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Sterilization & Disinfection Page 2

III. Filtration

Sterilization by filtration is employed mainly for thermolabile solutions. These may be sterilized by passage through sterile bacteria-retaining filters, e.g. membrane filters (cellulose derivatives, etc.), plastic, porous ceramic, or suitable sintered glass filters, or combinations of these. Asbestos-containing filters should not be used.

Appropriate measures should be taken to avoid loss of solute by adsorption onto the filter and to prevent the release of contaminants from the filter. Suitable filters will prevent the passage of microorganisms, but the filtration must be followed by an aseptic transfer of the sterilized solution to the final containers which are then immediately sealed with great care to exclude any recontamination.

Usually, membranes of not greater than 0.22 μm nominal pore size should be used. The effectiveness of the filtration method must be validated if larger pore sizes are employed.

To confirm the integrity of filters, both before and after filtration, a bubble point or similar test should be used, in accordance with the filter manufacturer’s instructions. This test employs a prescribed pressure to force air bubbles through the intact membrane previously wetted with the product, with water, or with a hydrocarbon liquid.

All filters, tubes, and equipment used “downstream” must be sterile. Filters capable of withstanding heat may be sterilized in the assembly before use by autoclaving at 121OC for 15 – 45 minutes depending on the size of the filter assembly. The effectiveness of this sterilization should be validated. For filtration of a liquid in which microbial growth is possible, the same filter should not be used for procedures lasting longer than one working day.

 Sterilization of  of  tissue culture media/ thermo labile liquids:

The various types of filters used for  clarifying or to remove the bacteria,fungi from the thermo labile liquids,media,solutions&buffers are as follows:

1.   EarthenwareCandlese.g.,Berkfeld,Chamberlandfilters.

2.   AsbestosPaperDisks e.g.,SeitzFilter.

3.   SinteredGlassFilters.

4.   MembraneFilters.

Str7 Str8 Str9 Str11 Str10
 Earthenware Candles Filter Holder  Sintered Glass Filter  Membrane Filter  Membrane Filter Incubated on NA

1. EarthenwareCandles

Berkfeld Filters:

Made  from  kieselguhr,  a  fossil  diatomaceous  earth  found  in  deposits  in Germany.Filters  are  of  coarse  type  owing  to  the  size  of  the  granules  forming  the substancethesubstanceof filter.

 

Madein threegradesofporosity:

V:Veil(the coarsest)donotallowtheSerratia marscens,thetestbacteriatopassthrough).

W:Wenig(thefinest).

N: Normal(theintermediate)

Filterscanbesterilizedbysteaming/autoclaving.Filtersshould be brushed with astiffnailbrushandthanboiledindistilledwater.Whencloggedwithorganic matterheatedtorednessinamuffle furnaceand allowedtocool slowly.

 ChamberlandFilters:

Madeupof unglazedporcelainandareproducedinvariousgradesof porosity, thefinestgradeallowsonly smallvirusessuchasFMDvirus, Circovirus.

Mostporous gradesL1a,L2,andL3are comparablewithV, N,and Wcandles respectively.

2. AsbestosPaperDisk  Filters. SeitzFilters:

Diskof Asbestosisinserted intoametalholder(14 cmin diameter-Largesize). Varioussizesareavailable.

Madein threegradesofporosity:

K  :Clarifying.

Do not allow Serratia marscens,thetest bacteriatopass.

N  : Normal.

EK:Specialgrade.

For sterilizationthe filterislooselyassembledwit hthe asbestosdiskinposition andthedeliverytubepassed througharubberbangwhenfilteringflaskifused. ThewholeassemblyiswrappedinKraft paperandsterilizedinautoclave. Plug thefiltrationflaskand the sidearmisfitted withanairfilter.

Beforeusingflushthediskwithsterilesalineandthenscrewdowntightlythe metalholder.

  1. SinteredGlassFilters:

Made  up  of  finely  ground  glass  fused  sufficiently  to  make  small  particles adhere,givinguniform averageporediameter(APD).

Manufacturedin threegradesof porosity:

Grade5:Finest.

Grade 3:Coarsest.Grade5/3: Specialgrade

Afterusesinteredglassfiltersarewashedwithrunningwaterinthereverse

direction. They  should  be  cleaned  with  warm  sulphuric  acid  +  potassium nitrate.

4.MembraneFilters:

Two typesof celluloseacetatemembranefilters are available:

-Oldertype(Gradocolmembrane)iscomposed ofcellulose nitratewhereasthe

-Modernmembranefiltersin usenowadaysaremadeup of cellulose acetate.

Gradocolmembranes:Madeindifferentgradeswithaverageporediameter rangingfrom3umto10nm. Usedtodeterminethesize ofmanyviruses.

 Modernmembrane filters(Celluloseacetate):DevelopedbyMillipore Filter Corporation inAmerica.

Chemical Sterilization

A. Ethylene Oxide (EtO) Gas

Ethylene Oxide gas was introduced in the 1950’s, and it is an effective, low temperature chemical sterilization method. It also takes longer than steam sterilization, typically, 16-18 hours for a complete cycle. Temperatures reached during sterilization are usually in the 50-60C range.

Ethylene Oxide (EtO) is an industrial chemical used in sterilizing medical items, fumigating spices, and manufacturing other chemicals.

Pure EtO is a colorless gas at room temperature and a mobile, colorless liquid at –47OC. Sold as a mixture with either carbon dioxide or fluorocarbon 12.

Ethylene oxide kills microorganisms by denaturing their proteins and subsequently modifying their molecular structure.

The active agent of the gas sterilization process can be ethylene oxide or another highly volatile substance. The highly flammable and potentially explosive nature of such agents is a disadvantage unless they are mixed with suitable inert gases to reduce their highly toxic properties and the possibility of toxic residues remaining in treated materials. The whole process is difficult to control and should only be considered if no other sterilization procedure can be used. It must only be carried out under the supervision of highly skilled staff.

The sterilizing efficiency of ethylene oxide depends on the concentration of the gas, the humidity, the time of exposure, the temperature, and the nature of the load. In particular, it is necessary to ensure that the nature of the packaging is such that the gas exchange can take place. It is also important to maintain sufficient humidity during sterilization. Records of gas concentration and of temperature and humidity should be made for each cycle. Appropriate sterilization conditions must be determined experimentally for each type of load.

After sterilization, time should be allowed for the elimination of residual sterilizing agents and other volatile residues, which should be confirmed by specific tests.

Bioindicator strains: spores of Bacillus subtilis (e.g. var. niger ATCC 9372) or of Bacillus stearothermophilus, (e.g. ATCC 7953).

There are some hazards associated with EtO use. Acute inhalation of high levels of EtO has resulted in nausea, vomiting, neurological disorders, bronchitis, pulmonary edema, and emphysema. Skin and eye contact with solutions of EtO has caused irritation of the eyes and skin in humans. Tests involving acute exposure of animals have shown EtO to have relatively high toxicity from oral and inhalation exposures.

A short-term effect of EtO in humans is mainly central nervous system depression and irritation of the eyes and mucous membranes. Chronic (long-term) exposure to ethylene oxide in humans can cause irritation of the eyes, skin, and mucous membranes, and problems in the functioning of the brain and nerves. Some human data show an increase in the incidence of leukemia, stomach cancer, cancer of the pancreas, and Hodgkin’s disease in workers exposed to EtO. EPA has classified EtO as a Group B1 hazard (probable human carcinogen).

EtO is not only present in sterilizers but also (in small concentrations) in the environment. Sources of environmental EtO include automobile exhaust and tobacco smoke.

Ethylene oxide (EtO) is a chemical agent that kills microorganisms, including spores. EtO gas must have direct contact with microorganisms on the items to be sterilized. Due to EtO being highly flammable and explosive in air, it must be used in an explosion-proof sterilizing chamber in a controlled environment.

Items sterilized by this process must be packaged with wraps and be aerated. The aeration time may be long and is needed to make sterilized items safe for handling and patient use.

 Note: There are also gas sterilizers available that use a mixture of EtO with carbon dioxide or chlorofluorocarbon (CFC) to represent it as nonflammable for use in healthcare facilities. In addition to safety concerns, this type of sterilization process requires an even longer aeration process compared to pure EtO sterilization.

In general, EtO gas is a reliable and safe agent for sterilization when handled properly.

 Application:

EtO is used to sterilize items that are heat or moisture sensitive.

 Disadvantages of EtO gas are that it can leave toxic residues on sterilized items and it possesses several physical and health hazards to personnel that merit special attention.

 Since EtO poses several health hazards, the alternative technologies that is currently available: a plasma phase hydrogen peroxide-based sterilizing agent .

B. Low Temperature Hydrogen Peroxide Plasma

Low temperature plasma sterilization was introduced to fill the gap between autoclave: high temperature steam sterilization (safest, fastest and least expensive) and EtO gas sterilization, which leaves toxic residuals. It is a low temperature, non-toxic, but fairly expensive sterilization method.

 In this process, hydrogen peroxide is activated to create a reactive plasma or vapor. Gaseous plasma is a new physical agent applied recently to sterilisation. High frequency energy initiates generation of the plasma from hydrogen peroxide vapours in a high vacuum and creates reactive species particles from the vapours that collide and kill microorganisms.

 Note: Plasma is ionized gas made up of ions and electrons and is distinguishable from solid, liquid, or gas. Plasma is often referred to as the fourth state of matter. The Hydrogen Peroxide Gas Plasma Sterilization system with an operating temperature range of 45-50C. Operating cycle times range from 45-70 minutes, depending on size of system.

 This sterilization system uses a combination of hydrogen peroxide and low temperature as plasma to quickly sterilize most medical instruments and materials without leaving any toxic residues. Hydrogen peroxide is a known antimicrobial agent that is capable of inactivating resistant bacterial spores. Sterilization by this method occurs in a low moisture environment.

 The Hydrogen Peroxide Plasma Process:

The process consists of two consecutive and equal sterilization phases.

Vacuum / Preplasma Stage:

When a low pressure is achieved in the vacuum stage, low temperature air plasma is generated. This helps in removing residual moisture from the chamber. The system is then vented to atmospheric pressure at the end of this stage.

 Sterilization Stage:

Pressure in chamber is reduced and an aqueous solution of hydrogen peroxide is injected and vaporized into chamber.

 The hydrogen peroxide diffuses throughout the chamber, surrounds the items to be sterilized, and starts the inactivation of the microorganisms.

After the pressure is reduced, applying radio frequency (RF) energy creates an electric field and thus forms low temperature plasma.

Free radicals are generated in the plasma by breaking apart the hydrogen peroxide vapor.

 Once the activated components react with the organisms and kill them, they lose their high energy and re-combine to form oxygen, water vapor, and nontoxic by-products.

This is half of the total sterilization process. The other half of the cycle is completed by repeating the above sterilization steps.

 At the completion of the second half cycle, the source of RF energy is turned off, vacuum is released, and chamber is returned back to atmospheric pressure by introduction of filtered air.

 Application:

This system is best suited to sterilize heat sensitive medical equipment .

C. Chlorine Dioxide

Chlorine Dioxide is a chemical liquid sterilization process. The best operating temperature range for this process is 25-30C, while using low concentrations of

ClO2. The process requires 6 hours of contact time to achieve sterilization. The presence of organic matter reduces activity. A processor converts a compound of

dilute chlorine gas with sodium chlorite to form ClO2 gas and this gas is then exposed to the equipment in a sterilizing chamber.

 Note: This alternative may corrode some materials and must be generated onsite.

Prehumidification of the ClO2 is also required.

D. Ozone

Ozone sterilizes by oxidation, a process that destroys organic and inorganic matter. It penetrates membrane of cells causing them to explode.

 In this process, a generator is used to convert oxygen to ozone, as a 6 to 12 percent concentration of ozone continuously flows through the chamber. Ozone penetration is controlled by vacuum pressure or by adding humidity. After the process is complete, oxygen is allowed to flow through the chamber to purge the ozone. The cycle time may be up to 60 minutes depending on the size of the chamber or load of items to be sterilized.

Ozone is formed by applying electrical energy to the oxygen molecule, which splits some portion of those oxygen molecules in half, into singlets of O. Therefore ozone molecules contain three atoms of oxygen and are unstable. Due to ozone gas being corrosive, and it being able to damage moisture sensitive equipment, there has not been much use of it in the medical industry.

Radiation Sterilization

Non ionising radiations–

Infra Red radiation ( rapid mass sterilization of syringes, etc)

Ultra Violet radiation (enclosed areas)

Ultraviolet rays with wavelengths shorter than 300 nm are extremely effective in killing microorganisms. The most effective sterilizing range for UV is within the C bandwidth (UVC). This range is called the germicidal bandwidth. UVC has been used in hospitals for decades to sterilize surgical instruments, water, and the air in operating rooms.

How UV Light Works

Germicidal ultraviolet (UVC) light kills cells by damaging their DNA.  The light initiates a reaction between two molecules of thymine, one of the bases that make up DNA. The resulting thymine dimer is very stable, but repair of this kind of DNA damage–usually by excising or removing the two bases and filling in the gaps with new nucleotides–is fairly efficient. Even so, it breaks down when the damage is extensive.

The longer the exposure to UVC light, the more thymine dimers are formed in the DNA and the greater the risk of an incorrect repair or a “missed” dimer. If cellular processes are disrupted because of an incorrect repair or remaining damage, the cell cannot carry out its normal functions. If the damage is extensive and widespread, the cell will die.

 Ionising – Gamma, X ray, cathode ray (plastics, syringes, oil, metal foils)

Gamma, Beta Sterilization

Mode of Action

Both, X rays and Gamma rays have wavelength shorter than the wavelength of ultraviolet light. X rays, which have wavelength of 0.1 to 40 nm, and gamma rays, which have even shorter wavelength, are forms of ionizing radiation, so named because it can dislodge electrons from atoms, creating ions. (Longer wavelengths comprise nonionizing radiation.) These forms of radiation also kill microorganisms and viruses and ionizing radiation damages DNA and produces peroxides, which act as powerful oxidizing agents in cells. This radiation can also kill or cause mutations in human cells if it reaches them.

Irradiation is an effective sterilization method, but it is limited to commercial use only. The product to be sterilized is exposed to radiation for 10 to 20 hours, depending on the strength of the source. The highest temperatures reached in gamma sterilization are usually 30-40C.

Gamma radiation is popular for sterilizing before shipment and it can be done through the packaging. A dose of 2.5 megarad is generally selected for many items. Ionizing radiation produces ions by knocking electrons out of atoms. These electrons are knocked out violently, and strike an adjacent atom and either attach themselves to it, or dislodge an electron from the second atom. The result is ionic energy that becomes converted to thermal and chemical energy.

This energy kills microorganisms by disruption of the DNA molecule, therefore preventing cellular division and propagation of biologic life.

The principal sources of ionizing radiation are beta particles and gamma rays.

 Beta particles, free electrons, are transmitted through a high-voltage electron beam from a linear accelerator. These high-energy free electrons will penetrate into matter before being stopped by collisions with other atoms. This means their usefulness in sterilizing an object is limited by the density, thickness of the object and by the energy of the electrons. These free electrons produce their effect by ionizing the atoms they hit, producing secondary electrons that kill microorganisms.

 Cobalt 60 is a radioactive isotope capable of breaking down to produce gamma rays. Gamma rays are electromagnetic waves that have the ability to penetrate a much greater distance than beta rays before losing their energy from collision. Because they travel with the speed of light, they must pass through a thickness measuring several feet before making sufficient collisions to lose all of their energy. Cobalt 60 is the most commonly used source for irradiation sterilization.

 Gamma radiation and electron beams are used to effect ionization of the molecules in organisms. Mutations are thus formed in the DNA and these reactions alter replication. These processes are very dangerous and only well-trained and experienced staff should decide upon the desirability of their use and should ensure monitoring of the processes.

Application:

The radiation can change the properties of some materials like plastics and have

adverse affects on glues or adhesives.

 Sterilization controls:

Radiation doses should be monitored with specific dosimeters during the entire process. Dosimeters should be calibrated against a standard source on receipt from the supplier and at appropriate intervals thereafter. The radiation system should be reviewed and validated whenever the source material is changed and, in any case, at least once a year.

The bioindicator strains proposed for validation of this sterilization process are: spores of Bacillus pumilus (e.g. ATCC 27142 ) with 25 kGy (2.5 Mrad) for which the D-value is about 3 kGy (0.3 Mrad) using 107-108 spores per indicator; for higher doses, spores of Bacillus cereus (e.g. SSI C 1/1) or Bacillus sphaericus (e.g. SSl C1A),  M. radiodurans  are used

  E-Beam Radiation

In this process, the E-beam generator delivers a high dose of electrons in a narrow beam at the items to be sterilized. The electrons from the E-beam generator have limited penetrating power, less than gamma radiation. For example, a 10MeV Ebeam will penetrate about 5 cm of a unit-density material.

  X-Ray Sterilization

This is a new developing process that is based on obtaining X-rays through conversion of electron beams. The X-rays produced have the same penetrating properties as the rays produced by Cobalt-60. But with this, treatment is faster, more flexible, and more environmentally friendly.

X-rays offer excellent product penetration in sterilization, thoroughly treating the surface and interior of a product.

   

Disinfection

 Disinfection is the killing of many, but not all microorganisms. It is a  process of reduction of number of contaminating organisms to a level that cannot cause infection, i.e. pathogens must be killed. Some organisms and bacterial spores may survive.

Disinfectants are chemicals that are used for disinfection. Disinfectants should be used only on inanimate objects.Antiseptics are mild forms of disinfectants that are used externally on living tissues to kill microorganisms, e.g. on the surface of skin and mucous membranes.

 The common disinfectants used in the medical & veterinary laboratories and hospitals are as follows:

 A. Glutaraldehyde

Glutaraldehyde, which has been a known disinfectant in the medical industry.

 Glutaraldehyde is an organic compound with the formula CH2(CH2CHO)2.A pungent colorless oily liquid, glutaraldehyde is used to disinfect medical and dental equipment. It is also used for industrial water treatment and as a preservative. It is mainly available as an aqueous solution, and in these solutions the aldehyde groups are hydrated.

 No carcinogenic properties.

 3.4% alkaline glutaraldehyde solution, has tuberculocidal and highlevel disinfection capabilities. It achieves high-level disinfection in 20 minutes at 25C and has up to a 28-day reuse life.

 2.4% alkaline glutaraldehyde solution, which has tuberculocidal and high-level disinfection capabilities. It achieves high-level disinfection in 45 minutes at 25C and has up to a 14-day reuse life.

 It is used to disinfect medical instruments and endoscopes. This solution can also be used in an automated reprocessor. (An automated reprocessor is the machine used to disinfect endoscopic and medical devices with a high level disinfectant solution.)

 Both the concentrations have been used as a cold liquid high-level disinfectant for heat sensitive equipment.

 Note: Glutaraldehyde products are being withdrawn from the European market due to concerns that it is toxic and harmful to health care staff in hospitals. Also, the U.S. market is requiring glutaraldehyde-free chemical solutions, which led to the formulation of the Cidex OPA solution. Cidex OPA solution is now known as the alternative to glutaraldehyde.

 B. Ethanol

The effectivity of ethanol as e.g. desinfectant or antiseptic agent depends on the concentration of ethanol-water-mixture: An ethanol percentage of 50-80% destroys the cell wall/membrane of bacteria by denaturing their proteins and dissolving their lipids (effective against most bacteria, fungi and some viruses; ineffective against bacterial spores). Therefore, the ethanol has to pass the bacterial membrane/wall to get into the bacteria – if you use 100% ethanol instead, the bacteria get ‘sealed’ and they will survive… An other mechanism is the high osmotic pressure of ethanol/water-mixtures; and the 70% has the highest one.

  C.Formaldehyde

Formaldehyde is used as a disinfectant and sterilant in both its liquid and gaseous states.

Formaldehyde is sold and used principally as a water-based solution called formalin, which is 37% formaldehyde by weight. The aqueous solution is a bactericide, tuberculocide, fungicide, virucide and sporicide.

 It is indicated that formaldehyde should be handled in the workplace as a potential carcinogen and set an employee exposure standard for formaldehyde that limits an 8-hour time-weighted average exposure concentration of 0.75 ppm . The standard includes a second permissible exposure limit in the form of a short-term exposure limit (STEL) of 2 ppm that is the maximum exposure allowed during a 15-minute period .

 Ingestion of formaldehyde can be fatal, and long-term exposure to low levels in the air or on the skin can cause asthma-like respiratory problems and skin irritation, such as dermatitis and itching. For these reasons, employees should have limited direct contact with formaldehyde, and these considerations limit its role in sterilization and disinfection processes.

 Mode of Action. Formaldehyde inactivates microorganisms by alkylating the amino and sulfhydral groups of proteins and ring nitrogen atoms of purine bases .

 Microbicidal Activity. Varying concentrations of aqueous formaldehyde solutions destroy a wide range of microorganisms.

Inactivation of poliovirus in 10 minutes required an 8% concentration of formalin, but all other viruses tested were inactivated with 2% formalin 72.

Four percent formaldehyde is a tuberculocidal agent, inactivating 104 M. tuberculosis in 2 minutes 82, and

2.5% formaldehyde inactivated about 107 Salmonella Typhi in 10 minutes in the presence of organic matter .

 The sporicidal action of formaldehyde was slower than that of glutaraldehyde in comparative tests with 4% aqueous formaldehyde and 2% glutaraldehyde against the spores of B. anthracis. The formaldehyde solution required 2 hours of contact to achieve an inactivation factor of 104, whereas glutaraldehyde required only 15 minutes.

  Although formaldehyde-alcohol is a chemical sterilant and formaldehyde is a high-level disinfectant, the health-care uses of formaldehyde are limited by its irritating fumes and its pungent odor even at very low levels (<1 ppm). For these reasons and others—such as its role as a suspected human carcinogen linked to nasal cancer and lung cancer . When it is used, direct exposure to employees generally is limited; however, excessive exposures to formaldehyde have been documented for employees of renal transplant units and students in a gross anatomy laboratory .

Formaldehyde is used in the health-care setting to prepare viral vaccines (e.g., poliovirus and influenza); as an embalming agent; and to preserve anatomic specimens; and historically has been used to sterilize surgical instruments, especially when mixed with ethanol. A 1997 survey found that formaldehyde was used for reprocessing hemodialyzers by  hemodialysis centers.

 If used at room temperature, a concentration of 4% with a minimum exposure of 24 hours is required to disinfect disposable hemodialyzers reused on the same patient .

 Paraformaldehyde, a solid polymer of formaldehyde, can be vaporized by heat for the gaseous decontamination of laminar flow biologic safety cabinets when maintenance work or filter changes require access to the sealed portion of the cabinet.

 Exercise:

  1. Draw well labelled diagrams of: Autoclave, Hot Air Oven, Seitz Filter, Sintered Glass Filter, Membrane Filter Assembly, & Syringe Filter.
  2. What is rectified spirit? Write its role in disinfection.
  3. Write the grades of Membrane filter used for filtration to remove the viruses.

  References

1. Medical Sterilization Methods – White Paper, Dec 2003

2. wwweducation.sterrad.com/c3/c3_types.htm

3. Malchesky, Paul S., Peracetic Acid and Its Application to Medical Instrument Sterilization, 1992, pg. 149.

4. William A. Rutala, Ph.D., M.P.H.1,2, David J. Weber, M.D., M.P.H.1,2, and the Healthcare Infection Control Practices Advisory Committee (HICPAC), Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008

5. Glutaraldehyde:An Effective Broad Spectrum Biocide, A series of articles published in International Hatchery Practice Magazine, 2005

6. www.steri.ee

7. www.thermproducts.com

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