritating. Acetic acid is typically applied by spraying, misting or immersing an item in a diluted solution. Household vinegar is a 4–5% soluton of acetic acid (by volume). Acetic acid has poor activity in organic material.

2.      Alcohols

a.      Examples are ethanol and isopropanol. Ethanol is considered virucidal; isopropanol is not effective against non–enveloped viruses.

b.      Alcohols are broad spectrum antimicrobial agents that damage microorganisms by denaturing proteins, causing membrane damage and cell lysis. Alcohols are used for surface disinfection, topical antiseptic and hand sanitizing lotions. Alcohols are considered fast–acting capable of killing most bacteria within five minutes of exposure but are limited in virucidal activity and are ineffective against spores.

c.       An important consideration with alcohols is the concentration used, with 70–90% being optimum. Higher concentrations (95%) are actually less effective because some degree of water is required for efficacy (to denature proteins). Alcohols evaporate quickly but leave behind no residue. The activity of alcohols is limited in the presence of organic matter. Alcohols are highly flammable, can cause damage to rubber and plastic, and can be very irritating to injured skin.

3.      Aldehydes

a.      Acts by denaturing proteins and disrupting nucleic acids. Aldehydes are non–corrosive to metals, rubber, plastic and cement. These chemicals are highly irritating, toxic to humans or animals with contact or inhalation, and are potentially carcinogenic; therefore their use is limited.

b.      Formaldehyde is used as a surface disinfectant and a fumigant and has been used to decontaminate wooden surfaces, bricks and crevices of electronic and mechanical equipment. Its use must occur in air tight building, which must remain closed for at least 24 hours after treatment. The efficacy of formaldehyde is dependent on relative humidity and temperature; optimum being humidity close to 70% and a temperature close to 57oF

c.       Formalin is 37% solution of formaldehyde in water

d.     Glutaraldehyde is primarily used as a disinfectant for medical equipment (e.g. endoscopes), but can provide sterilization at prolonged contact times. A 2% concentration is used for high–level disinfection. Its efficacy is highly dependent on pH and temperature, working best at a pH greater than 7 and high temperatures. It is considered more efficacious in the presence of organic matter, soaps and hard water than formaldehyde.

4.      Alkalis

a.      Alkaline agents work by saponifying lipids within the envelopes of microorganisms. The activity of alkali compounds is slow but can be increased by raising the temperature. Alkalis have good microbicidal properties, but are very corrosive agents and personal protection should be observed.

b.      Sodium hydroxide (lye, caustic soda, soda ash) is a strong alkali used to disinfect buildings but is highly caustic. Protective clothing, rubber gloves, and safety glasses should be worn when mixing and applying the chemical. Lye should always be carefully added to water. Never pour water into lye; a very violent reaction will occur as well as the production of high heat that can melt plastic containers. Sodium hydroxide is corrosive for metals.

c.       Ammonium hydroxide is effective disinfectant against coccidial oocysts however strong solutions emit intense and pungent fumes. This substance is not considered effective against most bacteria.

d.     Sodium carbonate (soda ash, washing soda) has been used in hot solution (180oF) for disinfecting buildings. It is more effective as a cleanser than a disinfectant since it lacks efficacy against some bacteria and most viruses. It has poor activity in the presence of organic material and can be deactivated by hard water. It can be irritating and requires protective clothing and is harmful to aquatic life.

e.      Calcium oxide (quicklime) becomes lime when mixed with water. This has biocidal effects on some bacteria and virus and is sometimes spread on the ground following depopulation of infected premises and has also been used to retard putrefaction of buried carcasses after depopulation.

5.      Biguanides

a.      Example is chlorhexidine

b.      Biguanidies are detrimental to microorganisms by reacting with the negatively charged groups on cell membranes which alters the permeability. Biguanidines have a broad antibacterial spectrum; however, they are limited in their effectiveness against viruses only. Biguanidines can only function in a limited pH range (5 – 7) and are easily inactivated by soaps and detergents. These products are toxic to marine life.

6.      Halogens

a.      Halogen compound are broad spectrum compounds that are considered low toxicity, low cost and easy to use. They do lose potency over time and are not active at temperatures above 110oF or at high pH (>9). Since these compounds lose activity quickly in the presence of organic debris, sunlight and some metals, they must be applied to thoroughly cleaned surfaces for disinfection.

b.      Chlorine compounds function through their electronegative nature to denature proteins and are considered broad spectrum, being effective against bacteria, enveloped and non–enveloped viruses, mycobacteria and fungi. Sodium hypochlorite is one of the most widely used chlorine containing disinfectant. Hypochlorites should never be mixed with acids or ammonia as this will result in the release of toxic chlorine gas.

c.       Iodine compounds function by denaturing proteins to interfere with the enzymatic systems of microorganisms. Iodines are often formulated with soaps and considered relatively safe. Iodine agents are inactivated by QACs and organic debris.

d.     Iodophors are iodine complexes that have increased solubility and sustained release of iodine. One of the more commonly used iodophors is povidone–iodine. They are good for general use and are less readily inactivated by organic matter than elemental iodine compounds. The dilution of iodophors actually increases the free iodine concentrations and antimicrobial activity.

7.      Oxidizing agents

a.      Function by denaturing the proteins and lipid of microorganisms.

b.      Hydrogen peroxide at a concentration of 5 – 20% acts best with bacteria, virus and fungi but has limited activity with Mycobacteria.

c.       Peracetic acid is a strong oxidizing agent and is a formulation of hydrogen peroxide and acetic acid.

d.     Potassium peroxymonosulfate and sodium chloride.

8.      Phenols

a.      Function by denaturing proteins and inactivating membrane bound enzymes to alter the cell wall permeability of microorganisms. They usually have a milky or cloudy appearance when added to water with a strong pine odor.

9.      Quaternary ammonium compounds (QACs)

a.      QACs are cationic detergents that are attracted to the negatively charged surfaces of microorganism, where they irreversibly bind phospholipids in the cell membrane and denature proteins impairing permeability.


Characteristics of a good disinfectant

1.      Should attack all types of microorganisms
2.      Be rapid in its action
3.      Should not damage materials being disinfected
4.      Should not be retarded in its action by organic matter
5.      Should dissolve easily in or mix with water to form a stable solution or emulsion.
6.      Should not penetrate material being disinfected
7.      Should not decompose when exposed to heat, light rays or unfavorable weather condition.
8.      Should not destroy body tissues or act as a poison when taken internally
9.      Should not have unpleasant odor or discolor the material being disinfected and be easily obtained at a comparatively low cost.


Phenol coefficient

This is the standard method of testing chemical agents. It is used for evaluating the effectiveness of disinfectant by comparing with phenol under identical condition. The larger the resulting value, phenol coefficient, the more active the agent under those conditions.

Coefficient is determined by taking the highest dilution of the disinfectant that gives evidence of killing the organism in 10 minutes (but not in 5 minutes). This is divided by the highest dilution of phenol giving the same result.

Example:

            1/1000 = disinfectant X
            1/90 = phenol

            Coefficient     =          1000/90
                                    =          11.1 for disinfectant X







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