Sterilization and Disinfection Lecturer:

Definition of Terms Physical Methods of Sterilization Sterilization TOTAL inactivation of ALL forms of microbial life o Most resistant are spores o Least resistant are viruses Unable to reproduce Disinfection Destruction of microorganism capable of causing infection by means of a germicidal agent Reduction in the number of pathogens to a level that is unlikely to cause infection Germicidal Agent/ Germicide Agent that kills a microorganism o Bactericidal destroys or kills a bacteria (e.g. UV Light) o Bacteriostatic inhibits the growth of the bacteria Antiseptic TOPICAL chemical agent applied to a body surface or living tissue that kills or inhibits the growth of pathogenic microorganisms Sanitization Use of disinfectant to lower microbial content of inanimate objects/utensils Antiseptic Applied to body surfaces Bacteriostatic in action Regarded as drug Used at the lowest concentration that results to desired effect Disinfectant Applied to inanimate objects Bactericidal in action Environmental poison Used at highest concentration

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PHYSICAL METHODS OF STERILIZATION Heat Most reliable; universally applicable Mechanism of thermal injury: o production of single strand breaks in DNA = 1 lethal event The sterilization of a bacterial population by heat is a gradual process Kinetics of death are exponential ( the # of survivors decreases with time) Loss of functional integrity of the membrane Denaturation or coagulation of proteins Oxidative damage Toxic effects of elevated levels of electrolytes A. Moist Heat Preferred over dry heat Vegetative forms are destroyed at 80 C in 5-10 minutes Spores : 4 minutes at 120C or 5.5 hours at 100 C Higher temperature, shorter time to kill Forms: 1. Steam under Pressure ( Autoclaving) o Most efficient o Allow application of moist heat above the normal atmospheric boiling point of water o Temperature of steam = 100C 121 C at 15 psi for 15-90 minutes 132 C at 27 psi for 4-20 minutes o Uses : To sterilize surgical bandages, instruments, media To decontamination of reusable supplies and equipment o Advantages: Minimal time required Most dependable sterilant for lab use o Disadvantages: Damages heat-sensitive materials Shielding dirt first be removed before autoclaving 2. Boiling o kills all vegetative forms of pathogenic organisms at 80 100 C for 10-30 minutes but NOT bacterial endospores o Advantage: minimal equipment required o Disadvantage: cumbersome (impractical for everyday use)

Sterilization Physical methods 1. Heat A. Moist a.Steam under pressure (autoclave) b. Boiling c. Tyndallization d. Pasteurization B. Dry a. hot air oven b. flame c. incineration 2. Dessication 3. Freezing 4. Radiation a. UV b. Ionizing 5. Filtration 6. Osmotic Pressure 7. Ultrasonic and sonic vibration Chemical methods based on mechanism of action A.Damage the cell membrane 1.Surface active disinfectants a. Cationic compounds Quaternary ammonium compounds Anionic compounds Soaps and detergents 2. Phenolic compounds 3. Alcohols B. Denatures proteins 1. Acids and alkalis C. Agents that modify functional groups or proteins and nucleic acids 1. heavy metals mercurials, silver nitrate, copper sulfate 2. oxidizing agents a. halogens : chlorine, iodine b. hydrogen peroxide 3. dyes triphenyl methane acridines 4. alkylating agents formaldehyde, glutaraldehyde, ethylene oxide

3.

Fractional Sterilization ( Tyndallization ) o used for sterilizing materials that would be damaged by autoclaving o Heating 80-100 C x 30 minutes for 3 consecutive days with IP in between o Not very reliable o Use : heat sensitive materials bacteriologic media, solutions of chemical

Pasteurization o Below boiling point (77 C) for up to 30 minutes o Milk and dairy products, food and beverages o Advantage: can be used on heat sensitive liquids and medical devices o Disadvantage: not a reliable sporicidal B. Dry Heat Higher temperature and longer exposure to heat Effectiveness dependent on penetration of heat through the material Hot air oven : most widely used 1. HOT AIR OVEN -160-180 C x 2-4 hours -used in : o anhydrous materials o oils, powder o lab glassware and instruments 2. RED HEAT FLAME -oxidation to ashes (burning) -used in: o inoculating loops o needles 3. INCINERATION -oxidation to ashes (burning T > 1000 C) -use in: o decontamination of waste items prior to disposal METHOD Hot Air Oven ADVANTAGES -Penetrates water insoluble materials - less corrosive to metals and sharp instruments -Rapid DISADVANTAGES -slow diffusion -not suitable to reusable plastics

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Dessication -Bacteria cannot grow in an environment without water Bacterial spores: R to drying or desiccation -used in: preservation of foods Freezing -Use : o preserve microorganisms and bacterial culture -Lyophilization : organism is frozen rapidly and dehydrated in high vacuum then stored under vacuum in sealed ampules in cold storage

E. Radiation 1. Ultraviolet Light (UVL) most effective bactericidal wavelength 240-280 nm: optimum of 260 emitted by UV lamps germicidal and reduce the number of microbes on exposed surfaces and in air poor penetrating power poses eye and skin hazard 2. Ionizing Radiation greater penetrating ability than UVL but potentially hazardous to human cells F. Filtration Mechanical sieving or physical separation of microorganism from the fluid makes use of cellulose ester membranes but the filter pore size or .22um allows small bacteria to pass through Osmotic Pressure used in: food preservation Ultrasonic and Sound Vibrations at high frequency in the upper audible and ultrasonic rangedisrupting cells range 9-100 kc/s passage of sound produce alternating pressure changes cause cavities in the fluid burst FACTORS AFFECTING DISINFECTANT POTENCY Concentration of chemical agent Time Temperature pH Nature of the medium Nature of organism CONCENTRATION OF CHEMICAL AGENT dependent on material being disinfected and organism to be destroyed higher conc : bactericidal weaker conc : bacteriostatic TEMPERATURE increase in T = increases killing increase in T speeds up the rate of chemical reaction pH the H ion conc influence both the organism and chemical agent determines the degree of ionization of the chemical agent NATURE OF THE MEDIUM (+) of extraneous materials chemical agent

G. H.

Red Heat Flame

Incineration Pasteurization

Tyndallization Boiling

-Reduce volume of waste by 95% -can be used on heat sensitive liquids and medical devices -R spores germinate killed on 2nd and 3rd days -minimal equipment required -minimal time required -most dependable -sterilant for lab use

- initial contact with flame produce a viable aerosol - accidental fire -requires transport of infectious waste -not reliably sporicidal -time consuming -not reliably sporicidal -cumbersome -not practical for everyday lab use -not reliably sporicidal -shielding dirt must first be removed -damage heat sensitive materials

Autoclave

the efficiency of

NATURE OF THE ORGANISMS (+) of special structures (spores)and the # of the organisms in the medium to be tested greatly affects disinfection EVALUATION OF DISINFECTANTS A. PHENOL COEFFICIENT TEST PHENOL standard reference materialbased on tube dilution proceduredetermine the ratio of the highest dilution that will kill the org within a specified time to the greatest dilution of phenol showing the same result 2 organisms used : Salmonella typhI & Staphylococcus aureus FILTER PAPER METHOD Filter paper soaked with disinfectant placed on the agar surface of a petri dish inoculated with a lawn of test organism clear area around the disk following incubation efficacy of disinfectant USE-DILUTION TEST Organism air dried onto a stainless steel surface exposed to disinfectant : following sterile water rinsing the entire surface placed in broth successful disinfection no bacterial growth in the broth most efficacious : disinfectants that completely kill microbes at the lowest dilutions

1.

Ethyl alcohol most effective at 50-70% skin disinfectant cannot destroy spores at normal T Isopropyl alcohol bactericidal activity slightly > ethanol less volatile greater toxic effect than ethanol Denatures proteins Acids and alkalis exert their action through free H and OH ions and alter the pH of the organisms environment Alcohol and acetone Agents that modify functional groups or proteins and nucleic acids

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II. A. B. III. A. B. 1. 2. C. D. A.

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Heavy Metals mercurials,silver nitrate, copper sulfate Oxidizing Agents Halogens : chlorine, iodine Hydrogen Peroxide Dyes triphenyl methane acridines Alkylating Agents formaldehyde, glutaraldehyde, ethylene oxide Heavy Metals poison enzyme activity forming mercaptides with the SH groups of cysteine residues 1. Mercurialsmerthiolate and mercurochromeantiseptics NOT reliable disinfectants Silver nitrate 1%- Sometimes placed in the eyes of NB to prevent gonococcal ophthalmia Copper sulfate is used to combat fungal diseases of plants and is also a common algicide

CHEMICAL METHODS BASED ON MECHANISM OF ACTION I. Damage the cell membrane A. Surface active disinfectants alter the energy relationships at interfaces reduction of surface tension Damage the lipids and or proteins of the semipermeable cytoplasmic membrane of the microorganismleakage of cellular materials needed to sustain life Denature microbial enzymes and other proteins by disrupting the hydrogen and disulfide bonds blocks metabolism 1. Cationic Compounds - Quaternary ammonium compounds distorts cellular membrane and loss of membrane permeability leakage of N and P containing compounds denatures proteins most effective at alkaline pH e.g. zephiran, benzalkonium chloride Anionic compunds - Soaps and detergents disrupts lipoprotein framework of the CM most effective at acid pH e.g. soaps and detergents

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B.

Oxidizing Agents inactivates enzymes converting functional SH grps to oxidized S-S for stronger agents also attack amino grps, indole grps and phenolic hydroxyl groups of tyrosine Halogens bactericidal; effective against sporulating org, Mycobacterium tuberculosis 1. Iodine BEST antiseptic because it kills spores, viruses, fungi,someendospores; It also denatures microbial proteins and is usually dissolved in an alcohol solution to produce a tincture Iodophores sterilizes of dairy equipment; combination of iodine and an anionic detergent that reduces surface tension and slowly releases the iodine Chlorine- liberates free chlorine; water disinfectant Hypochlorites- sanitizes dairy and food processing equipmenthousehold and hospitals *Chlorine gas reacts with water to form hypochlorite ions,which in turns denatures microbial enzymes. Na hypochlorite is the active agent in household bleach. Hydrogen peroxide- weak antiseptic; one used in cleaning wounds, disinfection of surgical devices and soft plastic contact lenses

C.

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Phenolic compounds cause leakage of cell contents and irreversible inactivation of membranebound oxidases and dehydrogenases includes phenols referencecresols alkyl phenols e.g. lysol and creolin kills bacteria, most fungi & some viruses ineffective against endospores they alter membrane permeability and denature proteins Alcohols disorganize lipid structure denatures cellular proteins

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Dyes limited in treatment of dermatologiclesions; stains bacteria 1. 2. Triphenylmethane brilliant green, malachite green, crystal violet Acridines wound antiseptics; retain antimicrobial activity in the presence of serum or pus

2. 3.

E. Alkylating agents 1. Formaldehyde (formalin)- preserve specimens and in preparation of vaccine ( whole-killed and toxoid) kill M. tuberculosis in sputum and

fungus in athletes foot; destroys all org including spores Glutaraldehyde- sporicidal and bactericidal; 10x more effective than formaldehyde and less toxic; cold sterilant for surgical instruments (resp) Ethylene oxide- gas sterilization of 50-1200 mg/L x 1-12 hrs; broad spectrum without heat or moisture; penetrate packaging material(polyethylenes tubes, electronic and medical equipment, biological and drugs);sterilizes heart and lung machines; potentially mutagenic and carcinogenic in humans (vapors are toxic to skin, eyes and mucous membranes)