Growth Control overview.doc

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University of Alberta
Microbiology (Biological Sciences)
Brian Lanoil

Control of Microbial Growth (Chapter 26 of 13 ed. Brock Biology of Microorganisms) OVERVIEW This chapter focuses on the control and destruction of microorganisms by physical, chemical, and biological agents. This is a topic of great importance because many microorganisms cause food spoilage and disease. It is therefore essential to kill or remove microorganisms from human environments to minimize their harmful effects. Principles of chemotherapy and explanations of how chemotherapeutic agents selectively damage target microorganisms while minimizing damage to the host are presented. OBJECTIVES 1) Compare and contrast the processes of disinfection, sanitization, antisepsis, and sterilization 2) Define D-values and Z-values 3) Compare the effectiveness of physical and chemical methods with respect to endospores and Gram positive vs. Gram-negative bacteria. 4) Discuss uses and limitations of various physical and chemical agents used to control microbial populations 5) Describe, in general terms, mechanisms of action and factors that influence effectiveness of antimicrobial drugs that inhibit or kill microorganisms. 6) Describe how the effective dose of an antimicrobial drug is determined. 7) Describe the classes of chemotherapeutic agents and the parts of prokaryotic metabolism/structures that they inhibit. 8) Discuss the various ways in which antimicrobial agents can damage pathogens while causing minimal damage to the host. 9) Discuss the increasingly serious problem of drug-resistant pathogens, why it is occurring and how it can be slowed. CHAPTER OUTLINE I. Definition of Frequently Used Terms A. Sterilization: destruction or removal of all viable organisms from an object or from a particular environment: e.g. autoclave or high energy ionizing radiation. B. Disinfection: killing, inhibition, or removal of microorganisms usually on inanimate objects: e.g. phenolics, boiling water, UV irradiation, hydrogen peroxide. Unlike sterilization, some organisms may remain behind following treatment with disinfecting agents. 1 C. Antisepsis: prevention of infection of living tissue by microorganisms: e.g. iodine, hexachlorophene (think prevention of “sepsis” which is a body-wide inflammatory response to a bacterial pathogen) D. Sanitization: reduction of the microbial population to a safe level as determined by public health standards: e.g. anionic detergents and soaps. The degree of sanitization in public areas (e.g. hospitals, daycares, swimming pools, etc.) is controlled by the government. E. -cide: a suffix indicating that the agent will kill the kind of organism in question (e.g., fungicide, bacteriocide) F. -static: a suffix indicating that the agent will prevent the growth of the type of organism in question (e.g., bacteriostatic, fungistatic) G. -lytic: a suffix indicating that the agent will lyse the microorganisms. II. Conditions Affecting Antimicrobial Activity A. Population size: larger populations take longer to kill than smaller populations B. Population composition: microorganisms differ markedly in their sensitivity to various agents. e.g., spore-forming bacteria, Gram-positive vs. Gram-negative bacteria C. Concentration or intensity of the antimicrobial agent: higher concentrations of chemicals or intensities of physical processes are generally more efficient, though the relationship is not always linear D. Duration of exposure: the longer the exposure, the greater the number of organisms killed E. Temperature: a higher temperature will usually (but not always) increase the effectiveness of killing F. Local environment: environmental factors, such as pH, viscosity, and concentration of organic matter can profoundly influence the effectiveness of a particular antimicrobial agent III. Physical Methods in Control Empirical decimal reduction times (D) are defined as the time required for a 90% reduction of viable cells. D values have been determined for many different bacteria, and are important for determining sterilization times. D values apply to all methods of microbial growth control including radiation exposure and chemical sterilization methods described below. A. Thermal Killing 2 1) Boiling water is effective against vegetative cells and eukaryotic spores 2) Autoclaving (steam under pressure) is effective against vegetative cells and most eukaryotic (fungal) spores. Usually, 15 min. at 2 atm. Pressure (121 ˚C) will be sufficient. Compare this to dry heat below. 3) Dry heat can be used to sterilize moisture-sensitive materials such as powders, oils, and similar items; less efficient than moist heat because it requires higher temperatures (over 150 ˚C) and longer exposure times (several hrs). 4) Decimal Reduction Time (D) = time necessary to kill 90% of the microorganisms in a suspension at a specific temperature and under defined conditions. 5) Z-values are the increase in temperature required to reduce the D value by one log unit. For example, if the D value is 30 min at 100 degrees, and 3 min at 120 degrees, the Z- value is 20 degrees. 6) Pasteurization – named after Louis Pasteur (15 sec at 71˚C or 15 min at 60˚C) reduces the total microbial population and thereby increases the shelf life of the treated material; it is often used for heat-sensitive materials that cannot withstand prolonged exposure to high temperatures (e.g. wine, beer, milk). It is effective against Gram-negative pathogens, but not many Gram-positive pathogens. 7) Tyndallization – named after John Tyndall (sequential boiling sterilization), a process that kills Gram positive bacteria by allowing spores to germinate following the first boiling to form new vegetative cells, and then boiling again to kill the newly germinated vegetative cells. B. Filtration 1) Removes microorganisms, rather than destroying them, from media that is heat sensitive (e.g. serum). 2) Membrane filters have defined pore sizes, usually 0.22 μm, that remove most bacteria, but not viruses. 3) High-efficiency particulate air (HEPA) filters are used in laminar flow biological safety cabinets to sterilize the air circulating in the enclosure. C. Ultraviolet and Ionizing Radiation 1) Ultraviolet radiation damages cells by causing the formation of thymine dimers in DNA. 2) Ultraviolet (UV) radiation is limited to surface sterilization because UV radiation does not penetrate glass, dirt films, water, and other substances. 3) Ionizing radiation such as X rays or gamma rays are even more harmful to microorganisms than ultraviolet radiation by their production of oxygen radicals 4) High levels produce mutations that result in cell death 5) Ionizing is effective and penetrates material. The World Health Organization has approved food irradiation and declared it safe. Concerns remain about effects of radiation on the chemistry of food, not about it becoming radioactive. 6) Many aerial borne bacteria (e.g. Deinococcus radiodurans) contain carotenoid pigments (red, orange, yellow colored), which protect them from UV irradiation, or have very active DNA repair systems. 3 IV. Chemical Methods of Control A. Phenolics were the first antimicrobial chemicals to be developed (by Joseph Lister of “Listerine” fame). Commonly used in laboratory and hospital as disinfectants. They act by denaturing proteins. B. Alcohols are widely used disinfectants and antiseptics. They will not kill endospores. They act by denaturing proteins and also by dissolving membrane lipids. C. Halogens are widely used antiseptics and disinfectants. Iodine acts by oxidizing cell constituents and iodinating cell proteins. Chlorine acts primarily by oxidizing cell constituents. D. Heavy metals (e.g. mercury) are usually toxic to the host. They act by covalently binding proteins and inactivating them. E. Aldehydes and lactones are used as chemical sterilants. They are highly reactive and covalently bind proteins and inactivated them. They are generally irritating to the skin. F. Sterilizing gases (e.g., ethylene oxide, betapropiolactone) are sterilants used for heat-sensitive materials such as plastic petri dishes and disposable syringes. They act by combining with proteins and inactivating them. G. Vapor-phase hydrogen peroxide has been used to decontaminate biological safety cabinets and spacecraft, medical equipment H. Quaternary ammonium compounds are cationic detergents used as disinfectants for food utensils and small instruments, and because of low toxicity, as antiseptics (at reduced concentrations) for skin and contact lenses. They act by disrupting biological membranes and also by denaturing proteins. I. Effectiveness of antimicrobial chemicals are rated relative to the “phenol-coefficient.” Phenol was the first antimicrobial developed in the late 1800’s by Joseph Lister. An index above 1 for an antimicrobial agent means that the agent is more effective at killing than phenol. Phenols and alcohols with longer, ergo more hydrophobic, side chains have progressively higher phenol coefficients. This is due to a greater detergent-like effect that causes disruption of the lipid cell membrane. V. The History of Chemotherapy (Biological control of microbial growth) A. The first person to mechanistically design chemicals that were toxic to bacteria, but not to the host, was Paul Ehrlich. He received the Nobel Prize in 1908 for his discovery of Salvorsan, an organo-arsenic compound. It was also called compound 666, which was literally his first success after 665 failures. Salvorsan was used for treatment of syphilis, which was treated prior with mercury. Mercury was as toxic to the host as it was to the pathogen. 4 B. Surprisingly, very little was done in the field of antimicrobial chemotherapy for the next 30 years. Gerhard Domagk made an exhaustive screening experiment on more than 1000 synthetic dyes against Streptococcus. Prontosil was the first of the many sulfa drugs. Domagk was awarded the Nobel Prize in 1939. C. Alexander Fleming (1928) accidentally discovered the antimicrobial activity of penicillin against Staphylococcus on a contaminated plate; however, follow-up studies convinced him that penicillin would not remain active in the body long enough to be effective. Howard Florey and Ernst Chain (1939) worked from Fleming’s published observations, obtained a culture from him, and demonstrated the effectiveness of penicillin. Together with Fleming, they were awarded the Nobel Prize in 1945. D. Selman Waksman (1943) established the basic foundation of the pharmaceutical industry. He was the first to demonstrate that antibiotic-producing bacteria of the genus Streptomyces resided in soil, and could be isolated and grown. He was awarded the Nobel Prize in 1952 for his discovery of streptomycin, which was used to treat tuberculosis (Mycobacterium tuberculosis). VI. Characteristics of Antimicrobial Drugs A. Selective toxicity with minimal side effects B. Therapeutic dose: the drug level required for clinical treatment of a particular infection C. Broad spectrum activity (activity against a wide variety of pathogens) is more desirable than narrow spectrum activity, but this is not crucial D. Drug can be -cidal (able to kill) or -static (able to reversibly inhibit growth) E. Chemotherapeutic agents can occur naturally, be synthetic or both (chemical modifications of naturally occurring antibiotics). Chemical modification of penicillins, has been very useful because many people have developed allergies to the original penicillin. Some modified penicillins are also effective against Gram negative bacteria. VII. Determining the Level of Antimicrobial Activity A. Dilution susceptibility tests: MIC, or minimum inhibitory concentration, is the lowest dose of antibiotic that prevents growth of a pathogen while the MLC, or minimum lethal concentration, is the lowest dose that kills the pathogen. This is determined by serial dilution of the antibiotic into test tubes followed by inoculation with the pathogen. The
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