sterilisation & disinfection ppt


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STERILISATION the complete removal or destruction of all forms of microbial life bacteria, viruses, fungi spores Probabilistic notion No absolute assurance that there is 0 microorganism


STERILISATION Sterility assurance level (SAL) used as measure of sterility SAL = probability of survival of a microorganism after sterilization process Expressed as log10 (probability of survival) SAL of 6 = < 1 chance in a million (10-6 ) that a particular item is contaminated SAL = 6 acceptable for critical item.


DISINFECTION Not all microbial forms Main difference with sterilization = the lack of sporocidal activity Categorized into 3 levels: High, Intermediate Low


OTHER DEFINITIONS Cleaning : the removal of adherent visible soil (blood, protein substance and debris), dust or other foreign material by manual or chemical process Sanitizing : process that reduces microbial population on object to a safe level Decontamination : process that removes pathogenic microorganisms from an object to make it safe to handle


OTHER DEFINITIONS Antiseptics = chemicals to prevent growth or destroy – Used on living tissues – Regulated by FDA Disinfectants = chemicals used for disinfection – Used for objects – Regulated by EPA AOAC (Association of Official Analytical Chemists) tests are used on antiseptics and disinfectants

Factors Affecting Effectiveness ofDisinfection : 

Factors Affecting Effectiveness ofDisinfection Cleaning – Residual particles harbor & shelter from disinfectant – Organic load restrict disinfectants effectiveness of alcohol, phenols, chlorine & iodines Nature of object: crevices, hinges, lumens more difficult to disinfect. Concentration of disinfectant: – Diluted during application – Lose potency with time

Factors Affecting Effectiveness ofDisinfection : 

Factors Affecting Effectiveness ofDisinfection Time of contact Physical and chemical environment: temperature, water hardness, pH


PHYSICAL AGENTS SUNLIGHT: bactericidal activity due to ultraviolet rays DRYING: 4/5th of weight of the bacterial cell is due to water, but this method is highly unreliable RADIATION: Non ionizing- like UV rays & IR rays Ionizing- like X rays, gamma rays & cosmic rays

Non ionizing radiations : 

Non ionizing radiations UV RAYS are used to sterilize internal Surfaces of safety cabinets, entry ways, Operation theaters etc. IR RAYS are used for rapid mass sterilization of pre packed items such as syringes, catheters etc.

Slide 11: 

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

UV disinfection: other applications : 

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)

Ionizing radiations : 

Ionizing radiations Gamma radiation emitted from a radioactive element, usually Co 60,provides a reliable means of sterilizing plastic and other materials that are heat sensitive. Used commercially for sterilization of packaged disposable articles such as syringes


APPLICATION OF HEAT Thermal death time (TDT) is the length of time required to kill all bacteria in a liquid culture at a given temperature Thermal death point (TDP) is the lowest temperature at which all bacteria in a liquid culture will be killed in 10 minutes Decimal reduction time (DRT) is the length of time in which 90% of a bacterial population will be killed at a given temperature (especially useful in canning industry


HEAT DRY HEAT Mechanism of action protein denaturation Oxidative damage Toxic effects of increased levels of electrolytes MOIST HEAT Mechanism of action Protein denaturation & coagulation Latent heat liberated when steam condenses on cooler surface Hydrolysis & breakdown of bacterial proteins


DRY HEAT Red heat : for inoculating wires, loops, points of Forceps etc Flaming : for scalpels, needles, culture tubes etc Hot air oven : for sterilizing glassware,forceps,scissors,scalpels,glass syringes,swabs,pharmaceutical products like liquid paraffin, fats & grease.


HOT AIR OVEN Electrically heated, and provided with a fan or a blower to ensure rapid & uniform heating Time the HOLDING PERIOD of 1hr at 160^C when the thermometer first shows that the oven has reached 160^C. Then the oven is allowed to cool gradually for about 2hrs before the door is opened.

Sterilization control for hot air oven : 

Sterilization control for hot air oven Spores of non toxigenic strains of clostridium tetani Paper strips impregnated with 106 spores are sterilized and inoculated in thioglycollate or cooked meat media and incubated for sterility test under strict anaerobic conditions for 5 days at 37^C. Browne’s tube which shows a green color after sterilization Thermocouples



Moist heat at below 1000C : 

Moist heat at below 1000C PASTEURISATION HOLDER METHOD : milk heated at 630C for 30 min FLASH METHOD : milk heated at 720C for 15 sec Both followed by cooling rapidly to 130C or lower VACCINE BATH Vaccines prepared from cultures of non sporing bacteria sterilized by heating in vaccine bath at a low temperature of 600C for 1 hr.

Moist heat at 1000C : 

Moist heat at 1000C BOILING Most vegetative forms almost immediately killed at 90-1000C but sporing bacteria require prolonged periods of boiling Hard water should not be used 2% sodium bicarbonate may be added to promote sterilization Koch or Arnold steam sterilizer is used


TYNDALLIZATION Means intermittent exposure at 1000C Principle : that one exposure kills vegetative organisms, between heatings the spores being in a favorable nutrient medium become vegetative forms which get killed during subsequent heating. Used for gelatin media, media containing sugars May fail to kill thermophilic,anaerobic and other bacteria whose spores do not germinate in a particular medium

Moist heat at above 1000C : 

Moist heat at above 1000C AUTOCLAVES


AUTOCLAVES Saturated steam under pressure. Cheap & nontoxic Penetrates fabric Method of choice for all items except those which are moisture or heat sensitive. 4 parameters of importance Pressure Temperature Time Steam Air must be removed and steam must reach the item for required time at required temperature


PRINCIPLE Water boils when its vapour pressure equals that of surrounding atmosphere. PV=nRT When pressure inside the closed vessel increases, the temp at which water boils also increases


MECHANISM When steam comes in contact with a cooler surface it condenses to water and gives up its latent heat to that surface (1600 ml steam at 100^C at atmospheric pressure condenses into 1 ml of water at 100^C and releases 518cal of heat) and moisture which together denature microbial proteins

Sterilization cycle : 

Sterilization cycle Sterilization cycle includes 1-warming of the chamber 2-vacuum extraction 3-pre-steam penetration time 4-steam penetration time 5-holding time 6-cooling time


STERILIZATION TIMES AUTOCLAVES 1150C, 10 lb/in2 for 45 min 1210C, 15 lb/in2 for 15-20 min 1340C, 30 lb/in2 for 3 min HOT AIR OVEN 1600C for 45 min 1700C for 18 min 1800C for 7.5 min

Types of autoclaves : 

Types of autoclaves According to structure Simple non jacketed autoclave Steam jacketed autoclave with automatic air and condensate discharge High pre-vacuum sterilizers According to function Simple lab autoclave Transportable bench top autoclave Large simple autoclave Downward displacement lab autoclaves Media preparators Multi purpose lab autoclave


USES Culture media Aqueous solutions Empty bottles and impervious containers Surgical instruments Wrapped dry goods and dressings

What should not be autoclaved? : 

What should not be autoclaved? Items containing solvents, volatiles or corrosive chemicals Radioactive material

Safety Procedures : 

Safety Procedures Follow manufacturers’ guidelines Do not open when chamber is pressurized Avoid standing directly in front of autoclave door when opening Place autoclave on preventive maintenance schedule Cool to below 80^Cbefore opening Bottles must not be overfilled


STERILIZATION INDICATORS AUTOMATIC PROCESS CONTROL Carries through the sterilization cycle according to a pre selected scheme for duration,temperature,and pressure of each stage RECORDING THERMOMETER Graphic record of temperature changes in chamber discharge channel avoiding errors in timing the holding period THERMOCOUPLE When kept inside the test article and attached to potentiometer, it indicates the temperature inside the test article during autoclaving


STERILIZATION INDICATORS CHEMICAL INDICATORS BROWNE’S STERILIZER has red solution which turns green when heated at 115^C for 25min(type 1),or 15min(type 2), or at 160^C for 60min(type 3) Stored at 20^C to avoid pre mature color change ADHESIVE TAPES Bowie-Dick autoclave tape test for steam penetration

Biological indicators : 

Biological indicators Organism : Bacillus stearothermophilus,a thermophile that requires to be cultivated 55-60^C.Its spores are killed at 121^C in 12min Preparation: culture grown aerobically on nutrient agar for 5days is suspended in sterile water to a concentration of one million spores per ml.small strips of filter paper soaked in the suspension are dried at room temp and packed in envelopes

Biological indicators : 

Biological indicators Procedure placed in the centre of the largest or most densely packed items and some in the coolest part After autoclaving, envelope is cut with a sterile scissors and strip transferred to a recovery medium,eg.thioglycolate broth with strict precautions against contamination Tube incubated for 7 days at 55^C and examined for growth. An unautoclaved spore strip is used as positive control and uninoculated tube of medium as negative control Results in terms of degree of heat resistance of preparation used




TECHNIQUE Forced passage of liquids through a filter of porosity small enough to retain any microorganism contained in them. A positive or negative pressure is necessary to draw the fluid through the filter into a sterile container e.g.. A filtering flask Used for sterilizing sera, solutions of sugars, vaccines, antibiotics used for culture media


TYPES OF FILTERS Earthenware candles Berkefeld,Mandler Chamberland Asbestos and asbestos paper discs,e.g.Seitz Sintered glass filters Cellulose membrane filters Gradocol and modern membrane filters Syringe,pressure,vacuum membrane filters High efficiency particle arresters(HEPA)




ALCOHOLS Ethyl alcohol and isopropyl alcohol are most frequently used Act by denaturing bacterial proteins Concentration required : 60-90% in water Protein slows its action,1%mineral acid or alkali enhances it Effective against both gram+ve and –ve bacteria, not sporicidal Isopropyl alcohol is preferred as it is better fat solvent, more bactericidal and less volatile Flammable Methyl alcohol is effective against fungal spores and used for treating cabinets and incubators affected by them Can be removed from disinfected articles by flaming


ALDEHYDES FORMALDEHYDE Active against the amino group of protein molecule Markedly bactericidal,sporicidal and virucidal Commercial formalin is 40% (w/v) solution of formaldehyde in water with 10%methanol to prevent polymerization Borax formaldehyde solution with 0.5%sodium tetraborate and 4%formaldehyde in water is used to disinfect clean metal instruments For gaseous disinfection,eg. For fumigating wards, sick rooms etc the atmosphere should have high relative humidity, over 60% and temp of at least 18^C


ALDEHYDE GLUTARALDEHYDE Markedly bactericidal,virucidal including HIV and HBV,mycobacteria and to a lesser extent spores Concentration required 2% Ability to penetrate organic material is poor Less corrosive Can be used to disinfect cystoscopes,endoscopes,corrugated rubber tubes, face masks, metal instruments,polythene tubes


PHENOLS Obtained by distillation of coal tar between temp 170-270^C active against gram+ve and –ve bacteria,mycobacteria,but little activity against spores and viruses Act by causing cell membrane damage, releasing cell contents and causing lysis Resistant to inactivation by organic matter Phenolic products like Lysol and cresol are good general disinfectants but are toxic to humans Related products chlorophenols and chloroxyphenols are less toxic, less irritant but inactive against pseudomonas

- LYSOL is used to disinfect linen- 3%lysol,0.5% phenol or 0.1% p-chloro-m-cresol is used for preserving sera and vaccines- hexachlorophene is toxic,chlorhexidine is a nontoxic skin antiseptic most active against gram+ve and fairly against gram-ve ones : 

- LYSOL is used to disinfect linen- 3%lysol,0.5% phenol or 0.1% p-chloro-m-cresol is used for preserving sera and vaccines- hexachlorophene is toxic,chlorhexidine is a nontoxic skin antiseptic most active against gram+ve and fairly against gram-ve ones


ETHYLENE OXIDE Colourless liquid with a boiling point of 10.7^C At normal temp and pressure, it is a highly penetrating gas with ethereal smell Acts by alkylating the amino,carboxyl,hydroxyl and sulphydryl groups in protein molecule and also by damaging DNA and RNA Highly inflammable, and highly explosive at a concentration of >3% in air, carcinogenic and mutagenic Explosive tendency eliminated by mixing with inert gases like CO2 and N2 to a concentration of 10% Active against most organisms including spores and viruses


ETO GAS TEST ORGANISM : Bacillus subtilis var.globigii USES : heat and moisture sensitive articles like plastic and rubber articles,blankets,pharmaceutical products,sutures,heart lung machines. Not for fumigating rooms since explosive Objects to be sterilized are put in a cabinet from which air has been removed using vacuum, and a non explosive mixture containing ETO gas is introduced to a pressure of 5-30 lb/in2 above atm pressure,temp to be maintained at 45-55^C and relative humidity 20-40%


HALOGENS IODINE Aqueous and alcoholic solutions used as skin disinfectants Actively bactericidal,virucidal,and fairly active against spores Iodine compounds with non ionic wetting or surface active agents called IODOPHORES are more active and efficient It inhibits protein synthesis and oxidizes –SH groups of amino acids


CHLORINE Reactions for free chlorine formation: Cl2 (g) + H2O <=> HOCl + Cl- + H+ HOCl <=> OCl- + H+ (at pH >7.6) Three different methods of application Cl2 (gas) NaOCl (liquid) Ca(OCl)2 (solid) Chloramine formation HOCl + NH3 <=> NH2Cl (monochloramine) + H2O NH2Cl + HOCl <=> NHCl2 (dichloramine) + H2O NHCl2 + HOCl <=> NCl3 (trichloramine) + H2O


CHLORINE Chlorine is used in free, hypochlorite as well as chloramine form Kills vegetative bacteria Kills viruses like HIV,HBV It is very effective against HBV and HIV viruses, therefore its use is recommended at 1:10 dilution for disinfection of blood spills. It is also recommended for disinfection of counter tops and work surfaces, hydrotherapy tanks, laundry. used for disinfection of water supplies and swimming pools not much useful against spores


DYES ANILINE DYES Brilliant green, malachite green and crystal violet React with acid groups in cell More active against gram+ve than gram-ve bacteria, no activity against tubercle bacilli Non irritant,non toxic Inhibited by organic material ACRIDINE DYES Proflavine,acriflavine,euflavine,aminacrine Impair DNA complexes of organisms and destroy reproductive capacity of the cell More active against gram+ve bacteria than gram-ve


OTHER CHEMICAL AGENTS METALLIC SALTS Salts of Ag,Cu and Hg,eg.merthiolate,a proprietary name for sodium ethylmercuriosalicylate is used for preservation of antitoxins and sera in 1:10000 dilution Protein coagulants and combine with free –SH groups of cell enzymes SURFACE ACTIVE AGENTS Alter energy relationship at interfaces producing a reduction of interfacial tension 4 groups : cationic,anionic,nonionic and amphoteric(Tego compounds)

Other chemical agents : 

Other chemical agents LIQUID PERACETIC ACID (STERIS ® ) Uses a solution of peracetic acid with H2O2 Peracetic acid disrupts and denatures proteins,sporicidal Extra oxygen rapidly inactivates many cell systems corrosive and irritant Sterilant for endoscopes HYDROGEN PEROXIDE PLASMA STERILIZATION (STERRAD ®) Radio frequency emissions applied to the H2O2 producing active radicals and reactive chemical species Plasma reverts to water and CO2 on removing energy Sporicidal,bactericidal,virucidal,fungicidal,mycobactercidal Not effective below 42^Cor in lipid contamination

Slide 55: 

* Type of Disinfectant: H = High level; I = Intermediate level; L = Low level


SPAULDING CLASSIFICATION Eagle H. Spaulding believed that how an object will be disinfected depended on the object’s intended use

Processing “Critical” Patient Care Objects : 

Processing “Critical” Patient Care Objects Classification: Critical objects enter normally sterile tissue or vascular system, or through which blood flows. Object: Sterility. Level germicidal action: Kill all microorganisms, including bacterial spores. Examples: Surgical instruments and devices; cardiac catheters; implants; etc. Method: Steam, ETO, hydrogen peroxide plasma, ozone or chemical sterilization

Chemical Sterilization of “Critical Objects” : 

Chemical Sterilization of “Critical Objects” Glutaraldehyde (> 2.0%) Hydrogen peroxide-HP (7.5%) Peracetic acid-PA (0.2%) HP (1.0%) and PA (0.08%) HP (7.5%) and PA (0.23%) Glut (1.12%) and Phenol/phenate (1.93%) Exposure time per manufacturers’ recommendations

Processing “Semicritical”Patient Care Objects : 

Processing “Semicritical”Patient Care Objects Classification: Semicritical objects come in contact with mucous membranes or skin that is not intact. Object: Free of all microorganisms except high numbers of bacterial spores. Level germicidal action: Kills all microorganisms except high numbers of bacterial spores. Examples: Respiratory therapy and anesthesia equipment, GI endoscopes, thermometer, etc. Method: High-level disinfection

High Level Disinfection of“Semicritical Objects” : 

High Level Disinfection of“Semicritical Objects” Exposure Time > 12 m-30m, 20oC Germicide Concentration Glutaraldehyde > 2.0% Ortho-phthalaldehyde (12 m) 0.55% Hydrogen peroxide* 7.5% Hydrogen peroxide and peracetic acid* 1.0%/0.08% Hydrogen peroxide and peracetic acid* 7.5%/0.23% Hypochlorite (free chlorine)* 650-675 ppm Glut and phenol/phenate** 1.21%/1.93% *May cause cosmetic and functional damage; **efficacy not verified

Processing “Noncritical”Patient Care Objects : 

Processing “Noncritical”Patient Care Objects Classification: Noncritical objects will not come in contact with mucous membranes or skin that is not intact. Object: Can be expected to be contaminated with some microorganisms. Level germicidal action: Kill vegetative bacteria, fungi and lipid viruses. Examples: Bedpans; crutches; bed rails; EKG leads; bedside tables; walls, floors and furniture. Method: Low-level disinfection

Low level disinfection for“non critical objects” : 

Low level disinfection for“non critical objects” Ethyl alcohol ≤ 10mn Isopropyl alcohol ≤10mn Chlorine 100 ppm ≤10mn Phenolic germicidal solution ≤10mn Iodophor germicidal solution Quaternary germicidal ≤10mn

Decreasing Order of Resistance of Microorganisms toDisinfectants/Sterilants : 

Decreasing Order of Resistance of Microorganisms toDisinfectants/Sterilants Prions Spores Mycobacteria Non-Enveloped Viruses Fungi Bacteria Enveloped Viruses


PRION STERILIZATION Critical/Semicritical-devices contaminated with high-risk tissue from high risk patients requires special prion reprocessing NaOH and steam sterilization (e.g., 1N NaOH 1h, 1210 C for 30 m) 1340C for 18m (prevacuum) 1320C for 60m (gravity) No low temperature sterilization technology effective


EVALUATING DISINFECTANTS PHENOL COEFFICIENT RIDEAL WALKER TEST : suspensions with equal numbers of typhoid bacilli are submitted to action of varying concentrations of phenol and of disinfectant to be tested Dilution of disinfectant sterilizing the suspension = phenol coefficient corresponding dilution of phenol CHICK MARTIN TEST : the disinfectant acts in presence of organic matter Both these fall short of simulating natural conditions DILUTION TEST Drying bacteria to surface followed by exposure to disinfectant and subsequent washing and inoculation in sterile broth

FILTER PAPER/DISC DIFFUSION METHODplacement of disinfectant impregnated filter paper on well inoculated agar : 

FILTER PAPER/DISC DIFFUSION METHODplacement of disinfectant impregnated filter paper on well inoculated agar



Slide 68: 

Chick-Watson Law: ln Nt/No = - kCnt where: No = initial number of organisms Nt = number of organisms remaining at time = t k = rate constant of inactivation C = disinfectant concentration n = coefficient of dilution t = (exposure) time Assumptions Homogenous microbe population: all microbes are identical “single-hit” inactivation: one hit is enough for inactivation When k, C, n are constant: first-order kinetics Decreased disinfectant concentration over time or heterogeneous population “tailing-off” or concave down kinetics: initial fast rate that decreases over time Multi-hit inactivation “shoulder” or concave up kinetics: initial slow rate that increase over time


CT CONCEPT Based on Chick-Watson Law disinfectant concentration and contact time have the same “weight” or contribution in the rate of inactivation and in contributing to CT “Disinfection activity can be expressed as the product of disinfection concentration (C) and contact time (T)” The same CT values will achieve the same amount of inactivation