The Open University of Sri Lanka Faculty of Health Sciences Department of pharmacy BPU 2223 Pharmaceutical Microbiology

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1 The Open University of Sri Lanka Faculty of Health Sciences Department of pharmacy BPU 2223 Pharmaceutical Microbiology Sterilization and Sterility Sterilization Sterileis thecondition being free from any living organisms. Sterilization is the process of killing or removing microorganisms from a product to ensure that it is sterile. Sterilization is an essential stage in the processing of any product designed for parenteral administration. Sterilization process involve the application of a biocidal agent or physical microbial removal process to a product or preparation with the object of killing or removing all microorganisms. These processes may involve elevated temperature, reactive gas, irritation or filtration through a microorganism-proof filter. Steam sterilization C, 15 lbs pressure for 15 min Dry heat sterilization C/ 1 hour or 140 C / 3 hours or suitable temperature and times Gas sterilization - Ethylene oxide, Chlorine dioxide Sterilization by radiation - Gamma sterilization, UV sterilization By filtration - Membrane filtration and Cartridge filtration The success of the process depends upon a suitable choice of treatment conditions. Eg. Temperature and duration of exposure. Sterility A specimen said to be sterile only when there is complete absence or viable microorganism from it. However, this absolute definition cannot currently be applied to an entire lot of finished articles, because of limitation in testing. Sterile Products For sterile products there is the added requirement that they must be free of viable microorganisms. This consequently means the product should be manufactured in a manner that reduces to the lowest likelihood the risk of microbial contamination. Thus a sterile

2 product should not contain viable bacteria, yeasts or fungi, nor other microorganisms such as rickettsiae, mycoplasmas or protozoa and viruses. Product requiring sterility 1. Medical devices Surgical gloves, clean room garments Specimen cups, wound care products, suture, Needles, syringes, catheters, drain bags, IV bags Implants, surgical tools, surgery supplies 2. Pharmaceuticals Injectable and inhalation drugs Nasal, ophthalmic and some topical products 3. In vitro diagnostics for healthcare related testing 4. Prepared media for microbiology testing Manufacture of the sterile product For production purposes an important distinction exists between sterile products which have been terminally sterilized and those which are not. Terminal sterilization involves the product being sealed in its container and then sterilized, usually by heat, but ionizing radiation or, less commonly, ethylene oxide may be employed. Such a product must be produced in a clean area. A product that cannot be terminally sterilized is prepared aseptically from previously sterilized materials or by sterile filtration and then filled into sterile containers. Strict aseptic conditions are required throughout. (figure 02)

3 Terminal sterilization Terminal sterilization is performed by heat (Moist heat and Dry heat), by radiation and by gasses and fumigants. Sterilization by heat Each cycle of sterilization must be recorded using time and temperature charts. One cycle can be decided into two phases as heating and cooling. - Heating phase: Determined for each load Sufficient time for the whole load - Cooling phase: Precautions to prevent contamination Sterilized cooling fluid/gas are used Sterilization by moist heat This method is suitable for water-wettable materials only. Eg: aqueous formulations Temperature, time and pressure must be monitored. Regular leak test must be performed when vacuum is part of the cycle. Materials should allow for removal of air and penetration of steam inside. All parts of the load should be in contact with steam and must be free from any contamination. Sterilization by dry heat Dry heat is suitable for non-aqueous liquids and dry powders. Air circulation in the chamber should be controlled and positive pressure in chamber must be controlled to prevent entry of non-sterile air. HEPA filtered air is supplied into the chamber. Sterilization by radiation Sterilization by radiation is suitable for heat-sensitive materials and products. But suitability of method must be confirmed on the material of packaging. Ultraviolet irradiation is not acceptable. During the procedure dose of radiation must be measured. Radiation sensitive colour discs can be used. Sterilization by gases Sterilization by gases is used only when no other method is suitable. On validation it must be proved that the gas has no damaging effect on the product. Sterilizing gas in direct contact with microbial cells is essential for effective sterilization. Time and conditions for degassing to remove residues must be identified. Eg: Sterilization by Ethylene dioxide produces residues of ethylene glycol and ethylene chlorohydrene. To remove these residues the sterilized articles must expose to C for 72 hours.

4 Ethyleneoxide Ethylene glycol Ethylene chlorohydrene Aseptic preparation Objectives of aseptic preparation is to maintain the sterility of a product, assembled from sterile components. Therefore operating conditions must be maintained to prevent microbial contamination. Careful attention is given to environment, personnel, critical surfaces, container/closure sterilization and transfer procedure to maintain sterility of sterilized products. Sterilization by filtration In this method the product to be sterilized is filtered through a sterile filter of 0.22 micrometer or less, into previously sterilized containers. Filtration removes bacteria and moulds but not all viruses or mycoplasmas. Double filter layer or second filtration is advisable, just before filling. Fiber shedding or asbestos filters are not allowed. Validation of process includes time taken to filter a known volume and pressure difference to be used across the filter. Same filter should not be used for more than one working day, unless such use has validated. The filter should not have any interaction with product towards removal of ingredients from the product or releasing any substances into product. Validation of all sterilization process needed to be done prior to use. Special attention should be paid on non-aqueous or oily solutions and when non-pharmacopoeia methods are employed. Before the method is adopted its suitability and efficacy demonstrated with desired conditions. All parts of the load Each types of load Physical measurements and biological indicators (where appropriate) Verified at least annually and after change Records maintained For effective sterilization the whole of the material should subjected to the treatment. Biological indicators are used appropriately and as an additional method of monitoring. In the use of biological indicators quality must be checked through positive control. Since this method is associated with the risk of contamination handling must be done carefully.

5 Sterilization control and sterility assurance A product to be labelled sterile must be free of viable microorganisms. To achieve this, the product, or its ingredients, must undergo a sterilization process of sufficient microbiocidal capacity to ensure a minimum level of sterility assurance. It isessential that the required conditions for sterilization be achieved and maintained through every operation of the sterilizer. the quality is assured by a combination of process monitoring and performance criteria; these may be considered under four headings: Bioburden determinations Environmental monitoring Validation and in-process monitoring of sterilization procedures Sterility testing Bioburden determination The term bioburden is used to describe the concentration of microorganisms in a material; this may be either a total number of organisms per millilitre or per gram, regardless of type, or a breakdown into such categories as aerobic bacteria or yeasts and moulds. Bioburden determinations are normally undertaken by the supplier of the raw material, whose responsibility it is to ensure that the material supplied conforms to the agreed specification, but they may also be checked by the recipient. Themaximum permitted concentrations of contaminants may be those specified in various pharmacopoeias or the levels established by the manufacturer during product development. The level of sterility assurance that is achieved in a terminally sterilized product is dependent upon the design of the sterilization process itself and upon the bioburden immediately prior to sterilization. However, the adoption of high standards for the quality of the raw materials is not, in itself, a strategy that will ensure that the product has an acceptably low bioburden immediately prior to sterilization. It is necessary also to ensure that the opportunities for microbial contamination during manufacture are restricted and that those organisms that are present initially do not normally find themselves in conditions conducive to growth. It is for these reasons that manufacturing processes are designed to utilize adverse temperatures, extreme ph values and organic solvent exposures in order to prevent an increase in the microbial load. For example, water is the most common, and potentially the most significant, source of contamination in the manufactured product, and maintenance of water at elevated temperatures is commonly employed as a means of limiting the growth of organisms such as Pseudomonas spp., which can proliferate during storage, even in distilled or deionized water. Precautions such as these ensure that chemically synthesized raw materials have bioburdens that are generally much lower than those found in natural products of animal, vegetable or mineral origin.

6 Environmental monitoring Environment / Sterile area Non-sterile products and sterile products must not be processed in the same area. The production of sterile preparations should be carried out in clean/sterile areas, entry to which should be through airlock for personnel and/or for goods. Clean area should be maintained to an appropriate standard of cleanliness and supplied with air that has passed through filters for an appropriate efficiency. The various operations of component preparation (such as containers and closures), product preparation, filling and sterilization should be carried out in separate area within a clean area. (figure 01) 01. Internal surfaces, fittings and equipment Contamination by both particulate and microbial must be prevented when sterile products are being manufactured. Therefore all the surfaces should possess smooth, impervious surfaces which will: Prevent the accumulation of dust or other particulate matter Allow for easy and repeated cleaning and disinfection The joints between ceiling/walls/floor should be coved to facilitate cleaning. A suitable flooring material is provided for by welded sheets of polyvinyl chloride (PVC). The preferred surface materials for walls are plastic, epoxy-coated plaster, plastic fiberglass or glassreinforced polyester. The final finish for floor, wall and ceiling is achieved using continuous welded PVC sheeting. Use should be made of well-sealed glass panels, especially in dividing walls, to ensure good visibility and satisfactory supervision. All fittings, such as doors, windows, power outlets and light fittings should be flush with the wall or ceiling surfaces and sealed to prevent entrainment of unclean air. Window should not be openable. Internal fittings such as cupboards, drawers and shelves must be kept to a minimum and made from stainless steel or a laminated plastic, which may be easily cleaned or disinfected. Stainless steel trolleys can be used to transport equipment and material within the clean areas. 02. Air supply Clean areas for the production of sterile products are classified according to the required characteristic of the air as grade A, B, C, and D. Grade A : equivalent to class 100 and ISO 5

7 - Local zone for high risk operations Eg : Product filling, stopper bowls, open vials, handling sterile materials, aseptic connections, transfer of partially stoppered containers to be lyophilized conditions usually provided by laminar air flow workstation Grade B : equivalent to class 100 and ISO 5 - Background environment for Grade A zone Eg : Cleanroom in which laminar flow workstation is housed Grade C : equivalent to class and ISO 7 - For carrying out less critical stages in manufacture of sterile products Eg : Preparation of solutions to be filtered Grade D : equivalent to class and ISO 8 - For carrying out less critical stages in manufacture of sterile products Eg : Handling of components after washing Each grade of cleanroom has specifications for viable and non-viable particles Non-viable particle Viable particle HEPA filtered air is used to achieve the necessary standards. This should be maintained at positive pressure throughout a clean or aseptic area, with the highest pressure in the most critical rooms and a progressive reduction through the preparation and changing rooms. The greatest risk of contamination of a pharmaceutical product comes from its immediate environment. Additional protection from particulate and microbial contamination is

8 therefore essential in both the filling area of the clean room and in the aseptic unit. This can be provided by a protective work station supplied with a unidirectional flow of filtered sterile air. 03. Clothing Because of the possibility of the shedding of fibers, cotton material is regarded as being unstable in the present context. Terylene, which sheds virtually no fibres, is suitable. Airbone particulate and microbial contamination is reduced when trouser suits, close-fitting at the neck, wrists and ankles, are worn. Clean suits for clean areas should be provided at least once daily, but fresh headwear, overshoes and power-free gloves are necessary for each working session. Special laundering facilities for this clothing are desirable. 04. Changing facilities Entry to clean or aseptic areas should be through a changing room fitted with interlocking doors. This acts as an airlock to prevent the influx of air from outside. This access route is intended for personnel only and does not constitute a means for regularly transferring materials and equipment into these area. Entry to aseptic area Black - Remove outer shoes, clothing Grey - Wash and dry hands, forearms. Put on sterile cap and mask. Rewash and dry hands and forearms White - Put on suit, over boots, gloves. Rinse gloved hands in antiseptic Aseptic - Commence work in aseptic area 05.Cleaning and disinfection Cleaning agent of alkaline detergents and ionic and nonionic surfactants are used to keep microbial contamination in a minimum level. Different types of disinfectants should be employed in rotation to help prevent development of resistance strains of microorganisms. In-use dilutions should not be stored unless sterilized. Disinfectants and detergents for use in grade A/B area must be sterile prior to use. Smooth, polished surfaces are cleaned most easily. Floors and horizontal surfaces should be cleaned and disinfected daily, walls and ceilings as often as required, but the interval should not exceed 01 month. Regular microbiological monitoring should be carried out to determine the efficacy of disinfection procedures.

9 06. Operation The number of persons involved in sterile manufacturing should be as minimum as possible, so as to avoid the inevitable turbulence and shedding of particles and organisms associated with operatives. All operations should be undertaken in a controlled and meyhodical manner. Containers made from fibrous materials such as paper, cardboard and sacking, are generally heavily contaminated and should not be taken into clean or aseptic areas where fibres or microorganisms shed from them could contaminate the product. Containers and closures for terminally sterilized products must be thoroughly cleaned before use and should undergo a final washing and rinsing process in apyrogenic distilled water (which has been passed through a bacteria-proof membrane filter) immediate prior to filling. Environmental monitoring methods Testing physical parameters as a means of measuring the performance of equipment/facilities is accepted practice. The physical tests performed are reproducible and accurate and can be used to validate the facilities/equipment s performance against accepted criteria. Testing microbiological parameters as a means of evaluating equipment/facilities performance is less accurate as the means of determination are highly variable. Microbiological levels in the environment are not uniformly distributed in a given area and fluctuate with time. It is therefore vital that test methodologies exist as part of the environmental monitoring program. Each test method selected for routine monitoring should be validated. If changes are made to test methods, variations may introduce unknown variability and comparisons between data are spurious. It is essential that test methodologies are defined and complied with so trends in contamination levels may be compared. It is important to emphasize that failure to comply with the methodologies may render the samples collected invalid. Each test emphasis on detecting patterns within the data obtained. A major consideration in the operation of clean room technology for aseptic dispensing is the monitoring of viable contamination within clean environments. It is important to stress that statistically microbiological monitoring is not as reliable as physical monitoring and that examination of trends or patterns of contamination must be carried out. 01. Microbiological methods It is preferable to use a growth medium with low selectivity which capable of supporting a broad spectrum of microoraganism including aerobes, anaerobes, fungi, yeast and moulds. The media types listed below have been found to be suitable. Nutrient agar

10 Tryptone Soya Agar (TSA) Blood agar Columbia agar with horse/sheep blood Incubation condition Following testing the sample should be incubated as soon as possible and should be held at room temperature with the medium uppermost until incubated or manipulated. If the medium is dropped or touched by an operator then this should be reported, the sample should be marked accordingly and treated as usual. Under no circumstance should samples that have been taken be refrigerated. In cubation of samples, inverted at C for at least for 2 days is suitable for the growth of bacteria. Incubation of samples, inverted at C for at least 5 days is suitable for the growth of mould and fungi. a. Settle plate methods Settle plate sampling is a direct method of assessing the likely number of microorganism depositing on to the product or surface in a given time. It is based on the fact that, in the absence of any kind of influence, airborne microorganisms, typically attached to larger particles, will deposit onto open culture plates. Microorganism are usually found in the air of occupied rooms rafted on to skin cells with very few present on their own. The average size of microbial particle will deposit, by gravity on to surface at a rate of approximately 1 cm/s. In settle plate sampling Petri dishes containing agar medium are opened and exposed for a given period of time, thus allowing microbe-bearing particle to deposit on to them. Petri dishes which are 90mm in diameter (approximate internal area 64 cm 2 ) are most commonly used. The number of microbe bearing particles deposited on to the agar surface of the plate over the period of exposure is determined by incubation of the plate and counting the number of microbial colonies, more commonly known as colony forming units (cfu). The microbial deposition rate may be reported as the number depositing in a given area per unit time. Sample location for settle plates in clean rooms should include areas where there is little air movement (dead spaces) or where airflows converge or are excessively turbulent. Areas where these conditions are most likely to occur are: Adjacent to doors At low level return air grilles Between HEPA s in clean rooms In corners of rooms

11 b. Active air sampling Control of viable particles or microorganisms, which are typical of certain types of manufacturing environments or products, is essential. Whilst there are finite limits and standard methods for the evaluation of non-viable particles within the pharmaceutical industry, as yet there is no definitive standardization of methods for the microbiological evaluation of airborne particles. Active air samplers all work on the principle of sucking or blowing a stream of air at a sufficient high velocity to cause any microorganisms in the sample to be impacted against a chosen medium. Areas where a microorganism controlled environment is necessary should be specified in a sampling plan and should be monitored. These include critical zone of clean air devices, transfer devices, background environments for clean air devices and changing rooms and areas where there is personnel activity or specific operations are carried out. Eg: Adjacent to bench area where trays that have been passed into the clean room are held before being transferred into a clean air device. The number and volume of sample taken in each area should be considered. The number of samples will depend on The size and The grade of the area being monitored In total, a minimum volume of 1000 liters (1m 3 ) should be sampled. This may be obtained from one sample or in larger areas from a number of samples. Sampling of air is done by means of samplers. Centrifugal sampler The air sample is drawn in to the sampling head by means of an impeller. The impeller then direct the air on to an agar strip fitted around the circumference of the sampling head.

12 c. Finger dab plates While this method does not actually measure environmental contamination, the data it generates is as important as those generated by other environmental monitoring techniques. Finger dab plates can be used to show a breakdown in operator aseptic technique where the operator touches a contaminated surface. Eg: Face when adjusting mask and contamination is transferred to the operators hand and then to products or materials that are handled in the critical work zone. They may also show a breakdown in the transfer process disinfection. Poor technique may lead to items being insufficiently disinfected and transferred in to the clean room or clean air device whilst still contaminated. The contamination may then be transferred to the operator gloved hands and then form there to product and materials. In addition finger dab plates can be used to evaluate operator training. Examine the plates for contamination before use. Sampling should take place at the end of a work session prior to carrying out any cleaning or tidying operations. The operator should lift the lid of the plate, with the opposite hand to that being tested, and keeping hold of the lid in the other hand, touch the agar surface with the tips of all fingers then the thumb (in the gap on the plate behind where fingers were tested) on the hand being tested. A firm and even pressure should be applied for approximately 5 to 10 seconds, taking care not to damage the agar surface. Replace the lid of the plate. Repeat the process for the other hand. Clean tested glove surface with a suitable before performing any other operations. d. Contact plates

13 Standard contact plates (RODAC: Replicate Organism Detection and Counting) of 55mm diameter (approximately 25 mm 2 internal area) should contain sufficient agar growth media to create a raised media surface, and may have a grid scored on the base. The convex agar meniscus allows direct application to test surfaces (e.g. walls, floors, equipment0 for hygiene control. Surface may become contaminated in a number of ways. Eg: Microorganisms settling out from the environment or from the direct touch by an operator. Once of the objectives of surface sampling is to determine the efficiency of routine cleaning procedures in removing contamination. Therefore, sampling should be done and after cleaning to determine the effectiveness of the cleaning procedure. The medium used may contain neutralizing agents, which inactivate any residual disinfectants on the surface to be tested and therefore enable comparative results before and after cleaning. Contact plates are best used for detecting microorganism that may be present on flat surfaces in relatively low numbers. 02. Physical methods Control of airborne contamination requires certain measures to be adopted, which are usually dictated by the type of product required at the end of the preparation process. Measurement and determination of the number and size of airborne particulate contamination is essential to ensure that a suitable environment is maintained for the preparation of aseptically prepared products. a. Non-viable particle counts Particles are significant because they can contaminate and also carry organism to sterile area. Therefore critical environment measurements are taken at a distance not more than 30cm from worksite, within airflow and during filling/closing operations. Assessment of particulate air makes the process difficult when process itself generate particles. Eg: powder filling

14 Appropriate alert and action limits should be set and corrective actions are defined if limits exceeded. b. Pressure differentials Positive pressure differential of Pascals should be maintained between adjacent rooms of different classification (with door closed). Most critical area should have the highest pressure. Pressure should be continuously monitored and frequently recorded. Alarms should sound if pressures deviate from standard level. Any deviations should be investigated and effect on environmental quality is determined. c. Air changes/ Air flow patterns/ Air flow velocity Uni-directional over critical areas (laminar flow) must be maintained to prevent generation of turbulent air. Sufficient velocity must be there to sweep particles away from filling/closing area. Clean up time/recovery time: Achieve particulate levels for the Grade A at rest state after a short clean-up period of 20 minutes after completion of operations. Maintain particle counts for Grade A in operation state whenever product or open container is exposed. Air speed of approximately 0.45m/s ± 20% at working position (guidance value) in laminar airflow work station should be maintained. d. Temperature and Relative Humidity Ambient temperature and humidity should not be uncomfortably high and a temperature of 18 C is suitable. Uncomfortable conditions could cause operators to generate particles. Validation and in-process monitoring of sterilization procedures There are several definitions of validation but, in simple terms, the word means demonstrating that a process will consistently produce the results that it is intended to. Thus, with respect to sterile products, validation would be necessary for each of the individual aspects of the manufacturing process, e.g. environmental monitoring, raw materials quality assessment, the sterilization process itself and the sterility testing procedure. Of these, it is the sterilization process that is likely to be subject to the most detailed and complex validation procedures, and these will be used to exemplify the factors to be considered. A typical validation procedure for a steam sterilization process is likely to incorporate most, or all, of the following features: The calibration and testing of all the physical instruments used to monitor the process, e.g. thermocouples, pressure gauges and timers. Production of evidence that the steam is of the desired quality (e.g. that the chamber temperature is that expected for pure steam at the measured pressure). The conduct of leak tests and steam penetration tests using both an empty chamber and a chamber filled with the product to be sterilized in the intended load conformation.

15 The use of biological indicators either alone or in combination with bioburden organisms to demonstratestrate that the sterilization cycle is capable of producing an acceptable level of sterility assurance under worst case conditions. The production of data to demonstrate repeatability of the above (typically for three runs). Comprehensive documentation of all of these aspects. There are different approaches to the demonstration of adequate sterility assurance in steam sterilization depending upon the thermostability and knowledge of the presterilization bioburden. Where the product is known to be stable, an overkill approach may be adopted in which biological indicators containing 10 6 test organisms are inactivated in half the proposed exposure time (thus achieving a 12 log reduction and a sterility assurance level of 10-6 in the full exposure period). For a marginally thermostable product the cycle could be validated on the basis of measurements of the worst case bioburden level and the heat resistance of the bioburden organisms; such an approach would necessitate rigorous control of the bioburden during routine manufacturing. Chemical indicators of sterilization are more convenient to use than biological indicators, but as they provide no direct measure of the efficacy of the process in terms of microbial killing they are considered to be less useful. Physical measurements of temperature, pressure, time, relative humidity, etc. are of such fundamental importance to the assurance of sterility that records of these parameters are retained for each batch of sterilized product. Sterility test Sterility test is a quality control test used as a part of product release for products required to be sterile. It is associated with significant statistical limitations and it will really detect gross contamination only. This test is applied to substances, preparations or articles which, according to the Pharmacopoeia, are required to be sterile. However, a satisfactory result only indicates that no contaminating micro-organism has been found in the sample examined in the conditions of the test. 1. Sampling The number of containers tested per batch and quantity tested from each container should be, as a minimum, in accordance with the pharmacopeial method followed. Sample from aseptic fills should be selected from at least the beginning, middle and end of the batch fill. Additionally, Standard Operating Procedures (SOPs) should define criteria for inclusion and collection of samples immediately after interruptions and operator interventions during the filling process. Samples from terminal sterilization cycles should be selected from at least the potentially coolest part of the load if such a location was identified during validation studies and from every load sterilized.

16 If an original test is declared invalid, then any samples used for the repeat sterility test should reflect the original samples in terms of sampling locations or aseptic processing times. 1. Parenteral preparations 2. Antibiotic solids 3. Ophthalmic preparations 4. Devices 5. Solids and liquids 2. Media

17 The media used should be in accordance with the pharmacopoeial method followed and should be capable of supporting growth of a range of low numbers of organisms in the presence of product. Soya-bean casein digest (SCD) and fluid thioglycollate media (FTM) should normally be used. a. Soyabean Casein Digest medium (SCD) Also known as Trypton Soy Broth (TSB) Suitable for the culture of both aerobic bactria and fungi A medium to be incubated at C (for bacteria) b. Fluid Thioglycollate medium (FTM) Primarily intended for the culture of anaerobic bacteria However, it will also detect aerobic bacteria A medium to be incubated at C Alternative media are permitted and may be appropriate if the nature of the product or method of manufacture could result in the presence of fastidious organisms (e.g. vaccines, bblood products, etc). Validation studies should demonstrate that alternative media are capable of supporting the growth of a wide range of microorganisms. Inactivators of antimicrobials may be incorporated in to growth media or rinse solutions as indicated by validation studies. 3. Methods There are three alternative methods available when conducting sterility tests. a. Direct inoculation The direct inoculation procedure involves introducing test samples directly in to nutrient media. b. Membrane filtration Membrane filtration id the technique recommended by most pharmacopoeias and involves filtration of fluids through a sterile membrane filter (pore size = 0.45 µm), any microorganism present being retained on the surface of the filter. After washing in situ, the filter is divided aseptically and portions transferred to suitable culture media which are then incubated at the appropriate temperature for the required period of time. Water-soluble solids can be dissolved in a suitable diluent and processed in this way. c. A sensitive method for detecting low levels of contamination in intravenous infusion fluids involves the addition of a concentrated culture medium to the fluid in its original container, such that the resultant mixture is equivalent to single strength culture medium. In this way, sampling of the entire volume is achieved.

18 4. Incubation period All test containers should be incubated at temperatures specified by the pharmacopoeial method for each test media for at least 14 days, regardless of whether filtration or direct inoculation test methodology is used. The temperature of incubators should be monitored and there should be records of calibration of the temperature monitoring devices. Test containers should be inspected at intervals during the incubation period and these observations recorded. If the product produces a suspension, flocculation or deposit in the media, suitable portions (Eg: 2-5%) of the contents of the containers should be transferef to fresh media under clean room conditions, after 14 days and re-inoculated for further 7 days. 5. Negative controls Negative product controls, which are similar in type and packaging to the actual product under test, should be included in each test session. These controls facilitate the interpretation of test results, particularly when used to declare a test invalid because of contamination in the negative product controls. A minimum of ten negative product control containers may be adequate to stimulate manipulations by the operator during a membrane filtration test. An equivalent number of sample to the test samples may be necessary to stimulate the manipulation of the product by the operator/s during a direct inoculation test. The negative control contamination rate should be calculated and recorded. 6. Positive control It is essential to show that microorganism will actually grow under the conditions of the test. For this reason positive controls have to be carried out; in these, the ability of small number of suitable microorganism to grow in media in the presence of the sample is assessed. The microorganism used for positive control test with a product containing or comprising an antimicrobial agent must, if at all possible, be sensitive to that agent, so that growth of the microorganism indicates a satisfactory inactivation, dilution or removal of the agent. The British Pharmacopoeia suggest the use of appropriate strains of following positive controls. Staphylococcus aureus, bacillus subtilis and Pseudomonas aeruginosa as aerobic organism Clostridium sporogenes as anaerobic organism Candida albicans and Aspergillus niger as fungi a. Growth promotion test

19 Challenge organism strains that are used to verify the fertility of each batch of standard test media should be selected from those reference strains specified by the pharmacopoeial method. Environmental or fastidious organisms may be used if alternative non-selective enrichment media have been selected for the sterility test. Media purchased from external vendors should be accompanied by certification of the growth promotion test performed on each batch of media. The test need not be repeated by the sterility testing laboratory provided there is documented control over the conditions used to transport media between the media manufacturer and the sterility testing laboratory. The media should be inoculated with CFU of challenge organisms. The challenge inoculum should be verified by concurrent viable plate counts. Growth promotion challenge organisms should show clearly visible growth in the test media within 3 days for bacteria and 5 days for fungi. There should be written instructions and protocols covering all procedures for the preparation, maintenance and cultivation of test organism strains. The identity (morphological and physiological properties) of the strains should be checked periodically. The growth promotion test may be performed concurrently with the product sterility test. b. Validation (bacteriostasis and fungistasis) test The test methodology should be validated by inoculation with CFU of challenge organism strains to the media/product container at the beginning of the test incubation period. The challenge inoculum should be verified by concurrent viable plate count. The preferred validation method involves addition of challenge organisms directly to the product prior to direct inoculation or membrane filtration. However where this is not practical due to inhibition or irreversible binding by the product, the challenge organisms should be added directly to the media containing the product in the case of direct test methodology or to the last rinse solution if membrane filtration methodology is used. The test is declared invalid if validation challenge organisms do not show clearly visible growth of bacteria within 3 days and fungi within 5 days in the test media containing product. In most cases, unless the sterile product causes turbidity in the media, visual recovery times should be comparable to those of the growth promotion test. Validation should be performed on all new products and repeated whenever there is a change in the experimental conditions. Although it is not a pharmacopoeial requirement, it is good laboratory practice to re-validate under the current experimental conditions every 12 months. Records of validation and/or re-validation tests should be maintained in the change control procedure protocol. 7. Antimicrobial agents

20 Where an antimicrobial agent comprises the product or forms part of the product, for example as a preservative, its activity must be nullified in some way during sensitivity testing so that an inhibitory action in preventing the growth of any contaminating microorganisms is overcome. This is achieved by following methods. a. Specific inactivation An appropriate inactivating (neutralizing) agent is incorporated in to the culture media. The inactivating agent must be non-toxic to microorganism as must any product resulting from an interaction of the inactivator and the antimicrobial agents. (table) b. Dilution The antimicrobial agent is diluted in the culture medium to a level at which it ceases to have any activity, for example phenols, cresols and alcohols. This methid applies to substances with a high dilution coefficient, R. c. Membrane filtration This method has traditionally been used to overcome the activity of antibiotics for which there are no inactivating agents, although it could be extended to cover other products if necessary, eg: these containing preservatives for which no specific or effective inactivators are available. Basically a solution of the product is filtered through a hydrophorbic-edged membrane filter which will retain any contaminating microorganisms. The membrane is washed in situ to remove any trace of antibiotic adhering to the membrane and is then transferred to appropriate culture media.

21 8. Results interpretation Record should include nature, size, composition and origin of each item in the sample, method of testing, nature and volume of media, neutralizing agent employed, results of test, results of negative control and results of positive control. Presence of growth is detected by periodic inspection by turbidity, centrifugation, microscopy of the sediment and subculture. (Picture) 9. Repeat test A test may be repeated only when it can be demonstrated that the test was invalid for causes unrelated to the product being examined. The European Pharmacopeia restricts criteria to one or more of the following conditions only: a. The data of the microbiolgical monitoring of the sterility testing facility show a fault; b. A review of the testing procedure used during the test in question reveals a fault; c. Microbial growth is found in the negative controls; d. After determination of the identity of the microorganisms isolated from the test the growth of this species or these species may be ascribed unequivocally to faults with respect to the material and/or the technique used in conducting the sterility test procedure. When conditions (a), (b) or (c) apply then the test should be aborted prior to the completion of the incubation period. For condition (d) to apply as the sole criterion used to invalidate a test, it is necessary to demonstrate that a micro-organism isolated from the product is identical to an isolate from the materials and/or the environment. This determination entails the use of a sensitive typing technique such as a molecular typing technique or other techniques similar to those used for epidemiological studies. However, if tests are performed competently in a clean room environment the chance of simultaneous adventitious contamination occurring in the environment, test sample and negative control is negligible. Provisions that allow repeat testing based on morphological or biochemical characterization of environmental and/ or product contaminants should not be permitted. It is possible for the environment to become contaminated by the samples under test, which may contain multiple micro-organisms that are difficult to differentiate without emplying sensitive typing techniques.

22 If contamination, which is established to be unrelated to the product, occurs in the original test, the test may be repeated with the same number of test samples as used in the original test, with negative product controls tested concurrently. If contamination is detected in the repeat test performed on the same number of test samples, the product does not comply with the test for sterility and the entire batch should be rejected. 10. Special modification for testing 1. Test for Mycobacterium tuberculosis in BCG vaccine 2. Surgical sutures 3. Devices 4. Dressings 5. Paraffin gauze 1. Test for Mycobacterium tuberculosis in BCG vaccine Virulent organism grows under the same conditions as the attenuated BCG Carriers of virulent organisms are not uncommon Accidental contamination would be dangerous Test involves. - Low speed centrifugation of 25ml of the product suspended in saline or preservative inhibitor - Centrifuge - Resuspend in saline - Inoculate into 10 tubes - Incubate at 37 C for 42 days 2. Surgical sutures Incubation period is 14 days. Founding of microorganisms require repeating of the test. Presence of microorganisms in the repeat test concludes the catgut is not sterilized externally. Leakage are tested simultaneously by immersion in an antiseptic dye solution before opening. In this test made more precise by applying and releasing a vacuum. Preservatives may automatically dilute off with the transfer of suture to the medium. 3. Devices Whole device should be incubated in an aerobic/ anaerobic media If not possible to incubate the whole device, - Crucial parts could be incubate - Device is rinsed with sterile medium and ringsings are tested

23 4. Dressings Whole dressing up to 500g is transferred to media or 2 50g portions from the innermost part of the sample are transferred to media