STERILIZATION & DISINFECTION

Control of Microbial Growth: Introduction In mid 1800s Semmelweiss and Lister helped develop aseptic techniques to prevent contamination of surgical wounds. Before then: Nosocomial infections caused death in 10% of surgeries. Up to 25% mothers delivering in hospitals died due to infection.

Definitions: Sterilization: Killing or removing all forms of microorganisms (including bacterial spores) in a material or an object. Autoclaving is the most commonly used method of sterilization.

Disinfection: is defined as killing most, but not all microorganisms in a location, however some bacterial spores may still remain behind. It involves reducing the number of pathogenic microorganisms to the point where they no longer cause disease. May use physical or chemical methods.

Phenol coefficient used to evaluate ability of disinfectant to kill MO (ratio of conc. Of phenol to disinfectant to cause same amount of killing.)

DefinitionsDisinfectant:

Applied to inanimate objects. Antiseptic: Applied to living tissue (antisepsis). Degerming: Mechanical removal of most microbes in a limited area. Example: Alcohol swab on skin. Sanitization: Use of chemical agent on food-handling equipment to meet public health standards and minimize chances of disease transmission. e.g: Hot soap & water.

Definitions Sepsis: Comes from Greek for decay or putrid. Indicates bacterial contamination. Asepsis: Absence of significant contamination. Aseptic techniques are used to prevent contamination of surgical instruments, medical personnel, and the patient during surgery. Aseptic techniques are also used to prevent bacterial contamination in food industry.

Control of Microbial Growth: DefinitionsBacteriostatic Agent: An agent that inhibits the growth of bacteria, but does not necessarily kill them. Suffix stasis: To stop or steady. Germicide: An agent that kills certain microorganisms. Bactericide:

An agent that kills bacteria. Most do not kill endospores. Viricide: An agent that inactivates viruses. Fungicide: An agent that kills fungi. Sporicide: An agent that kills bacterial endospores of fungal spores.

Disinfectant Performance Is dependent on Disinfectant concentrations Is dependent on length (time) of administration Is dependent on temperature during administration Microbe type (e.g., mycobacteria, spores, and certain viruses can be very resistant to disinfectionin general vegetative cells in log phase are easiest to kill) Substrate effects (e.g., high organic content interferes with disinfection. It is easier (and faster) to kill fewer microbes than many microbes

Chemical Antimicrobials

Agent

Mechanisms of Action

Comments

Alcohols

Denatures proteins; Disrupts lipidsMembrane Disruption; increased penetration Denature proteins; Disrupts lipids Disrupts cell membrane Oxidizing agent Disrupts cell membrane High/low pH Denature protein

Antiseptic & Disinfectant Ethanol and isopropylSoaps; detergents Antiseptic - benzalconium chloride, Cepacol; Disinfectant Disinfectant Irritating odor Antiseptic - Iodine (Betadine) Disinfectant - Chlorine (Chlorox) Mold and Fungi inhibitors; e.g., benzoate of soda Antiseptic & Disinfectant; Silver Nitrate

Detergents Quats (cationic detergent) Phenolics Halogens Organic acids and bases Heavy Metals

AldehydesEthylene Oxide Oxidizing agents

Denature proteinsDenaturing proteins Denature proteins

Gluteraldehyde - disinfectant (Cidex); Formaldehyde - disinfectantUsed in a closed chamber to sterilize Hydrogen peroxide antiseptic; Hydrogen peroxide disinfectan; Benzoyl peroxide antiseptic

Aqueous ethanol (60-95%) and isopropanol are used as disinfectants Effectively kill bacteria and fungi but not endospores nor nonenveloped viruses

Alcohols

Fast acting, no residue (evaporate away), no staining But not very penetrating and no residual activity (once gone gone) Exert their action by disrupting cell membranes (dissolving lipids) & denaturing proteins

Used to mechanically wipe microbes off skin before injections or blood drawing.

Quartenary Ammonium compounds Quats are cationic detergents that act by disrupting lipid bilayers Quats are bactericidal, fungicidal, viricidal (enveloped), and amoebicidal are most effective against Gram-positive bacteria but do not kill endospores are rapidly inactivated by organics including cotton and soap ZephiranBenzalkonium chloride CepacolCetylpyridinium chloride

PHENOLICS Carbolic acid 2-5% Powerful microbicidal, very corrosive General purpose disinfectant in hospital

Cresol, lysol Hexachlorophene, Triclosan, Lysol, soap

Two halogens are regularly employed as antimicrobials: Iodine and Chloride Chlorine: Used as a disinfectant (10% bleach) Chlorine: Hypochlorous acid (HOCl) is a product, formed in water, that is the active form of the disinfectant Chlorine: Applied in treatment of drinking water, swimming pool, and sewage

Halogens

Iodine:

Tincture of iodine (alcohol solution) was one of first antiseptics used. Combines with amino acid tyrosine in proteins and denatures proteins. Stains skin and clothes, somewhat irritating. Iodophors: Compounds with iodine that are slow releasing, take several minutes to act. Used as skin antiseptic in surgery. Not effective against bacterial endospores.

Betadine Isodine

Heavy Metals:Include copper, selenium, mercury, silver, and zinc. Oligodynamic action: Very tiny amounts are effective. A. Silver: 1% silver nitrate used to protect infants against gonorrheal eye infections until recently. B. Mercury Organic mercury compounds like merthiolate and mercurochrome are used to disinfect skin wounds. C. Copper Copper sulfate is used to kill algae in pools and fish tanks.

Hydrogen peroxide, is most common

Oxidizing Agents

It is not a terribly effective disinfectant or anticeptic This is because bacteria and body tissues contain enzymes (catalase) that inactivate hydrogen peroxide However, the oxygen released upon inactivation can help oxygenate deep wounds and thus kill strict-anaerobe contaminants, e.g., Clostridium tetani

Ozone and peracetic acid are also oxidizing antimicrobial agents They exert their effect by oxidizing cell macromolecules (e.g., proteins, DNA, etc.)

Aldehydes:Include some of the most effective antimicrobials. Inactivate proteins by forming covalent crosslinks with several functional groups. A. Formaldehyde gas: Excellent disinfectant. Commonly used as formalin, a 37% aqueous solution. Formalin was used extensively to preserve biological specimens and inactivate viruses and bacteria in vaccines. Irritates mucous membranes, strong odor. Also used in mortuaries for embalming.

Glutaraldehyde: Less irritating and more effective than formaldehyde. One of the few chemical disinfectants that is a sterilizing agent. A 2% solution of glutaraldehyde (Cidex) is: Bactericidal, tuberculocidal, and viricidal in 10 minutes. Sporicidal in 3 to 10 hours.

Commonly used to disinfect hospital instruments. Also used in mortuaries for embalming.

Glutaraldehyde is capable of effective sterilizationat room temperature, even against endospores, and even in the presence of organics, but achieving sterilization requries many hours of exposure

Glutaraldehyde

ETHYLENE OXIDE Highly inflammable, mixed with inert gases CO2, N Especially for heart lung machines, respirators, sutures, syringes, dental equipments (heat sensitive)

DYES Combine with nucleic acids Aniline dyes Brilliant green, malachite green, crystal violet

Acridine dyes Proflavine, acriflavine, euflavine, aminacrine

Skin & wound antiseptics Bacteriostatic, more active against GP bacteria

Physical disinfectant

Agent

Mechanisms of ActionDenatures proteinsDenatures proteins Denatures proteins Incineration of contaminants Oxidation & Denatures proteins

CommentsKills vegetative bacterial cells and viruses Endospores survive121C at 15 p.s.i. for 30 min kills everything Kills pathogens in food products Used for inoculating loop 170C for 2 hours; Used for glassware & instrument sterilization

Physical Antimicrobials

Moist Heat, boilingMoist Heat, Autoclaving Moist Heat, Pasteurization Dry Heat, Flaming Dry Heat, Hot air oven Filtration Cold, Lyophilization (also desiccation) Cold, Refrigeration

Separation of bacteria Used for heat sensitive liquids from liquid (HEPA: from air) Desiccation and low temperature Decreased chemical reaction rate Used for food & drug preservation; Does not necessarily kill so used for Long-term storage of bacterial cultures Bacteriostatic

Osmotic Pressure, Addition of salt or sugarRadiation, UV Radiation, X-rays Strong vis. Light

Plasmolysis of contaminants

Used in food preservation (less effective against fungi)Limited penetration Used for sterilizing medical supplies Line-drying laundry

DNA damage (thymine dimers) DNA damage

Moist Heat Moist heat kills microbes by denaturing enzymes (coagulation of proteins) Boiling (at 100C, I.e., at sea level) kills many vegetative cells and viruses within 10 minutes Autoclaving: steam applied under pressure (121C for 15 min) is the most effective method of moist heat sterilizationthe steam must directly contact the material to be sterilized Pasteurization: destroys pathogens (Mycobacterium tuberculosis, Salmonella typhi, etc.) without altering the flavor of the fooddoes not sterilize (63C for 30 seconds) Higher temperature short time pasteurization applies higher heat for a much shorter time (72C for 15 seconds) An ultra-high-temperature, very short duration treatment (140C for 3 sec.) is used to sterilize dairy products

Autoclave: Chamber which is filled with hot steam under pressure. Preferred method of sterilization, unless material is damaged by heat, moisture, or high pressure. Temperature of steam reaches 121oC at twice atmospheric pressure. Most effective when organisms contact steam directly or are contained in a small volume of liquid.All organisms and endospores are killed within 15 minutes.

Require more time to reach center of solid or large volumes of liquid.

Autoclave: Closed Chamber with High Temperature and Pressure

Physical Methods of Microbial Control:Moist Heat (Continued): Pasteurization: Developed by Louis Pasteur to prevent the spoilage of beverages. Used to reduce microbes responsible for spoilage of beer, milk, wine, juices, etc. Classic Method of Pasteurization: Milk was exposed to 65oC for 30 minutes. High Temperature Short Time Pasteurization (HTST): Used today. Milk is exposed to 72oC for 15 seconds. Ultra High Temperature Pasteurization (UHT): Milk is treated at 140oC for 3 seconds and then cooled very quickly in a vacuum chamber. Advantage: Milk can be stored at room temperature for several months.

Moist Heat - Temp at 1000C Boiling - 1000C for 10 min Kills all vegetative bacteria Water should be soft, deionized or distilled 2% sodium bicarbonate promotes the process Kills vegetative bacteria, hepatitis virus & some spores

Tyndallisation (intermittent sterilization) - 1000C, 30 min, 3 days Nutrient media & media containing sugars or gelatin I day all vegetative bacteria are killed. On II & III day spores that germinate are killed

Moist Heat - Autoclave

Moist Heat - Autoclave

Physical Methods of Microbial Control:Dry Heat: Kills by oxidation effects. Direct Flaming: Used to sterilize inoculating loops and needles. Heat metal until it has a red glow. Incineration: Effective way to sterilize disposable items (paper cups, dressings) and biological waste. Hot Air Sterilization: Place objects in an oven. Require 2 hours at 170oC for sterilization. Dry heat is transfers heat less effectively to a cool body, than moist heat.

Dry heat - FLAMING 2500C 3000C Points of forceps & Inoculation loops heat in bunsen flame till red hot Slow passage through flame to destroy vegetative bacteria on surface of scalpel blade, glass slides, mouths of test tubes

Flaming

Dry heat - INCINERATION 8700C - 9800C Complete burning to ashes Used for soiled dressings, animal carcasses, pathological material, disposables, non-reusable soiled bedding

Incineration

Dry heat - HOT AIR OVEN Holding temp & time: 1600C for 1 hr Used for glassware, forceps, swabs, water impermeable oils, waxes & powders Before placing in hot air oven Dry glassware completely Plug test tubes with cotton wool Wrap glassware in Kraft papers

Dont over load the oven Allow free circulation of air between the material

Dry heat Hot air oven

Asbestos Filter holder

Earthenware filter

RADIATION 2 types Non ionising Infra Red radiation ( rapid mass sterilization of syringes, etc) Ultra Violet radiation (enclosed areas)

Ionising Gamma, X ray, cathode ray (plastics, syringes, oil, metal foils) Sterilization controls Dosimeter measures radiation dose Colored discs M radiodurans, B pumilus

Ultraviolet irradiation: mechanism Physical process Energy absorbed by DNA pyrimidine dimers, strand breaks, other damages inhibits replication

UV

C G

A T

A T

T A

G C

C G

A T

DNA

UV disinfection: other applications Disinfection of air Surface disinfectant Hospital/food production

Industrial application Cooling tower (Legionella control) Pharmaceuticals (disinfection of blood components and derivatives)

Physical Methods of Microbial Control:Radiation: Three types of radiation kill microbes: 1. Ionizing Radiation: Gamma rays, X rays, electron beams, or higher energy rays. Have short wavelengths (less than 1 nanometer). Dislodge electrons from atoms and form ions. Cause mutations in DNA and produce peroxides. Used to sterilize pharmaceuticals and disposable medical supplies. Food industry is interested in using ionizing radiation. Disadvantages: Penetrates human tissues. May cause genetic mutations in humans.

Physical Methods of Microbial Control:Radiation: Three types of radiation kill microbes: 2. Ultraviolet light (Nonionizing Radiation): Wavelength is longer than 1 nanometer. Damages DNA by producing thymine dimers, which cause mutations. Used to disinfect operating rooms, nurseries, cafeterias. Disadvantages: Damages skin, eyes. Doesnt penetrate paper, glass, and cloth.

Physical Methods of Microbial Control:Radiation: Three types of radiation kill microbes: 3. Microwave Radiation: Wavelength ranges from 1 millimeter to 1 meter. Heat is absorbed by water molecules. May kill vegetative cells in moist foods. Bacterial endospores, which do not contain water, are not damaged by microwave radiation. Solid foods are unevenly penetrated by microwaves. Trichinosis outbreaks have been associated with pork cooked in microwaves.

Hands Spread Disease