Lect 11- sterilization by filteration.pdf
- 3. Sterilization by Filtration • Filtration involves the physical removal (exclusion) of all cells in a liquid or gas. It is especially important for sterilization of solutions which would be denatured by heat (e.g. antibiotics, injectable drugs, amino acids, vitamins, etc.).
- 4. Sterilization by Filtration • Portable units can be used in the field for water purification and industrial units can be used to "pasteurize" beverages. Essentially, solutions or gases are passed through a filter of sufficient pore diameter (generally 0.22 micron) to remove the smallest known bacterial cells.
- 6. Advantages 1. Removes rather than destroys microorganisms i.e. Both living and dead cells are removed 2. Does not affect the physical or chemical integrity of the sterilized material 3. Have an important role in sterility testing
- 7. Disadvantages 1. Bacterial toxins and viruses are not removed 2. Requires highly trained staff 3. Only for liquids or solutions 4. Filling after sterilization demands good aseptic technique in an aseptic area which in turn increases the cost. 5. No in-process controls and sterility tests must be carried on the final preparation.
- 8. Types 1. Depth filters: made of sintered glass or ceramic (finely ground porcelain) or asbestos/cellulosic fibers (fibrous e.g. Seitz filters). Retains or traps MO within the filter matrix.
- 10. Types 2. Screen (membrane) filters: either membranous (thin film of cellulose ester) or nucleopore (thin film of plastic). Retain MO on the surface i.e. particles larger than the pore size are mechanically sieved and retained on the surface of the filters.
- 12. Membrane Filter Depth Filter - Retain bacteria inside sharp angled streaks - Disposable - Need support - Clogging is easy - Stability to chemicals is variable - Faster filtration rate - Low absorption and adsorption of fluids and chemicals - By screening - Durable - Not - Less easy - Good - Slower - High
- 13. A typical set-up in a microbiology laboratory for filtration sterilization of medium components that would be denatured or changed by heat sterilization. The filter is placed (aseptically) on the glass platform, then the funnel is clamped and the fluid is drawn by vacuum into a previously sterilized flask.
- 16. Control devices for sterilization Are devices to insure a good sterility status 1.Indirect or in-process: monitoring the sterilization process by the use of physical, chemical, and microbiological indicators 2.Direct or after manufacturing: monitoring it microbiologically through sterility testing
- 17. Physical indicators 1. Dry heat: by recording temperature (thermocoupling) and time during the process 2. Moist heat: monitoring pressure (pressure gauge), time, and temperature
- 18. Physical indicators 3. Ethylene oxide: monitoring pressure, humidity (hygrometer), and temperature 4. Radiation: recording the absorbed radiation dose
- 19. Chemical indicators Using chemicals which melt or change color at specific temperature or radiation dose. 1. Browne's tube: ampoule containing certain ester which produces acid at certain temperature and change in color.
- 21. Chemical indicators 2. Witness tube: sealed tube containing a compound that melts at sterilization temperature e.g. sulfur at 115°C 3. Steri strips: stickers which change in color during sterilization forming letters writing the word STERILE
- 23. Microbiological indicators By the use of a highly resistant MO used to impregnate paper or metal foil strips, which is tested at the end of the sterilization process for viability. The most commonly known is Bacillus stearothermophilus for validation of sterilization process in an autoclave.
- 27. Microbiological indicators The efficiency of bacterial filters could be validated by filtering culture of small gram negative microorganisms as Pseudomonas diminuta.
- 28. Mathematical methods • An increase in D- and z-values indicate leakage, presence of highly resistant organisms, which may necessitate re- evaluation of the sterilization cycle and equipment.
- 29. ASEPTIC AREA
- 30. ASEPTIC AREA The main importance of aseptic technique is to prevent the access of microorganisms during preparation, e.g., aseptic filling, and during testing of pharmaceutical products e.g. in sterility testing. Aseptic room is a special area the design of which greatly reduces contamination because of the following:
- 31. ASEPTIC AREA 1. Kept under slight positive pressure 2. Sterile air supply by filtration through HEPA filters
- 33. ASEPTIC AREA 3. Air in the room is changed 10 to 20 times per hour thus continuously removing organisms 4. Air must be routinely analyzed 5. UV irradiation when out of work
- 35. ASEPTIC AREA 6. Smooth walls, ceiling and floor to prevent accumulation of particles 7. Windows are double glass 8. Doors are double with air lock
- 36. ASEPTIC AREA 9. Persons should be free from infection and must wear sterile clothes
- 37. ASEPTIC AREA 10.Entry should be through the following sequence: 1. black area: non sterile 2. gray area: intermediate for washing and changing 3. white area: aseptic area 11.Chemical disinfection
- 40. LAMINAR FLOW CABINET • It is considered to be a small scale aseptic area. Air is forced through HEPA filter and comes out in parallel streams without turbulence to replace non sterile air. Disinfection and UV also minimize contamination when the cabinet is out of work. The air streams may be horizontal or vertical but vertical type is preferable when dealing with pathogenic microorganisms.
- 46. STERILIZATION OF AIR • Sterile air (less than 2 colonies/cubic feet) is required for: • Aseptic area for aseptic filling of sterile pharmaceuticals • Biotechnology in fermentation processes
- 47. Methods of air sterilization 1. Electrostatic precipitation: Retain 95% of particles and can be used as a prefilter. Air particles are charged either +ve or -ve by passing air through high voltage plates then separated by passing through alternating +ve or -ve plates which attract particles having opposite charge.
- 48. Methods of air sterilization 2. Treatment with chemicals: by spraying of gases or fluids such as formaldehyde or lactic acid 3. UV irradiation: used mainly to maintain sterility of air or reduce the level of contamination in hospitals
- 49. Methods of air sterilization 4. Filtration: used for sterilization with efficiency 99.9%. Depends on the use of several types of filters: 1. Cotton or glass wool fibrous pads filters 2. Cellulose paper filters 3. Cartridge membrane filter 4. High efficiency particulate air (HEPA) filters
- 50. Microbial analysis of air 1. Settling plates: Petri dishes with agar left open and then incubated and colonies are counted. This method is slow and not very accurate.
- 51. Microbial analysis of air 2. Agar impinges: by the use of air sampler, where controlled volume of air is forced to pass through a slit, below which a revolving small nutrient agar plate is placed.
- 53. STERILITY TESTING • Required for all injectables and ophthalmics. It is considered as a final control on sterilization. It is not important except in case of filtration. • It is a destructive test i.e. samples tested are not reused. It tests that samples are free from bacteria and fungi, but not viruses.
- 54. STERILITY TESTING • Batch: group of articles which are exposed to the same conditions i.e. prepared, mixed, packed and sterilized together.
- 55. STERILITY TESTING • Sterilization efficacy is controlled by its inactivation factor (IF) where: IF: a/b • “a” is the initial number & “b” is the final number of contaminants.
- 56. STERILITY TESTING • The degree of sterility (DS) is controlled by: DS = IF/x • where x is the number of contaminant (Bioburden). • If x= 10 and IF = 107, then DS = 107/10 = 106 i.e. among 106 units one will be non sterile, while if x was 107, then DS = 107/107 = 1 i.e. All units are non sterile.
- 57. Sample size • Two percent of the batch with maximum 20 articles selected randomly
- 58. Culture media for sterility testing – Fluid Thioglycolate Medium (FTM) for detection of both aerobic and anaerobic bacteria. Incubated for 14 days at 30-35°C – Trypticase Soya Broth (TSB) which supports both fungi and aerobic bacteria. Incubated for 14 days at 20-25°C
- 60. 1-Direct Inoculation 1. In the direct inoculation method, the test articles are inoculated directly into tubes or bottles that contain an appropriate medium and are incubated for a period of 14 days. The advantages of the direct inoculation method is that it provides a means of sterility testing for materials that cannot be easily filtered.
- 61. 1-Direct Inoculation 2. If no growth is observed after the incubation period, the batch pass the test. 3. If visible growth is observed, the test is repeated. If again growth is observed, the material fails the test.
- 63. 2-Membrane Filtration • In the membrane filtration method, the test article is passed through a membrane filter, which is designed to retain microbial contaminants while permitting the passage of liquid test articles and inhibitors out of the test system.
- 64. 2-Membrane Filtration • After the test article passes through the filter, the membrane is rinsed with an appropriate sterile rinse fluid. The membrane filters would capture the microorganisms, if present. The filter units are then inoculated directly onto appropriate medium and are incubated for the same time as in the direct inoculation method (i.e., 14 days).
- 67. 2-Membrane Filtration Advantages of the membrane filtration method include: 1.Accommodation of large volume samples (up to 500 ml) 2.Removal of inhibitory substances that inhibit the growth of microorganisms by rinsing the filter membrane with a suitable agent
- 68. 2-Membrane Filtration Disadvantages: 1.High probability of contamination during the procedure 2.Needs specific equipment
- 69. Controls for sterility test Control for sterility of the media (negative control): • Carried out by incubating uninoculated tubes under the same conditions, which should show no growth at the end of the incubation period
- 70. Controls for sterility test Control for growth promoting ability of the medium (positive control): • Carried out by separately inoculating the media with different MO. All tubes should show visible growth within the incubation period at the appropriate temperature.
- 71. Problems created during sterility testing and how to solve 1. Products which gave turbidity with the medium: direct transfer into FTM and TSB and after incubation for the required time (turbidity will mask the growth if present), subcultures into fresh media are made and again incubate and watch for growth.
- 72. Problems created during sterility testing and how to solve 2. Immiscible liquids: centrifuge the oil and the deposit is tested for sterility. 3. Surgical dressings and sutures: if small, the whole article is transferred directly to the medium, whereas in case of large packages a suitable portion if the innermost part is cut and transferred to the medium.
- 73. Problems created during sterility testing and how to solve 4. Sterilized devices: if small, transfer directly. If large or complex, the most difficult part to sterilize is removed and transferred to the medium.
- 74. Problems created during sterility testing and how to solve 5. Products with antimicrobial activity: a. Dilution of the product b. Antagonism by constituents of the medium c. Antagonism by addition of sterile materials which antagonize or destroy the preservative or the antibiotic
- 75. Limitations of sterility tests Sterility test is limited in its scope because: • It is conducted on a fraction of the total batch • Doesn't detect viruses, existing parasitic bacteria or the majority of thermophilic and psychrophilic bacteria
- 76. Limitations of sterility tests • Doesn’t detect organisms that have been shocked by sublethal heat treatment (these organisms require special recovery conditions) • May not detect aged bacterial spores which have long germination period • Low degrees of contamination may be missed
- 77. Limitations of sterility tests Therefore, sterility test detects relatively gross contamination in a product.
- 78. Validation of aseptic area • By system suitability test (SST): carried by testing for microorganisms in everything: in air, machines, persons, walls and floors by swabbing
- 79. Validation of HEPA filter • Performed by using dioctylphthalate vapor or certain type of oil that is forced through the filter by a fan then tested on the other side
- 80. Tests for pyrogens and bacterial endotoxins in pharmaceuticals
- 81. Tests for pyrogens and bacterial endotoxins • Pyrogens are a group of poorly characterized bacterial endotoxins (or products) which cause an immediate rise in temperature upon injection into humans and could lead to shock.
- 82. Test for Pyrogens 1- Measures the rise in temperature of standardized rabbits upon intravenous injection of a sterile solution of the substance (or the inside of a container) to be tested, under standardized conditions.
- 83. Test for Pyrogens 2-‘LAL test’: It derives its name from the name of the reagent used: Limulus Amebocyte Lysate reagent, which is obtained from extracts of the horseshoe crab Limulus polyphemus. A series of dilutions of the standard endotoxin and a series of dilutions of the preparation being tested are placed in test tubes, and to each the LAL reagent is added.
- 85. Test for Pyrogens The contents of each of tube are mixed and incubated, with positive and negative controls for 60 minutes. A positive reaction is obtained when a gel is observed that remains firm when inverted 180°. Any other reaction is negative. The test may be carried out on a slide with incubation for 20 minutes, protected from loss of moisture.