The terms “antiseptic” and “disinfectant” are often confused and misused in micro- biology and medicine. Typically, “antiseptic” refers to an agent used to minimize, destroy, or remove microbial population on a living surface, such as the skin of a person who needs to be prepared for injection or a surgical procedure. A disinfectant, on the other hand, is a substance used to eliminate or minimize microbial presence on an inanimate surface, such as a work bench, glassware, or surgical instruments. It is noteworthy that both antiseptics and disinfectants can either be a microbicide or a microbistatic. In this chapter, we will treat the two entities together under the banner of control of microbial population. The microbial population in or on a surface, material, or product can be controlled, minimized, or eradicated either by physical means or by chemical means, some of which are summarized below.
PHYSICAL CONTROL OF MICROORGANISMS
Heat
Traditionally, heat application has been the most preferred means of controlling microbial population. This is achieved by any of the three means, namely, incinera- tion, dry heat, and moist heat. Incineration involves the use of very high tempera- tures, often (but not always) in a closed device, and is used for the disposal of medical waste and other biological material suspected of harboring dangerous microorganisms. The process kills all forms of all microorganisms. Dry heat, perhaps least efficient of the three methods, requires the application of a high temperature for an extended period of time. For example, a conventional convection oven will need at least 2 hours at 180°C to kill all microorganisms including endospores. This process is occasionally used for the sterilization of glassware and other heat-stable objects. In contrast, moist heat, by permitting faster penetration of heat into the objects, is more efficient. Boiling a material in water at 100°C for 5–10 minutes is sufficient to kill vegetative forms of most microorganisms. However, endospores do not die at this temperature. Raising the temperature to 121°C under moist conditions,which is achieved by the use of an autoclave at a pressure of 15 pounds per square inch (psi), can destroy all living organisms including endospores in 15 minutes. Autoclaves are often used in laboratories, hospitals, and pharmaceutical industries for the sterilization of media, glassware, surgical instruments, and other products that do not denature at this temperature and pressure.
Pasteurization is another form of heat application commonly used by beverage manufacturers to minimize microbial population in a product. This process is effec- tive in reducing only vegetative forms of the microbial population. It does not destroy endospores and its usefulness even in destroying viable Mycobacterium tuberculosis cells is questionable. There are two methods of pasteurization, low temperature hold (LTH) and high temperature short time hold (HTST). The LTH requires holding the products at 62.8°C for 30 minutes and the HTST works by holding the product at 71.7°C for 15 seconds. Both processes yield identical results. Pasteurization has played a great role in reducing a number of infectious diseases earlier associated with the consumption of milk and other beverages.
Filtration
Filtration involves physically removing microorganisms from a liquid or gas. This is achieved by passing the product through a membrane with a pore size < 0.2 µm in diameter. This size can block most bacteria, fungi, and protozoa, but not viruses. However, since viruses are intracellular obligate parasites, they are of a minimal concern perhaps with the exception of hepatitis A and a few similar viruses. This method is mostly used for the sterilization of heat-sensitive products. The membrane filters generally used for this purpose are made of cellulose acetate, cellulose nitrate, polycarbonate, teflon, or any other suitable material. The advent of high-efficiency particulate air (HEPA) filters has made it economically feasible to filter a large volume of gases. HEPA filters remove particulates that are larger than 0.3 µm in size. Since most microorganisms are larger than this size, they are easily removed. However, certain bacteria, such as mycoplasma, rickettsia, and chlamydia, which are less than 0.3 µm in size, and most viruses (except smallpox and Ebola viruses) can pass through HEPA filters. HEPA filters are currently in use for air handling in laminar flow safety cabinets and in other settings that require a germ-free environment.
Radiation
All forms of life, including viruses, which are at least technically not “living organ- isms,” are sensitive to radiation. Types of radiation that can destroy or seriously damage microorganisms and viruses are ultraviolet light and ionizing radiation. Carefully controlled radiation is used for the elimination of microorganisms and viruses from certain products and environment. The two types of radiation are described below.
Ionizing Radiation
Ionizing radiation triggers loss of electrons from the atoms. The common sources of ionizing radiation are gamma rays, having a wavelength of 10−3 to 10−1 nm, and X-rays, which have a wavelength of 10−1 to 10−2 nm. Ionizing radiation damages hydrogen bonds, double bonds, and ring structures of the molecules. Cell death and virus inactivation occurs due to destruction of nucleic acids (DNA and RNA). Most microorganisms and viruses die at an exposure of less than 1 Mrad (megarad, a unit of the measurement of absorbed radiation). However, endospores of Clostridium botulinum, polio, and vaccinia viruses can resist up to 1.5, 3.8, and 2.5 Mrad, respec- tively. Ionizing radiation is used for the sterilization of heat-sensitive materials or products that cannot be sterilized by chemical means. Laboratory petri dishes, dis- posable glass and plastic wares, and other products commonly used in healthcare facilities are sterilized using ionizing radiation. Ionizing radiation has a very high penetrating power and it does not leave any residue.
Ultraviolet Light
Ultraviolet light, having a wavelength of less than 400 nm, has a poor penetrating power. Therefore, ultraviolet light is mostly useful only for surface sterilization. Exposure to ultraviolet light having a wavelength of 260 nm for 1 minute is often sufficient to damage microbial DNA. Cell damage mostly results from mutation caused by formation of thiamine dimers in the DNA. Excellent results have also been obtained by exposure to ultraviolet light having a wavelength of 340 nm even though DNA does not sufficiently absorb ultraviolet light at this wavelength. Ultra- violet light is mostly used in safety cabinets and other closed environmental systems for killing microorganisms on the surfaces and in the ambient air in a small enclosed space.
CHEMICAL CONTROL OF MICROORGANISMS
Phenolic Compounds
Formerly popularly known as carbolic acid, phenol has been used as a common disinfectant by physicians and surgeons for decades. Joseph Lister, who is generally considered the father of antiseptic surgery, championed the use of carbolic acid for disinfecting surgical instruments. For decades phenol was considered to be the gold standard for determining effectiveness of a given disinfectant. However, this product is no longer in use because of its toxicity. Currently, several phenol derivatives including o-phenyl phenol, hexylresorcinol, hexachlorophene, and chloroxylenol are in use as antiseptics and disinfectants. The widely used household disinfectant Lysol is a mixture of phenolic compounds. Hexachlorophene is a popular antiseptic because of its long lasting residual effects. It is effective at a concentration of 1%–3%. Phenolic compounds act by denaturing proteins and disrupting the plasma membrane of the microbial cell.
Alcohols
Alcohols, especially ethyl alcohol (ethanol) and isopropyl alcohol (isopropanol), are bactericidal and fungicidal at a concentration of 65%–85% in water (volume by volume). However, they are not effective against endospores. A 70% concentration of ethanol or propanol is used as an antiseptic and less commonly as a disinfectant. Isopropyl alcohol is generally preferred over ethyl alcohol because it does not have the strong odor that is typical of the latter. The disadvantage of alcohols is that they evaporate rapidly, are highly inflammable, and irritate mucous membranes. Alcohols act by denaturing proteins and by dissolving lipids in the cytoplasmic membrane.
Halogens
Chlorine and iodine are most commonly used agents for microbial control. Chlorine compounds are generally used for disinfecting municipal drinking water and recre- ational water (swimming pools). Chlorine added to relatively clean water at a con- centration of 0.5 mg/L is usually sufficient to kill most microorganisms with the exception of Bacillus anthracis and Entamoeba histolytica, which can withstand a concentration up to ten times higher. It works best at neutral pH. Chlorine reacts with water to form hypochlorous acid, which actually is the active form. With a few exceptions noted above, almost all microorganisms die within 30 minutes. Chlorine has a residual effect but its corrosive nature can harm sensitive components. Iodine and iodophors are useful antiseptics. Under certain conditions iodine is preferred over chlorine for water treatment, albeit in a limited quantity. The effective concen- tration of iodine in an aqueous solution is 1%. Tincture iodine, once a popular antiseptic, is a mixture of 2% iodine in 70% ethanol. It is still one of the best anti- septics for use on skin. Iodophors on the other hand are water soluble, stable, and nonstaining. Iodophors are now widely used in hospitals for preoperative skin clean- ing and also as a disinfectant in hospitals as well as in laboratories. Iodine also has a residual effect, but tends to stain objects that come in contact with it. Halogens are powerful oxidizing agents and damage microbial cells by the oxidation of the sulfdryl group of enzymes.
Cationic Detergents (Quaternary Ammonium Compounds)
Quaternary ammonium compounds (quats), such as benzalkonium chloride, have lately emerged as popular antiseptics in clinical practice. Unlike to alcohol, which causes a burning sensation when applied to mucocutaneous surfaces, benzalkonium chloride is safe. It is often used for swabbing the urethral opening and vaginal areas in preparation for sample collection or surgical procedure. The effective concentra- tion is 0.1%–0.2% in aqueous solution. It inactivates microbial cells by damaging phospholipids in their cytoplasmic membrane.
Hydrogen Peroxide
A 3% solution of hydrogen peroxide is used as antiseptic. At this concentration it is lethal to most microorganisms. Vaporized hydrogen peroxide is occasionally used for the decontamination of safety cabinets, operating rooms and other enclosed places that need to be disinfected. Under certain circumstances it is a preferred disinfectant because it does not leave behind any residue or undesirable byproduct. The end products are oxygen and water, both of which are harmless. Hydrogen peroxide kill a wide-range of microorganisms including spore formers and it works at temperatures ranging from 4 to 80°C, without causing any material damage. Like halogens, hydrogen peroxide is also a powerful oxidizing agent. It produces hydroxyl- free radicals which impair proteins and DNA molecules.
Ethylene Oxide Gas
Ethylene oxide (EtO) gas is used for disinfecting temperature-sensitive equipment at a large scale. Items such as disposable petri dishes, syringes, components of heart or lung machines, sutures, and catheters are sterilized using this gas. Its effective concentration is 0.5–0.7 g/L. EtO is quite effective in sterilizing packaged products because it rapidly penetrates packaging materials including plastic wrapping. The materials to be sterilized are placed in a chamber similar to an autoclave with instru- ments to control EtO concentration, temperature, and humidity. A complete steriliza- tion is achieved in 5–8 hours at 38°C or in 3–4 hours at 54°C at a relative humidity of 40%–50%. Ethylene oxide is an explosive gas; therefore it is generally marketed as a mixture containing 15%–20% EtO in carbon dioxide. It is also highly toxic, a eye and skin irritant, highly inflammable, and carcinogenic. EtO kills microorgan- isms including spores by combining with cell proteins.
Aldehydes
A 3%–8% solution of formaldehyde is used for preserving tissues for histopathological examination. It is also extensively used in zoology laboratories for preserving small animals and animal organs for demonstration to students. A 37% solution (formalin) is occasionally used for vapor sterilization of a small room or laboratory suspected of contamination with pathogenic microorganisms. Another aldehyde, glutaraldehyde, is used as a high-level disinfectant at a concentration of 2%. It is often used for preserving tissues in preparation for histopathological examination. The disadvantages with aldehydes are their strong pungent odor; irritation of mucous membranes, especially the eyes and upper respiratory tract; and poor penetration. Formalin is also a carcinogen. Both glutaraldehyde and formaldehyde are alkylating agents.
Heavy Metals
A number of heavy metals, including gold, silver, copper, mercury, and zinc, have been in use as disinfectants or as antiseptics. A 1% silver nitrate solution has been widely used to prevent bacterial eye infection. Copper sulfate is generally used as an algicide in water supplies. Zinc is antifungal and is still used in ointments for topical applications. Mercury compounds, such as merthiolate (1:10,000 dilution), are used mostly in laboratories for preserving serum and other products meant for long-term storage. Another mercury salt, mercurochrome, was widely used as an antiseptic for several decades, but now its usage has been largely discontinued due to the fear of mercury toxicity. Gold, though too expensive to be used as a day-to- day disinfectant, has, nevertheless, powerful antimicrobial properties. Most heavy metals act by inactivating and precipitating vital proteins in microbial cells.
Dyes
Several dyes have strong antimicrobial properties. During the pre-antibiotics era, gentian violet solution was used for the treatment of thrush and yeast infections (vulvovaginitis) with excellent success. It is still recommended when everything else fails due to drug resistance. Other dyes with known antimicrobial properties include trypan red, malachite green, brilliant green, and certain acridine dyes. Dyes have a long-term residual effect, but stain the areas of application. Their mechanism of action involves interactions with nucleic acids. Most but not all dyes are bactericidal.
Ozone
Ozone is a strong oxidizing agent that reacts readily with most organic matters, and it is active against a wide range of microorganisms. It is more effective at lower temperatures. A minimum relative humidity of 60% is needed for disinfecting surfaces. It is generally used as a disinfectant in bottled drinking water. For that purpose, it is superior to chlorine because it is a stronger oxidizing agent and not much affected by the pH. Ozone is also preferred over chlorine for sewage treatment because of the risk of later reacting with organic molecules to form chlorinated compounds. It has little or no residual effect.
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