Chapter 10. STERILIZATION AND DISINFECTION I. H. SIDDIQUE

 

10.1 CHAPTER REVIEW:

The contributions of many scientists over the century have led us to practice procedures which help us to protect from many pathogenic and infectious agents. Physical methods which are used for destruction of pathogenic as well as non-pathogenic organisms are employed which include heat, freezing, high speed centrifugation, gaseous sterilization, radiation, filtration, and mechanical crushing. These physical methods of sterilization are explained in detail in the first section of this chapter.

The disinfectants were developed for the same purpose over a period of time to remove or destroy infectious and others organisms that are sensitive to these chemical agents. These chemical agents, their mechanism of action and their applications are described in the second section of this chapter.

10.2 Introduction.

Microorganisms are present everywhere and one must be constantly aware of the living invisible world. There is a strong need to kill bacteria when and where their presence is undesirable. Therefore, many situations such as preparation of surgical operations, microbiological studies, disinfection of infectious materials call forth the need and use of methods to destroy them.

The destruction of microorganisms may be achieved by physical and chemical means. In general, the former method is called sterilization while the later is termed disinfection. Sterilization is always aimed at both pathogenic and nonpathogenic bacteria, while disinfection, in its true sense, applies only to the pathogenic ones. A disinfectant is any agent, such as heat or a chemical (like iodine) that kills pathogenic microorganisms. The term generally applies to preparations, usually liquids, intended for use on inanimate objects, as distinguished from living tissues (antiseptics). Sterilization is achieved by any process that completely removes or destroys all living organisms in or on an object.

Many general terms, such as bactericide, are used to designate methods of destroying bacteria. Note these terms - germicide, algacide, fungicide, and virucide which are used to kill germs, algae, fungi and virus respectively. The term antiseptic is commonly applied when chemicals are used to inhibit the growth and multiplication of bacteria but do not necessarily destroy them (synonymous term - bacteriostatis). When no pathogenic bacteria are present, the term aseptic is used. Asepsis may be brought about by physical or chemical methods. Certain antiseptics or disinfectants that are used to prevent deterioration of foods, serums, and vaccines are known as preservatives. A deodorant is anything, usually a chemical, which removes or masks odors.

 10.3 General Information.

Before inhibition and destruction of microorganisms are discussed it is important to review some of the characteristics of microorganisms. All microorganisms are composed of complex organic materials, largely protein in nature; some vegetative cells have a protective substance such as wax closely associated with the cells, eg., the tubercle bacillus; some cell are capable of producing resistant forms, eg., all species of genera Bacillus and Clostridium produce thermostable spores, and certain protozoa produce cysts; and microorganisms differ in susceptibility to adverse changes in their environment.

10.4 Physical Methods for the control of microorganisms:

A. Heat in general is the most practical and efficient method of sterilization. The following types of heat treatments are used for this purpose. The heat used for this purpose may be in two forms: Dry heat and Moist heat.

l. Dry heat (incineration or burning) is effective in removing infectious materials and is used in the following ways:

• a. The platinum loop used to transfer cultures and disposal of animal carcasses with infectious diseases.
• b. Hot air sterilization. Glassware, metal wares, etc. are sterilized at 170`C (340`F) for 1 hour in an oven.
• c.Moist heat applied in the form of hot water or steam.

2. The moist heat is more effective and is used in the following ways:

a. Sterilization by boiling kills vegetative forms of pathogenic bacteria, fungi, and viruses but this is not a reliable method of sterilization. The addition of 2% sodium carbonate in boiling water hastens the destruction of spores and helps to prevent the rusting of instruments.

b. Sterilization by steam may be applied free-flowing or under pressure. Free-flowing steam has about the same sterilizing action as boiling water. However, steam under pressure or autoclaving is one of the most effective methods of sterilization. Steam under pressure is hotter than free-flowing steam, and the higher the pressure, the higher the temperature, eg. the temperature of steam is 121`C. The following table shows the temperature of steam at various pressures.

Pressure-Temperature Relations in Autoclave

(Figure based upon complete replacement of air by steam).

 

Pressure in Pounds Temperature 0C 0F

---

5 108 226

10 116 240

15 121 250

20 127 260

25 131 267

30 134 274

---

 

Source: Difco Manual, Difco Laboratories, Inc. Detroit, Michigan

 Usually steam under a 15 or 20 pounds pressure will kill all organisms and spores in 15-20 minutes. In order to maintain the needed temperatures at higher altitudes, the pressure must be increased-one pound for each 2,000 feet of increase in altitude. Moist heat has a greater penetrating power and results in protein coagulation. Operation of the autoclave will be explained in greater details in the laboratory.

c.Fractional Sterilization (Intermittent sterilization or tyndallization). This consists of exposing the material to free-flowing steam at the at the atmospheric pressure for 30 minutes on three successive days; between times it is stored under conditions suitable for bacterial growth. This can be used only to sterile media which cannot withstand autoclaving and can support spore germination.

d. Pasteurization: Consists of heating milk or other liquids at an increased temperature for a short time. This procedure destroys non-spore bearing pathogenic or other undesirable microorganisms without affecting the composition of the material being treated. Usually milk is exposed to 143`F (62`C) for 30 minutes in holding method or for 161`F (72`C) for 12 seconds in continuous flow or flash method for pasteurization.

C.Sterilization by other Physical Means

• l. Ionizing Rays - X-ray and gamma rays are much more powerful and penetrating than ultraviolet light. Although foods have been preserved experimentally by treatment with ionizing rays, their use in the food industry is limited. Cathode rays (electron beams) are also important; their sterilization power is being studied for commercial use. Although electrons do not penetrate readily, they have been effective in sterilizing thinly wrapped food products such as meat and other prepacked materials including sutures and plastic tubings.
• 2. Ultraviolet light - Since ultraviolet rays in the sunlight have been found to be an effective germicide, ultraviolet light is being used to control the spread of pathogenic organisms especially in operating rooms, laboratories etc. However, it does not have any penetrating power. Short wave lengths cover a range from partial visibility to total darkness-that a is wave length of 2000 A (angstroms) to 2800 A, with the maximum killing effect as 260 A (1A=1/10,000um). It inhibits the replication of DNA.
• 3. Filtration - Substances, such as serums, cannot be subjected to heat or chemical treatment (without decomposition) may be sterilized by this procedure. Sonic vibrations, trituration, Agitation - Supersonic or ultrasonic is used to indicate sound waves so high pitched that they are not audible. These waves have been demonstrated to coagulate protein solutions and destroy bacteria.
• 4. Cold temperatures - Cold temperatures and freezing cannot be used as a means of sterilization, since many bacteria survive at low temperatures for long periods of time. A technique known as lyophilization or freez-drying is used to preserve microorganisms. In this procedure, the organisms are frozen rapidly and then dehydrated in high vacuum directly from the frozen state. Repeated freezing and thawing may kill bacteria.
• 5. Osmotic pressure - when the concentration of the solution surrounding the bacterial cell is altered, it will result in plasmolysis. The use of brine or syrup in food preservation, exemplifies this principle.

10.5 CHEMICAL METHODS OF STERILIZATION

10.5.1 Introduction.

Disinfectants have lethal effects on all types of cells and organisms. Disinfectants act directly on cell structures. They do not require specific metabolic activities on the part of the organisms. Since many disinfectants are used for different purposes, it is important to know the relative germicidal activity of these disinfectants.

10.5.2 Standardization of Disinfectants.

Rober Koch (1881) devised a method in which dried anthrax spores were exposed to the disinfectant, washed, and transferred to a solid medium in order to ascertain whether all the spores had been killed. Later Kronig and Paul (1897) modified this procedure and used garnets instead of threads. After putting the organisms on the garnet and after drying, the garnets were exposed to the disinfectant, washed and plated to count the colonies. This test was further improved by Rideal and Walker (1903) in which similar quantities of organisms were subjected to the action of phenol and the germicide to be tested. Subcultures were made into broth every 5 minutes up to 15 minutes and the tubes incubated at 37 0 C for three days. In l931, Food and Drug Administration improved and adopted this method of testing which was later adopted in 1950 by the Association of Agricultural Chemists. This is a test of the germicidal activity of disinfectants as compared to phenol. The method is limited to phenol-like compounds and is less satisfactory for other agents, which may differ in their action. By using this method the phenol coefficient of a disinfectant can be determined.

10.2.2.1 Phenol Coefficient-

The phenol coefficient of a compound is the ratio of the minimal sterilizing concentration of phenol (under standard conditions) to that of the compound. Two organisms which are used in this procedure are Salmonella typhosa (Hopkin's strain) and Staphylococcus aureus (FDA strain 209). The results of the phenol coefficient can be used

• (1) to compare the germicidal activity of the disinfectant with that of phenol,
• (2) to compare relative germicidal efficiency of different phenol-like compounds, and
• (3) to serve as a means for determining the effective dilutions of a given disinfectant for use in practice. If phenol coefficient of a given disinfectant is greater than l, it will indicate that this disinfectant is stronger than phenol.

  10.5.2.2. Toxicity Index-

  The use of various chemicals to disinfect skin and other tissues prior to surgical operations and for the treatment of infections has prompted the development of methods which measure the toxicity of such antiseptics for use on tissues. An ideal antiseptic, obviously, would be one which would kill bacteria but would not injure tissue cells. Investigators have devised methods to measure the toxicity index of a disinfectant. This is expressed as the highest dilution of disinfectant required to prevent the growth of embryonic tissue divided by the highest dilution of the same disinfectant which will kill the test organism like S. aureus. The following table shows the efficiency of some germicides: 

 

TABLE I Comparative Efficiency of some Germicides Germicide Highest dilution which Highest dilution Toxicity Phenol prevents growth of which prevents Index A/B Coeffi- tissue (A) growth of S. cient (as aureus (B) reported) .TB 1.30" 3.60" 5.70" 7.20" Iodine 1800 20000 0.09 308 Hgcl2 45000 16000 2.8 246 Metaphen 76000 6000 12.7 92 Phenol 840 65 12.9 --- Merthiolate 176400 4000 35 76 Mercurochrome 10500 40 262 6 Iodine is the best of these germicides as it has high toxicity for germs and less to tissue.

10.5.2.3 Chemical Antagonism.

• The interference by a chemical agent with a normal reaction between a specific enzyme and its substrate is known as chemical antagonism. This may be due to antagonists of energy-yielding processes (example, poisons of respiratory enzymes) or antagonists of biosynthetic processes (example, analogous of the building blocks of proteins, amino acids).

  10.5.2.4 Characteristics of an Ideal Disinfectant -

  The most important characteristics of an ideal disinfectant are:

  l. Germicidal power - most commercial disinfectants, particularly the coal-tar products, are sold upon the basis of their phenol coefficient.

  2. Stability - should be relatively stable in the presence of organic matter.

  3. Homogeneity - should be homogeneous in composition.

  4. Solubility - dissolves in all proportions in water. Alcohol is a typical example.

  5. Non-toxic to higher life eg., arsphenamine in the treatment of infections.

  6. Non-corrosive - should not attack metal, injure fabrics, leave stains, or bleach color.

  7. Penetration - penetrates rapidly and efficiently.

  8. Economy - Low in cost.

  9. Power to remove dirt and grease dissolves or remove grease and all kinds of dirt efficiently.

  l0.Deodorizing power - combines with and destroys malodorous substances.

  10.5.2.5 Specific Chemical Agents

  One of the criteria for using an antibacterial agent is that it must be safe for the host under the conditions of its use. The following groups of chemicals are used as antibacterial agents.

• 1. Acids. Sulfuric acid and hydrochloric acid are toxic on tissues and therefore, their use is limited. Hydrogen-Ion is responsible for the killing power of the acids. Some organic acids such as acetic acid are used in food preservation. Another example is the preservation of silage by acids formed by organisms.
• 2. Alkali. Hydroxyl group (OH) is an active portion of the base for its antibacterial action. Sodium hydroxide (lye) is used in dairy industry. Note tubercle bacilli are resistant to this chemical.
• 3. Oxidizing agents. These agents Inactivate bacterial cells by oxidizing free sulfhydryl groups. Hydrogen peroxide, for example, is used in the treatment of deep wounds. These agents act on the catalase present in the tissues and liberate oxygen which inhibits the growth of the anaerobes eg. tetanus organisms.
• 4. Halogens. The halogens, eg. chlorine and iodine are most valuable in the processing of drinking water and 1ppm is enough to kill Salmonella typhosa and other members of the enteric group. Fluorides are also used as 3 ppm in drinking water to prevent dental caries. Iodine is incorporated with non-ionic detergents to retain its activity but to reduce its harmful effects, an example of such organic Iodine compound is Iodoform.
• 5. Heavy metals. Salts of metals such as mercury result in coagulation of proteins and are not used on human tissues in higher concentrations. In lower concentrations, they act by combining with sulfhydryl groups. Mercury is used in combination with organic compounds to reduce its toxic action. Examples of such preparations are merthiolate and mercurochrome. Except when used on clean skin surfaces these organic mercurials are of doubtful practical value; they are readily inactivated by extraneous organic matter.
• 6. Alcohols. Ethyl alcohol and isopropyl alcohol are commonly used. They act as protein coagulants and in a 70% concentration, are most effective. They are effective against most of the bacteria but are completely ineffective on spores. These are good wetting agents and spread readily resulting in diffused contact with the organisms. These are widely used in the hospitals for disinfection of thermometers etc., and an exposure for l0 minutes to alcohol is considered adequate.
• 7. Phenols. Phenol and phenolic compounds coagulate protein when used in 1-2% concentration; in lower concentrations, however, their mechanism of action may probably not the same as their action may be reversed by exposing cells to a component of yeast extract. Creosols which are derived from phenols, are superior and cheaper and are used in cleaning cages etc.
• 8. Detergents or synthetic Soaps. Those compounds have the property of concentrating at interfaces and are called detergents or surface active agents. They are both wetting and emulsifying agents, non-toxic to living tissues and are active in the presence of organic matter. These are used in cosmetics, dyeing, oils, food, and related industries. These agents either dissolve in the constituent of the cell membrane or combine with them chemically and thus change the surface of the cell so as to disrupt its essential functions. On the basis of their chemical structure and surface-tension reducing (surfactant) property,the detergents are divided into the following three main groups.
  • a. Anionic detergents.

    In this group the long-chain hydrocarbon has a negative charge when ionized and are more effective in the acid range. These are mainly effective against gram-positive organisms. Soaps (sodium salts of long-chain carboxylic acids), bile salts and sodium lauryl sulfate in which the surface tension reducing property resides in the negatively charged part of the molecule are included in this group.

    b. Cationic detergents-

    The fat-soluble moiety can be made to have a positive charge by combining it with a quaternary nitrogen atom. These are most active in an alkaline range and are equally effective against both gram-positive and gram-negative organisms. From disinfection point of view, the quaternary ammonium chloride derivatives (called quats) such as Zephiran and Diaperene chloride are very effective.

    c. Non-ionic detergents-

    They do not ionize in aqueous solution and probably do not interfere with the antibacterial active agents. They are excellent foaming and solubilizing agents and are often added with cationic formulations to make germicidal-detergent combinations. These are mainly complex ethers and polyglycerol soaplike compounds represented by Tween 80.

  9. Alkylating Agents- The following chemicals are alkylating agents and are employed for sterilization:

  • a. Ethylene oxide.

    The gas is applied in special autoclaves under carefully controlled conditions of temperature and humidity. Since pure ethylene oxide is explosive and an irritant, it is mixed with carbon dioxide or other diluent in various proportions. Ethylene oxide is used by commercial companies that dispense sterile packages of a variety of products.

    b. Beta-Propiolactone (BPL).

    At about 200 C this substance is a colorless liquid and is unstable at room temperatures. Aqueous solutions effectively inactivate some viruses, including poliomyelitis and rabies, and kill bacteria and bacterial spores. It appears to act by forming chemical compounds with cell proteins. Aqueous solutions of BPL can be used to sterilize biological materials such as virus vaccines, tissue for grafting and plasma.

  10.5.2.6 Mechanisms of Antiseptic and Disinfectant Action

  The major factors that determine how a disinfectant acts are concentration of disinfectants, time during which the disinfectant is allowed to act, temperature of disinfection, number and types of microorganisms present, and the nature of the material being disinfected. In order to kill an organism or prevent its growth the disinfectant must affect some vital part of the cell. Parts of the cell most susceptible to the action of a disinfectant are the protoplasmic membrane, and certain enzymes, the nucleus, or even structural proteins such as those found in the cell wall. Some of these mechanims of action are:

  • l. By oxidation of the microbial cell
  • 2. By hydrolysis
  • 3. Combination with microbial protein to form salt
  • 4. Coagulation of proteins
  • 5. Modification of the Permeability of the microbial plasma membrane
  • 6. Inactivation of vital enzymes of microorganisms
  • 7. Disruption of the Cell

  Note - Important features of a disinfectant is its ability to form combinations with microbial cells that are destructive to microbes.