Microbiology: A Systems Approach, 2nd 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 pH 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

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 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

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

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 gram-negative 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

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

Figure 11.16

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

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 pH can destroy or inhibit microbial cells Limited in applications due to their corrosive, caustic, and hazardous nature