Sterilization (Pharmaceutics)

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Sterilization V V S Narayana Reddy Karri Lecturer Dept of Pharmaceutics JSS College of Pharmacy, Ootacamund ( JSS University, Mysore ) Rocklands Udhagamandalam.


Definition Complete destruction of all microorganisms present in a system. The products free from living microorganisms are called “sterile products”


Spore a spore is a unit of asexual reproduction that may be adapted for dispersal and survival , for extended periods of time , in unfavorable conditions

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Things/products Chambers Rooms

What things to be sterilized.?:

What things to be sterilized.? Cotton Balls Gauze swab Ribbon Guaze Culture Media Surgical Instruments Infusion needles Parenteral Products Containers & Closures Glasswares Surgical Dressings

1. Physical Methods:

1. Physical Methods 1.1 Dry heat sterilization Heating at a temp of 160 0 C for two hours Oxidation of essential cell constituents Dry heating at 100 0 C kills all vegetative bacteria on one hour but it does not kill spores

Hot air oven:

Hot air oven Double walled chamber made of steel . Insulation material ( glass fibers or asbestos ) between the 2 walls to avoid heat loss. The door is also double walled . Two or more perforated shelves are fixed inside the oven to place the material to be sterilized An electric fan is fixed to circulate hot air in the oven in order to main require temperature. Heating elements are fitted on the bottom and are thermostatically controlled. A thermometer is fitted in the oven to note down the temperature.

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Precautions in hot air oven: Glass apparatus must be wrapped with clean cloth or filter paper. Containers must be plugged with cotton. Products to be sterilized should not be kept on the floor of the oven as it receives direct heat an becomes hotter Sufficient space b/w the articles should be maintained so that uniform distribution of heat will be happened. Advantages: Suitable for products liable (spoiled) to moist heat (powders) Suitable for sterilization of equipments such as all glass syringes etc., Disadvantages: Not suitable for surgical dressings Not suitable for medicaments, rubber and plastic equipments. Applications Used to sterilize glass wares, Injections, scissors, spatula, blades etc.

1. Physical Methods:

1. Physical Methods More efficient than dry heat (More penetration power) Very useful for killing spores Moist steam penetrates in to spores rupture it and escaping protoplasm 1.2 Moist Heat Sterilization

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Combinations of temp normally employed for sterilizing by heating in an autoclave

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Strong metallic chamber made of stainless steel A cover fitted with steam vent , pressure gauze and safety valve Rubber gasket is fitted on inner side of the lid, in order to make autoclave airtight Cover is closed with wing nuts and bolts . The electrically heated element is fitted at the bottom to heat the water to convert into steam. The perforated inner chamber is placed on the stand . The material to be sterilised is loosely packed into it. A sufficient quantity of water is poured into the chamber after removing the perforated chamber. The level of the water is adjusted in such a way that it does not touch the bottom of the perforated chamber . The material is packed in the perforated chamber . The lid is then closed with wing nuts and bolts. Autoclave

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The autoclave is switched on to heat the water. The vent is open and safety valve is set at the required pressure . When steam start coming out from the vent and it continues for 5 minutes, it is then closed. It indicates that air has been removed. The steam pressure start raising and it comes to the desired pressure i.e. 10 lbs/square inch with corresponding temperature 115°C or 15 lbs/ Sq.inch with corresponding temperature 121°C. Autoclave…

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Advantages of autoclaving Autoclaving destroys microorganisms more efficiently than dry heat and hence the material is exposed to a lower temper for a shorter period. It is used for sterilisation of a large number of official injections . Equipment or parts of rubber and plastic , such as, Nylon PVC can withstand the temperature and the pressure required sterilisation . A large quantity of material can be sterilised in one batch using a big autoclave. Disadvantages of autoclaving It is unsuitable for sterilisation of powders and oils. Applications The method is used for sterilisation of surgical dressings and surgical instruments. The containers and closures are sterilised by autoclaving. It is used for the sterilisation of a majority of official injections which can withstand the pressure of 15 lbs/ sq.inch for 30 minutes Autoclave…

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Other Methods of Sterilisation by Moist Heat Tyndallisation Pasteurisation Sterilisation of vaccines

a. Tyndallisation:

a. Tyndallisation This is a fractional sterilisation method. The method was official in B.P. 1932, for sterilisation of medicaments unstable at 115°C but able to withstand low temperature heating. The method was used for sterilisation of culture media . In this method, the solution to be sterilised is packed and sealed in its final container and heated at 80°C for one hour on each of three successive days . The first heating destroys the vegetative cells but not the bacterial spores. These bacterial spores germinate into the vegetative forms in the interval between the first and the second heating and are killed in the second heating . The third heating provides a safeguard against any spores which may not germinate until the second interval. The method was deleted from the B.P. 1932 by the 4th Addendum (1941) and replaced by " Heating with a Bactericide ”

b. Pasteurisation:

b. Pasteurisation It is a partial sterilisation method which is used to make milk safe and also to improve its keeping properties. The process kills only 97 to 99 per cent microorganism, but it does not kill bacterial spores . The methods used for pasteurisation of milk are as under: Holder method: The milk is heated at 62.8°C for 30 minutes in a steam jacketed stainless steel tank containing agitators . This provides correct exposure throughout the milk and prevents skin formation . Clean dry steams is blown to the space above the liquid to prevent the formation of skin and foam . The method kills all types of bacteria including mycobacterium tuberculosis.

b. Pasteurisation…:

b. Pasteurisation … (ii) Flash method: The milk is heated to 71.6°C for 15 seconds and then quickly cooled . The milk is heated by passing through narrow horizontal pipes inside large ones through which water passes in the opposite direction . The method is commonly used by most of the firms because it is a less time consuming process. It also needs less floor space and it is a continuous process.


Vaccine A vaccine is a biological preparation that provides active acquired immunity to a particular disease . A vaccine typically contains an agent that resembles a disease-causing micro-organism and is often made from weakened or killed forms of the microbe.

c. Sterilization of Vaccines:

c. Sterilization of Vaccines Incase of vaccine which is a suspension of dead bacteria , the organisms are killed in such a manner that its antigenic power is preserved. The suspension of microorganism is prepared in normal saline solution and transferred into a sealed container. Sterilization is carried out by immersing the container in a thermostatically controlled water bath at a temperature between 55° to 60°C for one hour . The strict aseptic precautions are observed to exclude any possibility of contamination. The vaccine is tested for sterility to confirm the proper sterilization of vaccine.

1.3. Radiation Sterilisation :

1.3. Radiation Sterilisation This is of two types: 1.3.1. Sterilisation by ultra violet light 1.3.2. Sterilisation by ionising radiation

1.3.1. Sterilisation by ultraviolet light :

1.3.1. Sterilisation by ultraviolet light Direct sunlight can destroy microorganism on account of its ultra violet rays of long wavelength. The sun emits both ultra violet rays of longer as well as shorter wavelength. The ultra violet rays of shorter wave length are absorbed by the earth's atmosphere. The antimicrobial activity of U.V. light depends on its wave length. It is maximum at 265 nm wave length.

1.3.1. Sterilisation by ultraviolet light.. :

1.3.1. Sterilisation by ultraviolet light.. Applications It is used for sterilisation of air to prevent cross infection in hospitals. It is used for sterilisation and maintenance of aseptic area in the pharmaceutical industry. It is used for sterilisation of thermolabile substances before packing It is also used for sterilisation of surfaces of working tables and rooms where aseptic technique is to be performed. The U.V. lamps should be installed in the ceiling and it should be shielded, in order to protect the worker from the harmful effects of U.V. radiation.

1.3.1. Sterilisation by ultraviolet light.. :

1.3.1. Sterilisation by ultraviolet light.. Disadvantages Ultra violet rays have a low penetration power , so it is not applicable to the sterilisation of packed pharmaceuticals. U.V. rays are less effective against organisms in the atmosphere or on surfaces if the relative humidity is high . The radiation is partially screened by dust or grease on the U.V. lamp. So regular cleaning of lamp is necessary. U.V. light is harmful for worker . The eyes and skin should be protected from direct ultra violet rays.

1.3.2. Sterilisation by ionising radiation:

1.3.2. Sterilisation by ionising radiation The ionising radiations are X-rays and Gamma rays . These are lethal to bacterial cell and destroy the nuclei of the cell. Gamma rays are produced from radio-isotonic source such as cobalt-60 or cesium-137 or of electrons energised by a suitable electron accelerator. A dose of 2.5 Mrads is generally accepted as adequate although other dosage levels may also be employed. The material to be sterilised is packed in the final container and then exposed to ionising radiation. Ionising radiations are much more efficient as a sterilising agent than ultra violet rays

1.3.2. Sterilisation by ionising radiation..:

1.3.2. Sterilisation by ionising radiation.. Advantages Gamma rays have a high penetration power and, so, materials can be sterilised after filling them in the final container. The temperature rise is negligible . The method is suitable for all types of materials such as dry, moist and frozen . Some bacterial and viral vaccines can be sterilised without any loss of its antigenic power.

1.3.2. Sterilization by ionising radiation..:

1.3.2. Sterilization by ionising radiation.. Disadvantages The plant used is very costly . A lot of capital is required in setting up the plant. The radiations are harmful to workers. It produces undesirable changes in many medicaments, such as, color, solubility and texture of the product. Applications The method is mainly used for sterilization of plastic syringes, hypodermic needles, scalpels, surgical blades and adhesive dressings . It is also used for sterilization of bone and tissue transplant, plastic tubing, catheters and sutures. It is used for sterilization of thermolabile medicaments .

2. Chemical Methods:

2. Chemical Methods These are of two types 2.1. Sterilisation by heating with a bactericide 2.2. Gaseous sterilisation

2.1. Sterilisation by heating with a bactericide :

2.1. Sterilisation by heating with a bactericide This method may be used for sterilising aqueous preparations which are unstable at higher temperatures. The method has a lower margin of safety and should be used only when moist heat sterilisation is not applicable. In this process the medicament is dissolved or suspended in a suitable solution of the following bactericide so as to achieve the concentration of bactericides.

2.1. Sterilisation by heating with a bactericide.. :

2.1. Sterilisation by heating with a bactericide.. The preparation is then transferred into the final container which is then sealed so as to exclude microorganisms and heated at 98-100°C for 30 minutes by heating it in boiling water. The bactericide used should possess the following properties: It should be non toxic . Compatible with medicaments. Stable during heating and storage.


Applications Applications The method is used for the sterilisation of injections which show some evidence of deterioration when heated at 115-116°C for 30 minutes (Autoclaving). The method cannot be used for the sterilisation of injections meant for intrathecal or peridural administration. Similarly this method is not suitable for intravenous injections when a single dose exceeds 15 ml.

3. Gaseous sterilisation :

3. Gaseous sterilisation In this case sterilisation is done with a chemical in gaseous state. In olden days, formaldehyde was very commonly used, but now it has been replaced by ethylene oxide .

3.1 Formaldehyde:

3.1 Formaldehyde It is an alkylating agent . In olden days , it was used in special chambers to sterilise catheters, syringes and other thermolabile hospital equipment. However nowadays, it is used for the fumigation of empty rooms after infectious diseases. Formaldehyde is inferior to ethylene oxide due to the following reasons: It has a weak penetration power. It is difficult to maintain a high concentration in the atmosphere. It requires a high humidity to be an effective sterilising agent. It is readily inactivated e.g. by proteins and other organic matter. It is irritating to the respiratory tract.

3.2 Ethylene oxide (C2H40) :

3.2 Ethylene oxide (C 2 H 4 0) It is a colourless gas at room temperature. It can be liquefied easily and the liquid boils at approximately 10.8°C . It is readily dissolved in water and organic solvents. It is highly inflammable so it is used by: Carbon dioxide is not so popular because it has a very high vapour pressure. The material to be sterilised is packed in the chamber and is treated with a sterilising mixture for the stated time.

3.2 Ethylene oxide (C2H40).. :

3.2 Ethylene oxide (C 2 H 4 0).. 2. Using ethylene oxide in the absence of air The exposure is carried out in an evacuated sterilizer at sub atmospheric pressure. Pure ethylene oxide or a mixture containing 90% ethylene oxide and 10% carbon dioxide is used as sterilizing agent. Ethylene oxide kills microorganisms by alkylation of protein molecules .

3.2 Ethylene oxide (C2H40).. :

3.2 Ethylene oxide (C 2 H 4 0).. Factors influencing efficiency of ethylene oxide These factors are as follows. 1. Temperature: Sterilisation can be done at room temperature but it takes a long time. It is very effective at the temperature of 40°C . 2. Concentration : It is very effective in concentration between 200 mg to 1 g/ litre . 3. Relative humidity : Some moisture is necessary to sterilise the material by ethylene oxide. Sufficient moisture is needed during exposure to ethylene oxide in order to provide the optimum humidity in the micro-environment of the organisms. Generally 40-50% moisture is necessary to have the maximum efficiency of ethylene oxide as a sterilising agent. 4. Power of penetration : Ethylene oxide has a high penetration power. It can penetrate through paper, fabrics and a number of plastics and rubbers. Ethylene oxide is a very superior sterilising agent because of the said factor. 5. Absorption : Many materials absorb ethylene oxide, and, so it causes reduction of the gas concentration in the chamber atmosphere. The sterilised articles cannot be used until the absorbed gas has escaped. The amount and the rate of absorption depend on the nature, thickness, surface area of the articles and their wrapping and the size of the load.

3.2 Ethylene oxide (C2H40).. :

3.2 Ethylene oxide (C 2 H 4 0).. Advantages 1. It is suitable for heat sensitive substances because sterilisation is effective at room temperature. 2. Ethylene oxide has a good penetration power; hence it can be used to sterilise the pre-packed articles. 4. The method is useful for sterilisation of moist-sensitive substances and equipment because only a low humidity is required. Disadvantages The method is very slow . The running costs are high . A lot of precautions are required to be taken to use ethylene oxide as it is highly inflammable and toxic. Certain toxic substances, such as, ethylene chlorhydrin are produced in certain materials. Applications 1. The method is used for sterilisation of thermolabile materials , such as, rubber and plastic items. 2. It is also used for sterilisation of delicate instruments or electric diagnostic instruments which are used in an operation theatre. 3. The method is applicable for sterilisation of powders packed in plastic or cellophane envelopes which are permeable to gases. 4. The method is suitable for sterilisation of instruments , metallic equipment, syringes and needles.

3. Mechanical Methods:

3. Mechanical Methods The solutions containing thermolabile medicament can be sterilised by filtration through bacteria proof filters. These filters retain the bacteria and the sterile filtrate is collected in sterilised receiver. To obtain a sterile filtrate it is necessary that the filter, receiver and all connecting parts likely to come into contact with the filtrate must be sterile. For successful sterilisation by filtration, the following points should be observed: The whole of the apparatus must be sterile . An aseptic technique should be followed in order to minimise the risk of contamination. The filter selected must be fine enough to obstruct the passage of all bacteria .

3.1. Ceramic filters :

3.1. Ceramic filters These are also called filter candles. These are made of porcelain or kieselguhr and are available in a range of pore size. These candles are numbered according to its pore size. Kieselguhr filters are usually softer than the porcelain type. The candle is placed in the solution to be sterilised and its opening is attached to the vacuum system. When vacuum is applied the pressure inside the candle is decreased. 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 sterile container. The filter becomes somewhat blocked with continuous use. This can be cleaned by scratching the outlet surface with a nail brush and passing water through it in, the reverse direction.

3. 2. Seitz filter :

3. 2. Seitz filter It consists of two parts. The lower part holds a perforated disc, on it a compressed asbestos sheet is placed. Two part are joined together with the help of nuts. As shown in Fig. There is a valve on the upper part through which vacuum is applied. The asbestos sheet is made up of asbestos fibres but may also contain cellulose and alkaline earth such as magnesium compounds. The asbestos pads are used once only and then discarded. Asbestos pads may yield alkali and cause precipitation of alkaloids' from aqueous solutions of their salts. Due to the fibrous nature of asbestos pads, it may shed fibres into the filtrate and also absorb drugs from solution. Hence, a few millilitres of filtrate should always be rejected.

3.3. Sintered glass/Metal filters:

3.3. Sintered glass/Metal filters These are made from borosilicate glass . The glass is finely powdered and particles of the required size are separated and is then packed into disc moulds. These moulds are heated until a suitable adhesion has taken place between the granules. These discs are fused to funnels of suitable shape and size. Sintered glass filters are available in different pore size and are numbered accordingly.

3.4. Membrane filters :

3.4. 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 100-150µ. Membrane filters are suitable for sterilising aqueous but are not suitable for organic solvents, such as, alcohols, ketones , or chloroform. Sterilisation by filtration involves four basic steps: 1. Filtration of the solution through one of the bacteria-proof filters which have been described above. 2. Aseptic distribution of the filtered solution into the previously strerilised final containers. 3. Aseptic closure of the containers. 4. Performing the sterility test. Sterility tests are required to be performed on injections sterilised by fitration .

3.4. Membrane filters… :

3.4. Membrane filters… Advantages The method is suitable for sterilisation 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. 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. Units may leak if carelessly handled. Aseptic technique is necessary. Highly trained staff is required, The process is only suitable for medicaments which are in solution form.




TESTS FOR STERILITY.. Principle: Bacteria or fungi if placed in a media which contains nutritive material , moisture and desired pH with favorable temp the organism will grow indicated by turbidity with respect to clear medium. Application: To detect the presence of viable bacteria or fungi in pharmaceutical preparations. Methods of testing: Membrane filtration method Direct inoculation method

1. Membrane filtration method:

1. Membrane filtration method

2. Direct inoculation method:

2. Direct inoculation method Specific quantity of sample is transferred in to the culture media. Observed for not less than 14 days for growth. Observation and results (at the end of the study) Observation Conclusion 1 No evidence of growth Passes test for sterility 2 Evidence of growth Fails test for sterility Re-testing with same no. of samples No evidence of growth Passes test for sterility 3 Evidence of growth Isolate and identify organism If identifiable Fails test for sterility (to that of first sample) If unidentifiable Conduct the test for sterility twice No evidence of growth in second-retest Passes test for sterility Evidence of growth in second-retest Fails test for sterility


ASEPTIC TECHNIQUES The methods which are used to prevent the access of microorganism during the preparation of parenteral products and their testing are called "Aseptic Techniques" When it is required ? Good aseptic techniques can only be applied if one knows the possible source of contamination . The various sources of contamination are: Atmosphere: which is contaminated with dust, droplet and drop-let nuclei becomes the breeding ground of microorganism. The hands are a major means of transmitting infection. Coughing, sneezing and spitting: can cause contamination at a considerable distance. The clothes which absorb dust particles are also a source of contamination. A handkerchief is the richest source of contamination. The hair , which is constantly exposed to atmospheric dust is source of contamination. The unsterile equipment ,

Design of Aseptic Room:

Design of Aseptic Room

Design of Aseptic Room:

Site: Stairs, lift and corridors ( airborne microbes ) Size: Depends on no of people working . (Preferably small-less cost) Windows: Source of contamination. Glass panes ( Lighting )+laminar flow ( Ventilation) Doors: Airlock with double door (1 meter apart). This prevent sudden inrush of air. When out door is opened indoor should be closed and vice-versa. Surface materials: Floors, walls, bench tops must be smooth ( easy cleaning ) and resistant to chemicals . Floors required frequent washing. Services Ventilation Electricity Gas supply Vacuum arrangement Disposal of waste Furniture: Least no of undesirable dust retaining cavities. Sack type Fume-Cupboard type Design of Aseptic Room

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Fume cupboard type Shack type Aseptic technique screen designs

Sterilization of surgical dressings:

Sterilization of surgical dressings Stages of sterilization Packed or wrapped in a muslin cloth or filter paper The packing contains perforated holes so that satisfactory steam penetrates. Sterilizer is closed and air inside sterilizer is replaced by steam. The final pack is exposed to 30-45 Min at 121 0 C to sterilize. Switch off the sterilizer and release the steam inside. The containers are labeled indicating the date of sterilization. Cotton Balls Gauze swab Ribbon Guaze

Sterilization of Transfusion Fluids:

Sterilization of Transfusion Fluids Stages of sterilization Transfusion fluids or I.V fluids are transferred in to washed and dried bottles. Bottles are loaded in sterilizer Bottles are exposed to desired temp and pressure. The steam is then cutoff . The steam in sterilizer is allowed to vent slowly. Bottles are taken out and cooled to less than 100 0 C. Bottles are then subjected to various quality control testes and then labeled.

Precautions for Handling of Sterilisation Equipment:

Precautions for Handling of Sterilisation Equipment By wearing sterilised gowns, face masks and gloves before entering into aseptic room. The hair should be properly covered with sterilised cap. The hands should be thoroughly washed with soap and antiseptic solution before handling the sterilised equipment. The working surface of the table or slab should be thoroughly cleaned with disinfectant before starting the aseptic work. The sterilised equipment should be transferred from one place to another in a trolley or in a crate so that the aseptic room is not opened time and again.

Factors affecting thermal destruction of microorganisms:

Factors affecting thermal destruction of microorganisms 1. pH: The majority of microorganisms are resistant to heat at pH of 6-8. So acidic/alkaline solutions are easier to sterilize. 2. Protective substances: Media containing high conc of proteins or carbohydrates, the microorganisms are not easily killed. Because protein forms a protective layer over bacteria cell. This problem is more important in the sterilization of glass wares and equipments such as tubes, syringes, containers etc. which are not cleaned properly after the use of proteins like blood or blood products, because some of the microorganisms may remain hidden inside. There fore it is necessary to properly clean equipments before sterilisation . 3. Antibacterial agents: The addition of antibacterial agents can reduce the sterilization temp form 115 0 C to 100 0 C. This method is very is useful for sterilization of thermo labile substances.

Factors affecting thermal destruction of microorganisms:

Factors affecting thermal destruction of microorganisms 4. Inhibitory medicaments: 5. The inactivation factor of the process: 6. Initial number of organisms:

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