Microbiology: A Systems Approach, Chapter 11 PDF

Microbiology: A Systems Approach, 2nd ed. Chapter 11: Physical and Chemical Control of Microbes 1  Outline and Learning Outcomes 11.1 Controlling Microorganisms 1. Distinguish among the terms sterilization, disinfection, antisepsis, and decontam...

Microbiology: A Systems Approach, 2nd ed. Chapter 11: Physical and Chemical Control of Microbes 1  Outline and Learning Outcomes 11.1 Controlling Microorganisms 1. Distinguish among the terms sterilization, disinfection, antisepsis, and decontamination. 2. Identify the types of microorganisms that are most resistant and least resistant to control measures. 3. Compare the action of microbicidal and microbistatic agents, providing an example of each. 4. Name four categories of cellular targets for physical and chemical agents.  11.1 Controlling Microorganisms General Considerations in Microbial Control – Sterilization: the destruction of all microbial life – Disinfection: destroys most microbial life, reducing contamination on inanimate surfaces – Antisepsis: destroys most microbial life, reducing contamination on a living surface – Decontamination: the mechanical removal of most microbes from an animate or inanimate surface 3  4 Figure 11.1 Relative Resistance of Microbial Forms Primary targets of microbial control: microorganisms that can cause infection or that are constantly present in the external environment Contaminants that need to be controlled – Bacterial vegetative cells and endospores (so resistant, the goal is sterilization) – Fungal hyphae and spores – Yeasts – Protozoan trophozoites and cysts – Worms – Viruses – Prions 5 6 Terminology and Methods of Microbial Control Sterilization – Removes all viable microorganisms including viruses – Material is said to be sterile – Usually reserved for inanimate objects – Mostly performed with heat – Sometimes chemicals called sterilants are used 7  Disinfection The use of a physical process or chemical agent (disinfectant) to destroy vegetative pathogens Does not destroy bacterial endospores Used only on inanimate objects Also removes toxins 5% bleach solution 8  Antisepsis Antiseptics applied directly to exposed body surfaces to destroy or inhibit vegetative pathogens Sepsis: the growth of microorganisms in the blood and other tissues Asepsis: any practice that prevents the entry of infectious agents into sterile tissues 9  The Agents Versus the Processes –cide: to kill – Bactericide: chemical that destroys bacteria (not endospores) – Fungicide: a chemical that can kill fungal spores, hyphae, and yeasts – Virucide: a chemical that inactivates viruses – Sporicide: can destroy bacterial endospores – Germicide and microbicide: chemical agents that kill microorganisms Stasis and static: to stand still – Bacteristatic: prevent the growth of bacteria – Fungistatic: inhibit fungal growth – Microbistatic: materials used to control microorganisms in the body 10  Decontamination Used when actual sterilization isn’t needed but need to decrease the risk of infection or spoilage (ex. food industry) Sanitization: any cleaning technique that mechanically removes microorganisms to reduce contamination to safe levels Sanitizer: compound such as soap or detergent that sanitizes Sanitary: may not be free from microbes but are safe for normal use Degermation: reduces the numbers of microbes on the human skin (ex. alcohol wipes) 11 Practical Concerns in Microbial Control 1. Does the application require sterilization, or is disinfection adequate? 2. Is the item to be reused or permanently discarded? 3. If it will be reused, can it withstand heat, pressure, radiation, or chemicals? 4. Is the control method suitable for a given application? 5. Will the agent penetrate to the necessary extent? 6. Is the method cost- and labor-efficient, and is it safe? 12  What is Microbial Death? When various cell structures become dysfunctional and the entire cell sustains irreversible damage If a cell can no longer reproduce under ideal environmental conditions 13  Factors That Affect Death Rate Figure 11.2 Factors that influence the rate at which microbes are killed by antimicrobial agents. (a) Length of exposure to the agent. During exposure to a chemical or physical agent, all cells of a microbial population, even a pure culture, do not die simultaneously. Over time, the number of viable organisms remaining in the population decreases logarithmically, giving a straight-line relationship on a graph. The point at which the number of survivors is infinitesimally small is considered sterilization. (b) Effect of the microbial load. (c) Relative resistance of spores versus vegetative forms. (d) Action of the agent, whether microbicidal or microbistatic.14  Factors that Influence the Action of Antimicrobial Agents The number of microorganisms (Fig. 11.2b) The nature of the microorganisms in the population (Fig. 11.2c) The temperature and pH of the environment The concentration of the agent e.g. The mode of action of the agent (Fig. 11.2d) The presence of solvents, interfering organic matter, and inhibitors e.g. 15 How Antimicrobial Agents Work: Their Modes of Action The Cell Wall – Block its synthesis – Digest it – Break down its surface – The cell becomes fragile and is lysed easily The Cell Membrane cellular synthetic processes (DNA, RNA) proteins. 16  The Cell Wall – Block its synthesis – Digest it – Break down its surface – The cell becomes fragile and is lysed easily 17 How Agents Affect the Cell Membrane Figure 11.3 Mode of action of surfactants on the cell membrane. Surfactants inserting in the lipid bilayer disrupt it and create abnormal channels that alter 18 permeability and cause leakage both into and out of the cell Protein and Nucleic Acid Synthesis Binding to ribosomes to stop translation e.g. chloramphenicol Bind irreversibly to DNA preventing transcription and translation e.g. formaldehyde and ethylene oxide Mutagenic agents e.g. Gamma, ultraviolet, or X radiation 19  Agents That Alter Protein Function Figure 11.4 Modes of action affecting protein function. (a) The native (functional) state is maintained by bonds that create active sites to fit the substrate. Some agents denature the protein by breaking all or some secondary and tertiary bonds. Results are (b) complete unfolding or (c) random bonding and incorrect folding. (d) Some agents react with functional groups on the20active site and interfere with bonding.  Outline and Learning Outcomes 11.2 Methods of Physical Control 5. Name six methods of physical control of microorganisms. 6. Compare and contrast moist and dry heat methods of control, and identify multiple examples of each. 7. Define thermal death time and thermal death point, and describe their role in proper sterilization. 8. Explain four different methods of moist heat control. 9. Explain two methods of dry heat control. 10. Identify advantages and disadvantages of cold treatment and desiccation. 11. Differentiate between the two types of radiation control methods, providing an application of each. 12. Outline the process of filtration and describe its two advantages in microbial control. 13. Identify some common uses of osmotic pressure as a control method.  11.2 Methods of Physical Control Heat as an Agent of Microbial Control – Generally, elevated temperatures are microbicidal and lower temperatures are microbistatic – Can use moist heat or dry heat 22 23 Practical Concerns in the Use of Heat: Thermal Death Measurements Temperature and length of exposure must be considered Higher temperatures generally allow shorter exposure times; lower temperatures generally require longer exposure times Thermal death time (TDT): the shortest length of time required to kill all test microbes at a specified temperature Thermal death point (TDP): the lowest temperature required to kill all microbes in a sample in 10 minutes 24  Common Methods of Moist Heat Control 1. steam under pressure, 2. nonpressurized steam, 3. pasteurization, and 4. boiling water. 25  Steam under pressure – Pressure raises the temperature of steam – Autoclave is used – Most efficient pressure-temperature combination for sterilization: 15 psi which yields 121°C 26 Steam sterilization with the autoclave. (a) A tabletop autoclave. (b) Cutaway section, showing autoclave components. 27 Figure 11.5  Nonpressurized Steam Intermittent sterilization or tyndallization Expose to free-flowing steam for 30-60 minutes, incubate for 23-24 hours, treat again; repeat for 3 days. 28  Pasteurization Used to disinfect beverages Heat is applied to liquids to kill potential agents of infection and spoilage, while retaining the liquid’s flavor and food value Special heat exchangers – Flash method: expose to 71.6°C for 15 seconds – Batch method: expose to 63°C to 66°C for 30 minutes Does not kill endospores or thermoduric microbes 29  Boiling Water For disinfection and not sterilization Expose materials to boiling water for 30 minutes 30 Dry Heat: Hot Air and Incineration Incineration – Ignites and reduces microbes to ashes and gas – Common practice in microbiology lab- incineration on inoculating loops and needles using a Bunsen burner – Can also use tabletop infrared incinerators. (see next) 31 Figure 11.6 Dry heat incineration. Infrared incinerator with shield to prevent spattering 32 of microbial samples during flaming.  Dry Oven Usually an electric oven Coils radiate heat within an enclosed compartment Exposure to 150°C to 180°C for 2 to 4 hours Used for heat-resistant items that do not sterilize well with moist heat 33 The Effects of Cold and Desiccation Cold temp. is to slow growth of cultures and microbes in food during processing and storage Cold does not kill most microbes; freezing can actually preserve cultures Desiccation: dehydration of vegetative cells when directly exposed to normal room air Lyophilization: a combination of freezing and drying; used to preserve microorganisms and other cells in a viable state for many years 34 Radiation as a Microbial Control Agent Radiation: energy emitted from atomic activities and dispersed at high velocity through matter or space For microbial control: – Gamma rays – X rays – Ultraviolet radiation 35  Modes of Action of Ionizing Versus Nonionizing Radiation Ionizing radiation: if the radiation ejects orbital electrons from an atom causing ions to form Nonionizing radiation: excites atoms by raising them to a higher energy state but does not ionize them 36 Figure 11.7 Cellular effects of irradiation. (a) Ionizing radiation can penetrate a solid barrier, bombard a cell, enter it, and dislodge electrons from molecules. Breakage of DNA creates massive mutations and damage to proteins prevents them from repairing it. (b) Nonionizing radiation enters a cell, strikes molecules, and excites them. The effect on DNA is mutation by formation of abnormal bonds. (c) A solid barrier cannot be penetrated by nonionizing radiation 37  Ionizing Radiation: Gamma Rays, X Rays, and Cathode Rays Cold sterilization Dosage of radiation- measured in Grays Exposure ranges from 5 to 50 kiloGrays Gamma rays, most penetrating; X rays, intermediate; cathode rays, least penetrating 38  Applications of Ionizing Radiation Food products Medical products 39 Figure 11.8 Foods commonly irradiated. Regulations dictate that the universal symbol for irradiation must be affixed to all irradiated materials. 40  Nonionizing Radiation: Ultraviolet Rays Wavelength approximately 100 nm to 400 nm Germicidal lamp: 254 nm Not as penetrating as ionizing radiation Powerful tool for destroying fungal cells and spores, bacterial vegetative cells, protozoa, and viruses 41 Figure 11.9 Formation of pyrimidine dimers by the action of ultraviolet (UV) radiation. This shows what occurs when two adjacent thymine bases on one strand of DNA are induced by UV rays to bond laterally with each other. The result is a thymine dimer (shown in greater detail). Dimers can also occur between adjacent cytosines and thymine and cytosine bases. If they are not repaired, dimers can prevent that segment of DNA from being correctly replicated or transcribed. Massive dimerization is lethal to cells. 42  Applications of Ultraviolet Radiation Usually disinfection rather than sterilization Hospital rooms, operating rooms, schools, food prep areas, dental offices Treat drinking water or purify liquids 43  Decontamination by Filtration: Techniques for Removing Microbes Effective for removing microbes from air and liquids Fluid strained through a filter with openings large enough for fluid but too small for microorganisms Filters are usually thin membranes of cellulose acetate, polycarbonate, and a variety of plastic materials 44  Figure 11.11 Figure 11.11 Membrane filtration. (a) Vacuum assembly for achieving filtration of liquids through suction. Inset shows filter as seen in cross section, with tiny assageways (pores) too small for the microbial cells to enter but large enough for liquid to pass through. (b Scanning electron micrograph of filter, showing relative size of pores and bacteria trapped on its surface.(21,930×). 45  Applications of Filtration Prepare liquids that can’t withstand heat Can decontaminate beverages without altering their flavor Water purification Removing airborne contaminants (HEPA filters) 46 HEPA: High-efficiency particulate air  Applications of Filtration Prepare liquids that can’t withstand heat Can decontaminate beverages without altering their flavor Water purification Removing airborne contaminants (HEPA filters) 47 HEPA: High-efficiency particulate air  11.3 Chemical Agents in Microbial Control Approximately 10,000 different antimicrobial chemical agents are manufactured Approximately 1,000 used routinely in health care and the home Occur in liquid, gaseous, or solid state 48  Choosing a Microbicidal Chemical Rapid action even in low concentrations Solubility in water or alcohol and long-term stability Broad-spectrum microbicidal action without being toxic to human and animal tissues Penetration of inanimate surfaces to sustain a cumulative or persistent action Resistance to becoming inactivated by organic matter Noncorrosive or nonstaining properties Sanitizing and deodorizing properties Affordability and ready availability 49  Factors that Affect the Germicidal Activity of Chemicals Nature of microorganisms being treated Nature of the material being treated Degree of contamination Time of exposure Strength and chemical action of the germicide 50 51 52  Germicidal Categories According to Chemical Group halogens, heavy metals, alcohols, phenolic compounds, oxidizers, aldehydes, detergents, and gases. 53 Halogen Antimicrobial Chemicals – Fluorine, bromine, chlorine, and iodine – Microbicidal and sporicidal with longer exposure – Chlorine compounds: liquid and gaseous chlorine, hypochlorites, chloramines Kills bacteria and endospores Also kills fungi and viruses Example: Household bleach – Iodine compounds: free iodine and iodophors 54  Phenol and its Derivatives Phenol coefficient: compares a chemical’s antimicrobic properties to those of phenol High concentrations: cellular poisons Lower concentrations: inactivate certain critical enzyme systems 55  Chlorhexidine Complex organic base containing chlorine and two phenolic rings Targets cell membranes and protein structure At moderate to high concentrations, it is bactericidal for both gram-positive and gram- negative bacteria but inactive against spores Advantages: Mildness, low toxicity, rapid action 56  Alcohols as Antimicrobial Agents Only ethyl and isopropyl alcohols are suitable for microbial control Mechanism of action depends in part upon its concentration – 50% and higher dissolve membrane lipids, disrupt cell surface tension, and compromise membrane integrity – 50% to 90% denatures proteins through coagulation; but higher concentration does not increase microbicidal activity – 100% (absolute alcohol) dehydrates cells and inhibits their growth Does not destroy bacterial spores at room temperature but can destroy resistant vegetative forms. More effective in inactivating enveloped viruses than nonenveloped viruses. 57  Hydrogen Peroxide and Related Germicides Germicidal effects are due to the direct and indirect actions of oxygen Oxygen forms hydroxyl free radicals ( HO) which are highly toxic and reactive to cells Bactericidal, virucidal, and fungicidal In higher concentrations is sporicidal 58  Chemicals with Surface Action: Detergents Act as surfactants Anionic (-) detergents have limited microbicidal power Cationic (+) detergents are more effective because the positively charged end binds well with the predominantly negatively charged bacterial surface proteins e.g. soaps are weak microbicides but gain germicidal value when mixed with agents such as chlorhexidine or iodine 59 Figure 11.13 The structure of detergents. (a) In general, detergents are polar molecules with a positively charged head and at least one long, uncharged hydrocarbon chain. The head contains acentral nitrogen nucleus with various alkyl (R) groups attached. (b) A common quaternary ammonium detergent, benzalkonium chloride. 60 Graph showing effects of hand scrubbing. Comparison of scrubbing over several days with a nongermicidal soap versus a germicidal soap. Germicidal soap has ersistent effects on skin over time, keeping the microbial count low. Without germicide, soap does not show this sustained effect. 61 Figure 11.15  Heavy Metal Compounds Hg, Ag, Au, Cu, As, and Zn have been used Only Hg and Ag still have significance as germicides Oligodynamic action: having antimicrobial effects in exceedingly small amounts Bind onto functional groups of proteins and inactivating them Drawbacks to using metals in microbial control: – Can be very toxic to humans – Often cause allergic reactions – Large quantities of biological fluids and wastes neutralize their actions – Microbes can develop resistance to them 62 Mercury, Silver, Gold, cupper, Arsenic and Zinc  Figure 11.16 Demonstration of the oligodynamic action of heavy metals. A pour plate inoculated with saliva has small fragments of heavy metals pressed lightly into it. During incubation, clear zones indicating growth inhibition developed around both fragments. The slightly larger zone surrounding the amalgam (used in tooth fillings) probably reflects the synergistic effect of the silver and mercury it contains. 63  Aldehydes as Germicides Organic substances bearing a–CHO functional group on the terminal carbon Glutaraldehyde and formaldehyde (formalin- aqueous solution)- most often used in microbial control accepted as a sterilant and high-level disinfectant. 64 Figure 11.17 Actions of glutaraldehyde. The molecule polymerizes easily. When these alkylating 65 polymers react with amino acids, they cross-link and inactivate proteins.  Gaseous Sterilants and Disinfectants Ethylene oxide (ETO) Propylene oxide Chlorine dioxide 66  Dyes as Antimicrobial Agents Primary source of certain drugs used in chemotherapy Aniline dyes (crystal violet and malachite green) are very active against gram-positive species of bacteria and various fungi Yellow acridine dyes (acriflavine and proflavine) sometimes used for antisepsis and wound treatment Limited applications because they stain and have a narrow spectrum of activity 67  Acids and Alkalis Very low or high pH can destroy or inhibit microbial cells Limited in applications due to their corrosive, caustic, and hazardous nature 68 69