SEMINAR 1 – PHYSICAL METHODS OF STERILISATION

NEED FOR STERILISATION?•Microorganisms are constantly present in the external environment and on the human body.•Microorganisms are responsible for contamination and infection.

HOW STERILISATION WORKS? Cell wall maintains integrity of cell

• When disrupted, cannot prevent cell from bursting due to osmotic effects Cytoplasmic membrane contains cytoplasm and controls passage of chemicals into and out of cell

• When damaged, cellular contents leak out Viral envelope responsible for attachment of virus to target cell

• Damage to envelope interrupts viral replication

STERILISATION – The processs by which an article, surface or medium is freed of all living microorganisms either in vegetative or spore state.

DISINFECTION – The destruction or removal of all pathogenic organisms or organisms capable of giving rise to infection.

ASEPSIS – The avoidance of pathogenic organisms involving the methods that prevents contamination of wounds and other sites by ensuring that only sterile objects and fluids come into contact them and risk of air-borne contamination is minimized. For eg- no touch technique in which the operator doen not touch any instruments with bare hands i.e. without cleaning hands and getting gloved.

ANTISEPSIS – The procedure or application of antiseptic solution or agent which inhibits growth of microorganisms while remaining in contact with them. For eg- scrubbing up. Antiseptic solution is butadiene.

PRINCIPLES OF STERILISATION All instruments should be thoroughly cleaned in such a way that all deposits of blood and debris are

removed before sterilisation. Sterilizing agent such as steam, heat, or gas should come in contact with each surface of all itemsto be

sterilised for specified period of time at specified temperature. All sterilizing equipments should me regularly serviced and maintained.

METHODS OF STERILISATIONPHYSICAL

Sunlight Drying – freeze drying, cold shock, osmotic shock Heat

1. Dry heat – flaming, incineration, hot air2. Moist heat – pasteurisation, boiling, steam at atmospheric pressure, steam under pressure

Filtration – candles, asbestos pads, membranes Radiation

CHEMICAL Alcohols- ethyl, isopropyl, trichlorobutanol Aldehydes – formaldehyde, glutaraldehyde Dyes Halogens Phenols Surface-active agents Metallic salts Gases – ethylene oxide, formaldehyde, beta propiolactone

SUNLIGHT – It possesses bactericidal activity. The action is due to its content of UV rays, most oif which are screened out by glass and presence of ozone in outer regions of atmosphere.

DRYING – Most of the bacteria grows in moisture environment and thus 4/5th of their weight is attributed to water. So, drying in air has deleterious effect on many bacteria.

1. FREEZE DRYING – At reduced temperature bacterial growth slows down and finally stops. Some bacteria like psychrophiles can grow even at 0o celcius, thus, for such bacterias freeze drying is employed which involves rapid freezing with subsequent drying. Freezing inactivates bacteria by damaging their outer membrane.

2. COLD SHOCK – The process in which organisms are suddenly chilled without freezing. It kills gram +ve and gram –ve bacteria but yeasts survive.

3. OSMOTIC SHOCK – In this bacteria are suspended in hypertonic solution of sucrose with EDTA and then it suspended in ice cold magnesium chloride solution.It kills the bacteria but some of the periplasmic enzymes are released.

Drying is not reliable method of sterilisation as bacterial spores remain unaffected by it.

HEAT – It is the most reliable method. It can be used either in dry form or moist form. Factors that influence sterilisation by heat are-a. Nature of heat – dry or moistb. Temperature and timec. No of microorganisms presentd. Characteristics of organisms i.e. their species, strain and sporing capacitye. Type of material from which organisms have to be eradicated.

DRY HEAT STERILISATIONThe killing effect is due to protein denaturation, oxidative damage and the toxic effect of elevated levels of electrolytes.It is suitable for glasswares, instruments specially sharp and paper wrapped articles, water impermeable oils, waxes and powders. But it cannot be used for water containing culture media.

ADVANTAGES DISADVANTAGESCan be used for sharp instruments. Cannot be used for water containing culture media,

plastic and rubber items.Instruments do not rust Process is time consuming.

1. FLAMING – Bunsen flame is used to destroy vegetative organisms present on the surface of instrument.It can be used for scalpel blades, glass slides, coverslips, mouths of culture tubes and bottle, inoculation wires and loops. Care is taken not to crack the glass.

2. INCINERATION – It is an excellent method for safe destruction of materials such as contaminated and pathological materials at a high temperature Eg- contaminated cloth, animal carcasses . Materials are reduced to ashes by burning. Instrument used is incinerator.

3. HOT AIR OVEN – It employs dry heat that kills by dehydration and oxidation. The oven is usually heated by electricity, with heating elements in the wall of chamber. A fan is fitted to ensure circulation and even distribution of air between the objects being sterilised and elimination of air pockets. In case there is no fan, the holding time has to be doubled for that particular temperature. Oven also has a temperature indicator, control thermostat and timer, open mesh shelving. Door interlocks are also fitted to ensure that heating cycle does not starts until door is shut and also that doors does not opens after the cycle until chamber temperature is fallen below 60o C. This method depends on combination of time and temperature. Various time-temperature combinations are:

TEMPERATURE HOLDING PERIOD160o C 120 minutes170o C 60 minutes180o C 30 minutes

Several measurements have been defined to quantify the killing power of heat. DRT (Decimal Reduction Time) / D value – It measures the rate of kill at a given temperature

required to reduce the no of viable organisms by 90%. Z value / Thermal death point – It measures the thermal resistance of the spore to the process

of measured as the no of degrees centigrade required to produce a 10-fold change in thermal death time.

Uses – Glassware, forceps, scissors, scalpels, all glass syringes, swabs, pharmaceutical products such as liquid paraffin, dusting powder, fats and Greece.

Disadvantages: 1. It does not penetrate grease, oil and powders, so equipments containg these substances can not be sterilised by hot air oven as hot air is a poor conductor of heat and thus has poor penetrating capacity.

3. High temperature damages fabrics and melts rubber.

STERILSATION CONTROL – Spores of nontoxigenic strain of Clostridium tetani are used as microbiological test for efficacy of dry heat sterilisation. Paper strips impregnated with 106 spores are placed in paper envelops and inserted into suitable packs. After sterilisation, strips are removed and inoculated into a thioglycollate or cooked meat media and incubated for sterility test under strict anaerobic conditions at 37o C for 5 days.

MOIST / STEAM HEAT STERILISATIONIt employs the steam generated by heating water. Steam is very efficient means of sterilisation as –

It has high penetrating capacity It gives up a large amount of heat to the surface with which it comes into contact and on which it

condenses as water.

MICROBIAL INACTIVATION BY MOIST HEATIN SPORULATING BACTERIAL IN NON-SPORULATING BACTERIA

Denaturation of spore enzyme Damage to cytoplasmic membraneImpairment of germination Breakdown of RNA

Damage to membrane Coagulation of proteinsIncreased sensitivity to inhibitory agents Damage to bacterial chromosome

Structural damageDamage to chromosomes

1. (PASTEURISATION) TEMPERATURE BELOW 100o C – Heat labile fluids are disinfected by holder method or flash process and then are allowed to cool quickly to 13o C. Uses- for serum or other body fluids containing proteins.HOLDER METHOD – Heating at 63o C for 30 minutes.FLASH PROCESS – Heating at 72o C for 15-20 seconds.Serum or body fluids containing coagulable proteins can be sterilised by heating for 1 hour at 56o C in a water bath on several successive days.Media such as Lowenstein-Jensen and Loeffler’s serum are rendered sterile by heating at 80-85o C for 30 minutes on 3 successive days in an inspissator.

2. (BOILING) TEMPERATURE AT 100 o C – Vegetative bacteria are killed almost immediately at 90-100 o C. The material, for which boiling is considered adequate, should be immersed in water and boiled for 10-30 minutes. The lid of steriliser should not be opened during sterilisation. Sterilisation is promoted by addition of 2% sodium bicarbonate to the water. Hard water should not be used.Disadvantages – It is not recommended for sterilisation of instruments used for surgical procedures.

3. (TYNDALLISATION / INTERMITTENT STERILISATION) STEAM AT ATMOSPHERIC PRESSURE – Free steam at normal atmospheric pressure i.e. 760 mmHg for 60 minutes is used to sterilize culture

media and kills all organisms except viruses and the spores of most resistant mesophilic and thermophilic bacteria.The steamer consists of tinned copper cabinet with the walls suitably lagged. The conical lid enables drainage of condensed steam and a perforated tray fitted above the water level ensures that the material placed on it is surrounded by steam. Steaming at 100o C is done for 20 min each on 3 successive days.PRINCIPLE- The first exposure kills all vegetative bacteria and spores which survived the heating process will germinate and are killed in subsequent exposure.

4. (AUTOCLAVES) TEMPERATURE ABOVE 100O C – Autoclaving is the most reliable method of sterilization. It can be used for all materials that are water-containing, permeable or wettable and not liable to be damaged by the process. The principle of autoclave or steam steriliser is that water boils when its vapour pressure is equal to the vapour pressure of surrounding atmosphere. Hence, when pressure inside a closed vessel increases, temperature at which water boils also increases. There are three major factors required for effective autoclaving:PressureTemperatureTimePressure is expressed in terms of psi (pounds per square inch) or kpa (kilo pascals) 1 kpa = 0.145 psiTemperature must be reached and maintained at 121o C to achieve required pressure.Time wrapped loads require a minimum of 20 minutes after reaching full temperature and pressure.Sterilization hold time – time for which entire load requires to be exposed to pure dry saturated steam at effective temperature to ensure sterilization.Heat penetration time – additional time required before the start of sterilization hold time to allow the penetration of heat into articles and heat them up to the effective temperature.Various combinations of temperature, pressure and holding times are used for sterilisation with PURE, DRY, SATURATED steam.PURE i.e. free from admixture with air or other non-condensable gasDRY i.e. free from suspended droplets of condensed waterSATURATED i.e. in free molecular balance with water from which it is formed

TEMPERATURE ABOVE ATMOSPHERIC PRESSURE HOLDING TIME (oC) (psi) (bar) (min) 115-118 10 0.7 30 121-124 15 1.1 15 134-138 30 2.2 3

Higher temperatures and greater pressures shorten the time required for sterilization.

FUNCTIONINGPreparation of load : Materials to be sterilised should be packaged in permeable wrappings of cloth, plastic, or kraft paper so that they are protected from contamination with environmental bacterial after their removal from autoclave. Autoclave should not be packed too fully so as to provide space between articles to permit a free passage of steam between them.

Condensation of steam : When steam makes contact with cooler articles in autoclave, it condenses into water over their surface and in their interstices. 1600ml steam at 100o C and at atmospheric pressure condenses into 1ml of water and releases 518 calories of heat. This condensation of steam into water is of critical importance.

It wets the microorganisms on the articles and so provides essential condition for their killing by moist heat.

It liberates a large amount of latent heat of steam, so, heat up the articles very rapidly to sterilizing temperature.

It causes greatest contraction in the volume of the steam and so promotes the ingress of fresh steam.

As the steam condenses into water, its contraction in volume is very great. Thus 1670 volumes of steam at 1 bar pressure will contract to form only 1 condensate. The negative pressure created by the contraction draws fresh steam on to the surface and into the interstices of the article. The cycle of

condensation, liberation of latent heat and drawing in of fresh steam is then repeated until article is heated upto steam temperature. Effective condensation takes place only if the steam is on the boundry between vapour and liquid phases and it must be pure, dry and saturated.

3 types of steam-Saturated steam – 98% Steam, 2% Water vapourDry steam – SuperheatedWet steam – Supersaturated :

Superheated steam – Steam above the phase boundry i.e. at a temperature too high for its pressure, behaves as a dry gas. Being unsaturated it fails to condense over the articles and thus moisturising and latent heat effects are lost. Moreover, temperature and pressure are no longer related so temperature cannot be controlled by regulation of pressure. Superheating may be caused

i) by overheating of the jacket of a jacketed autoclave or ii) by too great reduction in pressure as steam enters the autoclave or iii) by processing too dry load of textiles which will absorb an excessive amount of steam.

Supersaturated steam – If the steam is fed to autoclave by a long, poorly insulated, poorly drained or low velocity supply lines, it is liable to become wet due to condensation, forming suspended droplets of water as latent heat is lost before sensible heat. Wet steam delays the air removal from autoclave chamber.Moisture content of steam is inversely proportional to dryness fraction which measures the proportion of latent heat still available in it.

Air removal : Autoclave must contain pure steam free from admixture with residual air throughout the holding time of sterilization. Before holding time all air should be removed from the chamber. If air-steam mixture is left then the total pressure in the chamber will consist of sum of pressure of air and pressure of steam according to DALTON’S LAW. Thus steam’s pressure will be less and temperature will also be less than expected from pure steam. Moreover, bubbles of air trapped on article will form a insulating layer over solid surfaces and thus, will inhibit condensation and heat transfer.Reasons for presence of air in chamber during holding time includes :

i) Allowance of insufficient time for steam penetration into and removal of air from the loadii) Occurrence of leak in the chamberiii) Contamination of steam supply with air or other non condensable gas.

Drying the load : The articles of load will be moist at the end of their exposure to steam under pressure. They tend to dry by evaporation when the pressure of the steam is reduced (i.e. after holding time) and autoclave is opened. Drying may be promoted by the application of a vacuum before opening the autoclave that is done by the use of steam ejector working on venture principle.When the vacuum is used, air is reintroduced into the chamber but it passes through a filter which will hold back airborne bacteria that may contaminate the load.

TYPES OF AUTOCLAVES1. Simple laboratory autoclave2. Larger simple autoclaves3. Downward displacement laboratory autoclaves4. Porous load autoclaves5. Multi-purpose laboratory autoclaves6. High-security autoclaves

SIMPLE LABORATORY AUTOCLAVE – It is small, simple portable autoclave that operates like domestic pressure cooker for sterilisation of small metal or glass instruments.It consists of an upright metal tank heated below by electricity or gas and have a lid with a gasket enabling it to be fitted hermetically. The lid bears a manually operated tap for the discharge of air and steam, a pressure gauge and a pressurestat. A pressure-regulated (safety) valve is fitted that can be set to open when the pressure of steam rises above the chosen temperature for sterilization and to close when it falls below it, thus, pressure and temperature are held automatically at required levels. Before

the holding period, air is removed by turbulence and entraining in steam discharged freely through manually opened valve. A thermal cut-out device is fitted to shut off the source of heat in the event of autoclaving boiling dry.

LARGE SIMPLE AUTOCLAVE – It is the larger version of simple, pressure-cooker type autoclave. It is often horizontal with door at one end.The size of the chamber is too large to permit the effective removal of air, so the working temperature is not reached at the bottom of the chamber where the cooler air is layered and this failure goes undetected as thermometer is not fitted in a gravity discharge drain.

DOWNWARD DISPLACEMENT LABORATORY AUTOCLAVE – It is more sophisticated type of autoclave that removes air from the chamber and loads efficiently and also has other useful devices such as

Device that assist the drying of wrapped and porous loads Device that prevent the door from opening while chamber is under pressure Device that brings out the automatic control of the process

Pure steam is introduced from an external source into the upper part of chamber. It percolates downwards through the load and expels dense and air-steam mixture out through a discharge channel at the foot. A thermostatic steam trap is set on this channel to to open on arrival of air, air-steam mixture and close on arrival of pure steam at correct temperature. By this repeated cycle of opening and closing, the trap discharges a lot of air scavenged from the load and completes the removal of free air from the chamber in heating-up and air displacement period of about 10 min. A thermometer is fitted in the discharge line above the steam trap that indicates the temperature effectively in the lowest and coolest part of the chamber. When it shows the sterilizing temperature, sterilizing period begins. At the end of sterilizing period, the steam supply to chamber is cut off and the chamber with its load begins to cool through its door in jacketed autoclave and through its walls in unjacketed autoclave. Drying of load is effected by vapourizing the condensate by means of a vacuum drawn by venture steam injector or condenser. Drying takes 20-30min depending on nature of load.

MULTI-PURPOSE LABORATORY AUTOCLAVE – It has a essential feature of efficient assisted air removal and assisted cooling, door interlocks are provided to ensure that steam cannot enter the chamber until door is locked and door does not open if chamber gauge pressure exceeds 0.2 bar or until chamber temperature has fallen below 80o C.

Devices that are present in different autoclaves for different purposeAUTOCLAVES Device for air removal

from chamber and porous loads

Device for drying of wrapped and porous loads

Device for safe Handling

SIMPLE None None NoneDOWNWARD DISPLACEMENT

Balanced pressure steamtrap

Condenser or low vacuum venturi

Door interlock Thermocouple in Chamber

MULTI-PURPOSE Vacuum pulsing High vacuum pump Door interlock Thermocouple in Chamber

STERILISATION CONTROL – Spores of Bacillus stearothermophilus are used as test organism for determining the efficacy of moist heat sterilisation. It is an thermophilus organism with an optimum growth temperature of 55-60 oC and its spores requires an exposure of 12 minutes to be killed at 121 oC. Paper strips impregnated with 106 spores are dried at room temperature and placed in paper envelops. These envelops are inserted in different parts of load. After sterilisation, strips are inoculated into a suitable recovering medium and incubated for sterility tests at 55o C for 5 days.

FILTRATION – It helps to remove bacteria from heat labile liquids by forced passage through a filter of porosity small enough to retain any microorganisms contained in them. It can also be used for purification of water.There are variety of filters-

1. Candle filters Unglazed ceramic filters Diatomaceous earth filters

2. Asbestos filters3. Sintered glass filters4. Membrane filters5. Air filters6. Syringe filters

CANDLE FILTERSThey are made in the form of hollow candle and the fluid is forced by suction or pressure from the inside to outside or vice versa. They are manufactured in different grades of porosity. When they become clogged with organic matter they should be heated to redness in a furnace and allowed to cool slowly. They are of 2 types-Unglazed ceramic filters eg- Chamberland and Doulton filterDiatomaceous earth filters eg- Berkfeld and Mandler filter

ASBESTOS FILTERSThese are disposable, single use discs with high adsorbing capacity and tends to alkalinise filtered liquids. It has cariogenic potential due to which its use has been discarded. After use the disc is discarded. Eg- Seitz and Sterimat filter.

SINTERED GLASS FILTERSThese are made from finely ground glass fused sufficiently to make small particles adhere. They have low absorptive property and are brittle and expensive. After use they are washed with running water in reverse direction and cleaned with warm, strong sulphuric acid.

MEMBRANE FILTERSThese membranes are made up of variety of polymeric materials such as cellulose nitrate, cellulose diacetate, polycarbonate and polyester. They come in wide range of average pore diameter, 0.22 being most widely used. They are sterilised by autoclaving.These membranes are made in 2 ways-

1) Capillary pore membranes in which pores are produced by radiation that results in transparent membranes less than 10 microm thick with smooth surface and high tensile strength.

2) Labyrinthine pore membranes which are produced by forced evaporation of solvents from cellulose esters that results in thicker membrane and has 100-200 microm depth.

SYRINGE FILTERSThey are commonly fitted in syringe with the membrane of 13mm and 25mm diameter and fluid is forced through the filter by pressing down the piston. Uses – solutions of heat-labile sugars.

AIR FILTERSA principal use of this filtration is to render safe, the air withdrawn from an exhaust-ventilated safety cabinet which is being used for working with dangerous pathogens.Also it is used to decontaminate the air input into laminar flow cabinet used for work.Mainly HEPA (High Efficiency Particle Arresters) filter is used.

RADIATION – Radiation used for sterilisation is of 2 types-1. Ionizing radiation2. Non-ionizing radiation

IONIZING RADIATIONIt is effective for heat labile items and it has great penetrating properties. Its lethal action is due to its effect on DNA of nucleus i.e. breakdown of single stranded or sometimes double-stranded DNA and on other vital cell components. It is also called as Cold Sterilisation as there is no appreciable increase in temperature. Ionizing radiation includes X-rays, gamma rays and beta rays. The efficacy of ionizing radiation is influenced by certain factors-

Type of organisms Pre-irradiation treatment Oxygen effect Stage of sporulation

NON-IONIZING RADIATIONIn this type of radiation, electromagnetic rays with wavelengths longer than those of visible light are used. Non-ionizing radiation includes ultraviolet and infrared rays.

Ultraviolet rays – They are absorbed by proteins and nucleic acid and kills microorganisms by chemical reactions that they set up in bacterial cell. They has low penetrating capacity.

Infrared rays – It is another method of dry heat sterilisation. Infrared rays have no penetrating capacity.