In microbial research, sterilization is a preventive technique. It is defined as a process of intentionally killing, removing and deactivating all types of microbes including non-pathogenic, pathogenic and spore-producing microorganisms in order to proceed microbiological research work.
Decontamination is the process of cleaning laboratory materials from any form of hazardous materials like culture, chemicals or other materials which can support the growth of microorganisms. Once the experiment is completed or the experimental culture is contaminated it should be properly decontaminated and cleaned from the laboratory. The dispose or reuse of the material is depending on the materials usage and decontamination procedure. Glassware can be reused until it breaks.
Decontamination is the reduction or removal of chemical agents. Decontamination may be accomplished by removal of these agents by physical means or by chemical neutralization or detoxification. Decontamination of skin is the primary concern, but decontamination of eyes and wounds must also be done when necessary. Personal decontamination is decontamination of self; casualty decontamination refers to the decontamination of casualties; and personnel decontamination usually refers to decontamination of non-casualties.
- Neutralization of all agents
- Safety (compound to be both nontoxic and noncorrosive)
- Ease of application by hand
- Readily available
- Rapid action
- Nonproduction of toxic end products
- Stability in long�term storage
- Short�term stability (after issue to unit/individual)
- Affordability
- Nonenhancement of percutaneous agent absorption
- No irritability
- Hypoallergenicity
- Ease of disposal
Heat
Filtration
Radiation
Physical Methods of Controling Microorganisms: (A Historical Perspective)
Some Earliest Methods:
As early as the Stone Age, it is likely that humans were already using some physical methods of microbial control to preserve foods. Drying (desiccation) and salting (osmotic pressure) were probably among the earliest techniques. Ancient Egyptians dried perishable foods to preserve them. Scandinavians made holes in the centers of pieces of dry, flat, crisp bread in order to hang them in the air of their homes during the winter; likewise they kept seed grains in a dry place. Otherwise, both flour and grains would have molded during the long and very moist winters. Europeans used heat I the food-canning process 50 years before Pasteur's work explained why heating prevented food from spoiling.
Control in the Modern Age:
Today, physical agents that destroy microorganism are still used in food preservation and preparation. Such agents remain a crucial weapon in the prevention of infectious disease. Physical antimicrobial agents include various forms of heat, refrigeration, desiccation (drying), irradiation, and filtration.
Factors Effecting the Selection of Method:
When selecting methods of microbial control, consideration must be given to effects on things besides the microbes. For example, certain vitamins or antibiotics in solution might be inactivated by heat. Many laboratory or hospital materials, such as rubber and latex tubing, are damaged by repeated heating. There are also economic considerations; for example it may be less expensive to use presterilized, disposable plasticware than to repeatedly wash and re-sterilize glassware.
METHODS OF DECONTAMINATION
Three basic methods of decontamination are physical removal, chemical deactivation, and biological deactivation of the agent. Biological deactivation has not been developed to the point of being practical.
PHYSICAL REMOVAL
Several types of physical and chemical methods are at least potentially suitable for decontaminating equipment and material. Flushing or flooding contaminated skin or material with water or aqueous solutions can remove or dilute significant amounts of agent. Scraping with a wooden stick, i.e., a tongue depressor or popsicle stick, can remove bulk agent by physical means. For the decontamination of clothing only, adsorbents and containment materials (to be used on outer garments before their removal and disposal) have been considered. A significant advantage of most physical methods is their nonspecificity. Since they work nearly equally well on chemical agents regardless of chemical structure, knowledge of the specific contaminating agent or agents is not required.
Flushing with Water or Aqueous Solutions
When animal skin contaminated with GB was flushed with water (a method in which physical removal predominates over hydrolysis of the agent), 10.6 times more GB was required to produce the same mortality rate as when no decontamination occurred. In another study, the use of water alone produced better results than high concentrations of hypochlorite (i.e., 5.0% or greater, which are not recommended for skin). Timely copious flushing with water physically removes the agent and will produce good results.
Adsorbent Materials
Adsorption refers to the formation and maintenance of a condensed layer of a substance, such as a chemical agent, on the surface of a decontaminant as illustrated by the adsorption of gases by charcoal particles and by the decontaminants described in this section. Some NATO nations use adsorbent decontaminants in an attempt to reduce the quantity of chemical agent available for uptake through the skin. In emergency situations dry powders such as soap detergents, earth, and flour, may be useful. Flour followed by wiping with wet tissue paper is reported to be effective against GD, VX and HD.
M291 Resin
The current method of battlefield decontamination by the individual soldier involves the use of a carbonaceous adsorbent, a polystyrene polymeric, and ion exchange resins (M291). The resultant black resin is both reactive and adsorbent. The M291 Kit has been extensively tested and proven highly effective for skin decontamination. It consists of a wallet-like carrying pouch, containing 6 individual decontamination packets. Each packet contains a non-woven fiber-fill laminated pad impregnated with the decontamination compounds. Each pad provides the individual with a single step, non-toxic/non-irritating decontamination application, which can be used on the skin, including the face and around wounds. Instructions for use are marked on the case and packets. The individual decontamination pads are impregnated with the decontamination compound "Ambergard XE-555 Resin", which is the black, free-flowing, resin based powder. As the pad is scrubbed over the contaminated skin the chemicals are rapidly transferred into and trapped in the interior of the resin particles. The presence of acidic and basic groups in the resin promotes the destruction of trapped chemical agents by acid and base hydrolysis. Because the resin is black it maps out the areas that have been decontaminated.
CHEMICAL METHODS
Three types of chemical mechanisms have been used for decontamination: water/soap wash; oxidation; and acid/base hydrolysis.
HD (mustard) and the persistent nerve agent VX contain sulfur molecules that are readily subject to oxidation reactions. VX and the other nerve agents (GA, GB, GD, and GF) contain phosphorus groups that can be hydrolyzed. Therefore, most chemical decontaminants are designed to oxidize HD and VX and to hydrolyze nerve agents (VX and the G series).
Water/Soap Wash
Both fresh water and sea water have the capacity to remove chemical agents not only through mechanical force but also via slow hydrolysis; however, the generally low solubility and slow rate of diffusion of CW agents in water significantly limit the agent hydrolysis rate.
The predominant effect of water and water/soap solutions is the physical removal or dilution of agents; however, slow hydrolysis does occur particularly with alkaline soaps. In the absence of hypochlorite solutions or other appropriate means of removing chemical agents, these methods are considered reasonable options.
Oxidation/Hydrolysis
The most important category of chemical decontamination reactions is oxidative chlorination. This term covers the "active chlorine" chemicals like hypochlorite. The pH of a solution is important in determining the amount of active chlorine concentration. An alkaline solution is advantageous. Hypochlorite solutions act universally against the organophosphorus and mustard agents.
Both VX and HD contain sulfur atoms that are readily subject to oxidation. Current doctrine specifies the use of a 0.5% sodium or calcium hypochlorite solution for decontamination of skin and a 5% solution for equipment.
Hydrolysis
Chemical hydrolysis reactions are of two types: acid and alkaline. Acid hydrolysis is of negligible importance for agent decontamination because the hydrolysis rate of most chemical agents is slow, and adequate acid catalysis is rarely observed. Alkaline hydrolysis is initiated by the nucleophilic attack of the hydroxide ion on the phosphorus atoms found in VX and the G agents. The hydrolysis rate is dependent on the chemical structure and reaction conditions such as pH, temperature, the kind of solvent used, and the presence of catalytic reagents. The rate increases sharply at pH values higher than 8 and increases by a factor of four for every 10oC rise in temperature. Several of the hydrolytic chemicals are effective in detoxifying chemical warfare agents; unfortunately, many of these (e.g., NaOH) are unacceptably damaging to the skin. Alkaline pH hypochlorite hydrolyzes VX and the G agents quite well.
Three basic methods of decontamination are physical removal, chemical deactivation, and biological deactivation of the agent. Biological deactivation has not been developed to the point of being practical.
PHYSICAL REMOVAL
Several types of physical and chemical methods are at least potentially suitable for decontaminating equipment and material. Flushing or flooding contaminated skin or material with water or aqueous solutions can remove or dilute significant amounts of agent. Scraping with a wooden stick, i.e., a tongue depressor or popsicle stick, can remove bulk agent by physical means. For the decontamination of clothing only, adsorbents and containment materials (to be used on outer garments before their removal and disposal) have been considered. A significant advantage of most physical methods is their nonspecificity. Since they work nearly equally well on chemical agents regardless of chemical structure, knowledge of the specific contaminating agent or agents is not required.
Flushing with Water or Aqueous Solutions
When animal skin contaminated with GB was flushed with water (a method in which physical removal predominates over hydrolysis of the agent), 10.6 times more GB was required to produce the same mortality rate as when no decontamination occurred. In another study, the use of water alone produced better results than high concentrations of hypochlorite (i.e., 5.0% or greater, which are not recommended for skin). Timely copious flushing with water physically removes the agent and will produce good results.
Adsorbent Materials
Adsorption refers to the formation and maintenance of a condensed layer of a substance, such as a chemical agent, on the surface of a decontaminant as illustrated by the adsorption of gases by charcoal particles and by the decontaminants described in this section. Some NATO nations use adsorbent decontaminants in an attempt to reduce the quantity of chemical agent available for uptake through the skin. In emergency situations dry powders such as soap detergents, earth, and flour, may be useful. Flour followed by wiping with wet tissue paper is reported to be effective against GD, VX and HD.
M291 Resin
The current method of battlefield decontamination by the individual soldier involves the use of a carbonaceous adsorbent, a polystyrene polymeric, and ion exchange resins (M291). The resultant black resin is both reactive and adsorbent. The M291 Kit has been extensively tested and proven highly effective for skin decontamination. It consists of a wallet-like carrying pouch, containing 6 individual decontamination packets. Each packet contains a non-woven fiber-fill laminated pad impregnated with the decontamination compounds. Each pad provides the individual with a single step, non-toxic/non-irritating decontamination application, which can be used on the skin, including the face and around wounds. Instructions for use are marked on the case and packets. The individual decontamination pads are impregnated with the decontamination compound "Ambergard XE-555 Resin", which is the black, free-flowing, resin based powder. As the pad is scrubbed over the contaminated skin the chemicals are rapidly transferred into and trapped in the interior of the resin particles. The presence of acidic and basic groups in the resin promotes the destruction of trapped chemical agents by acid and base hydrolysis. Because the resin is black it maps out the areas that have been decontaminated.
CHEMICAL METHODS
Three types of chemical mechanisms have been used for decontamination: water/soap wash; oxidation; and acid/base hydrolysis.
HD (mustard) and the persistent nerve agent VX contain sulfur molecules that are readily subject to oxidation reactions. VX and the other nerve agents (GA, GB, GD, and GF) contain phosphorus groups that can be hydrolyzed. Therefore, most chemical decontaminants are designed to oxidize HD and VX and to hydrolyze nerve agents (VX and the G series).
Water/Soap Wash
Both fresh water and sea water have the capacity to remove chemical agents not only through mechanical force but also via slow hydrolysis; however, the generally low solubility and slow rate of diffusion of CW agents in water significantly limit the agent hydrolysis rate.
The predominant effect of water and water/soap solutions is the physical removal or dilution of agents; however, slow hydrolysis does occur particularly with alkaline soaps. In the absence of hypochlorite solutions or other appropriate means of removing chemical agents, these methods are considered reasonable options.
Oxidation/Hydrolysis
The most important category of chemical decontamination reactions is oxidative chlorination. This term covers the "active chlorine" chemicals like hypochlorite. The pH of a solution is important in determining the amount of active chlorine concentration. An alkaline solution is advantageous. Hypochlorite solutions act universally against the organophosphorus and mustard agents.
Both VX and HD contain sulfur atoms that are readily subject to oxidation. Current doctrine specifies the use of a 0.5% sodium or calcium hypochlorite solution for decontamination of skin and a 5% solution for equipment.
Hydrolysis
Chemical hydrolysis reactions are of two types: acid and alkaline. Acid hydrolysis is of negligible importance for agent decontamination because the hydrolysis rate of most chemical agents is slow, and adequate acid catalysis is rarely observed. Alkaline hydrolysis is initiated by the nucleophilic attack of the hydroxide ion on the phosphorus atoms found in VX and the G agents. The hydrolysis rate is dependent on the chemical structure and reaction conditions such as pH, temperature, the kind of solvent used, and the presence of catalytic reagents. The rate increases sharply at pH values higher than 8 and increases by a factor of four for every 10oC rise in temperature. Several of the hydrolytic chemicals are effective in detoxifying chemical warfare agents; unfortunately, many of these (e.g., NaOH) are unacceptably damaging to the skin. Alkaline pH hypochlorite hydrolyzes VX and the G agents quite well.
In the laboratory, sterilization can be achieved by
1. Physical Methods
- Heat
- Filtration
- Radiation
2. Chemical Methods
- Solids
- Liquids
- Gases
3. Fumigation
The most common terms relevant to sterilization and decontamination are
Antisepsis: It is a process of destructing or inhibiting the growth of microorganisms to prevent infection in the living being.
Antiseptic: It is a chemical agent applied to prevent further infection in skin or tissue. It should destruct the pathogenic flora, not the skin or tissue. E.g: hydrogen peroxide, alcohol, iodine, boric acid etc.
Biocide: An active chemical substance which is used to destroy microorganism by biological or chemical means. E.g: chlorine.
Disinfectant: A chemical which is used to clean microbial flora. It is preferably applicable to a larger surface area. The disinfectants are not active against spore-producing microorganisms. E.g: ammonium salts, formaldehyde, bleach, chloramine, chlorine oxide.
Germicide: A chemical substance that destroys pathogenic microorganisms. E.g: bisphenols.
Sanitizer: A chemical solution that is used to clean material and reduce the microbial contamination to an acceptable '"safe level". E.g: alkaline detergents.
Sanitization: The process of cleaning the experimental objects without sterilization. The chemical liquid which is used to clean the material and reduce the microbial contamination.
Sterilization by dry heat is the most efficient method of sterilizing laboratory glassware and surgical materials.
Decontamination by Dry heat:
Mode of Action :
Dry heat probably does most of its damage by oxidizing molecules. A simple analogy is the slow charring of paper in a heated oven, even when the temperature remains below the ignition point of paper.
Low Temperature
Mode of Action:
The effect of low temperatures on microorganisms depends on the particular microbe and the intensity of application. For example, at temperatures of ordinary refrigerators (0 ˚ C), the metabolic rate of some microbes is so reduced that they cannot reproduce or synthesize toxins. In other words, ordinary refrigeration has a bacteriostatic effect, but does not kill many microbes. Heat is much more effective than cold at killing microorganism.
Disadvantage:
Yet psychrotrophs do grow slowly at refrigerator temperatures and will alter the appearance and taste of foods after a time. For example, a single microbe reproducing only three times a day would reach a population of more than 2 million within a week.
Advantage by Medical Point of View:
Pathogenic bacteria generally will not grow at refrigerator temperature.
Uses of Cold temperature:
Refrigeration is used to prevent food spoilage. Freezing, drying, and freeze-drying are used to preserve both foods and microorganism, but these methods do not achieve sterilization.
Optimum Conditions:
Surprisingly, some bacteria can grow at temperatures several degrees below freezing. Most foods remain unfrozen until -2oC or lower. Rapidly attained subfreezing temperatures tend to render microbes dormant but do not necessarily kill them. Slow freezing is more harmful to bacteria; the ice crystals that form and grow disrupt the cellular and molecular structure of the bacteria. Thawing, being inherently slower is actually the more damaging part of a freeze-thaw cycle. Once frozen, one third of the population of some vegetative bacteria might survive a year, whereas other species might have very few survivors after this time.
Results of Low Temperature Treatment:
Many eukaryotic parasites, such as the roundworms that cause trichinosis, are killed by several days of freezing temperatures.
Conditions:
Many fresh foods can be prevented from spoiling by keeping them at 5 ° C (ordinary refrigerator temperature).
Limitations:
However, storage should be limited to a few days because some bacteria and molds continue to grow at this temperature. To convince yourself of this, recall some of the strange things you have found growing on left over of the back of your refrigerator. In rare instances strains of Clostridium botulinum have been found growing and producing lethal toxins in a refrigerator when the organism were deep within a container of food, where anaerobic conditions exist.
Freezing:
Uses of Freezing:
Freezing at -20 ° C is used to preserve foods in homes and in the food industry. Although freezing does not sterilize foods, it does significantly slow the rate of chemical reactions so that microorganism does not cause food to spoil. Frozen foods should not be thawed and refrozen. Repeated freezing and thawing of foods causes large ice crystals to form in the foods during slow freezing. Cell membranes in the foods are ruptured, and nutrients leak out. The texture of foods is thus altered, and they become less palatable. It also allows bacteria to multiply while food is thawed, making the food more susceptible to bacterial degradation.
Freezing can be used to preserve microorganisms, but this requires a much lower temperature than that used for food preservation. Microorganism are usually suspended in glycerol or protein to prevent the formation of large ice crystal (which could puncture cells), cooled with solid carbon dioxide (dry ice) to a temperature of -78 ° C, and then held there. Alternatively, they can be placed in liquid nitrogen and cooled to – 180 ° C.
Freeze Drying
Freeze-drying, or lyophilization, is the drying of a material from the frozen state. This process is used in the manufacture of some brands of instant coffee; freeze-dried instant coffee has a more natural flavor than other kinds. Microbiologists use lyophilization for long-term preservation rather than destruction of cultures of microorganisms. Organisms are rapidly frozen in alcohol and dry ice or in liquid nitrogen and are then subjected to a high vacuum to remove all the water while in the frozen state. Rapid freezing allows only very tiny ice crystal to form in cells, so the organisms survive this process. Organism so treated can be kept alive for years, store under vacuum in the freeze-dried state.
Drying
Drying can be used to preserve foods because the absence of water inhibits the action of enzymes. Many foods, including peas, beans, raisins, and other fruits, are often preserved by drying. Yeast used in baking also can be preserved by drying. Endospores presents on such foods cab survive drying, but they do not produce toxins. Dried pepperoni sausage and smoked fish retain enough moisture for microorganism to grow. Because smoked fish is not cooked, eating it poises a risk of infection. Sealing such fish in plastic bags creates conditions that allow anaerobes such as Clostridium botulinum to grow.
Drying also naturally minimizes the spread o infectious agents. Some bacteria, such as Treponema pallidum, which causes syphilis, are extremely sensitive to drying and die almost immediately on a dry surface; thus they can be prevented from spreading by keeping toilet seats and other bathroom fixtures dry. Drying of laundry in dryers or in the sunshine also destroys pathogens.