Study of methods of sterilization (Physical, Chemical and Mechanical)

The presentation is all about study of methods of sterilization includes all physical, chemical and mechanical method of sterilization

1. UNIT II 2.2 Study of methods of sterilization Presented by: Mohammad Abuzar( M. Pharm ) Assistant Professor School of Pharmacy AIKTC, New Panvel .

2. CONTENTS 2

3. Introduction Sterilization means the freeing of an article from all organism including viruses, bacteria and their spore, fungi and their spores both pathogenic and non-pathogenic. Sterile pharmaceutical products - Injections, ophthalmics , dressings, sutures, ligatures, containers and closures for sterile preparations, Syringes. Selection of sterilization technique depends on maximum acceptable risk of failing to achieve sterility and maximum level of product damage that is acceptable. Sterilization methods: Physical, chemical and mechanical. sterilization is required for culture media, suspending fluids, reagents, containers and equipment used in the laboratory. 3

4. 4 Methods of Sterilization Physical Chemical Mechanical Moist heat Dry heat Gaseous Filtration Radiation UV rays Gamma rays Electron accelerators Autoclave Hot air oven Ethylene oxide Formaldehyde Particulate filter Microbial filter Final filter Liquid Alcohol Phenol Aldehyde Halogens

5. 5 Dry heat sterilization The lethal effects of dry heat on microorganisms are due largely to oxidative processes Less effective than the hydrolytic damage which results from exposure to steam Dry heat sterilization usually employs higher temperatures in the range 160–180°C and requires exposure times of up to 2 hours depending upon the temperature employed Dry heat application is generally restricted to Glassware metal surgical instruments non-aqueous thermostable liquids thermostable powders The major industrial application is in the sterilization of glass bottles which are to be filled aseptically For the purposes of depyrogenation of glass, temperatures of approximately 250°C are used

6. Dry heat sterilization methods Depending on the application, different dry heating methods are employed Sunlight , drying , Red heat , Flaming , Incineration and Hot air oven Hot air oven - design Dry heat sterilization is usually carried out in a hot air oven Comprises an insulated polished stainless steel chamber, with a usual capacity of up to 250 litres Surrounded by an outer case containing electric heaters located in positions to prevent cool spots developing inside the chamber Sterilizer design A fan is fitted to the rear of the oven to provide circulating air, thus ensuring more rapid equilibration of temperature Shelves within the chamber are perforated to allow good airflow Thermocouples can be used to monitor the temperature of both the oven air and articles contained within 6

7. 7 Sterilizer operation Articles to be sterilized must be wrapped or enclosed in containers of sufficient strength Suitable materials are paper, cardboard tubes or aluminium containers Container shape and design must be such that heat penetration is encouraged in order to shorten the heating-up stage Articles must be carefully arranged within the chamber to avoid obscuring centrally placed articles from wall radiation or impending air flow The temperature variation within the chamber should not exceed ±5⁰C Following sterilization, the chamber temperature is usually allowed to fall to around 40°C Applications Glasswares like syringes, petridishes , test tubes, flasks, pepettes , spatula, swabs can be sterilized Chemicals such as powders which would clump or form into cake in presence of moisture Surgical instruments like forceps, scalpels, and scissors Oily fluid swhich are impermeable to water such as oils and fats

8. Advantages It is a continuous process It can be used for substances that would be harmed by moisture It is suitable for assembled equipments Provides sufficient time for penetration It is less damaging to glass and metal equipments than moist heat Disadvantages It requires long heating up times, high temp and long exposure time Thermo labile substances cannot be sterilized by this method Not suitable for surgical dressings, rubbers, plastics Not suitable for preparation containing water, alcohol or other volatile substances 8

9. 9 Dry heat sterilization Moist heat sterilization Less effective More effective Mechanism : destructive oxidation Mechanism : denaturation of enzymes Requires longer time for action Requires lesser time for action Heat resistance of the organisms is found to be more Heat resistance of the organisms is found to be less Conduction of heat is slow Conduction of heat is faster Latent heat of vapourisation No Latent heat of vapourization Differentiation between dry heat and moist heat sterilization WWW.PHARMANOTES.ORG Dry heat Vs Moist heat When water comes to boiling point it needs sufficient heat to convert into vapour , on condensation the vapour are released Hence moist heat is more efficient

10. Thermal sterilization / Sterilization by heat Moist heat sterilization / Steam sterilization A)Sterilization at temperature above 100⁰C (Saturated stream) Autoclaving / steam sterilization B)Sterilization at temperature of 100⁰C (Boiling water) Tyndallisation Sterilization by boiling water C)Sterilization at temperature below 100⁰C (Hot water below Boiling Point) Pasteurization Vaccine bath d)Heating with a bactericide 10

11. Moist Heat Sterilization Moist heat kills microorganisms primarily by Coagulating proteins (denaturation), which is caused by breakage of the hydrogen bonds that hold the proteins in their three-dimensional structure Breakage of DNA strands Loss of functional integrity of cell membrane 11

12. Sterilization at temperature above 100⁰C (Saturated stream) Autoclaving / Steam sterilization Reliable sterilization with moist heat requires temperatures above that of boiling water These high temperatures are most commonly achieved by steam under pressure in an autoclave Autoclaving is the preferred method of sterilization, unless the material to be sterilized can be damaged by heat or moisture The higher the pressure in the autoclave, the higher the temperature Under these conditions, steam at a pressure of about 15 psi (121 °C) will kill all organisms and their endospores in about 15 minutes Autoclaving is used to sterilize culture media, instruments, dressings, intravenous equipment, applicators, solutions, syringes, transfusion equipment and numerous other items that can withstand high temperatures and pressures Large industrial autoclaves are called retorts 12

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14. Stages of operations - autoclave 1)Air removal and steam admission Downward displacement or evacuation 2)Heating-up and exposure 3) Drying or cooling Dressings packs must be dried before removal from the chamber For bottled fluids cooling must achieved as rapidly as possible to minimize thermal degradation of the product Steam exhaust can be achieved by application of a vacuum 14

15. 15 Radiation Sterilization

16. 16 WWW.PHARMANOTES.ORG Types of sterilizing radiations Non-ionizing Ultra-violet rays Ionizing Electron beam (particulate radiation) Gamma rays Mechanism of action Major target is the DNA of microbial cells Ionizing radiations (gamma-rays and electrons) cause ionization and free radical production Ionizing radiations can also act indirectly, by the interaction of radiation with other atoms or molecules in the cell or surrounding the cell like water Non ionizing radiations (UV light) cause excitationRadiation sterilization Applications Sterilization of articles in the dried state; these include surgical instruments, sutures, prostheses, unit-dose ointments, plastic syringes and dry pharmaceutical products Prostheses - an artificial body part, such as a limb, a heart, or a breast implant

17. 17 Ultraviolet irradiation The optimum wavelength for UV sterilization is around 260nm A suitable source for UV light in this region is a mercury lamp giving peak emission levels at 254nm Sources are generally wall or ceiling-mounted for air disinfection, or fixed to vessels for water treatment Operators present in an irradiated room should wear appropriate protective clothing and eye shields Thymine dimers of DNA are formed when exposed to UV light

18. Repair of DNA damaged by UV radiations 18

19. 19 UV radiation sterilization   Ultraviolet light irradiation (UV, from a germicidal lamp) is useful for sterilization of surfaces and some transparent objects Many objects that are transparent to visible light absorb UV UV irradiation is routinely used to sterilize the interiors of biological safety cabinets between uses It is ineffective in shaded areas, including areas under dirt It also damages some plastics, such as polystyrene foam if exposed for prolonged periods of time.

20. MOA of ionizing radiation Radiation interacts with water, leading to the formation of free radicals that can diffuse far enough to reach and damage DNA OH• radical is responsible for 90% of DNA damage 20

21. Gamma-ray sterilizers Gamma-rays for sterilization are usually derived from a cobalt-60 ( 60 Co) source On disintegration emits radiation at two energy levels of 1.33 and 1.17MeV The isotope is held as pellets packed in metal rods The rods are replaced or re-arranged as the activity of the source either drops or becomes unevenly distributed Source is housed within a reinforced concrete building with walls some 2m thick, and it is only raised from a sunken water-filled tank when required for use Articles being sterilized are passed through the irradiation chamber on a conveyor belt or monorail system and move around the raised source, the rate of passage regulating the dose absorbed 21

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23. 23 Electron accelerators Two types of electron accelerators 1)Electrostatic accelerator Producing electrons with maximum energies of 5MeV A high energy electron beam is generated by accelerating electrons from a hot filament down an evacuated tube under high potential difference 2)Microwave linear accelerator Producing electrons with maximum Energies of 10MeV Additional energy is imparted to this beam in a pulsed manner by a synchronized travelling microwave Articles for treatment are limited to small packs Arranged on a horizontal conveyor belt The sterilizing dose is delivered more rapidly in an electron accelerator than in a 60 Co plant, with exposure times for sterilization usually amounting to only a few seconds or minutes

24. Gaseous sterilization The chemically reactive gases ethylene oxide (CH2)2O and formaldehyde possess broad-spectrum biocidal activity Sterilization processes using ethylene oxide sterilization are far more commonly used on an international basis Applications In the sterilization of re-usable surgical instruments Certain medical, diagnostic and electrical equipment Surface sterilization of powders An alternative to radiation sterilization in the commercial production of disposable medical devices 24 Gas used Concentration Temperature Ethylene oxide 800–1200mg/L 45–63°C Formaldehyde 15–100 mg/L 70–75°C

25. 25 Mechanism of action Alkylation of sulphydryl , amino, hydroxyl and carboxyl groups on proteins and amino groups of nucleic acids Limitations   Even at the higher concentrations and temperatures, the sterilization processes are lengthy Unsuitable for the re-sterilization of high-turnover articles Delays because of the need to remove toxic residues of the gases before release of the items for use Both gases are potentially mutagenic and carcinogenic Cause irritation of the skin

26. 26 Ethylene oxide sterilization   Ethylene oxide gas is highly explosive in mixtures of >3.6% v/v in air Usually supplied as mix containing 10% ethylene oxide + 90% carbon dioxide for sterilization purposes Pure ethylene oxide gas can be used below atmospheric pressure in sterilizer chambers from which all air has been removed Efficacy of ethylene oxide treatment depends upon achieving a suitable concentration in each article The gas diffuses readily into many packaging materials including rubber, plastics, fabric and paper Organisms are more resistant to ethylene oxide treatment in a dried state Hence the sterilization requires a humidity of 30–70%

27. Sterilizer design and operation Sterilizer consists of a leak-proof and explosion-proof steel chamber Capacity: 100 – 300 litre Surrounded by a hot-water jacket to provide a uniform chamber temperature Vacuum pump for evacuation of chamber Inlet for passage of sub-atmospheric pressure steam Port for the entry of preheated vaporized ethylene oxide from external pressurized canisters or single-charge cartridges 27 Operating cycle for ethylene oxide sterilization

28. 28 Sterilization using formaldehyde Low temperature steam formaldehyde (LTSF) Formaldehyde gas for use in sterilization is produced by heating formalin (37% w/v aqueous solution of formaldehyde) to a temperature of 70–75°C with steam Disadvantages Similar toxicity to ethylene oxide Low penetrating power Operating cycle for low-temperature steam and formaldehyde treatment

29. Sterilizer design and operation An LTSF (Low temperature steam formaldehyde) sterilizer is designed to operate with sub atmospheric pressure steam Air is removed by evacuation Steam is admitted to the chamber to allow heating of the load and to assist in air removal Release of formaldehyde by vaporization from formalin (in a vaporizer with a steam-jacket) Chamber temperature is maintained by a thermostatically controlled water-jacket Steam and condensate are removed via a drain channel and an evacuated condenser Two types of cycles: either a simple holding stage or through a series of pulsed evacuations along with steam and formaldehyde admission cycles At the end of the treatment period formaldehyde vapour is expelled by steam flushing and the load is dried by alternating stages of evacuation and admission of sterile, filtered air 29

30. Other gaseous sterliants Propylene oxide – bactericide β – propiolactone – virucidal and bactericide Glycols – aerial bactericide Methyl bromide – insecticide Methyl alcohol – fungal decontamination Factors affecting gaseous sterilization Concentration of the gases Temperature of load Effect of moisture Time of exposure The condition and accessibility of the organisms 30

31. 31 Mechanical Method Of Sterilization Mechanical Method Of Sterilization to the solution to be sterilized is pass through depth filter or screen filter which includes Particulate filter Microbial filter Final filter tration Filtration Filtration allows for the exclusion of organisms based upon size. There are many types of filtration techniques, but when sterilizing a system membrane filtration is used. To obtain a sterile filtrate it is necessary that the filter and all connecting parts likely to come into contact with the filtrate must be sterile. Contaminants are traps on the membrane filter due to their larger size than the pore size of the membrane. e.g. insulin, blood serum, and other products containing protein matters, heat-sensitive injections, biological products, etc. Filtration

32. 32 Some observations are essential for successful sterilization by filtration The whole apparatus must be sterile. An aseptic tech The assembly of the filtration unit should be so adjusted that there is minimal exposure of filtrate to the atmosphere. The filter selected must be fine enough to obstruct the passage of all bacteria. Four steps for sterilization by filtration Filtration of the solution through one of the bacteria-proof filters. Aseptic distribution of the filtered solution into the previously sterilized final containers. Aseptic closure of the containers. Performing the sterility test.

33. There are various types of filtrations used for various purposes viz. HEPA filter, sintered glass filters, colloidal or membranous filtrates, earthenware candles, etc. The various types of bacteria-proof filters used are as follows. Ceramic filters:   These are also known as filter candles , made of porcelain or kieselguhr and are available in a range of pore sizes. Kieselguhr filters are usually softer than the porcelain type. The candle is placed in the solution to be sterilized and its opening is attached to the vacuum system. When the vacuum has applied the pressure inside the candle is decreases. Due to the difference in pressure between the outside and inside of the candle, the solution moves into the candle. The filtrate is collected in a sterile container. The main disadvantage of ceramic filters is their tendency to absorb materials from aqueous solutions.

34. 34 Seitz filter:   It consists of two parts. The lower part holds a perforated disc and the upper part is a compressed asbestos sheet. Two parts are joined together with the help of nuts. There is a valve on the upper part through which a vacuum is applied. Due to the fibrous nature of asbestos pads, it may shed fibers into the filtrate and also absorb drugs from the solution. Hence, a few ml of filtrate should always be rejected and sintered glass disc may also be fixed in the filtration unit immediately after the Seitz filter. Sintered glass filters:   They are made from borosilicate glass. The glass is finely powdered and particles of the required sizes are separated and are then packed into disc moulds . These discs are fused to funnels of suitable shape and size. Sintered glass filters are available in different pore sizes and are numbered accordingly. For bacteria, proof filtration number 5 or 3 is used. The filtration is carried out under reduced pressure.

35. 35 Sintered metal filters They are the metallic counterpart of sintered glass filters. These are usually made from stainless steel. They have the advantage of having greater mechanical strength. Membrane filters: These are made of cellulose acetate or cellulose nitrate. These are fixed in metallic holders similar to those used with asbestos pads. The pore size in the membranes lies in the range of 100-150 µ. They are also called  millipores filters . They are suitable for sterilizing aqueous and oily solutions but are not suitable for organic solvents like alcohol, ketones, esters, or chloroform.

36. 36 Air filter: (HEPA filter) It is high-efficiency particulate air or originally called High-Efficiency Particulate Absorber (HEPA). It is used to describe filters that can trap 99.97 per cent of particles that are 0.3 microns. Air particles are circulated through the HEPA filter in four directions viz. Direct Impaction:  Large contaminants, such as certain types of dust, mould , and pollen, travel in a straight path, collide with fibre , and stick to it. Sieving:  The air stream carries a particle between two fibres , but the particle is larger than the gap, so it becomes ensured. Interception:  Airflow is nimble enough to reroute around fibres , but, thanks to inertia, particles continue on their path and stick to the sides of fibres . Diffusion:  Small, ultrafine particles move more erratically than larger ones, so they are more likely to hit and stick to fibres (Fig).

37. 37 HEPA Filter

38. 38 Advantages The method is suitable for the sterilization of thermolabile medicaments, such as blood products, insulin and enzymes. All types of bacteria i.e., living as well as dead, are removed from the preparation. Both clarification and sterilization are done side by side. It is an excellent method for the rapid supply of a small volume of a parenteral solution in an emergency. Disadvantages The method is not a reliable one and therefore a sterility test is necessary. The suspension and oily preparations cannot be sterilized by this method. There are chances of absorption of medicaments from a solution by the filter. Defects in the media are not immediately detectable. Highly trained staff is required. The process is only suitable for medicaments that are in solution form.

39. 39 W.B. Hugo and A.D. Russel: Pharmaceutical Microbiology, Blackwell Scientific publications, Oxford London. Prescott and Dunn., Industrial Microbiology, 4th edition, CBS Publishers & Distributors, Delhi. Pelczar , Chan Kreig , Microbiology, Tata McGraw Hill edn . Malcolm Harris, Balliere Tindall and Cox: Pharmaceutical Microbiology. Rose: Industrial Microbiology. Probisher , Hinsdill et al: Fundamentals of Microbiology, 9th ed. Japan Cooper and Gunn’s: Tutorial Pharmacy, CBS Publisher and Distribution. Peppler : Microbial Technology. I.P., B.P., U.S.P.- latest editions. Ananthnarayan : Text Book of Microbiology, Orient-Longman, Chennai Edward: Fundamentals of Microbiology. 12. N.K.Jain : Pharmaceutical Microbiology, Vallabh Prakashan , Delhi REFERENCES

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