Microbiology A Systems Approach 2 nd ed Chapter

Microbiology: A Systems Approach, 2 nd ed. Chapter 11: Physical and Chemical Control of Microbes

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

Figure 11. 1

Relative Resistance of Microbial Forms • Primary targets of microbial control: microorganisms that can cause infection or spoilage 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

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

Disinfection • The use of a physical process or chemical agent (disinfectant) to destroy vegetative pathogens • Does not destroy bacterial endospores • Usually used only on inanimate objects • Also removes toxins • 5% bleach solution

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

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, for example

Decontamination • Used when actual sterilization isn’t needed but need to decrease the risk of infection or spoilage (ex. food industry) • Sanitization: any cleansing 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)

Practical Concerns in Microbial Control • Does the application require sterilization, or is disinfection adequate? • Is the item to be reused or permanently discarded? • If it will be reused, can it withstand heat, pressure, radiation, or chemicals? • Is the control method suitable for a given application? • Will the agent penetrate to the necessary extent? • Is the method cost- and labor-efficient, and is it safe?

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 • Death begins when a certain threshold of microbicidal agent is met, and continues in a logarithmic manner

Figure 11. 2

Factors that Influence the Action of Antimicrobial Agents • The number of microorganisms • The nature of the microorganisms in the population • The temperature and p. H of the environment • The concentration of the agent • The mode of action of the agent • The presence of solvents, interfering organic matter, and inhibitors

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

Figure 11. 3

Protein and Nucleic Acid Synthesis • Binding to ribosomes to stop translation • Bind irreversibly to DNA preventing transcription and translation • Mutagenic agents

Protein Function Figure 11. 4

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

Heat Resistance and Thermal Death of Spores and Vegetative Cells

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

Common Methods of Moist Heat Control • 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

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 in a row

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

Boiling Water • For disinfection and not sterilization • Expose materials to boiling water for 30 minutes

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

Figure 11. 6

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

The Effects of Cold and Desiccation • 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

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

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

Figure 11. 7

Ionizing Radiation: Gamma Rays, X Rays, and Cathode Rays • • Cold sterilization Dosage of radiation- measured in Grays Exposure ranges from 5 to 50 kilo. Grays Gamma rays, most penetrating; X rays, intermediate; cathode rays, least penetrating

Applications of Ionizing Radiation • Food products • Medical products

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

Figure 11. 9

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

Figure 11. 10

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 • Pore size can be controlled and standardizes

Figure 11. 11

Applications of Filtration • Prepare liquids that can’t withstand heat • Can decontaminate beverages without altering their flavor • Water purification • Removing airborne contaminants (HEPA filters)

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 • Tinctures: solutions dissolved in pure alcohol or water-alcohol mixtures

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

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

Germicidal Categories According to Chemical Group • 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 • Topical antiseptic • Disinfectant

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

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 gramnegative bacteria but inactive against spores • Mild, low toxicity, rapid action

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

Hydrogen Peroxide and Related Germicides • Germicidal effects are due to the direct and indirect actions of oxygen • Oxygen forms hydroxyl free radicals which are highly toxic and reactive to cells • Bactericidal, virucidal, and fungicidal • In higher concentrations is sporicidal

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 • Soaps are weak microbicides but gain germicidal value when mixed with agents such as chlorhexidine or iodine

Figure 11. 14

Figure 11. 15

Heavy Metal Compounds • Hg, 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

Figure 11. 16

Aldehydes as Germicides • –CHO functional group on the terminal carbon • Glutaraldehyde and formaldehyde (formalinaqueous solution)- most often used in microbial control

Figure 11. 17

Gaseous Sterilants and Disinfectants • Ethylene oxide (ETO) • Propylene oxide • Chlorine dioxide

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

Acids and Alkalis • Very low or high p. H can destroy or inhibit microbial cells • Limited in applications due to their corrosive, caustic, and hazardous nature