Controlling Microbial Growth in the Environment 7:42 AM Chapter 9

Terminologies Sterilization: the removal or destruction of all microbes, including viruses and bacterial endospores, in or on an object. Aseptic: An environment or procedure that is free of contamination by pathogens. Disinfection: The use of physical or chemical agents known as disinfectants to inhibit or destroy microorganisms, especially pathogens. Does not guarantee elimination of all pathogens and Applies only to treatment of inanimate objects. Antisepsis: When a chemical is used on skin or other tissue and the agent is an antiseptic. 7:42 AM

Degerming is the removal of microbes from a surface by scrubbing, whether that surface is human skin or a table top. Sanitization is the process of disinfecting places and utensils used by the public to reduce the number of pathogenic microbes to meet acceptable public health standards. Pasteurization is the use of heat to kill pathogens and reduce the number of spoilage microorganisms in food and beverages. 7:42 AM

Suffixes –stasis or –static: Agents or techniques that inhibit the growth of microbes without necessarily killing them are indicated by the suffix. For example, refrigeration is bacteriostatic. –cide or –cidal: Agents or methods that destroy or permanently inactivate a particular type of microbe. For example, fungicides kill fungal hyphae, spores, and yeasts. 7:42 AM

Table 9.1 Terminology of Microbial Control 7:42 AM

Microbial Death Rates Microbial death: The permanent loss of reproductive ability under ideal environmental conditions. Microbial death rate Used to evaluate the efficacy of an antimicrobial agent Usually constant over time for any particular microorganism under a particular set of conditions. When the microbial death rate is plotted on a semilogarithmic graph, this constant death rate produces a straight line. 7:42 AM

Basic Principles of Microbial Control The modes of action of antimicrobial agents fall into two basic categories: Those that disrupt the integrity of cells by adversely altering their cell walls or cytoplasmic membranes Cell wall maintains integrity of cell. When damaged, cells burst because of osmotic effects Cytoplasmic membrane controls passage of chemicals into and out of cell. When damaged, cellular contents leak out. ***Non-enveloped viruses more tolerant of harsh conditions 7:42 AM

Action of Antimicrobial Agents, contd. Those that interrupt cellular metabolism and reproduction by interfering with the structures of proteins and nucleic acids. Protein function depends on 3-D shape Extreme heat or certain chemicals denature proteins Chemicals, radiation, and heat can alter/destroy nucleic acids Produce fatal mutants Halt protein synthesis through action on RNA 7:42 AM

The Selection of Microbial Control Methods A perfect antimicrobial method or agent would be: Inexpensive, Fast-acting, Stable during storage, Harmless to humans, and Effective against all types of microbes. No single method or agent meets all these criteria Several factors are considered when evaluating methods and agents. 7:42 AM

Factors Affecting the Efficacy of Antimicrobial Methods Site to be treated Harsh chemicals or intense heat cannot be used on human and animals tissues, as well as fragile objects. Relative susceptibility of the microorganisms. Generally, selected method should kill the hardiest microorganisms present, assuming that more fragile microbes will be killed as well. High - kill all pathogens, including endospores Intermediate - kill fungal spores, protozoan cysts, viruses, pathogenic bacteria Low - kill vegetative bacteria, fungi, protozoa, some viruses 7:42 AM

Degrees of resistance of microbes: 7:42 AM

Factors Affecting the Efficacy of Antimicrobial Methods The environmental conditions under which the antimicrobial agent is used, such as temperature and pH. For example, since chemicals react faster at higher temperatures, warm disinfectants generally work better than cool ones Figure 9.3 Effect of temperature on the efficacy of an antimicrobial chemical 7:42 AM

Methods for Evaluating Disinfectants and Antiseptics – Phenol Coefficient Phenol was an antiseptic used during surgery in the late 1800s. Phenol coefficient Evaluates efficacy of disinfectants and antiseptics by comparing to phenol an agent’s ability to control microbes Greater than 1.0 indicates agent is more effective than phenol Example: Chloramine (Chlorine + Ammonia). Phenol Coefficient – 133 against S. aureus 100 against Salmonella enterica 7:42 AM

Methods for Evaluating Disinfectants and Antiseptics - Use-dilution test Metal cylinders are dipped into broth cultures of bacteria Cylinders are dried Immerse cylinders into a different dilution of the disinfectants being evaluated. After 10 minutes, remove cylinders and incubate. The most effective agent is the one that entirely prevents microbial growth at the highest dilution. Current standard test in the U.S. New standard procedure being developed 7:42 AM

Methods for Evaluating Disinfectants and Antiseptics – Kelsey-Sykes Capacity Test Alternative assessment to the use dilution test Approved by the European Union Determines the capacity of a given chemical to inhibit bacterial growth. Test chemicals are added to broth cultures of bacteria, and the turbidity of the cultures is measured after 48 hours as an indication of growth inhibition. Can establish minimum time required for disinfectant to be effective 7:42 AM

Methods for Evaluating Disinfectants and Antiseptics - In-use test Provide accurate determination of an agent’s efficacy under realistic conditions Swabs taken from objects before and after application of disinfectant or antiseptic Swabs inoculated into growth medium and incubated Medium monitored for growth Accurate determination of proper strength and application procedure for each situation is important 7:42 AM

Summary of Microbial Control Methods 17 17

Physical Methods of Microbial Control Physical methods of microbial control include exposing the microbes to extremes of: Heat and cold Desiccation Filtration Osmotic pressure and Radiation 7:42 AM

Heat-Related Methods Effects of high temperatures Denature proteins Interfere with integrity of cytoplasmic membrane and cell wall Disrupt structure and function of nucleic acids Thermal death point: the lowest temperature that kills all cells in a broth in 10 minutes, and Thermal death time: the time it takes to completely sterilize a particular volume of liquid at a set temperature. Decimal reduction time (D) is the time required to destroy 90% of the microbes in a sample 7:42 AM

Heat-Related Methods – Moist Heat More effective than dry heat because water is a better conductor of heat than air. Used to disinfect, sanitize, and sterilize Denatures proteins and destroys cytoplasmic membranes Methods of microbial control using moist heat Boiling Autoclaving Pasteurization Ultrahigh-temperature sterilization 7:42 AM

Moist Heat – Boiling Kills vegetative cells of bacteria and fungi, protozoan trophozoites, most viruses Boiling time & atmospheric pressure is important. The higher the atmospheric pressure, the higher the boiling point Different elevations require different boiling times Rate of boiling not important – bec. heat escapes Boiling does not achieve true sterilization: Endospores, protozoan cysts, and some viruses can survive boiling 7:42 AM

Moist Heat - Autoclaving Pressure applied to boiling water prevents steam from escaping Boiling temperature increases as pressure increases Autoclaving – A device that consists of a pressure chamber, pipes, valves, and gauges that uses steam heat under pressure to sterilize chemicals and objects that can tolerate moist heat. Autoclave conditions – 121ºC, 15 psi, 15 min 7:42 AM

Moist Heat - Pasteurization A method of heating foods to kill pathogens and control spoilage organisms without altering the quality of the food. Used for milk, ice cream, yogurt, and fruit juices Not sterilization - Heat-tolerant microbes survive Can be achieved by several methods: Batch method at 63°C for 30 minutes, Flash pasteurization at 72°C for 15 seconds, and Ultrahigh-temperature pasteurization at 134°C for 1 second. 7:42 AM

Heat-Related Methods - Dry heat Used for materials that cannot be sterilized with moist heat like powders and oils Denatures proteins and oxidizes metabolic and structural chemicals Requires higher temperatures for longer time than moist heat Incineration is ultimate means of sterilization 7:42 AM

Refrigeration and Freezing Decreases microbial metabolism, growth, and reproduction Chemical reactions occur more slowly at low temperatures Liquid water not available Refrigeration between 0°C and 7°C halts growth of most pathogens Psychrophilic microbes can multiply in refrigerated foods Slow freezing at temperatures below 0°C more effective than quick freezing Organisms vary in susceptibility to freezing – many vegetative bacterial cells, bacterial endospores, and viruses can survive subfreezing temperatures for years. 7:42 AM

Physical Method of Microbial Control: Desiccation & Lyophillization Desiccation: Drying Used to preserve such foods as fruits, peas, and yeast. It inhibits microbial growth because metabolism requires liquid water. Lyophillization: freeze-drying. Preserves microbes and other cells for many years. Microbial culture is frozen in liquid nitrogen or frozen carbon dioxide and then remove water via a vacuum. Prevents the formation of large ice crystals, which would otherwise damage the culture; thus, enough viable cells remain to enable the culture to be reconstituted many years later. 7:42 AM

Filtration The passage of air or a liquid through a material that traps and removes microbes. Some membrane filters manufactured of nitrocellulose or plastic have pores small enough to trap the smallest viruses and even some large protein molecules. 7:42 AM

HEPA Filters HEPA (High-Efficiency Particulate Air) filters remove microbes and particles from air. 7:42 AM

Physical Methods of Microbial Control: Osmotic Pressure Use of high concentrations of salt or sugar in foods to inhibit growth Examples: Pickles; Salted fish jerky; Jams; etc Cells in hypertonic solution of salt or sugar lose water Fungi have greater ability than bacteria to survive hypertonic environments 7:42 AM

Physical Methods of Microbial Control: Radiation There are two types of radiation: Particulate radiation consists of high-speed subatomic particles freed from their atoms Electromagnetic radiation is atomic energy without mass traveling at the speed of light. 7:42 AM

Ionizing Radiation Electromagnetic radiation with wavelengths shorter than 1 nm Ejects electrons from atoms to create ions Ions disrupt hydrogen bonding, oxidize double covalent bonds, and create hydroxide ions which can produce effects leading to the denaturation of important molecules and cell death. Examples: electron beams, gamma rays, and X rays. Electron beams – effective at killing but do not penetrate well Gamma rays – penetrate well but require hours to kill microbes. 7:42 AM

Non Ionizing Radiation Wavelengths greater than 1 nm Excites electrons, causing them to make new covalent bonds Affects 3-D structure of proteins and nucleic acids Examples: Ultraviolet light, visible light, infrared light, and radio waves, Only UV light has enough energy to be a practical antimicrobial agent Causes pyrimidine dimers in DNA UV light does not penetrate well Suitable for disinfecting air, transparent fluids, and surfaces of objects 7:42 AM

Biosafety Levels Handling microbes demands safety. The CDC established four levels of guidelines for handling microbes in microbiology labs Each level has guidelines for which precautions to take and which organisms can be safely handled under those conditions. 7:42 AM

Biosafety Levels BSL-1 is suitable for nonpathogenic microbes, BSL-2 is intended for moderately hazardous microbes, and BSL-3 is designed for containment of true pathogens and includes specifications for the physical facility as well as procedures. BSL-4 is designed for the most dangerous pathogens and includes stringent guidelines for the design of the facility to contain the microbes. 7:42 AM

Chemical Agents in Microbial Control Disinfectants, antiseptics, sterilants, degermers, and preservatives Some desirable qualities of chemicals: Rapid action in low concentration Solubility in water or alcohol, stable Broad spectrum, low toxicity Penetrating Noncorrosive and nonstaining Affordable and readily available 35 35

Factors that Affect Germicidal Activity of Chemicals Nature of the material being treated Degree of contamination Time of exposure Strength and chemical action of the germicide 36 36

Nine major categories of antimicrobial chemicals used as antiseptics and disinfectants Surfactants Heavy Metals Aldehydes Gaseous Agents Enzymes Phenol and Phenolics Alcohols Halogens Oxidizing Agents

Phenol and Phenolics Compounds derived from phenol molecules that have been chemically modified by the addition of halogens or organic functional groups such as chlorine Intermediate- to low-level disinfectants Denature proteins and disrupt cell membranes Effective in presence of organic matter Remain active for prolonged time Commonly used in health care settings, labs, and homes Have disagreeable odor and possible side effects 7:42 AM

Alcohols Intermediate-level disinfectants – Denature proteins and disrupt cytoplasmic membranes Used either as 70–90% aqueous solutions. Example: Isopropanol (rubbing alcohol) or As a tincture - a combination of an alcohol and another antimicrobial agent. Ex. Tincture of Iodine Intermediate-level disinfectants – Effective as bactericidal, fungicidal, and virucidal against enveloped viruses; but Not effective against fungal spores or bacterial endospores 7:42 AM

Halogens Iodine, chlorine, bromine, and fluorine - four very reactive, nonmetallic chemical elements Believed to damage enzymes via oxidation or by denaturation Intermediate-level antimicrobial chemicals Used alone or in combination with other elements in organic and inorganic compounds. Ex. Iodophore, Betadine Widely used in numerous applications Iodine tablets, iodophores, chlorine treatment, bleach, chloramines, and bromine disinfection 7:42 AM

Oxidizing Agents Peroxides, ozone, and peracetic acid Kill by oxidation of microbial enzymes due to production of oxygen radicals High-level disinfectants and antiseptics Hydrogen peroxide can disinfect and sterilize surfaces Not useful for treating open wounds because of catalase activity Ozone treatment of drinking water by some MUDs in Canada and Europe more effective and not carcinogenic Peracetic acid is effective sporicide used to sterilize equipment. 7:42 AM

Surfactants Soaps have hydrophilic and hydrophobic ends “Surface active” chemicals. Reduce surface tension of solvents. Types: Soaps and detergents Soaps have hydrophilic and hydrophobic ends Good degerming agents but not antimicrobial Detergents are positively charged organic surfactants; disrupts cellular membranes Quaternary Ammonium Compunds Low-level disinfectants; not effective against mycobacteria, endospores, or nonenveloped viruses, and some pathogens Ideal for many medical and industrial applications 7:42 AM

Heavy Metals Arsenic, silver, mercury, copper, and zinc Heavy-metal ions denature proteins Low-level bacteriostatic and fungistatic agents 1% silver nitrate to prevent blindness caused by N. gonorrhoeae Thimerosal used to preserve vaccines Copper controls algal growth 7:42 AM

Aldehydes Compounds containing terminal –CHO groups Cross-link functional groups to denature proteins and inactivate nucleic acids Glutaraldehyde disinfects and sterilizes Formalin – 2% solution of glutaraldehyde and a 37% aqueous solution of formaldehyde used in embalming and disinfection of rooms and instruments 7:42 AM

Gaseous Agents Can be hazardous to people Often highly explosive Ethylene oxide, propylene oxide, and beta-propiolactone Microbicidal and sporicidal gases used in closed chambers to sterilize items that cannot be sterilized easily with heat, water-soluble chemicals, or irradiation. Ex. Plastic laboratory ware, artificial heart valves, mattresses, and dried foods. Denature proteins and DNA by cross-linking functional groups Used in hospitals and dental offices Disadvantages Can be hazardous to people Often highly explosive Extremely poisonous. Kills everything they contact without harming inanimate objects Potentially carcinogenic 7:42 AM

Enzymes Naturally produced by organisms to inhibit or destroy microbes. Ex. Lysozyme in human tear which digests peptidoglycan Scientists, food processors, and medical personnel are researching ways to use natural and artificial antimicrobial enzymes to control microbes in the environment, inhibit microbial decay of foods Enzymes to control microbes in the environment Lysozyme used to reduce the number of bacteria in cheese Prionzyme can remove prions on medical instruments 7:42 AM

Antimicrobials Antibiotics - produced naturally by microorganisms Semisynthetics - are chemically modified antibiotics; and synthetics, which are wholly synthetic antimicrobial drugs. Typically used for treatment of disease Some used for antimicrobial control outside the body 7:42 AM

Development of Resistant Microbes Little evidence that products containing antiseptic and disinfecting chemicals add to human or animal health Use of such products promotes development of resistant microbes When susceptible cells die, they reduce competition for resources, allowing any remaining resistant cells to proliferate Experts recommend limiting the use of such chemicals to food handling and health care involving high-risk patients and newborns 7:42 AM