Sterilization And Disinfection

l a i b o r Mic l o r t n o C Dr. R. Tan

Primary target of microbial control Microorganisms capable of causing infection or spoilage that are constantly present in the external environment and on the human body

Relative Resistance of Microbial Forms Highest resistance bacterial endospore

Moderate resistance protozoan cyst

(Bacillus & Clostridium)

some fungal spores some naked virus vegetative bacteria that have higher resistance ( M. tuberculosis, S.aureus, Pseudomonas)

Least resistance most bacterial vegetative cells ordinary fungal spores & hypae enveloped virus Yeasts Trophozoites

Definition of Frequently Used Terms Sterilization - process of destruction or removal of all viable microorganisms from an object or from a particular environment - total inactivation of all forms of microbial life in terms of the organism’s ability to reproduce Disinfection - refers to the use of physical process or chemical agent (disinfectants) that promotes killing, inhibition, or removal of pathogenic microorganisms (usually on inanimate objects) but not bacterial endospore Sanitization - is any cleansing technique that mechanically removes microorg. to reduce the level of contaminants / microbial population to a safe level as determined by public health standards

Sepsis – growth of microorganisms or the presence of microbial

toxins in the blood and other tissues

Asepsis - refers to any practice that prevents the entry of infectious agents into sterile tissues and thus prevents infection

Antisepsis – aseptic technique practiced in health care, range from sterile methods that uses chemical agents (antiseptics) which are applied directly to exposed body surfaces, wounds, and surgical incision to destroy or inhibit vegetative pathogens

Disinfectant - is an agent used to reduce the viability of a level that

microbial population below a threshold causes infection - Disinfectants are used on inanimate objects

Antiseptic - chemical agents that opposes sepsis or putrefaction either by killing microorganisms or by preventing their growth - Term commonly used for agents that are applied topically to living tissues This distinguishes them from antiseptics that serve the same purpose but are used on living tissues without causing toxicity.

suffix “cide” – meaninig to kill - added when a killing action is implied - a suffix indicating that the antimicrobial agent will kill or destroy a certain group of microorganism viricide – destroys virus fungicide – destroys fungi bactericide – destroys bacteria germicide – (or disinfectant) – agent that kills microorg. capable of producing an infection Suffix “static/stasis” – meaning to stand still - a suffix indicating that the agent will prevent the growth or multiplication of the type of organism but are not killed outright bacteriostatic - prevents the growth of bacteria fungistatic – prevents the growth of fungi

Dynamics of Sterilization & Disinfection microorganisms are not killed instantly when

exposed to a lethal agent The kinetics of death of a microbial population is exponential: the number of survivors decreases with time (exponential killing) because of the exponential form of survivor-time curve, the larger the initial number of cells to be killed, the more intense or prolonged is the treatment required for sterilization The rate of disinfection varies with the concentration of the disinfectant

Factors affecting disinfectant potency 1. Number of microorganisms - larger the initial number of cells to be killed, the more intense or prolonged is the treatment required for sterilization 2. Nature of microorganism - the efficacy of a chemical agent depends on these properties: specie , growth phase of culture, and presence of special structure (such as spores and capsules), and the number of the organisms in the test system 3. Temperature - the killing of bacteria by chemical agents increases with an increase in temperature - for each 10°C increase in temperature, there is doubling of the death rate

4. pH - Hydrogen ion concentration influences bactericidal action by affecting both the organism and the chemical agent - Some disinfectants are more effective at alkaline pH (glutaraldehyde) while others are more effective at acid pH (e.g. phenols)

5. Time - when bacteria are exposed to a specific concentration of a bacterial agent, even in excess, not all organisms die at the same time; rather, there is a gradual decrease in the number of living cells

6. Mode of action of the agents  damage cell membrane function  denatures protein  induce extensive nucleic acid damage

7. Concentration of the agent - the concentration required to produce a given effect varies with the disinfectant, the organism and the method of testing - high conc. → lethal to bacteria - low conc. → may stimulate, retard, or even kill the organism - An effective concentration must be used 8. Presence of exogenous materials - presence of organic matter (serum, blood, pus) alter disinfectant activity - alter disinfectant activity by:  surface absorption of the disinfectant by protein colloids  formation of a chemically inert or less active compound  binding of the disinfectant by active groups of foreign protein

Evaluation of Disinfectants Association of Official Analytical Chemists (AOAC)

methods Approved protocol for evaluation purposes Phenol Coefficient Test Phenol → reference standard agent official method used to test disinfectant potency designed to determine the ratio of the highest dilution of the germicide that will kill the test organism within a specified time to the greatest dilution of phenol showing the same result

Methods of Microbial Control Physical Agents Chemical Agents Chemotherapeutic Agents

• Heat • Freezing • Radiation • Filtration • Ultrasonic and SonicVibration

HEAT - most reliable and universally applied method of sterilization - whenever possible, should be the method of choice - 2 kinds of heat : 1. Dry 2. Moist Thermal Death Time - refers to minimum time required to kill all microbes at a specified temperature in a specified environment - time required inversely related to temperature of exposure - sterilization time is directly related to the number of organisms in a suspension Thermal Death Point - lowest temperature required to kill all microbes when

DRY HEAT Sterilization that requires higher temperature and

longer period of heating denotes air with a low moisture content that has been heated by flame or electric heating coil temp ranges from 160°C – several thousand °C MOA :  denaturation of proteins  oxidative damage  toxicity effects of elevated levels of electrolytes

Its use is limited primarily to sterilization of glasswares

and such materials as oils, jellies, and powders that are impervious to steam

Forms: 1. Direct flaming - bacteriological loop, needle, glass rods through the flame of a Bunsen burner 2. Incineration / cremation - burn to ashes - useful for decontaminating infected lab. Animals 3. Hot air oven - most widely used type of dry heat - oven heat set 1800C for 2-4 hours - temp. enough to kill all type of organism, including the sporeformer

MOIST HEAT  kills bacteria faster than dry heat  preferred because of its more rapid killing  temp ranges from 60-135°C  MOA:  denaturation and coagulation of proteins  production of single-stranded breaks of DNA  loss of functional integrity of membrane and

leakage of small molecules microorganism

Time required for sterilization

Most mesophilic nonsporeformers

60°C for 30min

S. Aureus & S. faecalis

60°C for 60min

Vegetative form of all bacteria, fungi & yeast Heat resistant sporeformers

80°C for 5-10min 120°C for 4min 100°C for 5.5min is required

Forms: 1. Boiling - can be relied only for disinfection and not for sterilization - 30 minutes boiling 1000C adequate to kill vegetative cell → Bacteria, Fungi, Protozoa - sporeformer 5½ hours 2. Free – Flowing steam - live steam → Arnold sterilizer - same sterilizing action with boiling

3. Steam Under Pressure - steam sterilization is carried out in an autoclave using pressured steam - the principal requirement : the whole of the material to be sterilized remain in contact with saturated steam at the required temperature for the necessary period of time - 1210C 15 – 20 minutes (15lbs steam pressure per sq. inch) - most dependable moist heat method of sterilization for hospital clinical lab. & research facilities - very effective against vegetative cells and most bacterial endospores - commonly used for sterilizing : 1. Hospital instruments ( surgical & medical )

4. Tyndallization - Fractional /Intermittent sterilization - free flowing steam 80-1000C 30 minutes for 3 consecutive days involves exposing the material to elevated temp (killing the vegetative cell) incubate at 37°C (to allow spores to germinate to form new vegetative cell) expose to elevated temp again (to kill the newly germinated vegetative cells) - for sterilization of certain liquid or semisolid materials that are easily destroyed by heat - it is used to sterilize heat-sensitive culture media containing such materials ac carbohydrates, egg or serum - kills both sporeformer & non-sporeformer bacteria

5. Pasteurization - applied to fresh beverages such as milk, milk products, beer and wine - heat is applied to these liquids to kill potential agents of infection and spoilage at the same time retaining the liquid`s flavor and food value - 63-66°C for 30 minutes followed by rapid cooling - the temperature does not sterilize the milk, but it does kill all disease producing bacteria commonly transmitted by milk - primary target: non spore forming pathogens (Salmonella sp., Campylobacter jejuni, Listeria monocytogenes, Brucella sp., Coxiella sp. and Mycobacterium sp.)

FREEZING Is not a reliable method of sterilization Primarily used in the preservation of bacterial cultures In freezing, the formation of ice crystals outside the

cell causes the withdrawal of water from the cell interior, resulting in an increased intracellular electrolyte concentration and denaturation of proteins The cell membrane is damaged, and a leakage of intracellular organic compounds ensues

Lyophilization ( Freeze-drying )  A process used for preserving biological material, by removing the water from the sample, which involves first freezing the sample and then drying it, under a vacuum, at very low temperatures

RADIATION - defined as energy emitted from atomic activities and dispensed at high velocity through matter or space -Sunlight possesses appreciable bactericidal activity and plays an important role in the spontaneous sterilization that occurs under natural conditions -2 types Ionizing radiation: Radiation that have sufficient energy to remove an electron completely from an atom and produce an electrical charge (ionization) Nonionizing radiation: Energy absorbed by the molecule cannot remove an electron completely, the excitation produced often leads to photochemical changes

Ionizing radiation -

Ex: electromagnetic rays: X-ray, alpha, beta & gamma rays uses short wavelength much higher energy content than UV rays has greater power penetration can penetrate a solid barrier, bombard a cell, enter it, and dislodge electrons from molecules

breakage of DNA creates massive mutations greater capacity to produce lethal effects - used for sterilization of cutgut , nylon sutures, plastic, syringe, catheter, prosthesis & plastic tubings

Non-ionizing radiation

- Ex. Ultraviolet light - effectiveness of UV light as a lethal and mutagenic agent is closely related to its wavelength (240-280nm); optimum at about 260nm which corresponds with the absorption maximum of DNA - Lethal effect on bacteria is attributed to absorption and resultant damage of DNA formation of abnormal bonds → (damage) killing of the organism - Energy of UV radiation is low - penetrating ability is very poor - primarily used to control airborne infections, where it is used for the disinfection of enclosed areas such as nurseries, hospital wards & OR

FILTRATION -an effective method to remove microbes from air and liquids - process of separating microorganism from contaminated solution - filtration sterilization is used to prepare liquids that cannot withstand heat (heat-labile), including serum and other blood products, vaccines, drugs, IV fluids, enzymes and culture media - useful for trapping microorganism only - types filters 1. Seitz – asbestose – cellulose 2. Sintered glass – glass filaments 3. Chamberland – unglazed porcelain 4. Berkefeld – diatomaceous earth 5. Membrane filter – cellulose ester -High efficiency particulate air (HEPA) filters are used to

ULTRASONIC AND SONIC VIBRATIONS

- Sound vibration at high frequency, in an upper audible and ultrasonic range (20-1000 kc), provide a useful technique for disruption & disintegration of the cell -The passage of sound thru a liquid produces alternating pressure changes, which, if the sound intensity is sufficiently great, causes cavities to form in the liquid - the cavities grow in size until they collapse violently leading to cell disintegration - No practical value in sterilization & disinfection - uses 1. research laboratories 2. treating sewage H2O

 destroys structural integrity of cell membrane (protein &

lipids)  interferes w/ normal membrane function  the net effect is the release of small metabolites from the cell and interfere with the active transport and energy metabolism  Agents:  Surface active agents • • • •

Cationic – most effective Anionic Non-ionic – not effective Amphoteric

 Phenolic compound  Alcohol

I.

Surface active agents

- Substances that alter the energy relationship at interfaces producing a reduction of surface or interfacial tension - Compounds that possess both water-attracting (hydrophilic) and water-repelling (hydrophobic) groups - They disrupt the integrity of cell membrane This results in the loss of  molecules from the cytoplasm and affects the proton motive force which provides energy for solute transport - The interface between the lipid-containing membrane of a bacterial cell and the surrounding aqueous medium provides a susceptible target site for agents of this type

Cationic -

-

Agents Quarternary ammonium compound QAC are any of a group of ammonium salts in which organic radicals have been substituted for all four hydrogens of the original ammonium cation This is the most important antibacterial surface-active agent Bactericidal for a wide range of organisms, gram (+) species are more susceptible Ex. Benzalkonium chloride ( Zephiran )  used

primarily in hand or face washes  must not be applied to areas which have not been fully rinsed as it is inactivated by organic compounds  Benzalkonium application many include disinfecting instruments and preserving drugs in low

Anionic Agents - Among the anionic detergents are soaps and fatty acids that

dissociate to yield a negatively charged ion - agents are most active in an acid pH - Causes gross disruption of the lipoprotein framework of the

cell wall - By combining an anionic agent with acid, a very effective

acid-anion surfactant sanitizer - display very rapid bactericidal action (within 30sec)

- Effective against gram (+) organisms but are relatively

ineffective among gram (-) species because of their lipopolysaccharide outer membrane

 The activity of all four Surface active agents may be

enhanced by incorporating other disinfectants such as the diguanide  Diguanides - have antimicrobial activity against vegetative bacteria,

-

yeasts, protozoans and enveloped viruses but not against spores, protozoan cysts and mycobacteria They function by disrupting cytoplasmic membrane to cause cell leakage and may enter cells to cause coagulation of the cell cytoplasm

-

It may be used together with surface active disinfectants at a concentration of 1 – 4% Ex. chlorhexidine which is more effective at pH 7 – 8  used

as a safe antiseptic to apply to prevent body infection and in oral rinses for treating sore gums and mouth ulcers and preventing plaque on teeth

II Phenolic compound - at low concentration, these compounds are rapidly bactericidal causing leakage of cell contents and irreversible inactivation of membrane-bound oxidases and dehydrogenases - Parent compound : Carbolic acid ( phenol ) - excellent for disinfecting feces, blood, pus, sputum & other proteinaceous material - primarily use for testing new bactericidal agent - It had been replaced as a practical disinfectant by less caustic and less toxic phenol derivative - Cresols - Xylenols - Diphenyl compound

- Phenolics are effective against bacteria, fungi and virus

Phenol derivatives: • Cresols - the simplest of the alkyl phenols - Ortho-, meta-, paracresols – are applicably more active than phenol - usually employed as a mixtureCresols obtained industrially by the distillation of coal tar -- usual source of these products - Cresols are used to dissolve other chemicals, as disinfectants and deodorizers, and to make specific chemicals that kill Insect pests - sold under the trade names: Lysol and Creolin • Xylenols - Dimethylphenols - important class of phenolics with great industrial importance - are used as pesticides and in the manufacture of antioxidants

Kills 99.9 % of germ 30 se s in cond s

• Hospital disinfectant-deodorant is highly effective against TB, MRSA, and HIV-1 • Tuberculocidal, virucidal, fungicidal, bactericidal • Minimizes concern over the spread of germs in public facilities • Prevents odors and growth of mold and mildew

• A general purpose Disinfectant • Kills most bacteria on surface • Destroys odors, cleans and disinfects against germs and against staph • Good septic tank disinfectant

• Diphenyl compound - the halogenated diphenyl compounds exhibit unique antibacterial properties - effective against gram + bacteria ( staph & strep ); offers no protection against gram-negative infections -The most impt, is the chlorinated derivative, Hexachlorophene • very useful as a topical anti-infective, anti-bacterial agent, often used in soaps, toothpaste and antiperspirant • also used in agriculture as a soil fungicide, plant bactericide • bacteriostatic skin cleanser • used as a preservative in cosmetic products • pHisoHex, was widely used as a very effective antibacterial skin cleanser in the treatment of acne

III Alcohol -Alcohols provide an insight into the interaction of organic solvents with lipid membranes - they disorganize lipid structure by penetrating into the hydrocarbon region -Alcohols are normally used at concentrations between 50%-90% and have optimal activity at 70% - 75% as the presence of water helps to denature membranes, dissolve cell membrane lipids causing cell lysis - In addition to their effect on the cell membrane, alcohols also denature proteins - effective against vegetative bacteria, fungi and viruses - have little sporicidal activity - usually used to disinfect work surfaces and, as antiseptics on skin - Antiseptic activity may be enhanced by incorporating other antimicrobial compounds: chlorhexidine (0.5%) iodine (1% to 2%)

Ethanol / Ethyl alcohol - Used as skin disinfectant because of their bactericidal action and ability to remove lipids from the skin surfaces - their action as disinfectant is restricted by their inability at normal temperatures to kill spores not relied for sterilization of instruments - Uses: - to sterilize skin before cutaneous injections - to disinfect thermometers - Most effective at 50-70% - effective against gram (+) , gram (-) , AF bacteria

Isopropyl alcohol - Most effective at 50-70% - most effective type of alcohol - bactericidal activity is slightly greater than ethanol - less volatile - for this reason, it had been recommended as replacement for the sterilization of thermometers -Toxic effect is greater and long lasting -Necrosis may result from absorption of vapors through the lungs during alcohol sponge bath

- In its native state, each protein possesses a characteristic conformation that is required for its proper function - Agents that alter the conformation of the proteins by denaturation cause an unfolding of the polypeptide chain so that the chain becomes randomly and irregularly looped or coiled - Agents: 1. Acid & alkalies - exert their antibacterial activity through their free H+ and OHions, through undissociated molecules or by altering the pH of organism’s environment

Benzoic acid Lactic acid Acetic acid Propionic acid 2. Alcohol 3. Acetone

used as food preservative

- The catalytic site of an enzyme contains specific functional groups that bind the substrate and initiate the catalytic events - Inhibition of enzyme activity results in one or more of these functional groups is altered or destroyed - Important functional groups of the cell wall, membrane and nucleic acids are also susceptible to inactivation -Agents: 1. Heavy metals 2. Oxidizing agents 3. Dyes 4. Alkylating agents

Heavy Metals – soluble salts of mercury, silver , arsenic and other heavy metals poison enzyme activity by forming mercaptides with the sulfhydryl groups of cysteine residue • Mercurials • Mercuric chloride – very toxic disinfectant, not used today • Metaphen, Merthiolate, Mercuchrome – less toxic, unreliable as skin disinfectant • Silver compounds • widely used as antiseptic ; either as soluble silver salts or as colloidal preparations • inorganic silver salts - efficient bactericidal agents but has irritant and caustic effects • Silver nitrate – highly bactericdal for gonococci and routinely used as prophylaxis against opthalmia neonatorum in neonates

Oxidizing agents • most useful antimicrobial agents: halogens (iodine, chlorine) hydrogen peroxide • Halogens - chlorine and iodine are among the most useful disinfectants - Almost exclusively bactericidal and effective against sporulating bacteria, virus, fungi and protozoa (1) iodine - exist principally in the form of I2 at pH value below 6 where maximal bactericidal action is manifested - iodine tinctures came into wide use as microbicides - Iodine quickly kills a broad range of microorganisms - destroys many microorganisms and viruses within 3-5 minutes - used in cleansing skin or disinfecting small wounds but presented with some problems - strong smell and can stain skin and clothing. - react to metals and are rather unstable - skin irritant

Betadine Solution - various mixtures and solutions of iodine, called Iodaphores, have been formulated that greatly lessen such disadvantages and dangers - enhance stability and microbe killing properties. -Contains 10% povidone-iodine - broad spectrum topical iodophor microbicide - available in "Swab Aid" pads, Swab Sticks and as a Surgical Scrub - It is a fast-acting, broad-spectrum antiseptic that kills bacteria (including antibiotic resistant organisms), as well as fungi/yeasts, viruses and protozoa - It is indicated for degerming skin, wounds and mucous membranes - widely use in hospitals, sanitation and water purification,

(2) chlorine - 3 types of Chlorine compound: hypochlorite, inorganic & inorganic chloramines - The active moiety of hypochlorites and chlorine is hypochlorous acid which is stable at a low pH, which is a strong oxidizing agent and an effective disinfectant - Water disinfectant - hypochlorites - most useful of the chlorine compound - widely used for sanitizing dairy products and food processing equipment - employed as sanitizers in most households, hospitals, and public buildngs - marketed as: Chlorox, Zonrox, Purex

• Hydrogen peroxide - antibacterial action is secondary to its oxidizing ability as well as formation of a more toxic free hydroxyl radical from the peroxide in an irondependent reaction - It is a weak acid - In a 3% solution, it is harmless but very weak antiseptic whose primary clinical use is in the cleansing of wounds - It has strong oxidizing properties and is therefore a powerful bleaching agent that is mostly used for bleaching paper

Dyes - some of the coal-tar dyes not only stain bacteria but are inhibitory at very high dilutions - within the usual pH range, the basic dyes are the most effective - their current medical use is limited primarily to the treatment of dermatologic lesions • Triphenyl methane dyes • aniline dye derivatives especially Crystal violet, Malachite green, Brilliant green • Highly selective for gram (+) organisms • Used in the laboratory in the formulation of selective culture media • Acidine dyes • Often referred as Flavines because of their yellow color • exert a bactericidal and bacteriastatic effect upon a number of organisms • Compounds of clinical use : proflavine, acriflavine

Alkalyting agent - The lethal effect results from their alkylating action on proteins - Glutaraldehydes and formaldehydes are the most commonly used aldehydes -They are active against bacteria and their spores, viruses, fungi and protozoa -Antimicrobial activity occurs as a result of cross-linking proteins and nucleic acids in fungi, protozoa and bacteria and capsid nucleic acid complexes in viruses -These effects are mediated predominantly via amine, sulfurhydryl and carboxyl groups on microbial surface proteins

Formaldehyde - One of the least agents acting on proteins - commercially available in: • aqueous solution containing 37% formaldehyde (Formalin) • paraformaldehyde a poymer (contain 91-99% formaldehde) - Formalin - used for preserving fresh tissues - major component of embalming fluids - when used in high concentration, it destroys all organisms, including spore - Used to inactivate virus in the preparation of vaccines - As a gas – used to decontaminate rooms, buildings, fabrics, and instruments

Glutaraldehyde - used as “cold sterilant” for surgical instruments - 10x more effective than formaldehyde as a bactericidal and sporocidal agent and less toxic Ethylene oxide - Employed in gaseous sterilization, especially on materials that would be damaged by heat (polyethylene tubings, electric & medical instruments,b iologicals and drugs) - Active against all types of bacteria, including spores & TB bacilli