Ch 9 - Controlling Microbial Growth in The Environment

Factors Affecting the Efficacy of Antimicrobial Methods Include:

- The Site to Be Treated - Relative Susceptibility of Microorganisms - The environmental conditions that pertain.

There are many types of chemical and physical microbial controls, but their modes of action fall into two basic categories:

1.) those that disrupt the integrity of cells by adversely altering their cell walls or cytoplasmic membranes 2.) those that interrupt cellular metabolism and reproduction by interfering with the structures of proteins and nucleic acids.

A biological indicator of sterility

A biological indicator of sterility uses endospores of bacteria such as Bacillus impregnated into tape. - After autoclaving, the tape is aseptically inoculated into sterile broth. - If no bacterial growth appears, the original material is considered sterile. - In a variation on this technique, the endospores are on a strip in one compartment of a vial that also includes, in a separate compartment, a growth medium containing a pH color indicator. - After autoclaving, a barrier between the two compartments is broken, putting the endospores into contact with the medium. - In this case, the absence of a color change after incubation indicates sterility

Alcohols

Alcohols are bactericidal, fungicidal, and virucidal against enveloped viruses; however, they are not effective against fungal spores or bacterial endospores. - Alcohols are considered intermediate-level disinfectants. - Commonly used alcohols include rubbing alcohol (isopropanol) and drinking alcohol (ethanol)

What does alcohol do?

Alcohols denature proteins and disrupt cytoplasmic membranes. - Surprisingly, pure alcohol is not an effective antimicrobial agent because the denaturation of proteins requires water; therefore, solutions of 70% to 90% alcohol are typically used to control microbes. - Alcohols evaporate rapidly, which is advantageous in that they leave no residue but disadvantageous in that they may not contact microbes long enough to be effective. - Alcohol-based antiseptics are more effective than soap in removing bacteria from hands but not effective against some viruses, such as diarrhea-causing noroviruses. - Swabbing the skin with alcohol prior to an injection removes more microbes by physical action (degerming) than by chemical action.

Chemical Methods of Microbial Control

Although physical agents are sometimes used for disinfection, antisepsis, and preservation, more often chemical agents are used for these purposes. - As we have seen, chemical agents act to adversely affect microbes' cell walls, cytoplasmic membranes, proteins, or DNA. - As with physical agents, the effect of a chemical agent varies with temperature, length of exposure, and the amount of contaminating organic matter in the environment. - The effect also varies with pH, concentration, and freshness of the chemical. - Chemical agents tend to destroy or inhibit the growth of enveloped viruses and the vegetative cells of bacteria, fungi, and protozoa more than fungal spores, protozoan cysts, or bacterial endospores. - The latter are particularly resistant to chemical agents

Biosafety Level 3 (BSL-3)

BSL3 is stricter, requiring that all manipulations be done within safety cabinets containing high efficiency particulate air (HEPA) filters. - BSL-3 also specifies special design features for the laboratory. - These include entry through double sets of doors and lower air pressure in the laboratory, such that an open door only allows air to move into, not out of, the room. - Air leaving the room is HEPA-filtered before being discharged. - BSL-3 is designed for experimentation on microbes such as bacteria of tuberculosis and anthrax and viruses of yellow fever and Rocky Mountain spotted fever

What are the 4 levels of biosafety?

Biosafety Level 1 (BSL-1) Biosafety Level 2 (BSL-2) Biosafety Level 3 (BSL-3) Biosafety Level 4 (BSL-4)

Biosafety Level 1 (BSL-1)

Biosafety Level 1 (BSL1) is suitable for handling microbes, such as Escherichia coli, not known to cause disease in healthy humans. - Precautions in BSL-1 are minimal and include handwashing with antibacterial soap and disinfecting surfaces

Bisphenolics

Bisphenolics are composed of two covalently linked phenolics. - An example is triclosan, which is incorporated into numerous consumer products, including garbage bags, diapers, and cutting board

Moist Heat Related Methods Include:

Boiling Autoclaving Pasteurization Ultra-High-Temperature Sterilization

Bromine

Bromine is an effective disinfectant in hot tubs because it evaporates more slowly than chlorine at high temperatures. - Bromine is also used as an alternative to chlorine in the disinfection of swimming pools, cooling towers, and other water containers

x-rays (ionizing radiation)

Consumers have been reluctant to accept irradiated food. - A number of reasons have been cited, including fear that radiation makes food radioactive and claims that it changes the taste and nutritive value of foods or produces potentially carcinogenic (cancer-causing) chemicals. - Supporters of irradiation reply that gamma radiation passes through food and cannot make it radioactive any more than a dental X-ray produces radioactive teeth, and they cite numerous studies that conclude that irradiated foods are tasty, nutritious, and safe. - X-rays travel the farthest through matter, but they have less energy than gamma rays and require a prohibitive amount of time to make them practical for microbial control.

Copper

Copper, which interferes with chlorophyll, is used to control algal growth in reservoirs, fish tanks, swimming pools, and water storage tanks. - In the absence of organic contaminants, copper is an effective algicide in concentrations as low as 1 ppm (part per million). - Copper, zinc, or mercury is used to control mildew in some paint.

Dessication

Desiccation, or drying, has been used for thousands of years to preserve such foods as fruits, peas, beans, grain, nuts, and yeast. - Desiccation inhibits microbial growth because metabolism requires liquid water. - Drying inhibits the spread of most pathogens, including the bacteria that cause syphilis, gonorrhea, and the more common forms of bacterial pneumonia and diarrhea. - However, many molds can grow on dried raisins and apricots, which have as little as 16% water content

Electron beams (ionizing radiation)

Electron beams are produced by cathode ray machines. - Electron beams are highly energetic and therefore very effective in killing microbes in just a few seconds, but they cannot sterilize thick objects or objects coated with large amounts of organic matter. - They are used to sterilize spices, meats, microbiological plastic ware, and dental and medical supplies, such as gloves, syringes, and suturing material.

Ethylene oxide

Ethylene oxide is frequently used as a gaseous sterilizing agent in hospitals and dental offices, and NASA uses the gas to sterilize spacecraft designed to land on other worlds lest they accidentally export earthly microbes. - Large hospitals often use ethylene oxide chambers, which are similar in appearance to autoclaves, to sterilize instruments and equipment sensitive to heat.

Filtration

Filtration is the passage of a fluid (either a liquid or a gas) through a sieve designed to trap particles—in this case, cells or viruses—and separate them from the fluid. - Researchers often use a vacuum to assist the movement of fluid through the filter. - Filtration traps microbes larger than the pore size, allowing smaller microbes to pass through. In the late 1800s, filters were able to trap cells, but their pores were too large to trap the pathogens of such diseases as rabies and measles. - These pathogens were thus named filterable viruses, which today has been shortened to viruses. - Now, filters with pores small enough to trap even viruses are available, so filtration can be used to sterilize such heat-sensitive materials as ophthalmic solutions, antibiotics, vaccines, liquid vitamins, enzymes, and culture media.

Fluorine

Fluorine in the form of fluoride is antibacterial in drinking water and toothpastes and can help reduce the incidence of dental caries (cavities). Fluorine works in part by disrupting metabolism in the biofilm of dental plaque.

Thimerosal (ethyl mercury)

For over 70 years, drug companies used thimerosal, a mercury-containing compound, to preserve vaccines. In 1999, the U.S. - Public Health Service recommended that alternatives be used because mercury is a metabolic poison, though the very small amount of mercury in vaccines is considered safe. - Today, the recommended children's vaccines are thimerosal free, except for influenza (flu) vaccines distributed in multidose vials. - A few adult vaccines also contain thimerosal, including the whole-cell pertussis vaccine and some vaccines against tetanus, flu, and meningococcal meningitis

Filtration in health care and laboratory settings

Health care and laboratory workers routinely use filtration to prevent airborne contamination by microbes. - Medical personnel wear surgical masks to prevent exhaled microbes from contaminating the environment, and cotton plugs are placed in culture vessels to prevent contamination by airborne microbes. - Additionally, high-efficiency particulate air (HEPA) filters are crucial parts of biological safety cabinets. - HEPA filters are mounted in the air ducts of some operating rooms, rooms occupied by patients with airborne diseases such as tuberculosis, and rooms of immunocompromised patients, such as burn victims and AIDS patients

Heavy-metal ions

Heavy-metal ions are ions of relatively high density metals, such as arsenic, zinc, mercury, silver, and copper, that are toxic at low concentrations. - Heavy-metal ions are antimicrobial because they combine with sulfur atoms in molecules of cysteine, an amino acid. - Such bonding denatures proteins, inhibiting or eliminating their function. - Heavy-metal ions are low-level bacteriostatic and fungistatic agents, and with few exceptions their use has been superseded by more effective antimicrobial agents

The Site To Be Treated

In many cases, the choice of an antimicrobial method depends on the nature of the site to be treated. - For example, harsh chemicals and extreme heat cannot be used on humans, animals, and fragile objects, such as artificial heart valves and plastic utensils. - Moreover, when performing medical procedures, medical personnel must choose a method and level of microbial control based on the site of the procedure because the site greatly affects the potential for subsequent infection. - For example, the use of medical instruments that penetrate the outer defenses of the body, such as needles and scalpels, carries a greater potential for infection, so they must be sterilized; however, disinfection may be adequate for items that contact only the surface of a mucous membrane or the skin. - In the latter case, sterilization is usually required only if the patient is immunocompromised.

Iodine

Iodine is a well-known antiseptic. - In the past, backpackers and campers disinfected water with iodine tablets, but experience has shown that some protozoan cysts can survive iodine treatment unless the iodine concentration is so great that the water is undrinkable. - Medically, iodine is used either as a tincture or as an iodophor, which is an iodine-containing organic compound that slowly releases iodine. Iodophors have the advantage of being long lasting and nonirritating to the skin. - Betadine is an example of an iodophor used in medical institutions to prepare skin for surgery and injections and to treat burns

Rubbing Alcohol (Isopropanol)

Isopropanol is slightly superior to ethanol as a disinfectant and antiseptic. - Tinctures of antimicrobial chemicals, which are solutions of the chemicals in alcohol, are often more effective than the same chemicals dissolved in water.

antimicrobial enzymes

Many organisms produce chemicals that inhibit or destroy a variety of fungi, bacteria, or viruses. Among these are antimicrobial enzymes, which are enzymes that act against microorganisms. - For example, human tears contain the enzyme lysozyme, which is a protein that digests the peptidoglycan cell walls of bacteria, causing the bacteria to rupture because of osmotic pressure and thus protecting the eye from most bacterial infections.

Moist Heat

Moist heat (which is commonly used to disinfect, sanitize, sterilize, and pasteurize) kills cells by denaturing proteins and destroying cytoplasmic membranes. - Moist heat is more effective in microbial control than dry heat because water is a better conductor of heat than air. - An example from your kitchen readily demonstrates this: you can safely stick your hand into an oven at 350°F for a few moments, but putting a hand into boiling water at the lower temperature of 212°F would burn you severely

formaldehyde

Morticians and health care workers use formaldehyde dissolved in water to make a 37% solution called formalin. - They use formalin for embalming and to disinfect hospital rooms, instruments, and machines. - Formaldehyde must be handled with care because it irritates mucous membranes and is carcinogenic (cancer causing)

Negative aspects of phenolics

Negative aspects of phenolics include their disagreeable odor and possible side effects; for example, phenolics irritate the skin of some individuals

microbial death rate

One technique for evaluating the efficacy of an antimicrobial agent is to calculate the microbial death rate, which is usually found to be constant over time for any particular microorganism under a particular set of conditions. - Suppose, for example, that a scientist treats a broth containing 1 billion (109) microbes with an agent that kills 90% of them in 1 minute. - The most susceptible cells die first, leaving 100 million (108) hardier cells after the first minute. - After another minute of treatment, another 90% die, leaving 10 million (107) cells that have even greater resistance to and require longer exposure to the agent before they die. - Notice that in this case, each full minute decreases the number of living cells 10-fold. - The broth will be sterile when all the cells are dead. When these results are plotted on a semilogarithmic graph—in which the y-axis is logarithmic and the x-axis is arithmetic—the plot of microbial death rate is a straight line; that is, the microbial death rate is constant

Ozone(O3)

Ozone (O3) is a reactive form of oxygen that is generated when molecular oxygen (O2) is subjected to electrical discharge. - Ozone gives air its "fresh smell" after a thunderstorm. - Some Canadian and European municipalities treat their drinking water with ozone rather than chlorine. - Ozone is a more effective antimicrobial agent than chlorine, but it is more expensive, and it is more difficult to maintain an effective concentration of ozone in water

Peracetic acid

Peracetic acid is an extremely effective sporicide that can be used to sterilize surfaces. - Food processors and medical personnel use peracetic acid to sterilize equipment because it is not adversely affected by organic contaminants, and it leaves no toxic residue

Autoclaving

Practically speaking, true sterilization using heat requires higher temperatures than that of boiling water. - To achieve the required temperature, pressure is applied to boiling water to prevent the escape of heat in steam. - The reason that applying pressure succeeds in achieving sterilization is that the temperature at which water boils (and steam is formed) increases as pressure increases. - Scientists and medical personnel routinely use a piece of equipment called an autoclave to sterilize chemicals and objects that can tolerate moist heat. - Alternative techniques (discussed shortly) must be used for items that are damaged by heat or water, such as some plastics and vitamins. - An autoclave consists of a pressure chamber, pipes to introduce and evacuate steam, valves to remove air and control pressure, and pressure and temperature gauges to monitor the procedure. - As steam enters an autoclave chamber, it forces air out, raises the temperature of the contents, and increases the pressure until a set temperature and pressure are reached.

Prions

Prions, which are infectious proteins that cause degenerative diseases of the brain, are more resistant than any cell or virus; it takes heating to 482°C for 4 hours to denature them

Refrigeration

Refrigeration halts the growth of most pathogens, which are predominantly mesophiles. - Notable exceptions are the bacteria Listeria, which can reproduce to dangerous levels in refrigerated food, and Yersinia, which can multiply in refrigerated blood products and be passed on to blood recipients

microbial death

Scientists define microbial death as the permanent loss of reproductive ability under ideal environmental conditions

synthetic detergents

Synthetic detergents are positively charged organic surfactants that are more soluble in water than soaps

Biosafety Level 4 (BSL-4)

The most secure laboratories are BSL4 facilities, designated for working with dangerous or exotic microbes that cause severe or fatal diseases in humans, such as Ebola, smallpox, and Lassa fever viruses. - BSL-4 labs are either separate buildings or completely isolated from all other areas of their buildings. - Entry and exit are strictly controlled through electronically sealed airlocks with multiple showers, a vacuum room, an ultraviolet light room, and other safety precautions designed to destroy all traces of the biohazard. - All air and water entering and leaving the facility are filtered to prevent accidental release. - Personnel wear "space suits" supplied with air hoses. - Suits and the laboratory itself are pressurized such that microbes are swept away from workers

What methods have scientists developed to measure the efficacy of antimicrobial agents?

These include the phenol coefficient, the use-dilution test, the KelseySykes capacity test, and the in-use test.

In-Use Test

Though phenol coefficient, use-dilution, and Kelsey-Sykes capacity tests can be beneficial for initial screening of disinfectants, they can also be misleading. - These types of evaluation are measures of effectiveness under controlled conditions against one or, at most, a few species of microbes at a time, but disinfectants are generally used in various environments against a diverse population of organisms that are often associated with one another in complex biofilms affording mutual protection. - A more realistic (though more time-consuming) method for determining the efficacy of a chemical is called an in-use test. - In this procedure, swabs are taken from actual objects, such as operating room equipment, both before and after the application of a disinfectant or an antiseptic. - The swabs are then inoculated into appropriate growth media that, after incubation, are examined for microbial growth. - The in-use test allows a more accurate determination of the proper strength and application procedure of a given disinfection agent for each specific situation.

Visible Light and Microwaves ((radio waves of extremely short wavelength) (Non-ionizing)

Visible light and microwaves (radio waves of extremely short wavelength) have little value in microbial control, though microwaves heat food, inhibiting microbial growth and reproduction if the food gets hot enough. - The more energetic microwaves produced by some commercial microwave ovens can kill fungal spores, preventing treated food from molding

Aldehydes

are compounds containing terminal —CHO groups. - Glutaraldehyde, which is a liquid, and formaldehyde, which is a gas, are highly reactive chemicals - Aldehydes function in microbial control by cross-linking organic functional groups, including amino, hydroxyl, sulfhydryl, and carboxyl, thereby denaturing proteins and inactivating nucleic acids

Halogens

are the four very reactive, nonmetallic chemical elements: iodine, chlorine, bromine, and fluorine. - Halogens are intermediate-level antimicrobial chemicals that are effective against vegetative bacterial and fungal cells, fungal spores, some bacterial endospores and protozoan cysts, and many viruses. - Halogens are used both alone and combined with other elements in organic and inorganic compounds. - Halogens exert their antimicrobial effect by unfolding and thereby denaturing essential proteins, including enzymes

thermal death time

e is the time it takes to completely sterilize a particular volume of liquid at a set temperature

Ideally, agents used for the control of microbes should be:

inexpensive, fast acting, stable during storage, and capable of controlling microbial growth while being harmless to humans, animals, and objects

Thermal death point

is the lowest temperature that kills all cells in a broth in 10 minutes

Sanitization

is the process of disinfecting plates and utensils used by the public to reduce the number of pathogenic microbes to meet accepted public health standards. - For example, steam, high-pressure hot water, and scrubbing are used to sanitize restaurant utensils and dishes, and chemicals are used to sanitize public toilets. - Thus, the difference between disinfecting dishes at home and sanitizing dishes in a restaurant is the arena—private versus public—in which the activity takes place

Degerming

is the removal of microbes from a surface by scrubbing, such as when you wash your hands or a nurse prepares an area of skin for an injection. - Though chemicals such as soap or alcohol are commonly used during degerming, the action of thoroughly scrubbing the surface may be more important than the chemical in removing microbes.

decimal reduction time (D)

is the time required to destroy 90% of the microbes in a sample. - This concept is especially useful to food processors because they must heat foods to eliminate the endospores of anaerobic Clostridium botulinum, which could germinate and produce life-threatening botulism toxin inside sealed cans

Pasteurization

is the use of heat to kill pathogens and reduce the number of spoilage microorganisms in food and beverages. - Milk, fruit juices, wine, and beer are commonly pasteurized.

sterilization

refers to the removal or destruction of all microbes, including viruses and bacterial endospores, in or on an object. - In practical terms, sterilization indicates only the eradication of harmful microorganisms and viruses; some innocuous microbes may still be present and viable in an environment that is considered sterile. - For instance, commercial sterilization of canned food does not kill all hyperthermophilic microbes; because they do not cause disease and cannot grow and spoil food at ambient temperatures, they are of no practical concern. - Likewise, some hyperthermophiles may survive sterilization by laboratory methods (discussed shortly), but they are of no practical concern to technicians because they cannot grow or reproduce under normal laboratory conditions

Why do scientists and health care professionals use the suffixes stasis or static?

scientists and health care professionals use the suffixes stasis or static to indicate that a chemical or physical agent inhibits microbial metabolism and growth but does not necessarily kill microbes. - Thus, refrigeration is bacteriostatic for most bacterial species; it inhibits their growth, but they can resume metabolism when the optimal temperature is restored. - By contrast, words ending in cide or cidal refer to agents that destroy or permanently inactivate a particular type of microbe; virucides inactivate viruses, bactericides kill bacteria, and fungicides kill fungal hyphae, spores, and yeasts. - Germicides are chemical agents that destroy pathogenic microorganisms in general.

Sterilization in an autoclave requires:

that steam be able to contact all liquids and surfaces that might be contaminated with microbes; therefore, solid objects must be wrapped in porous cloth or paper, not sealed in plastic or aluminum foil, both of which are impermeable to steam. - Containers of liquids must be sealed loosely enough to allow steam to circulate freely, and all air must be forced out by steam. - Since steam is lighter than air, it cannot force air from the bottom of an empty vessel; therefore, empty containers must be tipped so that air can flow out of them.

Osmotic Pressure

- Another ancient method of microbial control is the use of high concentrations of salt or sugar in foods to inhibit microbial growth by osmotic pressure. - Osmosis is the net movement of water across a semipermeable membrane (such as a cytoplasmic membrane) from an area of higher water concentration to an area of lower water concentration. - Cells in a hypertonic solution of salt or sugar lose water, and the cell shrinks. - The removal of water inhibits cellular metabolism because enzymes are fully functional only in aqueous environments. - Thus, osmosis preserves honey, jerky, jams, jellies, salted fish, and some types of pickles from most microbial attacks. - Fungi have a greater ability than bacteria to tolerate hypertonic environments with little moisture, which explains why jelly in your refrigerator may grow a colony of Penicillium mold but is not likely to grow the bacterium Salmonella

Antimicrobial drugs

- Antimicrobial drugs include antibiotics, semisynthetics, and synthetics. - Specifically, antibiotics are antimicrobial chemicals produced naturally by microorganisms. - When scientists chemically modify an antibiotic, the agent is called a semisynthetic. - Scientists have also developed wholly synthetic antimicrobial drugs. - The main difference between these antimicrobials and the chemical agents we have discussed in this chapter is that antimicrobial drugs are typically used for treatment of disease and not for environmental control of microbes. - Nevertheless, some antimicrobial drugs are used for control outside the body. - For example, nisin and natamycin are used to reduce the growth of bacteria and fungi, respectively, in cheese.

Biosafety Level 2 (BSL-2)

- BSL2 facilities are similar to those of BSL-1 but are designed for handling moderately hazardous agents, such as hepatitis and influenza viruses and methicillin-resistant Staphylococcus aureus (MRSA). - Access to BSL-2 labs is limited when work is being conducted, extreme precautions are taken with contaminated sharp objects, and procedures that might produce aerosols are conducted within safety cabinets

Disinfection (disinfectant)

- Disinfection refers to the use of physical or chemical agents known as disinfectants, including ultraviolet light, heat, alcohol, and bleach, to inhibit or destroy microorganisms, especially pathogens. - Unlike sterilization, disinfection does not guarantee that all pathogens are eliminated; indeed, disinfectants alone cannot inhibit endospores or some viruses. - Further, the term disinfection is used only in reference to treatment of inanimate objects

Non-ionizing Radiation

- Electromagnetic radiation with a wavelength greater than 1 nm does not have enough energy to force electrons out of orbit, so it is non-ionizing radiation. - However, such radiation does contain enough energy to excite electrons and cause them to make new covalent bonds, which can affect the three-dimensional structure of proteins and nucleic acids. - Ultraviolet (UV) light, visible light, infrared radiation, and radio waves are nonionizing radiation ** Of these, only UV light has sufficient energy to be a practical antimicrobial agent.

Gamma Rays (ionizing radiation)

- Gamma rays, which are emitted by some radioactive elements, such as radioactive cobalt, penetrate much farther than electron beams but require hours to kill microbes. - The U.S. Food and Drug Administration (FDA) has approved the use of gamma radiation for microbial control in meats, spices, and fresh fruits and vegetables. - Irradiation with gamma rays kills not only microbes but also the larvae and eggs of insects; it also kills the cells of fruits and vegetables, preventing both microbial spoilage and overripening.

Gluaraldehyde

- Hospital personnel and scientists can use 2% solutions of glutaraldehyde to kill bacteria, viruses, and fungi; a 10-minute treatment effectively disinfects most objects. When the time of exposure is increased to 10 hours, glutaraldehyde sterilizes. - Glutaraldehyde is less irritating and more effective than formaldehyde, but it is more expensive

Hydrogen peroxide (H2O2)

- Hydrogen peroxide (H2O2) is a common household chemical that can disinfect and even sterilize the surfaces of inanimate objects such as contact lenses, but it is often mistakenly used to treat open wounds. - Hydrogen peroxide does not make a good antiseptic for open wounds because catalase—an enzyme released from damaged human cells—quickly neutralizes hydrogen peroxide by breaking it down into water and oxygen gas, which can be seen as escaping bubbles. - Though aerobes and facultative anaerobes on inanimate surfaces also contain catalase, the volume of peroxide used as a disinfectant overwhelms the enzyme, making hydrogen peroxide a useful disinfectant. - Food processors use hot hydrogen peroxide to sterilize packages such as juice boxes.

Phenol and Phenolics

- In 1867, Dr. Joseph Lister (1827-1912) began using phenol (also known as carbolic acid) to reduce infection during surgery. - Phenolics are compounds derived from phenol molecules that have been chemically modified by the addition of organic functional groups or reactive atoms. - For instance, chlorinated phenolics contain one or more atoms of chlorine and have enhanced antimicrobial action and a less annoying odor than phenol. - Natural oils, such as pine and clove oils, are also phenolics and can be used as antiseptics - Phenol and phenolics denature proteins and disrupt cell membranes in a wide variety of pathogens. - They are effective even in the presence of contaminating organic material, such as vomit, pus, saliva, and feces, and they remain active on surfaces for a prolonged time. - For these reasons, phenolics are commonly used in health care settings, laboratories, and households

Refrigeration vs Freezing

- In many situations, particularly in food preparation and storage, the most convenient method of microbial control is either refrigeration (temperatures between 0°C and 7°C) or freezing (temperatures below 0°C). - These processes decrease microbial metabolism, growth, and reproduction because chemical reactions occur more slowly at low temperatures and because liquid water is not available at subzero temperatures. - Note, however, that psychrophilic (cold-loving) microbes can still multiply in refrigerated food and spoil its taste and suitability for consumption

Use-Dilution Test

- In this test, a researcher dips several metal cylinders into broth cultures of bacteria and briefly dries them at 37°C. - The bacteria used in the standard test are Pseudomonas aeruginosa, Salmonella enterica serotype Choleraesuis, and Staphylococcus aureus. - The researcher then immerses each contaminated cylinder into a different dilution of the disinfectants being evaluated. - After 10 minutes, each cylinder is removed, rinsed with water to remove excess chemical, and placed into a fresh tube of sterile medium for 48 hours of incubation. - The most effective agent is the one that entirely prevents microbial growth at the highest dilution. - The use-dilution test is the current standard test in the United States, though it was developed several decades ago before the appearance of many of today's more troubling pathogens, including hepatitis C virus, HIV, and antibiotic-resistant bacteria and protozoa. - Moreover, the disinfectants in use at the time the test was developed were far less powerful than many used today. - Some government agencies have expressed concern that the test is neither accurate, reliable, nor relevant; therefore, AOAC International—an association of chemists, microbiologists, food science personnel, and others—is developing a new standard procedure for use in the United States.

Gaseous Agents

- Many items, such as heart-lung machine components, sutures, plastic laboratory ware, mattresses, pillows, artificial heart valves, catheters, electronic equipment, and dried or powdered foods, cannot be sterilized easily with heat or water-soluble chemicals, nor is irradiation always practical for large or bulky items. - However, they can be sterilized within a closed chamber containing highly reactive microbicidal and sporicidal gases such as ethylene oxide, propylene oxide, and betapropiolactone - These gases rapidly penetrate paper and plastic wraps and diffuse into every crack. - Over time (usually 4-18 hours), they denature proteins and DNA by cross-linking organic functional groups, thereby killing everything they contact without harming inanimate objects - Despite their advantages, gaseous agents are far from perfect: - They are often highly explosive, and they are extremely poisonous, so workers must extensively flush sterilized objects with air to remove every trace of the gas (which adds to the time required to use them). - Finally, gaseous agents, especially beta-propiolactone, are potentially carcinogenic

Development of Resistant Microbes

- Many scientists are concerned that Americans have become overly preoccupied with antisepsis and disinfection, as evidenced by the proliferation of products containing antiseptic and disinfecting chemicals. - For example, you can now buy hand soap, shampoo, toothpaste, hand lotion, foot pads for shoes, deodorants, and bath sponges that contain antiseptics, as well as kitty litter, cutting boards, scrubbing pads, garbage bags, children's toys, and laundry detergents that contain disinfectants. - There is little evidence that the extensive use of such products adds to human or animal health, but it does promote the development of strains of microbes resistant to antimicrobial chemicals: - While susceptible cells die, resistant cells remain to proliferate. - Scientists have already isolated strains of pathogenic bacteria, including M. tuberculosis, P. aeruginosa, E. coli, and S. aureus, that are less susceptible to common disinfectants and antiseptics

chlorine

- Municipalities commonly use chlorine in its elemental form (Cl2) to disinfect drinking water, swimming pools, and wastewater from sewage treatment plants. Compounds containing chlorine are also effective disinfectants. - Examples include sodium hypochlorite (NaOCl), which is household chlorine bleach, and calcium hypochlorite (Ca(OCl)2). - The dairy industry and restaurants use these compounds to disinfect utensils, and the medical field uses them to disinfect hemodialysis systems. - Household bleach diluted by adding two drops to a liter of water can be used in an emergency to make water safer to drink, but it does not kill all protozoan cysts, bacterial endospores, or viruses. - Chlorine dioxide (ClO2) is a gas that can be used to disinfect large spaces; for example, officials used it to sterilize federal buildings contaminated with anthrax spores following the 2001 bioterrorism attack. - Chloramines—chemical combinations of chlorine and ammonia—are used in wound dressings, as skin antiseptics, and in some municipal water supplies - Chloramines are less effective antimicrobial agents than other forms of chlorine, but they release chlorine slowly and are thus longer lasting

Damage to Proteins and Nucleic Acids

- Proteins regulate cellular metabolism, function as enzymes in most metabolic reactions, and form structural components in membranes and cytoplasm. - A protein's function depends on its exact three-dimensional shape, which is maintained by hydrogen and disulfide bonds between amino acids. - When these bonds are broken by extreme heat or certain chemicals, the protein's shape changes. - Such denatured proteins cease to function, bringing about cellular death. - Chemicals, radiation, and heat can also alter and even destroy nucleic acids. - Given that the genes of a cell or virus are composed of nucleic acids, disruption of these molecules can produce fatal mutations. - Additionally, that portion of a ribosome that actually catalyzes the synthesis of proteins is a ribozyme—that is, an enzymatic RNA molecule. - For this reason, physical or chemical agents that interfere with nucleic acids also stop protein synthesis. - Scientists and health care workers have at their disposal many chemical and physical agents to control microbial growth and activity. - In the next section, we consider the factors and conditions that should be considered in choosing a particular control method, as well as some ways to evaluate a method's effectiveness.

Membrane Filters

- Scientists today typically use thin (only 0.1 mm thick), circular membrane filters manufactured of nitrocellulose or plastic and containing specific pore sizes ranging from 25 mm to less than 0.01 mm in diameter. - The pores of the latter filters are small enough to trap small viruses and even some large protein molecules. - Microbiologists also use filtration to estimate the number of microbes in a fluid by counting the number deposited on the filter after passing a given volume through the filter.

Lyophilization

- Scientists use lyophilization (lī-of′-i-li-zā′shŭn), a technique that combines freezing and drying, to preserve microbes and other cells for many years. - In this process, scientists instantly freeze a culture in liquid nitrogen or frozen carbon dioxide (dry ice); then they subject it to a vacuum that removes frozen water through a process called sublimation, in which the water is transformed directly from a solid to a gas. - Lyophilization prevents the formation of large, damaging ice crystals. Although not all cells survive, enough are viable to enable the culture to be reconstituted many years later.

How are scientists, food processors, and medical personnel using enzymes?

- Scientists, food processors, and medical personnel are researching ways to use natural and chemically modified antimicrobial enzymes to control microbes in the environment, inhibit microbial decay of foods and beverages, and reduce the number and kinds of microbes on medical equipment. - For example, food processors use lysozyme to reduce the number of bacteria in cheese, and some vintners use lysozyme instead of poisonous sulfur dioxide (SO2) to remove bacteria that would spoil wine. - One exciting development is the use of an enzyme to eliminate the prion that causes variant Creutzfeldt-Jakob disease (vCJD), also called mad cow disease. - The brain, spinal cord, placenta, eye, liver, kidney, pituitary gland, spleen, lung, and lymph nodes, as well as cerebrospinal fluid, can harbor prions. - Medical instruments contaminated by these highly infectious and deadly proteins may remain infectious even after normal autoclaving; boiling; exposure to formaldehyde, glutaraldehyde, or ethylene oxide; or 24 hours of dry heat at 160°C. - Until recently, harsh methods, such as autoclaving in sodium hydroxide for 30 minutes or complete incineration, were required to eliminate prions. - In 2006, the European Union approved the use of the enzyme Prionzyme to safely and completely remove prions on medical instruments. - Prionzyme is the first certified, noncaustic chemical to target prions.

aseptic

- The term aseptic describes an environment or procedure that is free of contamination by pathogens. - For example, vegetables and fruit juices are available in aseptic packaging, and surgeons and laboratory technicians use aseptic techniques to avoid contaminating a surgical field or laboratory equipment

Relative Susceptibility of Microorganisms

- Though microbial death rate is usually constant for a particular agent acting against a single microbe, death rates do vary— sometimes dramatically—among microorganisms and viruses. -Microbes fall along a continuum from most susceptible to most resistant to antimicrobial agents. - For example, enveloped viruses, such as HIV, are more susceptible to antimicrobial agents and heat than are nonenveloped viruses, such as poliovirus, because viral envelopes are more easily disrupted than the protein coats of nonenveloped viruses. - The relative susceptibility of microbes to antimicrobial agents is illustrated in - Often, scientists and medical personnel select a method to kill the hardiest microorganisms present, assuming that such a treatment will kill more fragile microbes as well.

UV light (non-ionizing)

- UV light with a wavelength of 260 nm is specifically absorbed by adjacent pyrimidine nucleotide bases in DNA, causing them to form covalent bonds with each other rather than forming hydrogen bonds with bases in the complementary DNA strand. - Such pyrimidine dimers distort the shape of DNA, making it impossible for the cell to accurately transcribe or replicate its genetic material. - If dimers remain uncorrected, an affected cell may die.

antisepsis (antiseptic)

- When a chemical is used on skin or other tissue, the process is called antisepsis, and the chemical is called an antiseptic. - Antiseptics and disinfectants often have the same components, but disinfectants are more concentrated or can be left on a surface for longer periods of time. - Of course, some disinfectants, such as steam or concentrated bleach, are not suitable for use as antiseptics.

Alteration of Cell Walls and Membranes

A cell wall maintains cellular integrity by counteracting the effects of osmosis when the cell is in a hypotonic solution. - If the wall is disrupted by physical or chemical agents, it no longer prevents the cell from bursting as water moves into the cell by osmosis. - Beneath a cell wall, the cytoplasmic membrane essentially acts as a bag that contains the cytoplasm and controls the passage of chemicals into and out of the cell. - Extensive damage to a membrane's proteins or phospholipids by any physical or chemical agent allows the cellular contents to leak out—damage that, if not immediately repaired, causes death. - In enveloped viruses, the envelope is a membrane composed of proteins and phospholipids that is responsible for the attachment of the virus to its target cell. - Damage to the envelope by physical or chemical agents prevents viral replication. - The lack of an envelope in non-enveloped viruses accounts for their greater tolerance of harsh environmental conditions, including antimicrobial agents

The use of silver nitrate in newborns

At one time, many states required that the eyes of newborns be treated with 1% silver nitrate (AgNO3) to prevent blindness caused by Neisseria gonorrhoeae (nī-se'rē-ă go-nor-rē'ī), which can enter babies' eyes while they pass through an infected birth canal. - Today, hospitals have replaced silver nitrate with less irritating antimicrobial ointments that are also effective against other pathogens. - Silver still plays an antimicrobial role in some surgical dressings, burn creams, and catheters

Boiling

Boiling kills the vegetative cells of bacteria and fungi, the trophozoites of protozoa, and most viruses within 10 minutes at sea level. - Contrary to popular belief, water at a rapid boil is no hotter than water at a slow boil; boiling water at normal atmospheric pressure cannot exceed boiling temperature (100°C at sea level), because escaping steam carries excess heat away. - It is impossible to boil something more quickly simply by applying more heat; the added heat is carried away by the escaping steam. - Boiling time is the critical factor. - Further, it is important to realize that at higher elevations, water boils at lower temperatures because atmospheric pressure is lower; thus, a longer boiling time is required in Denver than in Los Angeles to get the same antimicrobial effect. - Bacterial endospores, protozoan cysts, and some viruses (such as hepatitis viruses) can survive boiling at sea level for many minutes or even hours. - In fact, because bacterial endospores can withstand boiling for more than 20 hours, boiling is not recommended when true sterilization is required. - Boiling is effective for sanitizing restaurant tableware or disinfecting baby bottles

Ionizing Radiation

Electron beams, gamma rays, and some X-rays, all of which have wavelengths shorter than 1 nm, are ionizing radiation because when they strike molecules, they have sufficient energy to eject electrons from atoms, creating ions. - Such ions disrupt hydrogen bonding, oxidize double covalent bonds, and create highly reactive hydroxyl radicals. - These ions in turn denature other molecules, particularly DNA, causing fatal mutations and cell death

Phenol Coefficient

Joseph Lister introduced the widespread use of phenol as an antiseptic during surgery. - Since then, researchers have evaluated the efficacy of various disinfectants and antiseptics by calculating a ratio that compares a given agent's ability to control microbes to that of phenol under standardized conditions. - This ratio is referred to as the phenol coefficient. A phenol coefficient greater than 1.0 indicates that an agent is more effective than phenol, and the larger the ratio, the greater the effectiveness. - For example, chloramine, a mixture of chlorine and ammonia, has a phenol coefficient of 133.0 when used against the bacterium Staphylococcus aureus and a phenol coefficient of 100.0 when used against Salmonella enterica. - This indicates that chloramine is at least 133 times more effective than phenol against Staphylococcus but only 100 times more effective against Salmonella. - Measurement of an agent's phenol coefficient has been replaced by newer methods because scientists have developed disinfectants and antiseptics much more effective than phenol.

Pasteurization (Moist Heat Method)

Louis Pasteur developed a method of heating beer and wine just enough to destroy the microorganisms that cause spoilage without ruining the taste. - Today, pasteurization is also used to kill pathogens in milk, ice cream, yogurt, and fruit juices. - Brucella melitensis, Mycobacterium bovis, and Escherichia coli, the causative agents of undulant fever, bovine tuberculosis, and one kind of diarrhea, respectively, are controlled in this manner. **Pasteurization is not sterilization. - Thermoduric and thermophilic—heat-tolerant and heat-loving—prokaryotes survive pasteurization, but they do not cause spoilage over the relatively short times during which properly refrigerated and pasteurized foods are stored before consumption. - In addition, such prokaryotes are generally not pathogenic. - The combination of time and temperature required for effective pasteurization varies with the product. - Because milk is the most familiar pasteurized product, we consider the pasteurization of milk in some detail. - Historically, milk was pasteurized by the batch method for 30 minutes at 63°C, but most milk processors today use a high-temperature, short time method known as flash pasteurization, in which milk flows through heated tubes that raise its temperature to 72°C for only 15 seconds. - This treatment effectively destroys all pathogens. - Ultra-high temperature pasteurization heats the milk to at least 135°C for only 1 second, but some consumers claim that it adversely affects the taste.

Oxidizing Agents

Peroxides, ozone, and peracetic acid kill microbes by oxidizing their enzymes, thereby preventing metabolism. - Oxidizing agents are high-level disinfectants and antiseptics that work by releasing oxygen radicals, which are particularly effective against anaerobic microorganisms. - Health care workers use oxidizing agents to kill anaerobes in deep puncture wounds

What temperature kills all microbes?

Scientists have determined that a temperature of 121°C, which requires the addition of 15 pounds per square inch (psi) 8 of pressure above that of normal air pressure, destroys all microbes generally encountered in a laboratory or medical setting in a small volume in about 15 minutes. - Sterilizing large volumes of liquids or solids slows the process because they require more time for heat to penetrate. - Thus, it requires more time to sterilize 1 liter of fluid in a flask than the same volume of fluid distributed into smaller tubes. - Autoclaving solid substances, such as meat, also requires extra time because it takes longer for heat to penetrate to their centers.

How do scientists ensure that an autoclave has sterilized its contents?

Scientists use several means to ensure that an autoclave has sterilized its contents. - A common one is a chemical that changes color when the proper combination of temperature and time has been reached. - Often such a color indicator is impressed in a pattern on tape or paper so that the word sterile or a pattern or design appears. - Another technique uses plastic beads that melt when proper conditions are met.

Slow Freezing

Slow freezing, during which ice crystals have time to form and puncture cell membranes, is more effective than quick freezing in inhibiting microbial metabolism, though microorganisms also vary in their susceptibility to freezing. - Whereas the cysts of tapeworms perish after several days in frozen meat, many vegetative bacterial cells, bacterial endospores, and viruses can survive subfreezing temperatures for years. - In fact, scientists store many bacteria and viruses in low-temperature freezers at -30°C to -80°C and are able to reconstitute the microbes into viable populations by warming them in media containing proper nutrients. - Therefore, we must take care in thawing and cooking frozen food because it can still contain pathogenic microbes.

What are the 2 major type of microbial types of microbial control?

So far, we have seen that there are two major types of microbial control—sterilization, which is the elimination of all microbes, and antisepsis or disinfection, which each denote the destruction of vegetative (nonspore) cells and many viruses. - Modifications of disinfection include degerming, sanitization, and pasteurization. - Some scientists and clinicians apply these terms only to pathogenic microorganisms.

Dry Heat Related Methods

Some substances, such as powders and oils, cannot be sterilized by boiling or with steam. - Others, such as some metal objects, can be damaged by repeated exposure to steam. - For such materials, sterilization can be achieved by the use of dry heat, as occurs in an oven. - Hot air is an effective sterilizing agent because it denatures proteins and fosters the oxidation of metabolic and structural chemicals; however, in order to sterilize, dry heat requires higher temperatures for longer times than moist heat because dry heat penetrates more slowly. - For instance, whereas an autoclave needs about 15 minutes to sterilize an object at 121°C, an oven at the same temperature requires at least 16 hours to achieve sterility. - Scientists typically use higher temperatures—171°C for 1 hour or 160°C for 2 hours—to sterilize objects in an oven, but objects made of rubber, paper, and many types of plastic oxidize rapidly (combust) under these conditions. - Complete incineration is the ultimate means of sterilization. - As part of standard aseptic technique in microbiological laboratories, inoculating loops are sterilized by heating them in the flame of a Bunsen burner or with an electric heating coil until they glow red (about 1500°C). - Health care workers incinerate contaminated dressings, bags, and paper cups, and field epidemiologists incinerate the carcasses of animals that have diseases such as anthrax or bovine spongiform encephalopathy (mad cow disease).

Sulfactants

Surfactants are "surface active" chemicals. - One of the ways surfactants act is to reduce the surface tension of solvents such as water by decreasing the attraction among molecules. - One result of this reduction in surface tension is that the solvent becomes more effective at dissolving solute molecules. - Two common surfactants involved in microbial control are soaps and detergents. - One end of a soap molecule is hydrophobic because it is composed of fatty acids, and the other end is hydrophilic and negatively charged. - When soap is used to wash skin, for instance, the hydrophobic ends of soap molecules are effective at breaking oily deposits into tiny droplets, and the hydrophilic ends attract water molecules; the result is that the tiny droplets of oily material—and any bacteria they harbor— are more easily dissolved in and washed away by water. - Thus, soaps by themselves are good degerming agents though poor antimicrobial agents; when household soaps are antiseptic, it is largely because they contain antimicrobial chemicals

Environmental Conditions

Temperature and pH affect microbial death rates and the efficacy of antimicrobial methods. - Warm disinfectants, for example, generally work better than cool ones because chemicals react faster at higher temperatures. - Acidic conditions enhance the antimicrobial effect of heat. Some chemical disinfectants, such as household chlorine bleach, are more effective at low pH. - Organic materials, such as fat, feces, vomit, blood, and the intercellular matrix of biofilms, interfere with the penetration of heat, chemicals, and some forms of radiation, and in some cases these materials inactivate chemical disinfectants. - For this reason, it is important to clean objects before sterilization or disinfection so that antimicrobial agents can thoroughly contact all the object's surfaces.

Kelsey-Sykes Capacity Test

The Kelsey-Sykes capacity test is the standard alternative assessment approved by the European Union to determine the capacity of a given chemical to inhibit bacterial growth. - In this test, researchers add a suspension of a bacterium such as Psuedomonas aeruginosa or Staphylococcus aureus to a suitable concentration of the chemical being tested. - Then at predetermined times, they move samples of the mixture into growth medium containing a disinfectant deactivator. - After incubation for 48 hours, turbidity in the medium indicates that bacteria survived treatment. - Lack of turbidity, indicating lack of bacterial reproduction, reveals the minimum time required for the disinfectant to be effective.

Ultra-High-Temperature Sterilization

The dairy industry and other food processors can also use ultra-high temperature sterilization, which involves flash heating milk or other liquids to rid them of all living microbes. - The process involves passing the liquid through superheated steam at about 140°C for 1 to 3 seconds and then cooling it rapidly. - Treated liquids can be stored indefinitely at room temperature without microbial spoilage, though chemical degradation after months of storage results in flavor changes. - Small packages of dairy creamer served in restaurants are often sterilized by the ultra-high-temperature method.

UV light irradiation

The effectiveness of UV irradiation is tempered by the fact that UV light does not penetrate well. - UV light is therefore suitable primarily for disinfecting air, transparent fluids, and the surfaces of objects, such as barber's shears and operating tables. - Some cities use UV irradiation in sewage treatment. By passing wastewater past banks of UV lights, they reduce the number of bacteria without using chlorine, which might damage the environment

How is the effectiveness of germicides classified?

The effectiveness of germicides can be classified as high, intermediate, or low, depending on their proficiency in inactivating or destroying microorganisms on medical instruments that cannot be sterilized with heat. - High-level germicides kill all pathogens, including bacterial endospores. - Health care professionals use them to sterilize invasive instruments such as catheters, implants, and parts of heart-lung machines. - Intermediate-level germicides kill fungal spores, protozoan cysts, viruses, and pathogenic bacteria, but not bacterial endospores. - They are used to disinfect instruments that come in contact with mucous membranes but are noninvasive, such as respiratory equipment and endoscopes. - Low-level germicides eliminate vegetative bacteria, fungi, protozoa, and some viruses; they are used to disinfect items that contact only the skin of patients, such as furniture and electrodes.

quaternary ammonium compounds (quats)

The most popular detergents for microbial control are quaternary ammonium compounds (quats), which are composed of an ammonium cation (NH4 +) in which the hydrogen atoms are replaced by other functional groups or hydrocarbon chains. - Quats are not only antimicrobial but also colorless, tasteless, and harmless to humans (except at high concentrations), making them ideal for many industrial and medical applications. - If your mouthwash foams, it probably contains a quaternary ammonium compound. - Examples of quats are benzalkonium chloride (Zephiran) and cetylpyridinium (used in Cepacol mouthwash). - Quats function by disrupting cellular membranes so that affected cells lose essential internal ions, such as potassium ions (K+). - Quats are bactericidal (particularly against Gram-positive bacteria), fungicidal, and virucidal against enveloped viruses, but they are not effective against nonenveloped viruses, mycobacteria, or endospores. - The action of quaternary ammonium compounds is retarded by organic contaminants, and they are deactivated by soaps. - Some pathogens, such as Pseudomonas aeruginosa, actually thrive in quats; therefore, quats are classified as low-level disinfectants.

Radiation

There are two types of radiation: particulate radiation and electromagnetic radiation. - Particulate radiation consists of high-speed subatomic particles, such as protons, that have been freed from their atoms. - Electromagnetic radiation can be defined as energy without mass traveling in waves at the speed of light (3 * 105 km/sec). - Electromagnetic energy is released from atoms that have undergone internal changes. - The wavelength of electromagnetic radiation, defined as the distance between two crests of a wave, ranges from very short gamma rays; to X-rays, ultraviolet light, and visible light; to long infrared rays; and, finally, to very long radio waves. - Though they are particles, electrons also have a wave nature, with wavelengths that are even shorter than gamma rays. - The shorter the wavelength of an electromagnetic wave, the more energy it carries; therefore, shorter-wavelength radiation is more suitable for microbial control than longer-wavelength radiation, which carries less energy and is less penetrating. - Scientists describe all types of radiation as either ionizing or non-ionizing according to its effects on the chemicals within cells.

The most resistant microbes include the following:

• Bacterial endospores. The endospores of Bacillus and Clostridium are the most resilient forms of life. They can survive environmental extremes of temperature, acidity, and dryness and can withstand many chemical disinfectants. For example, endospores have survived more than 20 years in 70% alcohol, and scientists have recovered viable endospores that were embalmed with Egyptian mummies thousands of years ago. • Species of mycobacteria. The cell walls of members of the genus, Mycobacterium such as Mycobacterium tuberculosis, contain a large amount of a waxy lipid. The wax allows these bacteria to survive drying and protects them from most water-based chemicals; therefore, medical personnel must use strong disinfectants or heat to treat whatever comes into contact with tuberculosis patients, including utensils, equipment, and patients' rooms. • Cysts of protozoa. A protozoan cyst's wall prevents entry of most disinfectants, protects against drying, and shields against radiation and heat.