Conditions Influencing Antimicrobial Action Control by Physical Agents
Physical Agents Other Than Heat Control by Chemical Agents
Evaluating Disinfectants & Antiseptics Chemotherapeutic Agents Universal Precautions


Main reasons for practicing microbial control

  1. To prevent transmission of disease and infection

  2. To prevent decomposition and spoilage

  3. To prevent contamination

Microbes can be inhibited or destroyed by either physical or chemical means.

Some History

Some Terminology

Sterilization: Any process that completely removes or destroys all living organisms in or on an object. Something is either sterile or non-sterile. It is inaccurate to talk of sterilizing one’s hands because as long as any tissue or body part is living it cannot, by definition, be called sterile.

Disinfection: Any process that kills growing pathogenic organisms but not necessarily spore forms of pathogenic microbes. Term is commonly applied to processes used on inanimate objects. A disinfectant is any agent that kills pathogenic microbes.

Germicide: (Microbicide) is essentially the same as a disinfectant except that it is used on all kinds of microbes and in any place. Kills growing forms but not necessarily spore forms of microbes.

Bactericide: Similar to germicide but restricted to bacteria and does not affect their spores.

Fungicide: Kills fungi.

Virucide: Inactivates viruses

Sporocide: Kills spores of bacteria.

Antiseptic: A substance which opposes sepsis (infection), or arrests growth of microbes by killing or inhibiting them. Often is a diluted disinfectant.

Sanitization: Making objects free from pathogenic microbes and esthetically clean as far as organic material (saliva, mucus, feces) is concerned. A sanitizer is an agent that reduces microbial populations to safe levels as judged by health officials. A sanitizer usually kills 99.9% of bacteria present.

Bacteriostasis: Process of inhibiting (stopping or greatly slowing) the growth of bacteria. Freezing, drying, and the use of certain antibiotics are examples of bacteriostatic agents. A microbistatic agent inhibits the growth of microbes. A fungistatic agent inhibits the growth of fungi.

Antimicrobial Agent: Any agent that interferes with the growth and activity of microbes.

Remember to define microbial death, we refer to viability. The fundamental criterion is the ability of the microbe to propagate indefinitely when placed in a suitable environment.


  1. Characteristics of the microorganisms
    a. Kind of organism. In general, growing vegetative forms are more susceptible than spore forms. However, degrees of resistance exist among the various types of vegetative organisms. Mold and yeast spores are not as resistant as bacterial spores.

    b. Number of organisms. Large populations of microbes require more agent for killing. Clumping of the organism may prevent penetration of the agent to certain cells.
  2. Characteristics of the agent used for destruction
    a. Intensity and nature of a physical agent. For example, more heat will result in more killing. Moist heat is more effective for killing microbes than dry heat (at least moist heat provides more rapid killing than dry heat).

    b. Concentration and kind of chemical agent. A chemical may be bactericidal at one concentration and bacteristatic at a lower concentration. The “chemical nature” of a substance is important. For example, chlorine is of no value as a disinfectant in the form in which it occurs in table salt (NaCl) but is extremely effective in a free form either as a moist gas, an aqueous solution, or in the state in which it is present in bleaching solution (NaOCl). Water solubility is also important since some disinfectants must be ionized. Surface tension of disinfectant solutions is important. Low surface tension is best. In other words, it is important for the agent to be in a good wetting agent so that it will make contact with the microbe. Many substances help out in lowering surface tension. These care called surfactants (surface active agents) and include soaps, detergents, alcohols, etc. An alcoholic solution of iodine (tincture of iodine) spreads very well, comes into intimate contact with rough, dry surfaces and gets into narrow spaces where bacteria may escape solutions with higher surface tensions, such as aqueous solutions. An aqueous solution of iodine wets much less effectively.

    An important property of water-soluble surface-tension reducers in general, is that they are able to accumulate at surfaces and adhere to those surfaces. Water soluble surface tension reducing disinfectants such as cresol and its derivatives (e.g., hexachlorophene) and quaternary ammonium compounds (e.g., Zephiran, Ceepryn, etc.) tend to be absorbed and to remain in very thin films on the surfaces of hands and objects and to maintain disinfectant action there. Floors, surgeons’ and nurses’ hands and other surfaces if washed with appropriate and properly prepared solutions of surface-tension reducing disinfectants, may remain disinfected (not sterile) for some time – perhaps hours, afterward.

    Sometimes surfactants (e.g., Tween 80) are added to microbial cultural media when it is appropriate to increase the contact between microbial cells and the nutrients which are in the solution. For instance, the TB bacillus has a waxy coating which prevents effective contact with certain nutrients.
  3. Factors influencing the interaction between agent and microbe.
    a. Temperature. Temperature itself may be used to kill microbes but an increased temperature also may improve the efficiency of chemicals such as disinfectants. For example, a warm solution is likely to be more effective than a cold one. Warming lowers surface tensions and heat increases the speed of the chemical reactions involved in disinfection.

    b. Organic material. Blood, serum, mucus, pus, feces, foodstuffs, etc. are great obstacles in inhibiting and killing microbes for two main reasons:

              (1) Agents which coagulate protein will coagulate the organic material as a coating around the microbes and leave live microbes inside the coagulated mass, protected from disinfectant. Thus, when organic material is present it is necessary to mechanically break up the masses and possibly to use disinfectants which have less coagulative properties.

              (2) Many chemical disinfectants combine readily with extraneous organic material so that much of the disinfectant may be neutralized by this material. Thus large amounts of disinfectant may have to be applied.

    c. Time. The longer time an agent is applied, the more killing there should be. As a general rule, at least 10 minutes of boiling (at sea level) or one hour of contact with disinfectant may be used. Many circumstances will change these recommendations and neither boiling nor ordinary chemical disinfectants should be depended on to sterilize or to skills spores.

    d. pH. The acidity or alkalinity of a solution will affect the efficiency of a chemical agent. Bactericides that are acidic (anionic) in character (e.g., organic acids, phenol) are more effective at low pH. Cationic microbes tend to be inhibited by low pH and by anions. Acid solutions are more likely to be bactericidal if heated since heat tends to increase dissociation of acids.

    Examples of Modes of Action of Antimicrobial Agents
  1. Damage to microbial cell wall — For instance, lysozyme (from tears, saliva, etc.) attacks the gram positive cell wall. Penicillin inhibits wall synthesis.

  2. Alternation of cell permeability — For instance, phenol, detergents, soaps, quaternary ammonium compounds, etc. cause leaks in the cell, permitting loss of cellular constituents.

  3. Alteration of colloidal nature of protoplasm — For instance, high temperatures cause protein coagulation. 70% alcohol denatures protein thus disrupting the cell.

  4. Inhibition of enzyme activity — For instance, cyanide, fluoride, halogens, etc. may inhibit or inactivate enzymes. Interference with synthetic processes caused by antimetabolites such as sulfanilamide.


Heat is one of the most effective and reliable sterilizing agents. The vegetative cells of many fungi are killed by moist heat (60ºC for 5–10 minutes), while their spores must be exposed to about 5º–10ºC or higher for 5–10 minutes before they will be killed. Vegetative bacterial cells are usually killed in 5–10 minutes at temperatures of 60º–70ºC (moist heat) while bacterial spores require temperatures above 100ºC for extended periods of time. The susceptibility of viruses to heat is similar to that of the vegetative cells of bacteria.

Thermal Death Point (TDP) -- The lowest temperature at which suspension of bacteria is killed in 10 minutes.

Thermal Death Time (TDT) -- Shortest period of time required to kill a suspension of bacteria (or spores) at a given temperature and under specified conditions.

Note that the above terms express a time-temperature relationship to killing. It is absolutely necessary that the conditions be carefully controlled in these determinations. There is a great difference in the efficacy of moist and dry heat in killing microbes.

Dry Heat

Incineration: For infected laboratory animals, soiled dressings, sputum cups, garbage, etc. If infectious, materials should first be wrapped in paper.

Ovens: Dry heat dehydrates cells and kills by causing oxidation of intracellular constituents. Ovens are often used for sterilizing dry materials such as glassware, syringes and needles, powers, and gauze bandages. Petroleum and other oily substance must also be sterilized with dry heat in an oven because moist heat (steam) will not penetrate materials insoluble in water. To sterilize the materials in an oven, the oven must reach and maintain a temperature of 165º–170ºC (329º–338ºF) for 120 or 90 minutes respectively. This will destroy all organisms including spores.

Moist Heat

Autoclave: Heat in the form of saturated steam under pressure is the most practical and dependable agent for sterilization. Steam under pressure provides temperatures above those obtainable by boiling. Also, there is rapid heating, penetration, and moisture in abundance, which is expressed in pounds to the square inch. The actual amount of water present in the pressure chamber is usually small so that articles are not wet with much condensed stream when they are removed from the autoclave. 15–20 minutes at 121ºC, which is the temperature of stream, not air at 15 pounds pressure, is usually sufficient to kill all bacterial life, including spores. Of course, the amount and kind of material to be sterilized will influence the time required for sterilization. Autoclaving is used for objects that are not injured by moisture or by the high temperature. Bacteriological media, saline, and other solutions plus dressings, clothing, food and so on may be sterilized in the autoclave.

When sterilizing solutions, after autoclaving the pressure must be allowed to fall gradually so that the solutions will not boil. If the pressure falls rapidly, violent boiling occurs. Advantage is taken of this in autoclaving used for surgical instruments. They are immersed in water in a perforated tray. After autoclaving, the pressure is reduced suddenly. The water boils violently and washes the instruments clean.

NOTE: Machines are available for cleaning surgical instruments, syringes, etc. by extremely rapid (ultrasonic) vibrations. These can clean and dry hundreds of instruments every five to ten minutes. Remember, they do not sterilize.

Boiling water: Can never be trusted for absolute sterilization since maximum obtainable temperature is 100ºC (at sea level) and exposure to boiling water may not kill spores of inactivate certain viruses such as the hepatitis virus. Boiling water will kill most vegetative cells in 10 minutes. Boiling water is used when all that is desired is disinfection of materials like dishes, bedding, bedpans, etc. At altitudes over 5,000 feet, the boiling time should be increased by 50% or more because water there boils at temperatures of only about 95ºC or below.

Fractional Sterilization: Tyndallization (fractional sterilization) is used on materials which cannot be heated above 100ºC without being adversely affected. Either the autoclave or the Arnold Steam Sterilizer may be used for this process. Free flowing steam is used and (if the material can withstand 100ºC) the material to be sterilized is exposed to free flowing steam at 100ºC on 3 successive days with incubation periods in between. The incubation period is to give any spores a chance to germinate so that the emerging vegetative cells can be killed on the succeeding day.

Pasteurization: Pasteurization is not sterilization. The temperature selected for pasteurization is based on the thermal death time representative of the most resistant types of pathogen to be destroyed by the process. In practice, the heat treatment should be good enough to destroy the rickettsial organism, Coxiella burnetii, cause of Q fever, since this organism is the most resistant vegetative pathogen cell likely to be found in milk.

Low Temperature Holding (LTH) Method: Milk is exposed to 145ºF (62.8ºC) for 30 minutes in appropriately designed equipment.

High Temperature Short-time (HTST) Method: Milk is exposed to 161ºF (71.7ºC) for 15 seconds.

Both the above methods require equipment which is properly designed and operated. Precautions are observed to prevent recontamination after pasteurization. Finished product should be stored at low temperature to retard growth of these organisms which survived pasteurization.

Note: Low (cold) temperatures, however extreme, cannot be depended upon for disinfection or sterilization. Microbes have a unique capacity of surviving extreme cold.


Desiccation: Like low temperatures, desiccation primarily produces a static condition upon the growth and metabolism of microbes. Microbial species vary in their sensitivity to dehydration. Cells such as the gonococcus and meningococcus and the syphilitic spirochete are very sensitive to drying and will die in a matter of hours or even minutes when outside their hosts. Streptococci are more resistant and the TB bacillus is very resistant, remaining viable for months or longer. Lyophilization (freeze drying) may preserve the viability of microorganisms for many years.

Osmotic Pressure: Most microbes are inhibited by high concentrations of salt (10–15%) and sugar (50–70%). Thus, preserving foods by “salting” or using high sugar concentrations is generally effective. Microbes are inhibited (held in static condition) by plasmolysis – cells are dehydrated and thus unable to metabolize or grow. Salt concentrations above 1% are harmful to many bacteria but not to marine species (3.5%–4% salt in sea) or those found in the Dead Sea and Great Salt Lake of Utah (salinity is 29%). Many species actually require high salt concentrations (halphilies) or sugar concentrations (saccharophiles). In general, fungi have greater resistance to high osmotic pressure than bacteria.

Ultraviolet Light: Used for sterilization of air in some operating rooms and on smooth surfaces. It has virtually no power of penetration. UV lamps are used to suppress surface-growing molds and other organisms in meat packing houses, bakeries, storage warehouses, etc. Sunlight is a good inexpensive source of ultraviolet rays. UV works best at wavelengths of 260–270 nm. Depending on the dose, the effect may be inactivation, mutation or death. Thymine-thymine dimers are commonly formed. Many cells may revive themselves through photoreactivation or dark reactivation.

X-Rays: Penetrate well but require very high energy and are relatively costly and ineffective (rays are given off in all directions) for sterilizing. Their use is thus mostly for medical and experimental work and the production of microbial mutants. X-rays excite chemical groups in DNA and produce reactive radicals in H2O (ionizations) around the DNA.

Neutrons: Very effective in killing microbes but are expensive, hard to control and involve dangerous radiations. Thus neutrons are not practical.

Alpha Rays (particles): Are effective bactericides but have almost no power of penetration. Thus alpha rays are not practical.

Beta Rays (particles): Have slightly better penetration than alpha rays but are still not practical for use in sterilization.

Gamma Rays: High energy emissions from radioactive isotopes such as Co60. Resemble X-rays in many respects. The U.S. Army Quartermaster Corps has used gamma rays and other radiations to sterilize food for military use. X-rays or gamma rays must be applied in 2 mrad to 4 mrad doses to become a reliable sterilizing treatment of foods. (One mrad is 1/1000 of a rad). A rad is 100 ergs of absorbed energy per gram of absorbing material (retained by matter). Food exposed to effective radiation sterilization, however, undergo changes in color, chemical composition, taste and sometimes even odor. Only slowly are these problems being overcome.

Cathode Rays (electrons): Used mainly to kill microbes on surfaces on food, fomites and industrial articles. Have limited powers of penetration and thus have only minor usefulness for surgical sterilization. Cathode rays are being developed for general purposes such as food processing. This could revolutionize food canning and frozen good industries as well as surgical sterilization techniques. Pharmaceutical and medical products are adequately sterilized by treatment with a radiation dose of 2.5 mrad. The Association of the British Pharmaceutical Industry has reposted that benzylpenicillin, streptomycin sulfate and other antibiotics are satisfactorily sterilized by this method. Package radiation at dose levels of 2.5 mrad is common procedure for disposable syringes, needles and rubber gloves, tubing, etc.


Both Leeuwenhoek and Pasteur observed that microbes are removed by sedimentation from the atmosphere. Water on the earth’s surface also has its microbial flora reduced by filtration through layers of soil. These two processes: sedimentation and filtration are the basis for many pieces of equipment which are used for removing microbes from materials.

Filtration is used to sterilize thermolabile (destroyed by heat) fluids and solutions. Fluids and medications for hypodermic or intravenous use, culture media, especially tissue culture media and its components such as serum and various enzymes are often filter sterilized.

Various materials are used for filtration: an asbestos pad in the Seitz filter, diatomaceous earth in the Berkefeld filter and porcelain in the Chamberland-Pasteur filters, and sintered glass filters.

Filters do not act merely as mechanical sieves; porosity alone is not the only factor preventing the passage of organisms. Other factors such as an electric charge on the filter, the electric charge carried by the organisms, the nature of the fluid being filtered, all have a bearing on the efficiency of filtration.

However, the membrane (molecular) filter is a cellulose acetate filter which retains microbes primarily on the basis of the small uniform diameter of its pores. A bacteriological filter has a porosity of 0.45 + 0.02 micrometers; for virus studies filters with porosity of 0.01 + 0.002 micrometers are available. Fluid is usually forced through filter by applying a negative pressure to the filter flask by using a vacuum or water pump to impose a positive pressure above the fluid in the filter chamber thus forcing the fluid through.

Development of high-efficiency particulate air (HEPA) filters has made it possible to deliver clean air to an enclosure such as a cubicle or room. This type of air filtration together with a system of laminar air flow is now used extensively to provide dust and bacteria free air.


  1. Chemical Sterilizers

a. Ethylene oxide. A liquid at temperatures below 10.8ºC (51.4ºF). Above this temperature is vaporizes rapidly. The gas is applied in special autoclaves under carefully controlled conditions of temperature and humidity. Since pure ethylene oxide is explosive an irritating in use, it is generally mixed with carbon dioxide or Freon or other diluents in various proportions, e.g., 10%–90% ethylene oxide. This is a powerful sterilizing agent. Bacterial spores are effectively destroyed by this gas. The gas has very good penetrating ability.

Ethylene oxide has been used to sterilize many substances, including spices, biological preparations, soil, plastics, certain medical preparations, and contaminated lab equipment. It has been used to decontaminate certain spacecraft components. Beside its good penetration, this gas has a wide spectrum of activity (meaning it is effective against many different kinds of microbes). It is also effective at relatively low temperatures, and it does not damage materials exposed to it. One advantage is its relatively slow action upon microorganisms. The mode of action of ethylene oxide is believed to be alkylation reactions with organic compounds such as enzymes and other proteins. Alkylaton consists in the replacement of an active hydrogen atom in an organic compound with an alkyl group. In this reaction, the ring in the ethylene oxide molecule splits and attaches itself where the hydrogen was originally. The reaction would for instance, inactivate an enzyme. Ethylene oxide is usually measured in terms of milligrams of the pure gas per liter of space. For sterilization, concentrations of 450–1000 mg of gas per 1. are necessary. Concentrations of 500 mg per 1. generally are effective in 4 hours at about 130ºF (58ºC) and relative humidity of about 40%.

b. Beta-propiolactone: A colorless liquid at room temperature with a high boiling point (162.3ºC). It has a sweet but irritating odor. Although unstable at room temperature, it may be stored at 4ºC (refrigerated) for months without deterioration. Although beta-propiolactone is not flammable and does not penetrate as well as ethylene oxide, it is much more active than ethylene oxide. Aqueous solutions effectively inactivate some viruses, including polio and rabies and also kill bacteria and bacterial spores. The vapors, in a concentration of about 1.5 mg of the lactone per liter of air with a high relative humidity (75–80%) at about 25ºC kills spores in a few minutes.

Beta-propiolactone is not a substitute for ethylene oxide (because of its low penetrating power) but is used in place of formaldehyde for surface disinfection or sterilization. It is used to sterilize large enclosed spaces. Advantages over formaldehyde include more rapid antimicrobial action and faster removal from enclosure after application. Its mode of action is thought to be similar to that of ethylene oxide.

c. Formaldehyde: A gas that is stable only in high concentrations and at elevated temperatures. At room temperature it polymerizes, forming a solid substance. The important polymer is paraformaldehyde, a colorless solid which rapidly yields formaldehyde upon heating. Formaldehyde is also marketed in aqueous solution as formalin, which contains 37–40% formaldehyde. Formalin and paraformaldehyde are the two main sources of formaldehyde for gaseous sterilization. Vaporization of formaldehyde from either of these sources into an enclosed space for an adequate time will effect sterilization. Humidity and temperature have a pronounced effect on the microbicidal action of formaldehyde; in order to sterilize an enclosure, the temperature should be about room temperature (22ºC) and the relative humidity between 60–80%. One disadvantage is the limited ability of these vapors to penetrate covered surfaces.

  1. Disinfectants & Antiseptics

There is no one ideal antimicrobial chemical agent. There are too many variations in the use of these agents for any one to surface for all conditions or circumstances. Although an ideal chemical compound may never be found, the following traits should be aimed for in the preparation of new compounds and they should be considered in the evaluation of disinfectants. The following are the traits of an “ideal” disinfectant.

a. Phenolic compounds: Phenol (“Carbolic acid”) is an efficient bactericide in a 2–5% solution but is very corrosive to animal tissue and if used in the pure form, is so expensive that it is rarely used as such. 2–5% solutions are used to disinfect sputum, urine, feces, and contaminated instruments or utensils, etc. Spores and viruses are more resistant to phenol than vegetative bacteria. Antimicrobial activity is reduced at alkaline pH and in the presence of organic material.

Many phenol derivatives are useful disinfectants. Most of these act in a manner similar to phenol (i.e., probably by combined coagulative, toxic and dissolving action). Proteins are denatured and membranes are damaged. Exact mechanisms are not clear and differ with compounds and probably with different species of microbes. Many of these cpds. Are surface tension reducers and tend to remain absorbed as thin films on surfaces. Their action is thus longer than that of alcohols or halogens, which tend to volatilize quickly. Two general classes of phenol derivative are noted here: the cresols, which are like phenol with methyl (CH3) groups attached, and biophenols, which are composed of two phenol groups or phenol derivatives joined through some other radical.

(1) Cresols: More effective than pure phenol for most general purposes. Saponated solution of cresol (1–5%) is an example. The soapy solution of cresol has a far lower surface tension than does the aqueous solution. This disinfectant is used for feces, soaking contaminated instruments and general cleaning and disinfection. Cresols, like phenol are corrosive to living tissues. Several preparations similar to saponated cresol (i.e., Lysol) are on the market. One great advantage of Lysol over the cresols commonly used, is that it does not foam when used in 2% solution for scrubbing and washing surfaces.

(2) Hexylresorcinol: Marketed as a solution of glycerin and water. Is a strong surface tension reducer. Has good bactericidal activity. Hexylresorcinol preparations are used as general antiseptics.

(3) Hexachlorophene (G–11): A crystalline substance insoluble in water and soluble in alcohol, acetone and dilute alkalies. Highly germicidal. Most effective against gram positive cells. Used for skin antisepsis, many creams, soaps. Oils contain hexachlorophene. Also widely used for impregnating materials as protection against the fungi and bacteria that cause mildew and rot. Various proprietary combination of hexachlorophene with soap and other ingredients include: Gamophe, Hex-O-San, pHisoHex, Surgi-Cen, Surofen, etc. Skin washes and shampoos include pHisoHex, Foster, pHisoderm, Hexagerm, etc. In some hospitals, hexachlorophene has taken the place of topically used alcohol. For nurses, dentists and others who must wash their hands many times a day, or for patient’s body care, hexachlorophene containing preparations are less unpleasant to use than many more irritating, but perhaps more effective antiseptics. In recent years, warnings have been issued against hexachlorophene compounds because of a possible toxic effect produced by these compounds.

Orthophenylphenol: Used in proprietary mixtures as for instance O-Syl. Chlorothymol is another phenol derivative that has disinfectant properties. These bis-phenols are said to be effective after surgical washup because they tend to sustain the disinfectant action on the skin. Opinions are divided on this. The use of any disinfectant should be a supplement to, and not substitute for cleanliness and clean techniques.

NOTE: Phenol derivatives, as mentioned, are often mixed with soaps. Several of these appear to be effective. However, many have limited usefulness. For instance, those that contain hexachlorophene. Hexachlorophene has relatively little effect on gram negative cells (e.g., those that cause typhoid and various types of infant enteritis) and thus has limited use in hospital nurseries and similar places. Soap, is present in excess, many actually coat bacteria, displace the disinfectant and thus protect the bacteria!!

b. Halogens:

(1) Iodine. Iodine is the most effective bactericide of the halogens and is effective against all kinds of bacteria. In alcoholic solution (tincture) it is used for cuts, abrasions, preparation of skin for surgery and disinfection of clinical thermometers. Aqueous solutions of iodine are sometimes used but their surface tension is higher. However, they appear to be effective and the irritating effect of alcohol is avoided. A 2.5% solution (aqueous) is satisfactory for use on skin and in cuts. The most commonly available solution of iodine is a 2% solution in 70% alcohol.

Iodine is very poisonous and can cause serious burns of skin unless used properly. The 2% solution should be applied in one coat, allowed to air dry, then covered with sterile gauze. When used for surgery, iodine solution should not be allowed to run down the sides of the body so that it can concentrate under the patient. If the patient is lying on a rubber mat on an operating table, this concentration of iodine on the back can cause serious burns.

Iodophors are organic compounds which contain iodine loosely bound to a surfactant. Iodine is released slowly from these compounds. They are effective for a good period of time. Wescodyne and Ioclide are two representative iodophors. These are not irritating to the skin (except in cases of iodine hypersensitivity) but may cause a slight, temporary tan discoloration if used in strong solutions. These products are probably sporicidal under certain conditions of use. Other iodophors are Betadine, Hi-Sine and Iosan.

The mechanism of iodine’s antimicrobial activity is unclear however, the action seems to involve halogenation of tyrosine units of enzymes and other cellular proteins requiring tyrosine for activity. Iodine is also an oxidizing agent, which may account for its antimicrobial action.

(2) Chlorine. Chlorine gas, although highly effective, is very toxic and requires special apparatus for its use. It is widely used to disinfect municipal water supplies and swimming pools.

Calcium hypochlorite (CaOCl, chloride of lime) is used in 1–5% aqueous solution and is an excellent general disinfectant. It releases chlorine. 5–70% solutions are used for sanitizing dairy equipment and eating utensils in restaurants.

Sodium hypochlorite (NcOCl) solutions (5.25%) are available in all grocery stores as laundry bleach. (Clorox and Purex or crystals of Comet are good examples) This is a useful, convenient form of chlorine and is highly efficient disinfectant and deodorant. Unless diluted it is irritating to skin and mucus membranes and is used mainly for laundry, floors and objects of all sorts. It can be used to disinfect drinking water, to deodorize and many other purposes.

Chloramines (organic compounds of chlorine) are used as disinfectants, sanitizing agents or antiseptics. Azochloramine and dichloramine toluol, unstable organic compounds of chlorine are more convenient for many purposes than the hypochlorites. Their action is slower thus providing longer release of chlorine. They are used for general sanitization in dairies, restaurants, and similar places.

(c) Alcohols:

The rapid application of ethyl alcohol to the skin prior to hypodermic injection probably accomplishes nothing more than cleansing the area and removal of some of the surface bacteria. The time is undoubtedly too short for disinfection. An alcohol rinse or soak following scrubbing of the hands before surgery, probably reduces the numbers of bacterial inhabitants to some degree. The use of ethyl alcohol for “cold sterilization” of instruments, syringes or needles should not be practice, except for special purposes because spore forming organisms or resistant viruses may be present. If thermometers are adequately wiped or washed with soap and water to remove all organic material, and if these thermometers are then completely immersed for a minimum of 10 minutes in 70% ethyl alcohol, they will probably not transmit the bacterial pathogens commonly occurring in the mouth or rectum, especially if the alcohol contains 0.5% to 1% iodine or one of the quaternary ammonium compounds. They are not necessarily sterile.

Higher alcohols, propyl; butyl; amyl; etc. are more germicidal than ethyl alcohol. There is a progressive increase in germicidal power as the weight of alcohols increases. However, those alcohols with a molecular weight greater than propyl are not miscible in all proportions with water and thus are not commonly used as disinfectants. Propyl and isopropyl alcohols (40–80%) are useful skin disinfectants. Isopropyl alcohol (“rubbing alcohol”) is often used as 70% solution and is as effective as ethyl alcohol for ordinary purposes, especially if fortified with iodine or another disinfectant. It is cheaper and more easily obtainable than ethyl alcohol. Alcohols are protein denaturants and lipid solvents and dehydrating agents. Part of their effect is due to cell membrane damage. Alcohol concentrations above 60% have antiviral activity.

(d) Detergents: Surface-tension depressants, or wetting agents, employed mainly for cleansing surfaces are called detergents. Few if any household detergents are effective disinfectants. On the basis of chemical structure, surfactant detergents are divided into three groups:

(1) Anionic surfactants. Surface tension reducing property is in the negatively charged part of the molecule – the anion. This group includes the “soaps” and some organic (alkyl) sulfates as sodium lauryl sulfate.

Household soap is a good detergent or cleansing agent because of its surfactant properties. It is an effective emulsifier of fats and oils. It thus aids the mechanical removal of bacteria, especially from oil surfaces like the skin. Actually, many of the advertised detergents are not soap, but surface tension reducers. Tide, Cheer and Electra Sol for example. They have cleaning and emulsifying properties like those of soap buy are not disinfectants. An important ingredient in many of them is the potent surfactant alkyl benzene sulfonate (“ABS”). This refused to be decomposed by microbes in sewage disposal plants. Thus rivers and lakes were polluted with this detergent, as evidenced by excessive foaming. The manufactures of detergents have been urged to stop using “microorganism-resistant” chemicals. Today the “foaming cleaner” actually foams less and is probably inferior to the original product. It can, however, be readily decomposed by sewage microbes (i.e., “biodegradable”) and can be safely disposed of by running the washing machine water into the sewer. Polyphosphates also came to be used in many commercial available products. Phosphates of course have been implicated in the eutrophication of lakes and streams, resulting in algae blooms, etc. Actually soap is usually bactericidal to a degree, especially against certain microbes like Treponema pallidum, the syphilitic spirochete. The disinfectant value of soap depends to great extent on the chemical nature of its fatty acid radical (i.e., what kind of fat it was made from).

(2) Nonionic surfactants. Mainly complex ethers and polyglycerol soaplike compounds e.g., Tween 80. These do not ionize and do not have significant antimicrobial activity.

(3) Cationic surfactants. The quaternary ammonium chloride derivatives commonly called “quats”. Included are Zephiran, Ceepryn, Phemerol chloride, Diaparene Chloride, C.T.A.B. and Roccal. The most important detergents when disinfection is the consideration are the cationic quats. These combine both disinfectant and surface-tension reducing properties in the cation. Actually the quats are poor cleansing agents – but they are goods surfactants and their value is in the disinfectant ability.

Quats are used mainly for general external purposes as disinfectants and sanitizing agents. They are only slightly injurious to animal tissues, they are effective against many microbes in very high dilutions, they are stable, they have no odor, they do not stain, they are not corrosive, they dissolve easily in water, and they are inexpensive. Their mode of action is not precisely known – however, they do denature proteins and probably act mainly by disrupting the cell membrane.

In practice, the cationic qauats should never be mixed with anionics such as soaps because of the incompatible charges of the ions. For this reason quats are less effective in “hard” waters and iron-rich waters; however, they tend to retain their activity in the presence of organic matter like pus, blood or feces. Quats act against many microbes, including both gram positive and gram negative bacteria, many fungi and protozoa and viruses. Viruses which contain many lipids are more susceptible than other viruses.

(e) Acids & Alkalies: The killing action of mineral acids (e.g., HCl and H2SO4) is a function of the degree of dissociation and thus of the final hydrogen ion concentration. Organic acids are somewhat different in that not all their germicidal activity can attributed to H-ions. Organic acids ionized to relatively low degree thus the high germicidal action of some of them must be due to the nature of the molecule. Acidic solutions have many applications for antimicrobial purposes.

Disinfecting ability of alkalies is also dependent on dissociation and the resulting concentration of hydroxyl ions. However, one additional factor relates to the metallic ion of the alkai, which may be toxic and thus may contribute to the hydroxyl-ion effect. Strong alkalies are generally more effective against gram-negative bacteria and viruses than against gram-positive bacteria or protozoa. Acid-fast bacteria (e.g., Mycobacterium tuberculosis, M. leprae) are very resistant to alkalies.

Lye is a preparation of sodium hydroxide that has some application as a disinfectant. Lime is calcium oxide, Ca(OH). In dilute form it is called whitewash. Lime preparations have little, if any activity as disinfectants.

Strong acids and alkalies are sporidical, but their use is limited because of the corrosive and caustic nature they have.

(f) Compounds of Heavy Metals: The ability of extremely small amounts of certain metals e.g., mercury, silver and copper, to exert a lethal effect upon bacteria is called oligodynamic action. Their effectiveness is due to the high affinity of certain cellular proteins for the ion; large amounts are accumulated in the cell from a dilute solution. These metals and their compounds combine with and denture cellular proteins.

(1) Bichloride of mercury. It was formerly used widely in dilution of between 1:1000 and 1:5000, but has generally been replaced  by other more efficient disinfectants.

(2) Organic mercury compounds. Several organic compounds of mercury are used for disinfection of skin and for superficial applications. Included are Mercurochome, Merthiolate,Metaphen, and phenylmercuric nitrate. These organic forms are less irritating and less coagulative than mercuric chloride. They act mostly bacteriostatically. No doubt the artificial red color makes some people feel that these compounds must be doing some good, but many bacteriologists question the actual benefit derived from the application of these disinfectants to a wound.

(3) Silver nitrate. Most often used as a 1% solution (aqueous) in the eyes of newborn babies to prevent gonorrheal (and other) infections (ophthalmia neonatorum). The excess silver nitrate solution must be carefully removed from the eyes by washing with physiological saline after instillation. If allowed to remain, the silver nitrate could cause serious irritation to the membranes of the eye. In some cities, penicillin has been substituted for or combined with silver nitrate, with promising results. The use of one or the other is generally required by law for newborn infants.

(4) Organic silver compounds. Argyrol is an example of an organic (protein) compound of silver. It is used sometimes in 5–20% aqueous solutions for treating infections of mucous membranes of the eye, nose, and urethra. In 5–20% it is non-irritating to mucus membranes. Other organic silver compounds sometimes used are Silvol, Neo-Silvol and Protargol.

(5) Copper sulfate. Much more effective against algae and molds than against bacteria; 2 ppm (parts per million) in water is sufficient to prevent algal growth; used in swimming pools and open water reservoirs; used in form of Bordeaux mixture as a fungicide for prevention of certain plant diseases.

(g) Dyes: Many dyes are not toxic to microorganisms. Generally gram-positive bacteria are more susceptible to them than gram-negative. Dyes (e.g., crystal violet) are sometimes used to treat certain infections and are also included in growth media to make them selective. The specific mode of action of dyes is not known. However, their action is probably through a combination with cellular macromolecules. For example, acridine dyes are known to bind to nuclei acids. They are strong mutagenic agents.

(h) Pine Oil: Derived by steam distillation from weathered pine boles and roots, this product is emulsified in water the soap or resin. It is effective against many gram-negative bacteria such as Salmonella sp., but has very limited bactericidal activity against certain gram-positive pathogens. It is used for janitorial or household purposes on floors, walls, and bathrooms and has a pleasant odor. Several products containing it are available in household supply stores.

(i) Miscellaneous: Hydrogen peroxide is an antiseptic used in 3% solution for a mouth wash or gargle, and another oxidizing agent, potassium permangante (KNnO4) is also used. But since peroxides are strongly mutagenic, their used in the mouth should be discouraged; instead plain salt water is highly recommended as a gargle. Warm salt water is very effective in oral hygiene.

Many natural (e.g., botanical) substances have been used for ages as antiseptic or disinfectant materials, even before people really knew why they were using these substances. Many drug type substances have been isolated from plants. These substances have a variety of uses in medicine, one of which may be to treat infections. Other substances are used as pain killers, sedatives, etc. Some of these chemicals are rather nonspecific in their action on microbes while others are quite specific. For instance, quinine from cinchona bark is used for controlling malaria (protozoan parasite), oil from seeds of the chaulmoogra tree is used for slowing the progress of leprosy (Mycobacterium leprae) and material from ground pumpkin seeds is used to treat schistosomiasis (blood fluke infection).

Fungi are especially productive in this sense. Fungi produce a wide range of chemicals that are important e.g. hallucinogens, aflatoxin, antibiotics, etc.


Laboratory techniques for evaluating antimicrobial chemical agents involve one of the three procedures. The agent is tested against a selected microbe called the “test organism”.

(1) Liquid water-soluble substances appropriately diluted are dispensed into sterile tubes, to which are added a measured amount of the test organism. At specific intervals, a transfer is made from this tube into tubes of sterile media that are then incubated and observed for growth. In this type of procedure one must determine whether the inhibitory action is bactericidal or bacteriostatic. This technique can also be used to determine the number of organisms killed per unit time by doing a plate count on samples taken at intervals.

One test based on this principle is the AOAC phenol-coefficient method, sometimes called the “FDA method”. (AOAC = Association of Official Agricultural Chemists; FDA = Food & Drug Administration). This procedure is suitable for testing disinfectants miscible with water exerting their antimicrobial action in a manner similar to that of phenol. The test organism is a specific strain of either Salmonella typhosa or Staphylococcus aureus. The temperature at which the test is performed, the method of making subcultures, the composition of the subculture medium, the size of the test tubes, and all other details are spelled out in the official procedure and must be followed if results are to be valid. Briefly the test is done as follows:

To a series of dilutions of the disinfectant being tested (5 ml per tube), 0.5 ml of 24 hour broth culture of test organism is added. At the same time, similar additions, in the same amounts, are made to a series of dilutions of phenol. All tubes (disinfectant + organisms and phenol + organisms) are placed in 20ºC water bath. At intervals of 5, 10, 15 minutes, subcultures are made with loop transfer needle into sterile tubes of medium. The inoculated subculture tubes are incubated and later examined for growth. The greatest dilution of disinfectant killing the test organism in 10 minutes but not in 5 minutes is divided by the greatest dilution of phenol showing the same result. The number obtained by this division of the phenol coefficient of the substance tested.

If a phenol coefficient is greater than 1, the disinfectant tested is better than phenol under the conditions of the test. If less than 1, the disinfectant is not as good as phenol.

(2) Chemical agent is incorporated into agar or broth medium, inoculated with test organism, incubated, then observed for (a) decrease in amount of growth, or (b) complete absence of growth.

(3) Agar plate inoculated with test organism, then chemical agent placed on this medium. After incubation, plate is observed for zone of inhibition around the chemical agent. (If liquid, the agent may be impregnated into absorbent paper disks).

No single test method is appropriate for evaluating all germicidal chemicals for all applications recommended. One should exercise care in selecting a test method for a specific chemical agent, so results obtained will be meaningful and reproducible and lend themselves to some degree of practical interpretation. The ultimate criterion for the effectiveness of a germicidal agent is its performance under practical conditions. But the lab test should give a reliable index of its practical value.


Chemotherapeutic agents are chemical substances used for treatment of infectious diseases caused by the proliferation of malignant cells. Those substances which are naturally occurring, i.e. produced by microbes or other plants and animals, are called antibiotics. Other substances which are prepared by synthesis in the chemical laboratory (e.g., sulfa drugs) are not generally called antibiotics although they are, of course, chemotherapeutic agents. Some antibiotics are now prepared synthetically, but most of them are produced commercially by biosynthesis. Antitoxins and other substances produced by the bodies of infected animals are not considered to be chemotherapeutic agents; and compounds used for killing or inhibiting microbial growth in virto are classified not as chemotherapeutic agents but as disinfectant, antiseptics, or germicides.

A good chemotherapeutic agent:

  1. Will destroy or prevent the activity of a parasite without injuring host cells or with only minor injury to host cells.

  2. Will be able to contact the parasite by penetrating cells and tissues of host in effective concentrations.

  3. Will leave the host’s natural defense mechanisms (e.g., phagocytosis) unaltered. This is critical since many chemotherapeutic agents inhibit cells (rather than kill) and thus the natural defenses such as phagocytosis and the antibody response have a chance to overcome the invading parasite.

Chemotherapy (treating infections, diseases or malignant cells with chemical agents) is used in human and veterinary medicine and in horticulture. The term chemotherapy was coined by Paul Ehrlich, discoverer of the first antibacterial chemotherapeutic agent, active against syphilis, namely salvarsan (Cpd 606, or diosydiaminoarsenobenzene dihydrochloride).

About 1935, Domagk, who investigated the poisonous action of a certain aniline (coal tar) dye, Prontosil, discovered the sulfas. Porntosil, in staining bacteria, eventually killed them. Sulfanilamide (from which virtually all sulfonamide drugs are derived) was found to be the active moiety of Prontosil. Although not a dye, sulfanilamide is derived from coal tar, like many dyes and acts in much the same way as Prontosil, i.e., by metabolite antagonism.

The group of “sulfa drugs” includes sulfathiazole, sulfadiazine, sulfamerazine and many others. One of the main drawbacks with these drugs at first was their toxic side effects, but forms are not available that avoided these difficulties to a great extent (sulfisoxazole, sulfaethylthiadiazole, etc.)

Sulfonamides are very useful in treating infections by meningococcus and Shigella, respiratory infections caused by streptococci and staphylococci, and urinary infections due to gram-negative organisms. They are useful in preventing rheumatic fever, bacterial endocarditis, wound infections and urinary tract infections following surgery or catheterization. Usually sulfonamides are ineffective against viruses, rickettsias, fundi and protozoa. Since the end of WWII, sulfonamide drugs have been superseded by antibiotics in many clinical situations, but they still have important uses.

Antibiotics are chemotherapeutic agents derived from living organisms, generally microorganisms that have an inhibitory effect on parasitic microorganisms. To be truly useful, they should have all the qualities of any good chemotherapeutic agent plus the following traits:

  1. The ability to destroy or inhibit many different species of pathogenic microbes. This is what is meant by a “broad-spectrum” antibiotic.

  2. They should prevent the ready development of resistant forms of the parasites.

  3. They should not produce undesirable side effects in the host, such as sensitivity or allergic reactions, nerve damage, or irritation of the kidneys and gastrointestinal tract.

  4. The should not eliminate the normal microbial flora of the host, because doing so may upset the “balance of nature” and permit the normally nonpathogenic strains, or particularly pathogenic forms normally restrained by the usual flora, to establish a new infection. For instance, the broad-spectrum antibiotics may eliminate all the normal bacterial flora except for Monilia from the intestinal tract. Under these conditions, the Monilia may establish an infection that is not controlled by antibiotic therapy.

Universal Precautions

Universal precautions (UP) are a set of infection control practices, developed by the Centers for Disease Control and Prevention (CDC) in which health care workers (HCWs) appropriately utilize barrier protection (gloves, gowns, masks, eyewear, etc.) for anticipated contact with blood and certain body fluids of all patients.

  1. The hands and skin must be carefully washed when contaminated with blood or certain body fluids.

  2. Particular care is taken to prevent injuries caused by sharp instruments.

  3. Resuscitation devices should be available where the need is predicable.

  4. HCWs with exudative lesions or weeping dermatitis should refrain from patient care until the condition resolves.

Blood and Body Fluids to Which UP Apply
Blood is the single most important risk source of HIV, HBV and other blood-borne pathogens in the occupational setting. Thus prevention of transmission must focus on reducing the risk of exposure to blood and other body fluids or potentially infectious materials containing visible blood.

  1. UP should be used when exposure to the following body fluids may be anticipated:
  1. Note: Blood, semen and vaginal secretions have been shown to transmit HIV. The others, with the exception of fluids containing visible blood, remain a theoretical risk.


  1. UP reduce the risk of parenteral, musous membrane and skin exposure to blood-borne pathogens such as, but not limited to HIV and HBV.

  2. For several reasons, focusing precautions only on diagnosed cases, misses the vast majority of persons who are infected (many of whom are asymptomatic or sub-clinical) and who may be as infectious as the diagnosed cases. Persons who have seen a physician and have been diagnosed with acute or active disease, represent only a small proportion of all persons wit infection. Infectivity always preceded the diagnosis, which often is made once symptoms develop.