Lab experiment sterility testing
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This document describes sterility testing methods to ensure pharmaceutical preparations are sterile. There are two recommended sterility testing methods: direct inoculation and membrane filtration. In direct inoculation, samples are directly inoculated into fluid thioglycolate medium and tryptic soy broth, which are then incubated. In membrane filtration, samples are filtered through a 0.25 micron membrane and the membrane is placed in the media and incubated. After incubation, turbidity indicates a non-sterile sample while transparent media indicates a sterile sample. These sterility testing methods help validate that pharmaceutical products are free of microbes.
Lab experiment sterility testing
- 2. Experiment: Sterility Testing To Ensure the Sterility of Pharmaceutical Preparations Materials Required • Media (1. FTG: Fluid Thioglycolate Medium (used for the detection of aerobic and anaerobic bacteria), 2. TSB: Tryptic Soy Broth (used for the detection of aerobic bacteria, yeast and mold) • Standard organisms for growth promotion test: For the validation of FTG: (Staphyloccocus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 09027, Clostridium sporogens ATCC 11437). For the validation of TSB: (Bacillus subtilis ATCC 6633, Candida albicans ATCC 10321, Aspergillus niger ATCC 16404)
- 3. • Glass Vials (screw capped vials) • Filtration assembly • 0.25 micron filters (membrane filters) • Incubators • Test samples (Normal saline, Eye drops) Lab Utilities/Supplies Required
- 4. FTG (Fluid Thioglycolate Medium) Components Quantity g/L L-Cystein 0.5g Nacl 2.5g Dextrose 1g Yeast extract 5g Pancreatic digest of casein 15g Sodium thioglycolate/glycolic acid 3.5g Resazurin sodium salt 10g Agar 0.75g Distilled water Volume make up to 1L pH = 7.1+ - 0.2 Incubation conditions: 32.5°C±2.5°C for 14 days, under aerobic as well as anaerobic conditions
- 5. TSB (Tryptic Soy Broth) Components Quantity g/L Pancreatic digest of casein 17g Peptic digest of soybean meal 3g Dibasic potassium phosphate 2.5g Nacl 5g Dextrose 2.5g Distilled water Volume make up to 1L pH = 7.3+ - 0.2 Incubation conditions: 22.5°C±2.5°C for 14 days, under aerobic conditions
- 6. Principle (Introduction/Theoretical Background) Sterilization of pharmaceutical preparation such as injection, ampule water (water for injection WFI), normal saline, ointments, eye drops, some time antibiotics, is necessary to avoid any type of harmful effects in the users. For maintenance of sterility these products are either prepared under sterile conditions or are terminally sterilized . Sterilization during manufacturing is achieved in sterile manufacturing areas or reactors which have the controlled air filtration faculty with HEPA filter and installed UV lamps etc, while terminal sterilization refers to the manufacturing of product under normal clean conditions and then passing it through sterilization method by ultrafiltration of UV irradiation etc.
- 7. Once the sterile product is manufactured it is tested for sterility in the sterile laboratory (usually attached with the manufacturing area) (Design of sterile laboratory and manufacturing area is a topic of class assignment). There are two recommended methods of sterility testing (these two methods are usually agreed upon by all the pharmacopeia including USP (US pharmacopeia), BP (British Pharmacopeia), EU Pharmacopeia, Japnes Pharmacopeia etc) 1. Direct inoculation 2. Membrane filtration method
- 8. Direct Inoculation In this method the recommended quantity of the sample is directly inoculated in the two recommended media (FTG and TSB), and the media are incubated under recommended culture conditions for each media. Membrane filtration method In this method the recommended quantity of the sample is first passed through the membrane filter (usually 0.25 micron filter) on a filtration assembly, the solid or powder samples such as antibiotics are first dissolved in some sterile diluent and then are passed through the filtration assembly. Later the membrane filter is removed from the assembly, and is cut in to two halves with sterile scalpel, one half is inoculated in the FTG, while the other half is inoculated in the TSB. The media are incubated under recommended conditions. After incubation the appearance of turbidity or growth in the media is observed
- 9. Sample specifications No of articles in a batch Articles to be tested <100 4 articles >100 <500 10 articles >500 <1000 20 articles > 1000 2% of the articles Recommendations for liquid samples Quantity in a vial Quantity to be tested <1 ml Whole sample >1 ml <40 ml Half sample >40 ml <100 ml 20 ml of the samples > 100 ml 10-20% of the sample
- 10. Recommendations for solid samples Quantity in a vial Quantity to be tested <50 mg Whole sample >50 mg <300 mg Half sample >300 mg <5g 150 mg > 5 g 500 mg
- 11. Procedure All the sterility testing activities are performed under aseptic conditions in a sterile lab Direct Inoculation Method 1. Prepared the recommended media (FTG and TSB), adjust pH, dispense in screw capped bottles (15-20 ml broth in each vial) and autoclave 2. Take the sample vial to be tested, agitate the container, and aseptically transfer the recommended quantity of the sample to the recommended media 3. Incubate the media under specified conditions (FTG: 32.5± 2.5°C, for 14 days, under aerobic and anaerobic conditions, TSB: 22.5±2.5°C for 14 days under aerobic conditions)
- 12. Membrane Filtration Method 1. Prepare the recommended media (FTG and TSB), adjust pH, dispense in screw capped bottles (15-20 ml broth in each vial) and autoclave 2. Take filtration assembly , adjust 0.25 micron membrane filter in it, wrap the assembly in aluminum foil and autoclave it 3. Take the sample vial to be tested, agitate the container, and aseptically pass the recommended quantity of the sample through the filtration assembly 4. Remove the membrane filter from assembly, divide it in two equal halves with sterile cutter or scissor and transfer one half in the FTG and other in the TSB 5. Incubate the media under specified conditions (FTG: 32.5± 2.5°C, for 14 days, under aerobic and anaerobic conditions, TSB: 22.5±2.5°C for 14 days under aerobic conditions)
- 13. Results Interpretation 1. Turbidity in the media after incubation is the indicative of microbial growth and contamination and product is considered as non sterile 2. No turbidity or transparent media after incubation is the indicative of sterility and product is considered as sterile 3. Proper negative and positive controls are used in both the methods