Millennia before “animalcules” were viewed by van Leuwenhoek (see Chapter 1, Scope of Microbiology), and before the germ theory of disease and Koch’s postulates had been accepted, references to germicidal agents had been made in the ancient writings of the Egyptians. Their embalmers applied spices, vegetable oils, and gums to their dead, using techniques that have kept them in a superior state of preservation even today. Persians, Greeks, and Romans had legal documents requesting the use of bright copper and silver vessels for the storage of public drinking water. The armies of Alexander the Great boiled their drinking water and buried their wastes. Very early on, salting, spicing, and smoking of foods were practiced as means of preservation. Moreover, the use of wine and vinegar in wound dressings dates back to the time of Hippocrates. Iodine was used to treat wounds before pus formation was understood because putrefaction and disease were believed to be associated.

Food preservation techniques have evolved along with culture and technology, with drying and freezing markedly influenced by the climates in which we live. Although humankind invented many methods of food preservation, fermentation was more or less discovered, not invented. Not to be upstaged by past authors who emphasized the impact of religion on science, Benjamin Franklin wrote that “…wine [is] constant proof that God loves us, and loves to see us happy.” Pickling, curing (actually dehydration), the use of honey and sugars to make jams and jellies, and canning have historically impacted food-processing technologies as well.

IMPACT

Ignaz Semmelweis (see Chapter 1), in 1847 at the maternity clinic of Vienna’s General Hospital, requested that interns who had performed autopsies wash their hands with chlorinated lime before examining expectant mothers. After hand-washing procedures had been instituted the incidence of death due to post delivery infection fell to about 1%.

Joseph Lister (see Chapter 1) instituted aseptic surgical techniques by the use of carbolic acid on surgeons’ hands, the operating field, the surgical instruments used, and the hospital environment in general. Protective surgical gloves were developed to protect both patients and surgeons, which greatly reduced infection during and after surgical procedures.

Food preservation by canning procedures was pioneered in the late 1700s by the French and later perfected by the British, who instituted the use of tin cans in 1810. Coupled with Pasteur’s work on the relationship between microbes and food spoilage/sickness, pressure canners led to canning at temperatures in excess of 100° C (212° F) and in the 1920s this canning procedure showed a significant reduction in the survival of the spores of Clostridium botulinum.

FUTURE

Improvements in disinfection techniques along with food preservation processes are essential to the future of physical and chemical methods of control of microorganisms in our environments. For example, better control of biofilms and of new and/or emerging resistant strains of microbes such as methicillin-resistant Staphylococcus aureus (MRSA) will all benefit from renewed attention.

The use of minimal food-processing techniques results in increased susceptibilities to resistant microbial strains. Therefore, in addition to developing better technologies for the improvement of disinfection and antisepsis, much greater shelf life of food is being emphasized because of the movement of population centers from the countryside to the city and the coming of the space age. Current conventional, thermal, and nonthermal methods are being investigated as potential preservation technologies for future space mission products, where minimization of mass and volume, water use, power requirements, crew size, time, and resistance to breakthrough contaminants are only some of the factors essential to the success of mission products and products having greater shelf lives.

Introduction

Under natural conditions humans and a large number of different microorganisms share the environment. Although the majority of microorganisms are harmless others can cause infection and disease, food spoilage, and water contamination. It is neither possible nor desirable to remove all microorganisms from the environment, but the control of microbial growth is necessary under many circumstances.

Microbial Control

General Considerations in Microbial Control

The control of microorganisms in the environment is a never-ending concern in healthcare, in the laboratory environment (see Chapter 4, Microbiological Laboratory Techniques), as well as in various industries, especially the food industry. Microbial control can be achieved by physical methods, chemical agents, or a combination of both. Under ideal circumstances the methods used for microbial control should be inexpensive and fast-acting. Several factors need to be considered before deciding which method is most appropriate in a given circumstance. These factors include the following:

• Site to be treated: The nature of the site or item to be treated will determine the method of treatment. It needs to be determined whether or not the item is treatable with harsh chemicals, heat, or radiation, or a combination thereof, without undue damage to the item.

• Environmental conditions: Environmental conditions have an impact on the effectiveness (efficacy) of the antimicrobial method. For example, warm disinfectants generally work better than cold ones, because chemical reactions usually occur faster at warm temperatures, therefore reducing the time of exposure. Furthermore, certain chemical agents will perform better under acidic or alkaline conditions. Items contaminated with organic substances such as feces, vomit, blood, and so on need to be cleaned before disinfection or sterilization is performed for optimal results. Biofilms notoriously prevent the penetration of chemicals into all layers of the biofilms, and physical methods may have to be used before chemical methods can be effective.

• Susceptibility: Susceptibility of the microorganisms needs to be considered for any of the treatments (see later).

The methods used in the control of microbes belong to the general measures of decontamination. In the world of microbiology, contaminants are microorganisms present at a place and a time when they’re undesirable for human health. Decontamination is defined as the reduction or removal of chemical or biological agents so that they no longer present a hazard. The prerequisite for any decontamination procedure is adequate precleaning of the item to be decontaminated. Organic material such as feces, blood, and soil may inactivate disinfectants and protect microorganisms from the decontamination process; therefore, the actual physical removal of microorganisms by scrubbing is as important as the antimicrobial effect of disinfectants.

In addition to everyday management of microbial control, emergency departments and emergency medical services are responsible for the management of potential biological and chemical disasters caused by accident or by terrorist activities. Details on this topic are provided in the section First Responders to Natural Disasters and Bioterrorism in Chapter 24 (Microorganisms in the Environment and Environmental Safety).

TABLE 19.1

Terminology in the Control of Microbial Growth

Term	Definition
Asepsis	Technique to prevent the entry of microorganisms into sterile tissues
Antisepsis	Destruction of pathogens on living tissue
Commercial sterilization	Sufficient treatment with heat to kill Clostridium botulinum endospores; used in the food industry
Decontamination	Destruction, removal, or reduction of the number of undesirable microbes
Degermination	Removal of microbes from a limited area (i.e., area of skin being prepared for injection)
Disinfection	Destruction of vegetative pathogens
Sanitization	Treatment to reduce microbial counts on eating and drinking utensils to achieve safe public health levels
Sterilization	The complete destruction of all forms of microbial life to include endospores and prions

Sterilization

Sterilization occurs when all forms of microbial life including the most resistant forms, the bacterial endospores and prions, are eliminated. Items to be sterilized can be surfaces, equipment, foods, medications, as well as biological culture media. Sterilization can be achieved by application of heat, pressure, chemicals (liquid and gas), irradiation, or filtration. Extreme heat is the most common method used to kill microorganisms. Filtration is also an effective method to use for the removal of microbes from liquids or air. In the case of prions, the sterilization procedure typically utilizes both chemical and physical approaches, as prions are destroyed by autoclaving the contaminated items in a broth of 1 N NaOH.

Commercial Sterilization

Unfortunately for the food industry, temperatures used to kill all living microbes will also result in degrading the quality of food during the canning process. Instead, commercial sterilization is used, which applies just enough heat to destroy the endospores of Clostridium botulinum, an organism capable of producing a toxin deadly to humans.

Disinfection

Disinfection is the destruction of vegetative microorganisms via chemical or physical methods. Disinfection does not destroy bacterial endospores and can be achieved by chemicals, ultraviolet radiation, boiling water, or steam. Usually the term disinfection is used when inert substances or surfaces are treated chemically, and any disinfection of living tissue is called antisepsis and the chemical used is an antiseptic.

Degermination

Degermination is the mechanical removing of most of the microbes in a limited area. An example of degerming is the cleaning of skin with alcohol before receiving an injection.

Sanitization

Medical instruments require sterilization, but in other areas of life, microbes do not have to be completely eliminated, just reduced in sufficient numbers to prevent infection, disease, and the transmission of infection and disease. Sanitization is a process that intends to lower microbial counts to safe public health levels and therefore minimize the chances of disease transmission between users. For example, sanitization is used for glassware and other tableware in restaurants, institutions, as well as in the home environment.

Pasteurization

Pasteurization was introduced by Louis Pasteur in 1857 (see Chapter 1, Scope of Microbiology), using heat to kill vegetative bacteria and therefore reducing the number of microorganisms that have the potential to spoil food. To this day, pasteurization is used in the food industry in the preparation of milk, fruit juices, wine, and beer. Pasteurization does not kill all the microorganisms but reduces their numbers, or eliminates dangerous pathogens enough to prevent foodborne diseases and food spoilage.

Microbicidal Agents

The term microbicidal agent describes an agent that is designed to destroy microbes. They can be further subgrouped into bactericidal agents that kill bacteria, fungicidal agents that lead to fungal death, and virucides that inactivate viruses.

Microbiostatic Agents

The term microbiostatic indicates that microbial growth is at a standstill. Therefore a bacteriostatic agent prevents further bacterial growth, and fungistatic agents inhibit fungal growth.

Microbial Death

Microbial death is the permanent loss of the reproductive ability of microbes, as well as the permanent loss of all other vital activities. Lethal agents do not always alter the appearance of a microbial cell, even if the motility of the organism is gone. Therefore, the term microbial death can be used only if reproduction cannot occur even if the organism is given the optimal growth conditions.

The destructive forces of chemical or physical agents act on the individual cells and if exposed intensively and long enough, the cell structures become dysfunctional and the cells show irreversible damage. In general, cells in a given culture/environment vary in their susceptibility to antimicrobial agents depending on their level of metabolic activity. Young, rapidly dividing cells have the tendency to die more rapidly than older, less active cells. When microorganisms are killed by any method of control, they have the tendency to display exponential death curves. In other words, they die at some fractional rate per unit time. If 50% of microorganisms in a population die every minute, after 2 minutes 25% will still be alive, after 3 minutes 12.5% are still alive, and so on (Figure 19.1). It is therefore safe to say that the total number of organisms present when the disinfection process began determines the time required to eliminate all microbes. The effectiveness of an agent is influenced by other factors besides time, such as:

FIGURE 19.1 Exponential death of microbes.
As this graph demonstrates, the microbial population shows an exponential death curve as the method of microbial control is applied.

• The number of microorganisms, which dictates the amount of time required for the destruction of all contaminants.

• The nature of the organism(s) to be destroyed. Biofilms, for example, include a number of different organisms and species. Other contaminants can include vegetative organisms as well as spores. Any target population that contains more than one organism can present a broad spectrum of resistance.

• The temperature and pH of the environment have an influence on the effectiveness of the antimicrobial agent.

• The overall concentration of the microbial control agent: Most are more active at higher concentrations.

• The presence of other materials, such as organic matter, solvents, and other substances, can inhibit or interfere with the actions of antimicrobial agents.

Microbial control requires many considerations to find the most appropriate method to achieve the desired control in a given environment. Several questions need to be answered before a given method and/or agent is selected for microbial control. Some of these questions are as follows:

• Is the goal disinfection or sterilization?

• Will the item be discarded or is it reused?

• If dealing with a reusable item, which temperatures, pressures, chemicals, or types of radiation are acceptable?

• Will the treatment leave an undesirable residue?

• Is the agent able to penetrate to the necessary extent, that is, removal of biofilms?

• Is the process cost and labor efficient, in addition to being safe?

Physical Control

The optimal growth of most microorganisms depends on the most favorable environmental conditions for each species; most grow best in a narrow range of temperature, pH, osmotic pressure, and atmospheric conditions (Table 19.2). For example, obligate anaerobic bacteria will be killed instantly by exposure to oxygen. If the nature of the contaminant is known, the best agent of control can be determined rather easily and selectively. However, under most circumstances the exact nature of the microbe is unknown and rather stringent methods of decontamination will have to be employed.

TABLE 19.2

Optimal pH for the Growth of Some Bacteria

Bacteria	Minimal pH	Optimal pH	Maximal pH
Thiobacillus	1.0	2–2.8	4–6
Escherichia coli	4.4	6–7	9.0
Clostridium sporogenes	5.4	6–7.6	9.0
Pseudomonas aeruginosa	5.6	6.6–7	8.0
Nitrobacter	6.6	6.6–8.6	10.0

The nature of the contaminated item(s) or areas needs to be considered to efficiently and effectively remove the unwanted organisms without destroying the item. In the case of the food industry, nutrients might be inactivated by improper treatment (see Food Preservation, later in this chapter). A summary of physical methods for the control of microbial growth is provided in Table 19.3.

TABLE 19.3

Summary of Physical Methods Available for the Control of Microbial Growth

Method	Mechanism	Comments	Use
Dry Heat
Flaming	Burning to ashes	Sterilization	Inoculating loops
Incineration	Burning to ashes	Sterilization	Dressings, wipes, animal carcasses
Hot air sterilization	Oxidation	170° C/2 h	Glassware, needles, glass syringes
Moist Heat
Boiling	Denaturation	Kills vegetative cells but not spores and prions	Equipment, dishes
Autoclaving	Denaturation	Sterilization	Media, linens, equipment
Tyndallization	Denaturation	Intermittent sterilization; free-flowing steam (30–60 min) for 3 d in a row	Substances that cannot withhold the high temperature of an autoclave
Pasteurization
LTLT (low temperature, long time)	Denaturation	63° C for 30 min	Milk, milk products
UHT (ultrahigh temperature)	Denaturation	138° C for a fraction of a second	Milk, juices
Low Temperatures
Refrigeration	Slows down microbial growth	Bacteriostatic for most bacteria	Foods, drugs
Deep freezing	Stops most microbial growth	Preservation	Foods, drugs, cultures
Desiccation, Lyophilization, Osmotic Pressure
Desiccation	Inhibits microbial growth	Loss of water; preservation	Foods
Lyophilization	Inhibits growth	Loss of water; preservation; combination of drying and freezing	Storage of cultures, vaccines, cells
Osmotic pressure	Plasmolysis	Loss of water	Food preservation
Ionizing Radiation
	DNA destruction	Electron beams, gamma rays, x-rays	Sterilizing medical and dental supplies
Nonionizing	DNA damage	Ultraviolet light, visible light, infrared radiation, microwaves, radio waves	Disinfecting air; transparent fluids; germicidal lamps

Temperature

Each species of microorganism has an optimal temperature range for growth, and any deviation from this will either slow down growth or stop growth altogether (Figure 19.2). For example, human pathogens prefer normal body temperature for optimal growth. In response to an infection, the human immune system (see Chapter 20, The Immune System) will increase the body temperature in an attempt to kill the invading microorganism. Microorganisms involved in the spoilage of food also generally prefer a moderate temperature for growth. Refrigeration commonly slows their growth, and appropriate cooking will kill the vegetative organisms. In other words, the majority of microbes that humans are most interested in controlling will be sensitive to abrupt changes in their environmental temperature.

The most common physical method of microbial control is heat, either dry or moist. Enough heat denatures proteins, and therefore enzymes necessary for microbial growth will be inactivated and the microbe will cease to exist in its vegetative form.

Heat resistance among different microbes exists, is variable, and is expressed as the thermal death point (TDP). This point is the lowest temperature at which all the microorganisms in a particular liquid will be killed within 10 minutes. Another factor to be considered is the length of time required to kill the microorganisms in a particular liquid at a given temperature. This period of time is referred to as the thermal death time (TDT). TDP and TDT are valuable guidelines used to determine the level of treatment required to kill a given population of microorganisms, especially bacteria. Another concept, the decimal reduction time (DRT), is related to bacterial resistance and indicates time in minutes in which 90% of the bacterial population in question will be killed at a given temperature.

Dry Heat

One of the simplest methods of dry heat sterilization is to expose the object to direct flaming or electric heating. If contaminated objects, such as wound dressings, are expendable, incineration is usually recommended, because it is the most rigorous of all heat treatments. Direct exposure to intense heat ignites substances and reduces them to ashes and gas. Incineration of microbial samples on inoculating loops or needles is a common practice in microbiology laboratories, but although it is fast and effective it is also limited to metals and heat-resistant glass materials. The method of flaming inoculating loops and needles also produces a potential hazard to the operator through the possible splattering of contaminants when the loop is placed into the flame. For this reason tabletop infrared incinerators (Figure 19.3) have replaced the traditionally used Bunsen burners in many laboratories. However, many clinical laboratories and also educational facilities are now using sterile, disposable, plastic inoculation loops and needles.

FIGURE 19.3 Tabletop infrared incinerator.
These are sometimes used instead of Bunsen burners, when there may be a risk of dangerous aerosols being produced if a loop is sterilized in the open flame of the burner.

Other than incineration, dry heat is less efficient than moist/wet heat sterilization because it requires longer times and/or higher temperatures. Furthermore, not all objects that require sterilization can withstand direct flaming, and the specific times and temperatures must be determined for each type of material to be sterilized. Higher temperatures and shorter times can be used for heat-resistant materials. To accomplish sterilization with dry heat in an oven, the objects must be exposed to temperatures of 160° C to 170° C (320° F–338° F) for a period of 2 to 4 hours. Because of the time required for this type of sterilization and other factors it is often not reasonable as the method of choice.

The advantage of hot air sterilization is that it can be used on powders and other heat-stable items that would be adversely affected by steam, such as occurs with rusting of steel objects.

Moist Heat

Moist heat requires special equipment but is a more effective way to control or kill microbial growth because it requires lower temperatures and shorter periods of time than dry heat to achieve the same goals. Moist heat to control microbial growth is applied and used in several ways, for example, steam under pressure, flowing heat, boiling water, pasteurization, and ultrahigh-temperature sterilization. Both pasteurization and ultrahigh-temperature sterilization are used as methods for microbial control in the food industry and are discussed in the section Food Preservation, later in this chapter.

Steam Under Pressure

The most dependable procedure to destroy all forms of microbial life with moist heat is steam sterilization under pressure. True sterilization using moist/wet heat requires temperatures higher than can be reached by boiling water. To achieve these higher temperatures pressure is applied to boiling water to increase the boiling point, and the temperature will proportionally increase with increasing pressure. A widely used instrument for pressurized steam sterilization is the autoclave (Figure 19.4).

FIGURE 19.4 Autoclave.
The autoclave is basically a large pressure cooker that increases the temperature of the steam in the chamber above the normal boiling point of water. It accomplishes this by raising the boiling point of the water through an increase in the pressure in the chamber.

In general, an autoclave consists of a pressure chamber, pipes to supply and release the steam, valves to remove air in order to control the pressure, and temperature as well as pressure gauges to monitor the procedure. Autoclaves generally use steam heated to 121° C (250° F), which can be achieved under a pressure of approximately 15 pounds per square inch (psi) above the normal air pressure. The average time of autoclaving is about 20 minutes, but the duration of the process varies according to the items to be sterilized, and how full the chamber is. Autoclaving is an effective choice for sterilization of heat-resistant materials such as glass, cloth, metallic instruments, liquids, paper, heat-resistant plastic, and many types of culture media. The autoclave is also suitable to sterilize items that are not being used or reused, but discarded. Proper autoclave treatment will inactivate all fungi, bacteria, viruses and even most heat-resistant bacterial spores. However, it will not necessarily eliminate all prions, and the procedure is also ineffective for the sterilization of substances that repel moisture, such as oils and waxes; or for powders, which will absorb the moisture and cause the particles to cake together.

Other than instrumentation to ensure that the autoclaving process displays the pertinent information abut the process, heat-sensitive indicator tape is often placed on packages of products to be sterilized before autoclaving. The chemical in the tape will change color when the desired heating conditions have been met. In addition, biological indicators distributed throughout the autoclave can be used to verify the performance of the autoclave.

Flowing Steam

Certain substances cannot withstand the high temperature of the autoclave, but can be subjected to intermittent sterilization, a process called tyndallization, named after John Tyndall, the inventor of this process. This procedure is designed to reduce the level of sporulating bacteria. The technique requires a chamber to hold the materials and a reservoir for boiling water. The items are exposed to free-flowing steam for 30 to 60 minutes. The procedure is based on the assumption that some spores will survive the procedure and germinate given the appropriate environment. Furthermore, it is assumed that this germination will result in less resistant vegetative cells and, after incubation and subsequent additional exposure to steam, more of the resistant cells will be eliminated. . The procedure is repeated for 3 days in a row. Although many resistant spores will no longer germinate, the most resistant ones still may survive. Intermittent sterilization is often used to process heat-sensitive culture media containing sera, egg, other sensitive proteins, or carbohydrates.

Boiling Water

Boiling items in water for 10 to 15 minutes will kill most vegetative bacteria and viruses, but it is ineffective against bacterial and fungal spores, as well as prions. On the other hand, although boiling water cannot sterilize, it is an effective, simple way to quickly decontaminate and disinfect items in the home and even in clinical environments. Boiling in water for 30 minutes will kill most nonsporing pathogens including some resistant species such as tuberculosis bacilli and staphylococci. Boiling is a recommended method for disinfecting unsafe drinking water and it is also a useful method to sanitize and disinfect materials needed for food preparation, utensils, bedding for humans, as well as materials used in the care of infants.

Refrigeration and Freezing

Another method used in the control of microbial growth is the use of cold temperatures. This method is only bacteriostatic for many microbes, and certainly cannot be applied to disinfect or sterilize items. It is, however, a convenient method to control microbial growth for limited times during food preparation and storage:

• Refrigeration halts the growth of many pathogens because they are predominantly mesophiles, yet some pathogens are perfectly capable of reproduction in a refrigerated environment.

• Slow freezing forms ice crystals that can puncture cell membranes and therefore is generally more effective in inhibiting microbial metabolism than quick freezing; however, many bacterial cells, endospores, and viruses can survive ultralow temperature for years. Because they are able to become viable microbes given the appropriate temperature and medium, many bacteria and viruses can be stored in ultralow-temperature freezers for later use in the laboratory environment. Bacteria, viruses, and tapeworms can survive in frozen food for days and months, which needs to be considered when thawing and using frozen foods (later in this chapter).

Desiccation and Lyophilization

Microbial metabolism requires water; therefore desiccation (drying) inhibits microbial growth. This method has been used over the years by many cultures for the preservation of a variety of foods.

Lyophilization is a technique that combines (1) freezing and (2) drying to preserve microbes, other cells, and materials for many years. It is successfully used by scientists to preserve organisms for later use. For the preservation of microbes the culture is instantly frozen in liquid nitrogen or dry ice and then subjected to a vacuum treatment that removes the water by transforming water instantly from the solid state into a gas. Therefore, lyophilization prevents the formation of ice crystals that otherwise would damage the cells. Although not all cells will survive this treatment, enough will be viable to restart the cultures many years later.

Osmotic Pressure

Subjecting microorganisms to hypertonic environments will lead to the loss of water within the cell, which interrupts the cell’s metabolism. The use of osmotic pressure by high salt or sugar concentrations has long been used for food preservation. However, this method of microbial growth control is not always effective against molds which have a greater ability to grow in hypertonic environments than do bacteria.

Radiation

Radiation is also a method used in the control of microbial growth and one of the most controversial ones. By definition, radiation is the propagation of energy through space in the form of electromagnetic waves. Radiation has a variety of effects on cells, depending on the type of radiation used. These types include ionizing radiation, and nonionizing radiation. The effects of the different kinds of radiation greatly depend on the following factors:

• Time of exposure

• Distance from the source

• Shielding

Ionizing Radiation

Gamma rays, electron beams, and x-rays have wavelengths of about 1 nanometer (nm) and cause ionization. This energy has the potential to ionize atoms or molecules through atomic interactions. Ionizing radiation has enough energy to disrupt hydrogen bonding, for oxidation of double covalent bonds, and so create highly reactive hydroxide ions. These ionized molecules can denature other molecules including DNA molecules in biological organisms. The DNA damage can cause mutations and cell death.

• Electron beams are highly energetic and very effective at killing microbes within just a few seconds. However, because they do not penetrate deep into matter they cannot be used to sterilize thick objects, or objects that are covered with a large amount of organic matter. They can be used for the sterilization of microbiology plastic ware, and medical supplies including gloves, syringes, and suturing material.

• Gamma rays penetrate deep into material but may require more time to sterilize large masses. Irradiation with gamma rays not only kills microbes, but also the larvae and eggs of insects. Gamma rays are often used for sterilization of disposable medical equipment, such as needles, syringes, cannulas, and intravenous sets. This type of radiation requires bulky shielding for the safety of the operators. Irradiation with x-rays or gamma rays does not make the materials radioactive. Irradiation is used by the United States Postal Service to sterilize mail in the Washington, DC area.

• X-rays penetrate the deepest but they have less energy than gamma rays and require too much time to be useful for the control of microbial growth.

Nonionizing Radiation

Unlike ionizing radiation, nonionizing radiation, a type of electromagnetic radiation with a wavelength greater than 1 nm, does not have enough energy to remove an electron from an atom. But nonionizing radiation has enough energy to cause the formation of new covalent bonds, which affects the three-dimensional structure of proteins and nucleic acids. Nonionizing radiation includes the spectrum of ultraviolet (UV) light, visible light, infrared radiation, microwaves, and radio waves. UV light, with a wavelength of about 260 nm, is the only form of nonionizing radiation with enough energy to kill microbes in a practical setting. UV light damages DNA by causing bonds to form between adjacent pyrimidine bases, resulting in the inhibition of DNA reproduction in cells.

A disadvantage of the use of UV light as a means of disinfection is the poor penetration capacity of UV light, and organisms must be directly exposed to the rays to cause their destruction. Therefore, UV radiation is used primarily to disinfect air, transparent fluids, and the surfaces of objects. UV or “germicidal” lamps are commonly found in hospital rooms, nurseries, operating rooms, and cafeterias. UV light is also used to disinfect vaccines and other medical products. In addition, some cities use UV irradiation in sewer treatment plants by passing wastewater past UV light sources in order to reduce the number of bacteria without the use of chlorine.

Microwaves do not affect microbes directly; however, moisture-containing foods are heated by microwave action and this heat will kill most vegetative pathogens. The uneven distribution of moisture in solid foods reduces the ability of microwaves to kill the microorganisms.

Filtration

Filtration is the mechanical separation of solids from fluid or gas by means of filters, which have various pore sizes. The separation of solids from liquid is based on the molecular size of the solutes. In microbiology filters must have pores small enough to retain microorganisms. Filtration traps only particles that are larger than the pores of the filter, and in the early years of microbiological studies filters were able to trap bacteria, but the pores were too large to trap the pathogens causing diseases such as rabies and measles. These pathogens were then named “filterable viruses” (an example being the 1918 influenza virus) and are now known simply as viruses. Today, filters are produced with pores small enough to sterilize—that is, eliminate all microbes, both bacteria and viruses—heat-sensitive materials such as ophthalmic solutions, antibiotics, vaccines, liquid vitamins, enzymes, sera, and culture media.

Liquids usually flow through filters by gravity, such as in coffeemakers, but in the laboratory environment a vacuum is often used to assist the movement of fluid through the filter (Figure 19.5). Filters originally were constructed from porcelain, glass, cotton, asbestos, and other special materials. Today’s scientists use membrane filters manufactured from nitrocellulose or plastic with specific pore and filter sizes. The pore sizes of these membrane filters range from 25 µm to less than 0.01 µm in diameter; these pore sizes are small enough to trap small viruses and even some of the larger protein molecules (Table 19.4).

TABLE 19.4

Membrane Filters

Pore Size (µm)	Microbes Retained
5	Multicellular algae and fungi
3	Yeasts and large unicellular algae
1.2	Protozoa and small unicellular algae
0.45	Largest bacteria
0.22	Largest viruses and most bacteria
0.025	Larger viruses, mycoplasmas, rickettsias, chlamydias, and some spirochetes
0.01	Smallest viruses

FIGURE 19.5 Filtration.
When supplements for media are not heat stable, the supplements may be sterilized by filtration through a membrane. This photograph shows a simple vacuum flask setup in which the liquid to be sterilized is pulled down through a funnel with a filter by a vacuum created in the lower collection flask. Filter pore size may vary, but the two sizes used frequently when filtering for bacteria/fungus and spores are 0.22 and 0.45 µm.

Chemical Control

In addition to the physical factors used to control microbial growth, chemical factors can also be employed both to kill microbes (bactericidal) and/or to inhibit growth (bacteriostatic). Different types of chemical agents are used to control microbial growth on both nonliving objects and living tissues. They are used to control microbes on surgical instruments and kitchen countertops, to clean toilets, and to prevent microbial growth in food and pharmaceutical products, just to name a few.

Disinfectants and Antiseptics

Antimicrobial chemicals are available in the form of liquids, gases, or solids. Few chemicals accomplish sterility; therefore most of the chemical methods of microbial control are aimed at disinfection and antisepsis. Chemicals specifically designed to be used on nonliving surfaces are referred to as disinfectants and those to be used on living tissues are referred to as antiseptics. Each category is designed for a specific task, and because no single chemical control agent is suitable for all circumstances, the selection of the appropriate agent is crucial for safety and quality control purposes. Chemical disinfectants and antiseptics have various characteristics that must be considered before any of them is used for a particular purpose.

Not all chemical agents are used to kill all microbes, because there may be circumstances in which the complete elimination of all microbes may not be desirable; instead, the inhibition of growth may be the desired effect. For example, the complete elimination of microorganisms might include the destruction of a population of normal flora, thus opening the potential for repopulation by a pathogenic microbe.

Although many antimicrobial agents have similar mechanisms of action they often vary in their effectiveness against different types of microbes. In these cases the groups of disinfectants/antiseptics may be further classified as bactericides, fungicides, algicides, or virucides. No matter what the chemical agent of choice, unless an aggressive protocol is used, a disinfectant/antiseptic typically does not sterilize an object because some resistant organisms or spores survive the treatment.

Factors Influencing Antimicrobial Effectiveness

Chemical disinfectants are commonly used in laboratory and healthcare settings for the decontamination and disinfection of benches, furniture, equipment, floors, and bathrooms, to mention a few. In addition to the nature of the chemical agent itself, other conditions influence the effectiveness of antimicrobial chemical agents as well. These factors include the following:

• Population size: Most agents kill organisms at a constant rate, and therefore require a longer period of time to kill or reduce a larger population.

• Population composition: As previously mentioned, the effectiveness of a chemical agent can vary greatly depending on the nature of the organisms being targeted. Because of differences in susceptibility, conditions in which there are a number of populations of different organisms may significantly decrease the overall effectiveness of the agent being used.

• Duration of exposure: As most agents kill at a constant rate, the longer the population of microbes is exposed to an agent, the more microbes are killed. Typically an exposure time long enough to reduce the probability of survival to 10^–6 is required to achieve sterilization.

• Local environment: A bacterial population is surrounded and affected by various environmental factors that may alter the effectiveness of chemical agents. The formation of a biofilm is a good example of a population condition that could dramatically decrease the activity of a chemical agent.

• Concentration of the chemical agent: During the initial period of exposure, an increase in the concentration of a chemical agent will typically lead to a rise in the killing rate against the target organisms. Beyond a certain point, however, a concentration increase will not alter the killing rate. Sometimes a chemical agent is actually more effective at lower concentrations. For example, ethyl alcohol is more effective at 70% concentration than at 95% concentration because of the fact that as the alcohol denatures proteins, the higher concentration will cause the proteins to coagulate rapidly throughout the entire cell wall/membrane thus preventing a more complete penetration of the alcohol deeper into the cell. A lower concentration allows the denaturing process to proceed more slowly thus allowing a deeper penetration into the cell.

• Temperature: An increase in temperature often increases the effectiveness of a chemical agent, provided the chemical stability of the compound is not exceeded. It is frequently possible to use lower concentrations of a chemical agent if it is used at higher temperatures.

• Organic matter: The presence of organic matter such as feces, vomit, or sputum can decrease the effectiveness of chemical disinfectants.

In addition, microorganisms have a range of resistances to chemical disinfectants, and the following points should be considered before choosing a particular one:

• Type of microorganisms, numbers, possible presence of spores

• Physical situation, such as surface type and suspension

• Noncorrosive or nonstaining properties with regards to the surface being treated

• Available contact between disinfectant and microorganism

• Possible interaction between disinfectant and materials

• Allowable contact time

• Concentration of the chemical: Remember, it is essential to follow the manufacturer’s recommendations for dilution of a concentrate

Evaluating Disinfectants

Formal testing of the effectiveness of chemical agents is a somewhat complicated process regulated by the U.S. Environmental Protection Agency (EPA) and the U.S. Food and Drug Administration (FDA). The EPA is concerned primarily with the testing and regulation of disinfectants, whereas the FDA oversees the regulation of chemical agents that are used on humans or animals. As stated previously, antiseptics and disinfectants are used in different ways to control microbial growth. The efficiency of a particular disinfectant or antiseptic can be determined by the use-dilution test and/or the disk-diffusion method.

Use-dilution Test

The use-dilution test is a bioassay method for testing the effectiveness of disinfectants and antiseptics on microorganisms. In the past, disinfectants and antiseptics were tested by the so-called phenol coefficient test, a procedure that compared the activity of a disinfectant with that of phenol, and has been replaced by the use-dilution test (Association of Analytical Communities). The test organisms used for this testing procedure are Salmonella enterica, Staphylococcus aureus, and Pseudomonas aeruginosa.

Disk-diffusion Method

In the disk-diffusion method a filter paper disk impregnated with a chemical disinfectant or antiseptic is placed on an agar plate on which a bacterial culture has been spread-plated. The chemical will diffuse from the disk into the agar around the disk. The size of the area of chemical infiltration around the disk is a function of the solubility of the chemical in agar, and of its molecular size. Microbes on the agar will not grow in the area around the disk if the organism is susceptible to the chemical. The area of no growth around the disk is called the “zone of inhibition” (Figure 19.6).

Because several conditions may affect the disk-diffusion susceptibility test, conditions must be constant from test to test, so that only the size of the zone of inhibition is variable. These conditions include the following:

• Agar used

• Amount of organism used

• Concentration of chemical used

• The incubation conditions, such as time, temperature, and atmosphere

The amount of organism needs to be standardized according to a turbidity standard, either a visual approximation or by means of a spectrophotometer.

Antimicrobial Agents

A wide variety of chemical compounds is available to meet the varied requirements for control of microbial growth. The main groups of compounds include phenols and phenolics, halogens, alcohols, surfactants, quaternary ammonium compounds, heavy metals, and aldehydes (Table 19.5).

TABLE 19.5

Chemicals Used in the Control of Microbes

Chemical Compound	Effectiveness	Advantages	Disadvantages	Preferred Use
Halogens
Chlorine	Kills most vegetative cells, some viruses and fungi	Good deodorant, inexpensive	Reduced by organic material; irritating odor and residue; solutions somewhat unstable	Purification of drinking water; disinfectant in food industry and hospitals
Iodine	Kills vegetative cells, some spores and viruses, if used in high concentrations	Useful as skin disinfectant; can be used over a wide pH range	Irritating odor and residue, except with iodophors; toxic if ingested	Wound dressing, preoperative preparation, sterilization of dairy equipment
Alcohols	Kill vegetative cells and many viruses and fungi	Unaffected by organic compounds; no residue; stable and easily handled; rapid effect	Flammable	Skin and surfaces
Phenols and phenolics	Kill vegetative cells and some fungi; only moderately effective against spores	Stable to heating and drying; unaffected by organic compounds	Pure phenol is harmful to tissues; disagreeable odor; expensive; corrosive	In combination with halogens and detergents, make excellent disinfectants
Surfactants	Disrupt bacterial attachment to surfaces	Inexpensive, usually nontoxic	Usually dislodge, not kill, bacteria	Surfaces including skin and counters
Quaternary ammonium compounds	Kill bacteria (including staphylococci; Mycobacterium tuberculosis) and enveloped viruses	Stable in presence of organic compounds; easy to handle; no irritation residue	Hard water, detergents, and fibrous material can interfere with activity; can rust metals; low concentrations can support some bacterial growth	Small metal instruments
Heavy metals	Kill some vegetative cells and viruses	Fast and inexpensive; no special equipment required	Inactivated by organic compounds and chemical antagonists	Rarely used except as preservatives and in fungal and protozoan infections
Alkylating agents
Aldehydes	Readily kill staphylococci, TB, and viruses; spores killed on prolonged exposure	Wide range of activity; noncorrosive	Prolonged exposure may be required; irritating to tissue, can be toxic	Instrument disinfection
Ethylene oxide (ETO)	Kills all microorganisms, including spores	Rapidly penetrates packing material	Extremely toxic, must be aerated; absorbed by porous materials, pure ETO is explosive	Sterilization of heat-sensitive materials and unwieldy objects in hospitals

Phenols and Phenolics

Phenol, also known as carbolic acid, and phenolics (substances chemically related to phenol) are disinfectants that denature proteins and therefore disrupt the cell membranes of a wide variety of pathogens. Joseph Lister introduced and started the use of phenol in 1867 to reduce infection during surgeries. Although often replaced by the use-dilution test, the efficacy of phenol remains a standard against which the actions of other antimicrobial agents can be compared.

Phenol and phenolics have been shown to be effective even in the presence of organic contaminants such as vomit, feces, saliva, and pus. Furthermore, these chemicals remain active on surfaces for prolonged periods of time. Although they are effective agents for microbial control, phenolics do have an intense odor and can irritate the skin and mucous membranes of some individuals. Phenol itself is still used for general disinfection of drains, cesspools, and animal quarters, but rarely as a medical germicide. Phenol derivatives such as cresols, combined with soap, are used for low-level disinfection in the hospital environment. One percent to 3% emulsions of lysol and creolin are commonly used household disinfectants. Several phenolics are major ingredients in disinfectant aerosol sprays used in hospitals and as laboratory disinfectants. One of the most widely used phenolics is 5 chloro-2-(2,4-dichlorophenoxy)phenol (triclosan), an antibacterial compound that is added to many household products.

Halogens

Halogens are reactive nonmetallic elements: iodine, chlorine, bromine, and fluorine. The mechanism of action of the halogens in controlling microbes is the oxidation of various cellular materials, including enzymes. This denaturation of enzymes is permanent and stops all metabolic activities of the microbes. Fluorine and bromine are hazardous to handle and are no more effective than iodine and chloride, and therefore the choices for germicidal preparations are iodine and chlorine. In fact, a common example of a microbicidal halogen compound is bleach. Both chlorine and iodine are routinely used in germicidal preparations, because they are not only microbiostatic, they are microbicidal and even sporicidal given longer exposure times.

Chlorine

Chlorine and its compounds have been used as disinfectants for more than 200 years. Chlorine not only kills bacteria and their endospores, but also fungi and viruses. Gaseous and liquid chlorine are used for large-scale disinfection of drinking water, swimming pools, sewage, and industrial and agricultural wastewaters. Furthermore, hypochlorite, a chlorine compound, is used to disinfect food equipment in dairies, restaurants, canneries, and more. Common household bleach is an effective all-around disinfectant, deodorizer, and even stain remover.

Iodine

Iodine, when dissolved in water, forms a brown-colored solution that rapidly penetrates microorganisms and interferes with their metabolism. With proper exposure times and concentration, iodine kills all classes of microorganisms. Weak iodine solutions (1% to 5%) are used as a topical antiseptic before surgery and also in the treatment of burned or infected skin. Disinfection of nonliving objects is done with stronger solutions, up to 10%.

Alcohols

Alcohols are bactericidal, fungicidal, and virucidal, but are ineffective against fungal spores and bacterial endospores. Commonly used alcohols include rubbing alcohol (isopropanol) and drinking alcohol (ethanol). The common antiseptic isopropyl alcohol (isopropanol) is slightly superior to ethanol as a disinfectant and antiseptic. The mechanism of action of alcohols in controlling microbes is the denaturing of cellular proteins and the dissolving of lipid membranes. Because the denaturation of proteins requires water, pure alcohol is not as effective an antimicrobial agent as the 70% or 90% alcohols that are commonly used to control microbial growth.

Surfactants

Surfactants are surface-active chemicals that reduce the surface tension of solvents so that the solvent becomes more effective at dissolving solute molecules. Two common surfactants in the control of microbes are soaps and detergents. These compounds do not directly affect microbial cells but instead reduce or prevent their attachment to surfaces. For these reasons, soaps and detergents are good degerming agents, but poor antimicrobial agents. Surgical and household soaps that are antiseptic contain antimicrobial chemicals.

Quaternary Compounds

Quaternary compounds or “quats” are the most popular detergents for microbial control. They are cationic detergents that not only act as emulsifiers and wetting agents but are also microbicidal. These compounds contain positively charged quaternary nitrogen and a long hydrophobic chain that can disrupt microbial membranes and may denature some proteins. In addition, quats are colorless, tasteless, and harmless to humans, except in high concentrations.

Quats are bactericidal, fungicidal, and virucidal against enveloped viruses, but are ineffective against naked viruses, mycobacteria, or endospores. Organic compounds and soaps eliminate the action of quaternary ammonium compounds. Some pathogens such as P. aeruginosa will thrive in the presence of these compounds; therefore, surfactants are classified as low-level disinfectants.

Heavy Metals

Heavy metal compounds (so called because of their relatively high atomic weight), such as the elements mercury, silver, gold, copper, arsenic, and zinc, have been used in microbial control for centuries. Their mechanism of action involves altering the three-dimensional shape of proteins, thereby interfering with or eliminating the appropriate function of cellular proteins.

For many decades, a large number of states in the United States required that the eyes of newborns be treated with cream containing 1% silver nitrate to prevent possible blindness caused by Neisseria gonorrhoeae, which newborns could acquire during passage through the birth canal. Although silver is still used in some surgical dressings, burn creams, as well as catheters, it has largely been replaced by other antimicrobial treatments that have been proven to be less irritating but still effective against pathogens.

Copper is used to control algal growth in fish tanks, swimming pools, reservoirs, and water storage tanks. Copper interferes with chlorophyll and is an effective algicide in the absence of organic contaminants.

MEDICAL HIGHLIGHTS

Thimerosal in Vaccines

Pharmaceutical companies have used thimerosal, a mercury-containing compound (an organomercurial), for decades to preserve some vaccines and other products, beginning in the 1930s. Mercury is a metabolic poison; however, no harmful effects have been reported from thimerosal at doses used in vaccines. However, concern about the theoretical potential for neurotoxicity of even low levels of organomercurials has grown over the years. Furthermore, an increased number of thimerosal-containing vaccines have been added to the infant immunization schedule. As a result, concerns about the use of thimerosal in vaccines and other products have been raised. Because of public pressure, in July 1999 agencies of the U.S. Public Health Service, the American Academy of Pediatrics, and vaccine manufacturers agreed that as a precautionary measure thimerosal should be reduced or eliminated in vaccine preparation and storage. With the exception of some influenza vaccines, none of the vaccines in the United States used for the protection of preschool children against 12 infectious diseases contain thimerosal.

Alkylating Agents

Alkylating agents constitute a diverse group of chemicals, often toxic because of their ability to inactivate nucleic acids and proteins. Agents used in the control of microorganisms include aldehydes and ethylene oxide.

• The aldehydes most often used in microbial control are glutaraldehyde, a liquid, and formaldehyde, a gas. Glutaraldehyde is a yellow acidic liquid and is one of the few chemicals that are accepted as a sterilant and high-level disinfectant. A 2% solution of glutaraldehyde will kill bacteria, viruses, and fungi within a 10-minute period of time for most objects. Spores and fungi can be killed within 3 hours, and an exposure time of 10 hours will achieve sterilization. Glutaraldehyde is less irritating and more effective than formaldehyde, but also more expensive. Formaldehyde is an irritating, strong-smelling gas that readily dissolves in water to form a solution called formalin. Although formalin is an intermediate to high-level disinfectant, it is extremely toxic (carcinogen), which limits its clinical usefulness.

• Ethylene oxide (ETO) is colorless, exists as a gas at room temperature, and is used as a sterilizing agent in hospitals and dental offices. It provides an effective method to sterilize and disinfect plastic materials and delicate, heat-sensitive instruments in hospitals and industries. Because of their toxicity and explosive properties, gaseous agents can be extremely hazardous to the people using them. Specially designed ETO sterilizers with safety features should be used by trained personnel only.

Food Preservation

Food preservation refers to a variety of techniques used in handling/preparing and treating food to avoid or slow down food spoilage, and to prevent foodborne illnesses. At the same time, food preservation processes should result in the maintenance of nutritional value, density, texture, and flavor.

Food poisoning is a common and usually mild illness; however, at times it can turn deadly. Symptoms include nausea, vomiting, abdominal cramps, and diarrhea, which occur within 48 hours of consuming a contaminated food or drink. Foodborne diseases (also see the Healthcare Application tables in Chapter 1, Scope of Microbiology) are caused by either the contaminating microbe itself or by its toxins. Infectious diseases can be spread through food or beverages and are a problem for millions of people in the United States and around the world. The Centers for Disease Control and Prevention (CDC) estimates that 76 million people annually suffer from foodborne illnesses in the United States alone, causing approximately 325,000 hospitalizations and more than 5000 deaths. According to the National Institutes of Health (NIH) the annual cost of all foodborne diseases in the United States is 5 to 6 billion dollars in direct medical costs and lost productivity. An increase in reported food poisoning cases has been noted by the CDC, and it is estimated that nearly 25% of the U.S. population suffers from some sort of food poisoning each year. It has been suggested that this increase is due to the mass production and distribution of processed foods, including raw vegetables, fruits, and meats. Any type of safety shortcuts and improper handling will lead to contamination of large masses of food with dirt, water, or animal wastes.

Safe food preservation involves preventing the growth of bacteria, fungi, and other microorganisms. It also involves stopping fat oxidation, which results in spoilage (rancidity), as well as processes that will inhibit the natural aging and discoloration that occur during food processing. Methods to prevent these processes include canning, pickling, drying, freeze-drying, pasteurization, smoking, addition of chemical additives, and irradiation.

HEALTHCARE APPLICATION
Total Number of Foodborne Disease Outbreaks by Etiology: 2005 Summary Statistics *

Etiology	Number of Outbreaks	Number of Cases
Bacterial	188	4348
Chemical	40	151
Parasitic	6	739
Viral	170	5018
Multiple	6	525
Unknown etiology	572	9398
Total:	982	20,179

^*Data assembled by the Centers for Disease Control and Prevention (CDC). Current updates from the CDC can be viewed at http://www.cdc.gov/foodborneoutbreaks/.

Pasteurization

Pasteurization (also see Chapter 1, Scope of Microbiology) is a method by which heat is applied to liquids to kill potential pathogens, as well as microbes that could lead to food spoilage, without changing the flavor and food value of the material. Pasteurization is designed to achieve a mathematical “log reduction” in the number of viable organisms to such a point that they are unlikely to cause disease. Although pasteurization does eliminate most pathogens, it does not sterilize, and heat-loving and heat-tolerant bacteria survive the procedure. These swimming organisms do not cause food spoilage over a relatively short time period, if appropriately refrigerated, and they are generally not pathogenic.

Effective pasteurization varies with the product and the time and temperature required for effectiveness, all of which must be adjusted accordingly. Pasteurization uses temperatures below the boiling point to avoid damage to the food. Today, two types of pasteurization are typically used: high temperature short time (HTST) and ultrahigh temperature (UHT).

• HTST pasteurization can be achieved in two ways: by batch or continuous flow. In the batch process the fluid is held at 63° C for 30 minutes and then quickly cooled to about 4° C. In the continuous flow process, liquid is guided between metal plates or pipes that are heated from the outside by hot water.

• UHT pasteurization is done by holding the liquid at a temperature of 138° C for a fraction of second; products are accordingly labeled and can be stored at room temperature without microbial spoilage. After months the food prepared by this method may lose some of its flavor.

In the United States, pasteurization methods are standardized and controlled by the U.S. Department of Agriculture (USDA). Different standards are applied for different products, usually depending on the content and intended use.

LIFE APPLICATION

Milk: Pasteurized or Raw?

Since its adoption in the early 1900s, pasteurization has resulted in the reduction of foodborne illnesses due to contaminated milk. By carefully processing milk and milk products many illnesses can be prevented. However, some groups of people firmly believe that the processed milk destroys the natural nutritive properties of milk and may make it harmful for human consumption. Although pasteurization will change some proteins within milk through denaturation, it also will prevent disease from organisms that contaminate raw milk—milk cannot be harvested without some contamination by pathogens, because our environment is not sterile. According to the U.S. Department of Agriculture, drinking untreated (raw) milk or eating raw milk products is equivalent to playing “Russian roulette” with your health. Annually the Centers for Disease Control and Prevention reports contain many cases of foodborne diseases due to consumption of raw milk or raw milk products.