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.

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

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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