Physical and Chemical Control of Microbes Chapter 11 Physical and Chemical Control of Microbes

Controlling Microorganisms General Considerations in Microbial Control The methods of microbial control belong to the general category of decontamination procedures, in that they destroy or remove contaminants Most decontamination methods employ either physical agents, such as heat or radiation, or chemical agents such as disinfectants and antiseptics

Microbial Control Methods

Relative Resistance of Microbial Forms Highest resistance Bacterial endospores Moderate resistance Protozoan cysts Fungal sexual spores (zygospores) Naked viruses Mycobacterium tuberculosis, Staphylococcus aureus, and Pseudomonas species Least resistance Most bacterial vegetative cells Ordinary fungal spores and hyphae Enveloped viruses Yeasts Trophozoites

Terminology and Methods of Microbial Growth Sterilization is a process that destroys or removes all viable microorganisms, including viruses Bactericide is a chemical that destroys bacteria except for those in the endospore stage Fungicide is a chemical that can kill fungal spores, hyphae, and yeasts Virucide is any chemical known to inactivate viruses, especially on living tissues A sporicide is an agent capable of destroying bacterial endospores

Terminology and Methods of Microbial Growth Bacteriostatic agents prevent the growth of bacteria on tissues or on objects in the environment Fungistatic chemicals inhibit fungal growth Germicde, also called a microbicide, is any chemical agent that kills pathogenic microorganisms Disinfection refers to the use of a physical process or a chemical agent to destroy vegetative pathogens but not bacterial endospores Sepsis is defined as the growth of microorganisms in the body or the presence of microbial toxins in blood and other tissues

Terminology and Methods of Microbial Growth Asepsis refers to any practice that prevents the entry of infectious agents into sterile tissues and thus prevents infection Antiseptics are applied directly to exposed body surface, wounds, and surgical incisions to destroy or inhibit vegetative pathogens Sanitization is any cleansing technique that mechanically removes microorganisms to reduce the level of contaminants Degermation is a process usually involves scrubbing the skin or immersing it in chemicals, or both

Factors that affect Death Rate The number of microorganisms The nature of the microorganism (vegetative vs. endospore) The temperature and pH of the environment The concentration of the agent

How Antimicrobial Agents Work The mode of action of the agent (destructive or inhibitory) Damage to cell wall Damage to cell membrane (surfactants) Inhibit protein and nucleic acid synthesis Alteration of protein function The presence of solvents, interfering organic matter, and inhibitors

Mode of Action of Surfactants on the Cell Membrane Surfactants inserting in the lipid bilayer disrupt it and create abnormal channels that alter permeability and cause leakage both into and out of the cell

Mode of action affecting protein function Some agents denature the protein by breaking all or some secondary and tertiary bonds, resulting in complete unfolding or random bonding and incorrect folding Some agents react with functional groups on the active site and interfere with bonding

Physical Methods for Controlling Microorganisms Moist Heat: Use of hot water or steam in disinfection to kill vegetative cells or in sterilization to kill spores. Mode of action: denaturation of proteins, destruction of membranes, and DNA For Sterilization Autoclave uses steam under pressure (15 psi/121C/10-40 minutes) Applied on heat-resistant material that steam can penetrate Not recommended for oils, powders, heat-sensitive material Intermittent sterilization uses free-flowing unpressurized steam at 100C applied for 30-60 minutes on 3 successive days Used for substances that cannot be autoclaved, especially media and certain canned foods

Sterilization using Steam under Pressure

Physical Methods for Controlling Microorganisms For Disinfection Pasteurization is application of heat less than 100C to liquids; employs the flash method to heat liquid to 71C for 15 seconds Targets non-spore forming milk-borne pathogens, such as Salmonella and Listeria, and reduces spoilage by lowering the overall microbial count; also applied to beer and wine Only reduces microbial content; many thermourics survive Boiling water at 100C used to destroy pathogens (not spores) on household supplies; materials may become recontaminated

Physical Methods for Controlling Microorganisms Dry Heat: Use of hot air with low moisture content to sterilize Mode of action: depending on the temperature, combusts, dehydrates, or coagulates proteins Incineration Open flame: Materials are placed for a few seconds in an 800C to 1800C flame of a Bunsen burner Furnace: Incinerator chamber equipped with 600C - 1200C flame that burns materials to ashes Used for decontamination of hospital and industrial wastes; may add pollutants to atmosphere Dry Oven: Closed chamber heated to 150C - 180C for 2 – 4 hours Used for sterilizing metals and glass, but too vigorous for liquids, plastics, paper, cloth

Dry Heat Incineration Infrared incinerator with shield to prevent spattering of microbial samples during flaming

Physical Methods for Controlling Microorganisms Cold Temperatures Refrigeration (0C - 15C) or freezing (below 0C) is not a reliable mode of disinfection It is only microstatic, meaning it slows the growth rate of most microbes Refrigeration is used for preserving foods, media, and cultures Drying/Dessication is the gradual withdrawal of water from cells by exposure to room air, leading to metabolic inhibition Effective in preservation, but not reliable for pathogens, and not really an effective method for infection control

Radiation/Irradiation Use of energy in the form of waves (electromagnetic) and particles that can be transmitted through space A method of cold sterilization

Radiation/Irradiation Ionizing radiation uses high-energy, short waves and particles that can dislodge electrons from atoms This causes direct damage to DNA by the formation of breaks and mutations Types are gamma rays, X rays, cathode rays (high speed electrons) used in irradiation and used to sterilize heat sensitive medical materials in packages; also to sterilize and increase storage time of fresh products; very penetrating Somewhat more expensive and dangerous than other methods

Radiation/Irradiation Nonionizing radiation uses moderate energy, medium-length ultraviolet waves that excite, but do not ionize, atoms Rays act on DNA molecule, forming dimers between adjacent pyrimidines, and produce toxic products UV lamps disinfect air in medicine and industry; treat water, sera, vaccines, drugs; disinfect solid surfaces Does not penetrate most solids and can damage human tissues (skin, eyes)

Formation of pyrimidine dimers by the action of ultraviolet (UV) radiation

Sterilization by Filtration Filtration involves the physical removal of microbes from liquids and air by trapping them in fine filters The fluid passes through tiny pores into a container Can be used to remove viruses Filters can sterilize heat-sensitive liquids (vaccines, serum, drugs, media, water) Can remove microbes from air in hospital rooms, isolation units, and “clean rooms”

Chemical Control of Microorganisms General uses: Disinfectants, antiseptics, sterilants, preservatives, sanitizers, degermers Physical states: Solutions come in aqueous form (chemicals dissolved in water) and tincture form (chemicals dissolved in alcohol); solutions expressed as dilutions, percents, and parts per million Halogens Chlorine: Forms are Cl2, hypochlorites, chloramines Denaturation of proteins by disrupting disulfide bonds; can be sporicidal with adequate time Elemental chlorine (1-2 ppm) disinfects water by destroying pathogenic vegetative pathogens Hypochlorites (chlorine bleach) are used extensively for disinfection and sanitization Chloramines are clinical disinfectants and antiseptics and alternative water disinfection agents Chemical action may be retarded by high levels of organic matter

Water is passed through a chlorination tank

Chemical Control of Microorganisms Iodine: Forms are free iodine (I2) and iodophors (iodine complexed to organic polymers) Interfere with protein interchain bonds, causing denaturation; can be sporicidal Weak solutions are topical antiseptics; iodine tincture is for a high degree of surgical asepsis and disinfection Iodine solutions stain and corrode; stronger solutions are too irritating and toxic to use on tissue Iodophors are aqueous solutions with 2-10% iodine (Betadine, Povidone); they are milder medical and dental degerming agents, disinfectants, and ointments

Use of providone-iodine sponge

Chemical Control of Microorganisms Phenolics Phenols act on microbes by disrupting cell membranes and precipitating proteins They are bactericidal, fungicidal, and virucidal, but not sporicidal Phenol is an older toxic disinfectant; 1-3% cresol in soap is a common housekeeping disinfectant and cleaner Orthophenyl phenol (Lysol) is a milder phenolic used in air sprays and hospital disinfectants Triclosan is an antibacterial additive to soaps, detergents, and numerous household products Most phenolics are to toxic for use as antiseptics and are relatively insoluble

Chemical Control of Microorganisms Chlorhexidine is available as Hibiclens and Hibitane It is a surfactant and protein denaturant, with bactericidal, some antiviral and antifungal effects, but not sporicidal An aqueous or alcohol solution is a skin degerming agent for preoperative scrubs, skin cleaning, and burns Product is relatively mild, nontoxic, and fast-acting

Chemical Control of Microorganisms Alcohols include ethyl and isopropyl, usually in solutions of 50-95% Act as a surfactant to dissolve membrane lipids and coagulate proteins; work on bacterial vegetative cells and fungi, but are not sporicidal Ethyl alcohol (70-95%) is a germicide in the clinic, laboratory; used at home for degerming, low-level disinfection, and sanitization Isopropyl alcohol has similar uses, though it is more toxic and less safe

Chemical Control of Microorganisms Hydrogen peroxide is available in weak (3%) to strong (25%) solutions that produce highly active hydroxyl-free radicals and damage proteins and DNA molecules; also decomposes to water and O2 gas, which can be toxic to anaerobes; strong solutions are sporicidal 3% hydrogen peroxide is used for skin and wound antisepsis, care of mucous membrane infections; and disinfection of utensils 6-25% can be used to sterilize equipment Decomposes in presence of light and catalase

Chemical Control of Microorganisms Detergents and Soaps Cationic detergents are known as quaternary ammonium compounds or quats They are surfactants that alter cell permeability Their action is limited to some bacteria and fungi; they are not sporicidal Benzalkonium and cetylpyridinium chlorides are quats used in environmental disinfection, as cleaners in clinics and the food industry, and as presevatives Soaps are not very microbicidal but function in the mechanical removal of grease and soil on skin, utensils, and environmental surfaces Soaps do not destroy the tubercle bacillus or hepatitis B virus and are inactivated by large quantities of organic matter

Detergents

Chemical Control of Microorganisms Heavy metal compounds include mercury and silver solutions and tinctures; work in high dilutions (oligodynamic action) and precipitate proteins; are germicidal but not sporicidal Organic mercurial tinctures (thimerosal, nitromersol) are skin antiseptics and preservatives Silver nitrate solutions are used as a topical antiseptic; silver sulfadiazine ointment can prevent burn infections; colloidal silver preparations are used for mouth and eye rinses Metal solutions are highly toxic, cause allergies, and are neutralized by organic substances

Chemical Control of Microorganisms Aldehydes include glutaraldehyde and formaldehyde or formalin solutions They kill cells by alkylation of amino and nucleic acids, with wide microbial action Glutaraldehyde in 2% solutions (Cidex) is used as a sterilant for heat-sensitive instruments in the medical and dental office, and for some types of environmental disinfection Formalin in 1-8% is limited to disinfection of some instruments, rooms, and as a preservative Glutaraldehyde is somewhat unstable; formaldehyde is toxic and irritating

Chemical Control of Microorganisms Gases and aerosols include ethylene oxide (ETO), propylene oxide, and betapropiolactone, which inactivate nucleic acids and proteins All are sporicidal, but only ETO is an approved sterilant Ethylene oxide is added to a special chamber for sterilizing plastic supplies and to treat spices and dried foods Propylene oxide can disinfect food (nuts, starch) Betapropiolactone is used to disinfect rooms and some biological materials Toxic and has poor penetration ETO is somewhat dangerous because of its explosiveness and toxicity, and it is slow-acting