STERILIZATION Part 1 COURSE: PHARMACEUTICAL MICROBIOLOGY II COURSE CODE: 203

Difference between sterilization & disinfection :  Sterilization is defined as the process where all the living microorganisms, including bacterial spores are killed.  Disinfection is the process of elimination of most pathogenic microorganisms (excluding bacterial spores) on inanimate (nonliving) objects.  Sterilization is an absolute condition while disinfection is not. The two are not synonymous.

Methods of sterilization:  Sterilization can be achieved by physical, chemical and physiochemical means.  Physical methods include – heat (dry or moist heat) - ultraviolet light - ionising radiations - filtration through a bacteria proof filter  Chemical methods involves the use of - liquid and - gaseous sterilizing agents.

Methods of sterilization:

Methods widely applied to Pharmaceutical Preparations:  Heat  Filtration  Combined physical and chemical method involving heat in the presence of a bactericide Methods mainly used for Surgical materials and Equipment :  Ionising radiations  Gaseous sterilization Liquid sterilizing agents > antiseptics and disinfectants.

Methods for pharmaceutical preparations : The British Pharmacopoeia has five methods for ensuring that injections are sterile :  Dry heat  Moist heat  Moist heat in the presence of a bactericide  Filtration through a bacteria-proof filter  Aseptic technique during preparation

Pharmaceutical Importance of Sterilization :  Moist heat sterilization is the most efficient biocidal agent. In the pharmaceutical industry it is used for: Surgical dressings, Sheets, Surgical and diagnostic equipment, Containers, Closures, Aqueous injections, Ophthalmic preparations and Irrigation fluids etc.  Dry heat sterilization can only be used for thermo stable, moisture sensitive or moisture impermeable pharmaceutical and medicinal. These include products like; Dry powdered drugs, Suspensions of drug in non aqueous solvents, Oils, fats waxes, soft hard paraffin silicone, Oily injections, implants, ophthalmic ointments and ointment bases etc.

 Gaseous sterilization is used for sterilizing thermolabile substances like; hormones, proteins, various heat sensitive drugs etc.  U.V light is perhaps the most lethal component in ordinary sunlight used in sanitation of garments or utensils.  Gamma-rays from Cobalt 60 are used to sterilize antibiotic, hormones, sutures, plastics and catheters etc.  Filtration sterilizations are used in the treatment of heat sensitive injections and ophthalmic solutions, biological products, air and other gases for supply to aseptic areas. They are also used in industry as part of the venting systems on fermentors, centrifuges, autoclaves and freeze driers. Membrane filters are used for sterility testing. Pharmaceutical Importance of Sterilization :

Physical method of sterilization: HEAT  Heat is considered to be most reliable method of sterilization of articles that can withstand heat. Those articles that cannot withstand high temperatures can still be sterilized at lower temperature by prolonging the duration of exposure.  There are two types of heat – dry heat and moist heat.  Moist heat is heat along with moisture.  Dry heat kills microorganisms by oxidation.  Moist heat kills microorganisms by coagulation of proteins that leads to denaturation of proteins.

Sterilization by Heat Factors affecting sterilization by heat are:  Nature of heat: Moist heat is more effective than dry heat  Temperature and time: temperature and time are inversely proportional. As temperature increases the time taken decreases.  Number of microorganisms: More the number of microorganisms, higher the temperature or longer the duration required.  Nature of microorganism: Depends on species and strain of microorganism, sensitivity to heat may vary. Spores are highly resistant to heat.

Sterilization by Heat Factors affecting sterilization by heat are:  Type of material: Articles that are heavily contaminated require higher temperature or prolonged exposure. Certain heat sensitive articles must be sterilized at lower temperature.  Presence of organic material: Organic materials such as protein, sugars, oils and fats on poorly cleaned equipments increase the time required.  Other factors: whether or not the devices /articles were properly loaded into the sterilizer, whether or not the sterilizing agent is properly delivered into the system, the sterilizer’s condition and maintenance protocol and whether or not the correct sterilization method and cycle were used.

Sterilization by Heat Susceptibility of microorganisms to heat can be expressed by :  Thermal death point (TDP) is the lowest temperature at which all the microorganisms in a particular liquid suspension will be killed in 10 minutes.  Thermal death time (TDT) is the minimum length of time for all bacteria in a particular liquid culture to be killed at a given temperature.  Decimal reduction time (DRT or D-value) is the time in minutes in which 90% of a population of bacteria at a given temperature will be killed. It is related to bacterial heat resistance.

Sterilization by DRY HEAT Examples of Dry heat sterilization are: 1. Incineration -This is a method of destroying contaminated material by burning them in incinerator. Articles such as soiled dressings; animal carcasses, pathological material etc should be subjected to incineration. This technique results in the loss of the article, hence is suitable only for those articles that have to be disposed. 2. Red heat- Articles such as bacteriological loops, straight wires, tips of forceps and searing spatulas are sterilized by holding them in Bunsen flame till they become red hot.

Sterilization by DRY HEAT Examples of Dry heat sterilization are: 3. Flaming - This is a method of passing the article over a Bunsen flame, but not heating it to redness. Articles such as scalpels, mouth of test tubes, flasks, glass slides and cover slips are passed through the flame a few times. 4. Hot air oven

Sterilization by DRY 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, convection and to a lesser extent by conduction.  The first stage in the design of a heat sterilization process is choice of suitable temperatures and times.  This depends on the need to obtain a sterile product which is influenced by the stability of the material/preparation.  Using dry heat for 1½ hours at 100°C will destroy all vegetative bacteria, 3 hrs at 140°C for most resistant spores and 1½ hours at 115°C for mould spores.

Sterilization by DRY HEAT  Pharmacists must consider the stability of their products and should not expose them to conditions greatly in excess of those needed to produce sterility e.g. the B.P. recommends 150°C for 1 hr for oily solutions.  There is no objection to the use of high temperatures where harmful effects cannot result e.g for glass vessels and containers and for these the B.P. specify not less than 1 hr at not lower than 160°C.

 Dry-heat sterilization is accomplished by thermal (heat) conduction, convection and radiation.  Initially, heat is absorbed by the exterior surface of an item and then passed to the next layer.  Eventually, the entire object reaches the temperature needed for sterilization.  Death of microorganisms occurs with dry heat by oxidation that leads to slow destruction of protein. Sterilization by DRY HEAT

HOT AIR OVEN

The design of the oven must satisfy the following requirements:  Every article inside must receive the correct exposure, wherever it is placed.  The sterilizing temperature must be reached quickly and maintained with little variation.  Hot air oven: This method was introduced by Louis Pasteur. Articles to be sterilized are exposed to high temperature (160° C -180 ° C) for duration of 1 -2 hours in an electrically heated oven. Since air is poor conductor of heat, even distribution of heat throughout the chamber is achieved by a fan. The heat is transferred to the article by radiation, conduction and convection. HOT AIR OVEN (Sterilization by dry heat)

Parts of HOT AIR OVEN i) An insulated chamber made of aluminium or stainless steel surrounded by an outer case containing glass-fibre insulation and electric heaters. ii) A fan (to allow circulation of hot air) iii) Perforated Shelves ( perforated to allow circulation of hot air) iv) Thermocouples/ thermostat v) Temperature sensor/ thermometer vi) Door locking controls with asbestos gasket that provides a tight seal. vii) vents (on top of the oven).

Parts of HOT AIR OVEN

Asbestos Gasket

 Hot Air Oven.avi Hot Air Oven.avi HOT AIR OVEN

Operation of HOT AIR OVEN  i) Articles to be sterilized are first wrapped or enclosed in containers of cardboard, paper or aluminum. Mouths of flasks, test tubes and both ends of pipettes must be plugged with cotton wool. Articles such as petri dishes and pipettes may be arranged inside metal canisters and then placed.  ii) Then, the articles must be placed at sufficient distance so as to allow free circulation of air in between them and to ensure uninterrupted air flow.  iii) Oven may be pre-heated for materials with poor heat conductivity.

 iv) The door can then be shut and the heater and the fan is switched on.  v) When the thermometer shows that the oven air has reached sterilizing temperature, heating is continued for the required period of time.  vi) The temperature is allowed to fall to 40 0 C, prior to removal of sterilized material ; this prevents breakage of glasswares. Operation of HOT AIR OVEN

1. Glasswares – Glasswares that are regularly sterilized by dry heat includes flasks, beakers, tubes, containers (e.g ampoules), pipettes, petri dishes and all glass syringes. At first they must be thoroughly degreased by washing in hot water and detergent and rinsing well, followed by a final three rinses in apyrogenic distilled water. New or very dirty articles should be soaked first in chromic acid (H 2 CrO 4 ) cleaning solution overnight. Then they are dried in a drying oven at about 65°C. Pharmaceutical Applications of sterilization by DRY HEAT

2. Other equipments include:  some articles of porcelain (such as mortars, pestles, evaporating basins) and  Metals (such as beakers, dishes of stainless steel, scissors, scalpels and ointment tubes). Pharmaceutical Applications of sterilization by DRY HEAT

3. Oils and similar anhydrous materials: Dry heat sterilization is of particular importance when contact with moisture must be avoided e.g.  Powders  Vehicles used for oily injections (e.g fixed oils, ethyl oleate and other fatty acid esters)  Ingredients of ointment bases (e.g liquid, soft and hard paraffins, beeswax)  Medical lubricants (e.g. glycerol).

4. Powders  Dusting powders fall into two groups : medical and surgical powders.  Medical powder is used to treat superficial skin conditions and so sterility is not essential.  Surgical powders must be sterile because they are used in body cavities and major wounds or on burns.  They must be free from dangerous pathogens and so must be sterilized by maintaining the powder at not less than 160°C for at least an hour. Pharmaceutical Applications of sterilization by DRY HEAT

The following substances present special problems that complicate their sterilization by heat :  Starch – Starch does not flow easily because its particles tend to stick together and this is made worse if the moisture content is high. However, if it is dried at 100°C for about an hour and then powdered before sterilization (at 150 °C for 1 hr) its flow properties are enhanced. Pharmaceutical Applications of sterilization by DRY HEAT

 Sulphonamides – The main problem with sulphonamide which is used as a diluent in penicillin is to produce a free-flowing powder without discoloration. A number of factors are involved such as particle size, moisture content and other added substances (e.g Kaolin and zinc oxide gave a more free-flowing powder). The usual method is to use crystals of suitable fineness, to dry these in a thin layer at 100 °C and then to sterilize by maintaining at 150 °C for 1 hr. Pharmaceutical Applications of sterilization by DRY HEAT

 Lactose – Lactose has been used occasionally as a diluent for penicillin. Since penicillin must be kept dry to avoid decomposition, the lactose should be dried in an oven at 105 °C, then sterilized and finally mixed with the antibiotic. Pharmaceutical Applications of sterilization by DRY HEAT

Advantages of dry-heat sterilization  It is an effective method of sterilization of heat stable articles.  It is the only method of sterilizing oils and powders.  Provided sufficient time for penetration is allowed, it is suitable for assembled equipment, e.g. all glass-syringes. In moist-heat sterilization, steam or water must be in contact with every surface and this is not always possible for e.g on the closely fitted adjacent surfaces of the barrel and plunger of an assembled syringe.  It is less damaging to glass and metal equipment than moist heat. Repeated exposure of glass to moisture at high temperatures can produce clouding and rusting is a serious risk when instruments are sterilized by wet methods.

Disadvantages of dry-heat sterilization  Due to high temperatures, long exposure and very long heating up times, most of the medicaments, rubbers and plastics are too much thermolabile for sterilization by dry heat.  It is unsuitable for surgical dressings.  Dry-heat sterilization takes longer than steam sterilization, because the moisture in the steam sterilization process significantly speeds up the penetration of heat and shortens the time needed to kill microorganisms.