METHOD OF STERILIZATION.
The use of physical method to control micro-organism
- Heat
Moist heat readily kills viruses, bacteria and fungai. Exposure to boiling water for 10mins is sufficient to destroy vegetative cells and eukaryotic spores. Thermal death point (TDP) this is the lowest temperature at which a microbial suspension is killed in 10mins.
Thermal death time (TDT)
This is the shortest time needed to kill all organisms in a microbial suspension at a specific temperature and under defined condition.
Decimal reduction time (D) or D value
This is the time required to kill 90% of the micro-organisms or spores in a sample at a specified temperature. D values are used to estimate the relative resistance of a micro-organism to different temperatures through calculations of the Z value.
Z value
This is the increase in temperature required to reduce D to 1/10th its value or to reduce it by one log cycle when log D is plotted against temperature. The food processing industry makes extensive use of the D and Z values. After a food has been canned, it must be heated to eliminate the risk of botulism arising from Clostridum botulilum spores.
Moist heat sterilization must be carried out at temperatures above 1000 C in other to destroy bacterial spores and this requires the use of saturated steam under pressure. Steam sterilization is carried out with an autoclave, a device somewhat like a fancy pressure cooker. Many substances such as milk are treated with controlled heating at temperatures well below boiling, a process known as pasteurization.
Dry heat
Sunlight: The microbicidal activity of sunlight is mainly due to the presence of ultra violet rays in it. It is responsible for spontaneous sterilization in natural conditions. In tropical countries, the sunlight is more effective in killing germ due to combination of ultraviolet rays and heat. By killing bacteria suspended in water, sunlight provides natural method of disinfection of water bodies such as tanks and lakes. Sunlight is not sporicidal, hence it does not sterilize.
Heat: Heat is considered to be most reliable method of sterilization of articles that can withstand heat. Heat acts by oxidative effects as well as denaturation and coagulation of proteins. Those articles that cannot withstand high temperatures can still be sterilized at lower temperature by prolonging the duration of exposure.
Factors affecting sterilization by heat
Nature of heat: Moist heat is more effective than dry heat
Temperature and time: temperature and time are inversely proportional. As temperature increases the time taken decreases.
Number of microorganisms: More the number of microorganisms, higher the temperature or longer the duration required.
Nature of microorganism: Depends on species and strain of microorganism, sensitivity to heat may vary. Spores are highly resistant to heat.
Type of material: Articles that are heavily contaminated require higher temperature or prolonged exposure. Certain heat sensitive articles must be sterilized at lower temperature.
Presence of organic material: Organic materials such as protein, sugars, oils and fats increase the time required.
- Low temperatures
This is the use of freezing or refrigeration to inhibit microbial growth and reproduction. This is important in food micro biology. Freezing items at -20o C or lower stop microbial growth because of the low temperature and the absence of liquid water. Freezing is a very good method for long term storage of microbial samples when carried out properly and many laboratories have a low temperature freezer for culture storage at -30 or -700C. Frozen food contained many micro-organisms and hence, it should be prepared and consumed promptly after thawing in other to avoid spoilage and pathogen growth.
- Filtration
Filtration is an excellent way to reduce microbial population in solutions of which sensitive materials and it can be used to sterilize solvent. The filter simply removes micro-organism rather than destroying contaminating micro-organisms.
There are two types of filters-depth filters and membrane filters.
Depth filters are those that remove microbial cells by physical screening or entrapment and also by adsorption to the surface to the filter material.
Membrane filters with pore size about 0.2mm in diameter are used to remove most vegetative cells but not viruses from solutions. Membrane filters remove micro-organisms by screening them out much as a sieve separate large sand particles from small ones. This filters are used to sterilize pharmaceuticals, culture media, oils, antibiotics and other heat sensitive solutions.
- Radiation
Ultraviolent (UV) radiation around 260mm is quite lethal but does not penetrate glass, dirt films and water. Because of this disadvantage, UV radiation is used as a sterilizing agent only in a few specific situations. Pathogens and other micro-organisms are destroyed when a thin layer of water is passed under the lamps.
Ionizing radiation is an excellent sterilizing agent and penetrate deep into objects. It will destroy bacteria endospores and vegetative cells both prokaryotic and eukaryotic. Gamma radiation from a cobalt 60 source is used in the cold sterilization of antibiotics, hormones, sutures and plastic disposable suppliers such as syringe. Gamma radiation used to sterilize and pasteurize heat and other food can work with them and communicate efficiently.
The use of chemical method of sterilization to control microorganisms
Chemical sterilization is the elimination of all viable microorganisms and their spores using liquid or gaseous compounds.The method use for chemical sterilization varies with the form of chemical used. Liquid sterilization involves submerging equipment in a chemical fluid for enough time to kill all viable microorganisms and their spores. It is used to sterilize heat sensitive device which can be immersed. Gas sterilization involves exposing equipment to chemical gases in an enclosed heated or pressurized chamber.
Types of chemical sterilants
Liquid sterilants include glutaraldehyde, ortho-phthaldehyde, peracetic acid, hydrogen peroxide, and hypochlorite. Evaporating chemical sterilants may be toxic, so it is a good idea to place them in covered containers. Two of these liquids are found in many households and used as disinfectants. Hydrogen peroxide for home use is 3.0% concentration. 7.5% to 25% hydrogen peroxide concentration is needed for sterilization. Another disinfecting liquid is bleach, which is a type of hypochlorite solution. Submerging items in bleach for short times kills many pathogens (disinfects) but only submersion for 24-32 hours results in sterilization
At times, liquid sterilizing agents are impractical or ineffective for sterilizing items. Gaseous chemicals are more effective sterilants because they are able to permeate small openings and crevices easily. Gas chemicals also sterilize faster than liquids because they usually are combined with high heat. Gas residue is also easier to remove from sterilized articles, but requires much more expensive equipment.
classification of chemical sterilization
- Based on consistency- Liquid (alcohol, phenol)
Gaseous (formaldehyde vapor, ethylene oxide)
- Based on spectrum of activity-High level
Intermediate level
Low level
- Based on mechanism of action- Action of membrane (alcohol, detergent)
Denaturation of cellular proteins (alcohols, phenols)
Denaturation of group of enzyme (H2O2, Halogen)
Damage to nucleic acids (ethylene oxide,formaldehyde)
Liquid and Gaseous chemical agents
Liquid includes: alcohols, aldehhdes, phenolics, halogens, heavy metals, surface active agents and dyes. Gaseous includes: formaldehyde, ethylene oxide and plasma
Alcohol
Alcohols work through the disruption of cellular membranes, solubilization of lipids, and denaturation of proteins by acting directly on S-H functional groups. Ethyl and isopropyl alcohols are the two most widely used alcohols for their biocidal activity. These alcohols are effective against lipid-containing viruses and a broad spectrum of bacterial species, but ineffective against spore-forming bacteria. They evaporate rapidly, which makes extended contact times difficult to achieve unless the items are immersed.
The optimum bactericidal concentration for ethanol and isopropanol is in the range of 60% to 90% by volume. Their cidal activity drops sharply when diluted below 50% concentration. Absolute alcohol is also not very effective. They are used to clean instruments and wipe down interior of Biological Safety Cabinets and bottles, etc. to be put into Biological Safety Cabinets. Alcohols are generally regarded as being non-corrosive. Alcohol refers to two water-soluble chemical compounds—ethyl alcohol and isopropyl alcohol—that have generally underrated germicidal characteristics. FDA has not cleared any liquid chemical sterilant or high-level disinfectant with alcohol as the main active ingredient. These alcohols are rapidly bactericidal rather than bacteriostatic against vegetative forms of bacteria; they also are tuberculocidal, fungicidal, and virucidal but do not destroy bacterial spores.
Mode of action.
The most feasible explanation for the antimicrobial action of alcohol is denaturation of proteins. This mechanism is supported by the observation that absolute ethyl alcohol, a dehydrating agent, is less bactericidal than mixtures of alcohol and water because proteins are denatured more quickly in the presence of water. Protein denaturation also is consistent with observations that alcohol destroys the dehydrogenases of Escherichia coli, and that ethyl alcohol increases the lag phase of Enterobacter aerogenes and that the lag phase effect could be reversed by adding certain amino acids. The bacteriostatic action was believed caused by inhibition of the production of metabolites essential for rapid cell division.
Microbicidal Activity.
Methyl alcohol (methanol) has the weakest bactericidal action of the alcohols and thus seldom is used in healthcare . The bactericidal activity of various concentrations of ethyl alcohol (ethanol) was examined against a variety of microorganisms in exposure periods ranging from 10 seconds to 1 hour. Pseudomonas aeruginosa was killed in 10 seconds by all concentrations of ethanol from 30% to 100% (v/v), and Serratia marcescens, E, coli and Salmonella typhosa were killed in 10 seconds by all concentrations of ethanol from 40% to 100%. The gram-positive organisms Staphylococcus aureus and Streptococcus pyogenes were slightly more resistant, being killed in 10 seconds by ethyl alcohol concentrations of 60%–95%. Isopropyl alcohol (isopropanol) was slightly more bactericidal than ethyl alcohol for E. coli and S. aureus .
Aldehydes: (Formaldehyde, Paraformaldehyde, Glutaraldehyde)
Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group, and probably damages nucleic acids. It kills all microorganisms, including spores. Examples: Formaldehydeand Gluteraldehyde
Application: 40% Formaldehyde (formalin) is used for surface disinfection and fumigation of rooms, chambers, operation theatres, biological safety cabinets, wards, sick rooms etc. Fumigation is achieved by boiling formalin, heating paraformaldehyde or treating formalin with potassium permanganate. It also sterilizes bedding, furniture and books. 10% formalin with 0.5% tetraborate sterilizes clean metal instruments. 2% gluteraldehyde is used to sterilize thermometers, cystoscopes, bronchoscopes, centrifuges, anasethetic equipments etc. An exposure of at least 3 hours at alkaline pH is required for action by gluteraldehyde. 2% formaldehyde at 40o C for 20 minutes is used to disinfect wool and 0.25% at 60o C for six hours to disinfect animal hair and bristles.
Disadvantages: Vapors are irritating (must be neutralized by ammonia), has poor penetration, leaves non-volatile residue, activity is reduced in the presence of protein. Gluteraldehyde requires alkaline pH and only those articles that are wettable can be sterilized.
Formaldehyde – and its polymerized solid paraformaldehyde have broad-spectrum biocidal activity and are both effective for surface and space decontamination. As a liquid (5% concentration), formaldehyde is an effective liquid decontaminant. Its biocidal action is through alkylation of carboxyl, hydroxyl and sulfhydryl groups on proteins and the ring nitrogen atoms of purine bases. Formaldehyde’s drawbacks are reduction in efficacy at refrigeration temperature, its pungent, irritating odor, and several safety concerns. Formaldehyde is presently considered to be a carcinogen or a cancer-suspect agent according to several regulatory agencies.
Paraformaldehyde – is a solid polymer of formaldehyde. Paraformaldehyde generates formaldehyde gas when it is depolymerized by heating to 232 to 246°C (450 to 475°F); the depolymerized material reacts with the moisture in the air to form formaldehyde gas. This process is used for the decontamination of large spaced and laminar-flow biological safety cabinets when maintenance work or filter changes require access to the sealed portion of the cabinet. A neutralization step, heating ammonium carbonate, is required prior to ventilation of the space. Formaldehyde gas can react violently or explosively (7.0 – 73% v/v in air), when exposed to incompatibles, therefore, only individuals that have specific training and have been approved by the Dept. of Environmental Health & Safety are permitted to use this gas.
Glutaraldehyde – is a colorless liquid and has the sharp, pungent odor typical of all aldehydes, with an odor threshold of 0.04 parts per million (ppm). It is capable of sterilizing equipment, though to effect sterilization often requires many hours of exposure. Two percent solutions of glutaraldehyde exhibit very good activity against vegetative bacteria, spores and viruses. It is ten times more effective than formaldehyde and less toxic. However, it must be limited and controlled because of its toxic properties and hazards. It is important to avoid skin contact with glutaraldehyde as it has been documented to cause skin sensitization. Glutaraldehyde is also an inhalation hazard. The NIOSH ceiling threshold limit value is 0.2 ppm.
Cidex, a commercially prepared glutaraldehyde disinfectant is used routinely for cold surface sterilization of clinical instruments. Glutaraldehyde disinfectants should always be used in accordance with the manufacturer’s directions.