Sterilization

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1. Sterilization A complete story

2. Prepared By Zisan Ahmed Ziko Roll : 141003 ID No : 1410333 Dept. Of Pharmacy Jessore University Of Science & Technology

3. Sterilization Sterilization is the process of killing or removing bacteria and all other forms of living microorganisms and their spores from preparations and articles.

4. Sterile/Sterile products : A products is said to be sterile when it is free from all living microorganisms and passes the sterility tests.

5. Other terms Used in relation to sterilization Antiseptics : These are the substance used to inhibit the growth or destroy germs. They are used on the skin. Bactericides : These are the substance used to kill bacteria. Bacteriostatic agents : These are the agents which inhibit the growth of bacteria. Disinfectants : These are the agents which are used to killed bacteria and other infectious agents from non living articles. it is 2 types, 1. Broad spectrum : It works over large variety of microorganisms. 2. Narrow spectrum : It works over smale variety of microorganisms.

6. Fungicides : These are the agents which kill fungi. Germicides : These are the substance which kill germs. Mycocides : These are the agents which kill molds. Viricides : These are the agents which kill viruses.

7. Application of sterilization Food : Sterilization has helped to reduce food poisoning. Medicine and surgery : Surgical instruments and medications that enter in the body must be sterilized to a high sterility assurance level. Example : Artificial pacemaker. In pharmaceuticals : This is also used in the manufacture of parenteral pharmaceuticals. Research and laboratory : The success of research facility is often dependent upon an efficient sterilization process. Vaccine and biological productions : The success of vaccine and biological production is often dependent on efficient sterilization process.

8. Differences between sterilization and Disinfection Sterilization Disinfection It is the method of complete removal of microorganisms. It only reduces the number of microorganisms. These methods are well defined and need to followed strictly to remove the microorganisms completely. It does not require a very strict protocol to be followed. It kills not only the bacteria but also their spores. It does not kill bacterial spores. A sterile object has no viable microorganism present. Disinfected object ha lesser load of the microorganism. Sterilization is used in surgical operations or in lab where sterile condition is necessary. Disinfection is commonly used in daily life.

9. Difference between Antiseptic and disinfectants Antiseptics Disinfectants Antiseptics are designed to kill microorganisms on the skin. Disinfectants are designed to kill microorganisms on inorganic objects. It don’t have the power to kill all microorganisms and bacteria on surfaces. It have the power to kill all microorganisms and bacteria on surfaces. Alcohol, anilides etc act as antiseptics. Vantocil, formaldehyde etc act as disinfectants. Antiseptics do not cause skin crosion and inflammation. Disinfectants can cause skin crosion and inflammation. It is used in surgery and hand washing. It is used in household cleaning.

10. Thermal death time The thermal death time may be defined as the time required to kill a specific type of microorganisms at a given temperature under specific conditions.It not only depends on controllable factors like temperature, pH etc but also depends on some uncontrollable factors like contaminating microorganisms and their resistance to heat.

11. Decimal reduction time It is one of the functions to indicate the efficiency of sterilization process. This is the time in minutes required reduce the number of viable organisms by 90 percent.The order of death microorganisms can be calculated freo the equation, K = 1/t (log N – log N) Where K is the constant.

12. Factors affecting the Thermal destruction of Microorganisms pH Inhibitory medicaments Antibacterial agents Protective substance Initial number of organisms The inactivation factor of the process

13. 1. pH : pH has a tremendous effect of thermal destruction of microorganism. The thermal resistance of most micro organisms is highest in the pH range of 6-8. In the pH acidic or alkaline solutions are easier to sterilize. 2. Inhibitory medicaments : Solutions of certain medicaments are harmful to bacteria. It’s happen for the direct toxic effect of the drug. Example : Staphylococcus aures are killed within 24 hours at room temperature by a number of medicaments. 3. Antibacterial agents : The addition of antibacterial agents in injections can reduce the sterilizing temperature from 115 C to 100 C. It is quite useful in relatively thermolabile substance and sterilization methods.

14. 4. Protective substances : Micro organisms are less easily killed in media containing high concentration of organic substance such as proteins and carbohydrate .The reason is uncertain but it is believed, protein forms a protective coat on the cells.So the microorganisms are more capable to sterilize those products which contains protein or carbohydrate. The apparatus like tubes,syringes,containers are not thoroughly cleaned after using proteins or carbohydrate . Because some of the microorganisms which are protected from steam may remain hidden inside the equipment.

15. 5. Initial number of organisms : When a population of living organism is heated at a deadly temperature ,the cells are not killed at once they are killed gradually as the exposure is prolonged. If a certain fraction of the initial number is destroyed in a particular time interval then the same fraction of the survivors will be destroyed in the succeeding interval of time equal direction and will continue till all the organisms are killed and the preparation is sterilized.

16. 6. Inactivation factor of the process : The inactivated factor is the degree to which the viable population of organisms is reduced. Inactivation factor = 10 t/D Where, t = exposure time in minutes , D = Decimal reduction time for the same temperature and conditions.

17. Methods of sterilization Methods of sterilization have been divided into 3 types : Physical methods Chemical methods Mechanical methods

18. Physical methods Dry heat sterilization Moist heat sterilization Sterilization by radiation

19. Chemical methods Gaseous sterilization Sterilization by disinfectants

20. Mechanical methods Filtration through the bacteria proof filter

21. Physical methods

22. Dry heat sterilization : Substance which get destroyed by moist heat may be sterilized by dry heat. It is a function of time temperature combination.It is the simplest and most economical method of sterilization.It means without moisture all microorganisms including spores can be destroyed by the heat.Sterilization by dry heat is usually carried out in an apparatus known as hot air oven in which heat is transferred from its source to the load by radiation, convention and to a lesser extent conduction. During this process the microorganisms are killed by oxidation. It is less effective than moist heat because higher temperature and longer periods of exposure are required. Such as exposure at 160 C for 1 hour is required for dry heat sterilization. The economy must be considered during the time temperature relationship.

23. Dry heat sterilization depends upon, Temperature and time Number of microorganisms Nature of heat Type of material Apparatus

24. Advantages It is the most suitable method of sterilization for moisture sensitive material. Example : Oily substance and dry powders It is suitable for assembled equipment providing sufficient time for penetration. Example : All glass,syringes,test tube etc It is less damaging to glass and metal equipment than moist heat. The method is economically safe.

25. Disadvantages : It requires high temperature and long exposure time. Most medicaments ,rubber and plastic articles which are thermolabile get destroyed by this method. Preparations containing H 2 O, alcohol, other volatile substances can not be sterized,alcohol,other volatile substances can not be sterilized by the method because the liquid may evaporate at high temperature. It is unsuitable for surgical dressing because the natural moisture of the fiberes quickly vaporizes which leads to deterioration and ultimately charring may take place. Drastic nature may appear in high temperature.

26. Hot air oven Hot air oven : Hot air oven or dry heat sterilizer consists of a metallic chamber of aluminium or stainless steel, which is electrically heated and thermostatically controlled.

27. The chamber of the oven has double walls which are separated from each other by thick layer of glass- fibre insulation . The hollow flanged door is also filled with glass fibre insulation. The inner side of the door is fitted with asbestos gasket that provides a tight seal to prevent heat loss. Depending on the size of the oven 3-4 perforated salves are provided in the chamber which can be removed as and when desired. A good quality of thermometer is fitted in front of the chamber for nothing the temperature the process. A fan is provided for air circulation in the oven. A vent is fitted at the top of the oven . A on-off switch is provided along with green and red indicators. The heating elements are fitted on the lower side of chamber. Heat is transferred from the source to the articles by conduction, convection and radiation . For getting good results the oven should be properly loaded so that all the articles are exposed to uniform heat for required time. Care should be taken that the oven is not opened in between the sterilization operation.

28. Moist heat sterilization Moist heat sterilization is also known as steam sterilization. Microorganism can be exposed to moist heat by using Hot water Boiling water Steam at atmospheric pressure Steam under pressure

29. Moist heat is believed to destroy microorganisms by causing protein coagulation or denaturation. It can kill microorganisms at lower temperature and in shorter times than dry heat. For example, all vegetable bacteria are destroyed by 1 hour at 80 C and very few will survive 10 minutes at this temperature. The spores of pathogens, however require 30 minute at 115 C will also destroy easily the vegetative forms of moulds , yeasts and yeast spores.

30. Advantages of Moist Heat Sterilization The penetrating power of steam is much more than that of dry heat. So microorganisms are killed more efficiently in lesser time at lower temperature than dry heat. In large size autoclaves large quantities of materials can be sterilized in one batch. Solutions packed in sealed containers as ampoules are readly sterilized by this method. Bulk solutions,glassware,surgical dressings,rubber gloves and surgical instruments are effectively sterilized by this method.

31. Disadvantages of Moist Heat Sterilization : This method is unsuitable for materials which can not withstand the heating at 115 C or more. This method is not useful for oils,fats,oinments , powder,oily injections and other preparations through which steam cannot penetrate.

32. Why moist heat sterilization is more efficient than dry heat sterilization ?? Moist heat sterilization is powerful than dry heat sterilization because, Penetrating power : The penetrating power of steam is much more than that of dry heat. So microorganisms are killed more efficiently in lesser time at lower temperature than dry heat. Thermal capacity : the thermal capacity of steam is much greater than that of dry heat. Denaturation : In presence of moisture, denaturation or coagulation of essential proteins present in microorganisms takes place at lower temperature.

33. Difference between Dry Heat sterilization and Moist Heat sterilization Moist Heat sterilization Dry Heat sterilization The moist heat sterilization have water and steam. The dry heat sterilization have no use of water and steam. Sterilization with coagulation of protein. Sterilization with oxidation. This process is under pressure. This process is on direct flame. This process takes less time. This process takes more time. Moist heat are boiling and autoclave. Dry heat are flame and incineration.

34. Why steam is used under pressure to reduced the time of sterilization ? We know that sterilization by steam under pressure is called autoclaving. This is a process of heating in an autoclave in steam saturated. Steam under pressure is allowed to penetrate through the materials. This is because pressure itself has no sterilizing power. Hence steam is under pressure to obtain temperatures high enough to destroy micro-organisms quickly and greater the pressure applied, higher the temperature obtained and lesser the tone required .That’s why steam under pressure is applied for sterilization.

35. How micro organism is killed by moist heat sterilization ? Autoclaving : Autoclaving is the process of heating in an autoclave in which saturated steam under pressure is allowed to penetrate through the materials for at least 15 minutes at a minimum temperature of 121 C . The amount of time begins when the temperature of the material being sterilized reaches at 121 C. Horizontal large scale autoclave : For larger scale this type of autoclave are used which may measure upto 20 ft in length and 12 inch to 6 ft diameter. The construction and and working procedure are same as the portable autoclave.

36. Heating with a Bactericide : This method is used for sterilizing aqueous solutions and suspensions that are destroyed by high temperature of autoclaving . A bactericide in suitable is added to the solutions to be sterilized which is distributed in final container and sealed. The sealed container are then heated at 100 C at 30 minute in a steam sterilizer or water bath. Commonly used bactericides include benzalkonium chloride 0.01% , chlorocresol 0.2% etc . Sterilization by boiling water at atmospheric pressure : The method is most useful for sterilizing instruments like syringes, needles, knives, scissors and other surgical instruments .They should be completely dipped in boiling water and should be boiled at least 20 minutes.

37. A. Tyndallization or fractional sterilization : At 80 C for 1 hr 3 days are needed for those substance which are destroyed above 115 C. B. Pesterurization : Pesterurization are used to make milk safe and improve its keeping properties. The original method was developed by pesteur . He discovered that the responsible microorganisms could be killed by heating for a few minutes at 50-60 C Moist heat sterilization at 55 to 60 C : This method is quite useful for sterilization of certain vaccines which get destroyed at a temperature higher than 60 C .The sealed containers containing the vaccine are immersed in a water bath which is thermostatically controlled they are heated at 55-60 C for 15-60 minutes, depending on the nature of microorganism to be killed. By this method the antigenic properties of the vaccines are protected but the bacteria are killed.

38. A short note on autoclave . Autoclaving : Autoclaving is the process of heating an autoclave in which saturated steam under pressure is allowed to penetrate through the materials for at last 15 minutes at a minimum temperature of 121 C .

39. Construction Autoclave is an apparatus used for sterilization by steam under pressure. A portable or bench autoclave is quite similar to pressure cooker. It is a hollow cylindrical vessel of about 15 liters capacity made of aluminium or stainless steel, fitted with a lid which can be firmly secured.In one type of autoclave the liod is fitted with eight screw clamps, a pressure gauge an air vent and adjustable safety valve . The externally fitted lid has the advantage that whole capacity of the vessel can be used but at the same time it has disadvantage also that even if one clamp is damaged or left loose, the pressure imposed on others may lead to explosion . Therefore it is essential that all the clamps should be secured and carefully maintained . The autoclave is electrically heated, the electric elements are fitted at the bottom of the autoclave. It is provided with a perforated metallic basket fitted with legs, in which the material to be sterilized is placed.

40. Working : The working of this type of autoclave is very simple. The perforated metallic basket is taken out of the vessel and a bucket full of water is put in the vessel so that the heating elements get completely immersed in water. The material to be sterilized is loosely packed in the basket which is then kept in autoclave. Care should be taken that the basket should not touch the water. After sometimes the water in autoclave starts boiling and steam generated in the body of autoclave. When the steam is passed freely through the autoclave the vent for minute , close the vent. As the pressure inside the autoclave increases the temperature also increases and reaches to the sterilization point controlled by thermostat which is generally , 10 pounds per square inch at 115 C for 30 minutes 15 pounds per square inch at 121 C for 20 minutes

41. Sterilization by radiation or cold Sterilization Sterilization by radiation is known as cold sterilization because no heat is used in this method.The exact mechanism is not clear to kill microorganisms. According to one theory alteration of chemicals presents in microorganisms takes place with the formation of new compounds which destroy the microorganisms. According to another theory the vital structure of cells,such as nucleoprotein are destroyed by the microorganisms are UV, Gamma, Infra-red radiation. It is two types , Electromagnetic waves : Infra-red radiations, ultraviolet light , X-ray, gamma rays Stream of minute particles of matter : Alpha radiation , Beta radiation.

42. Chemical method

43. A) Gaseous sterilization : Gaseous sterilization is a spetial type of chemical sterilization in which the chemical used as gases or vapours are not the liquids or solids dissolved in a solution. Many gases like sulphur sioxide,chlorine,ozone,formaldehyde,beta-propiolactone and ethylene oxide have bacterial effects. Now a days ethylene oxide has become the most widely used gaseous sterilizing agent in pharmacy and medicine. The gases used for chemical sterilization are as follows, Ethyline oxide : It is a colorless gas at ordinary temperature . the mode of action of ethylene oxide to kill the microorganisms is based on the process of alkylation of essential substances present in a protein molecules. Formaldehyde : It has been used commonly for fumigating the rooms and blanket in the hospital. Beta- propiolactone : It is quite active at low concentration.

44. Sterilization by disinfectants : Disinfectants are mainly used for the sterilizing the surfaces used for aseptic work. In emergency they may be used for sterilizing the surgical instruments like forceps ,scissors ,knives blades etc. For this reason the required instrument need to wash with sterilized water. The commonly used disinfectants include alcohol,iodin,chlorine etc.

45. The application of sterilization by gas : it sterilizes surgical instruments such as catheters, needles etc. Polythene bags can be sterilized by it. It is used for fumigating the room. It kills bacteria and all bacterial spores. It is used in alcoholic solution. It sterilizes the laboratories. They also used in disinfectants include alcohol, iodine, chlorine etc

46. Ethylene Oxide Sterilization : Ethylene oxide is a colorless gas at ordinary temperature . It is a simple cyclic ether having the formula is H 2 C CH 2 O It is highly imflammable when mixed with air. If 3% ethylene oxide mixed with carbon dioxide it becomes non- imflammable and can be safety used for sterilization purpose. But a mixture of 90% carbon dioxide with 10% ethylene oxide are non explosive.Concentration of ethylene oxide used for sterilization range from 200 to 1000 mg/ litre . In general the concentration of ethylene oxide used should be 450 mg/ litre of sterilizer chamber space must be exposed from 6-16 hours at temperatures of 49-60 C .

47. Advantages of Ethylene Oxide sterilization : Thermolabile substances can be sterilized at room temperature or at highly raised temperature. It has great penetrating power .For this reason it is useful for sterilizing surgical instruments such as catheters, needles etc. It do not damage the moisture sensitive substances and equipments rather it needs moisture for killing the microorganisms. Powder packed in polythene bags can be sterilized by it.

48. Disadvantages of Ethylene Oxide sterilization : Since it is a slow process therefore it requires long exposures. Costly equipment is required for sterilization. Since it is highly inflammable it requires special precautions.

49. Mechanical sterilization

50. Sterilize by filtration : Sterilize by filtration is one of the oldest methods of sterilization used in pharmaceutical industries for small scale filtration operations. This method is very useful for thermolabile solutions and can also be used for other solutions. The microorganisms are physically removed by absorption on the filter medium . The bacterias are entrapped in the pores of the filter and are removed from the solutions .These pores is very slow for this vacuum or pressure are employed to enhance the filtration, In practice the filter, accessories , receiving vessels must be sterilized.

51. Types of filters : Seitz filter Sintered glass filters Berkefeld glass filters Pasteur Chamberland filters Millipore filters Membrane filters.

52. Membrane filters : Membrane filters have become very common among all ultrafiltration methods . They are made of cellulose ,polyvinyl chloride, nylon and other cellulose derivatives . Their pore size is 8µ to 22 µ . However for bacterial filtration 0.22 to .45 µ are recommended.

53. Advantages : Bacteria are removed by sieving . Because the membranes used are very thin so absorption of medicament is negligible . A new disc is used for every disk of operation . Filtration is quite rapid. They do not liberate particles or chemical substance to filtrate .

54. Disadvantages : Fine pores may be clogged easily for which a prefilter may be used to remove colloidal matter. They are chemically less resistance. They are soluble in certain organic solvents like ketones and esters. They are very brittle .

55. Aseptic technique : Aseptic technique by itself is not a process of sterilization but is a procedure that does not allow the bacteria to introduce into the preparation from possible sources of contamination such as ingredients, solvents, mixing equipment , final containers , the working area the operators hand and other contact parts .

56. Aseptic area or room When over any work is to be done optically , it must be performed in an area free from microorganism known as aseptic area.  It is a doubled door room which is made of wood . The upper slanting part is made of glass . A handle is also provided to lift the slanting glass . 1 or 2 U.V. lamps is fitted to the working place . which makes the inside area sterile . A florescent tube is provided to light the area. At the time of operation the lights needed to switched on about 15-30 minutes before to make this whole area sterile . The operator should scrub his hands with alcohol and wear sterilized gloves, headgear ,mask and clothing. The whole are should be air tight .