Introduction
Sterility testing ensures that pharmaceutical preparations meant for parenteral, ophthalmic or implant use are completely free from viable microorganisms. Any contamination in sterile products can cause serious infections. Sterility tests check whether microorganisms grow when the product is placed in suitable culture media under favourable conditions.
The principle is simple: if microorganisms are present in the test sample, they will grow in nutrient media and cause turbidity. If no growth occurs, the sample is considered sterile.
Factors Affecting Sterility Test
The probability of detecting microorganisms depends on:
- The number of microbes present in the sample
- The species of contaminating organisms
- The quantity of sample used
- Proper test conditions and handling
External surfaces of vials, ampoules and containers must be disinfected before the test. If the product is packed under vacuum, sterile air is introduced to avoid contamination during testing.
Cultural Media Used in Sterility Testing
Three types of media are commonly used:
1. Fluid Thioglycollate Medium (FTM)
Used mainly for detecting anaerobic bacteria, but can also detect aerobic species. The medium contains an oxygen indicator that becomes pink upon exposure to air. Before use, not more than the upper one-tenth of the medium should appear pink.
2. Alternative Thioglycollate Medium (ATM)
Used for turbid, viscous products and for medical devices with narrow lumens. It supports growth under anaerobic conditions.
3. Soyabean Casein Digest Medium (SCDM)
Supports growth of both fungi and aerobic bacteria. It is used for samples where a wide spectrum of microbial growth must be detected.
Suitability of Culture Media
Before performing the sterility test, culture media must pass the following evaluations:
Sterility Test of Media
FTM/ATM is incubated at 30–35°C, and SCDM at 20–25°C for at least 14 days. The media should remain clear without microbial growth.
Growth Promotion Test
Each batch of sterilized media is inoculated with about 100 viable cells of test organisms listed in pharmacopeia. The media should support visible growth within the recommended time.
Test for Bacteriostasis and Fungistasis
The product may inhibit microbial growth. This test checks whether the test sample interferes with growth of the standard microorganisms. If inhibition occurs, a suitable neutralizer or dilution must be used.
Methods for Sterility Testing
Sterility testing is performed using one of the following two methods:
- Method A: Membrane Filtration (preferred method)
- Method B: Direct Inoculation
Method A: Membrane Filtration
This is the preferred method for most pharmaceutical preparations, especially:
- Oils and oily solutions
- Ointments that can be solubilised
- Heat-sensitive liquids
- Large-volume samples (100 ml or more)
Apparatus
The membrane filtration assembly includes:
- A sterile filtration unit
- Membrane filter (0.45 µm pore size)
- Sterile receiving container
Cellulose nitrate filters are used for aqueous, oily and weakly alcoholic solutions; cellulose acetate filters are used for strong alcohol-containing samples.
Dilution Fluids
- Fluid A: Peptone-water solution adjusted to pH 7.1 ± 0.2
- Fluid B: Fluid A with 1% polysorbate 80 (for oily samples)
Procedure
The required amount of sample is filtered under vacuum. The membrane is divided aseptically:
- One half placed into SCDM (for aerobic microorganisms)
- Other half placed into FTM (for anaerobic microorganisms)
Both media are incubated for at least 14 days (FTM at 30–35°C and SCDM at 20–25°C).
Special Cases
- Oils: Filter directly through dry membrane
- Ointments: Dilute to 1% w/v using sterile diluent
- Solids: Dissolve in sterile diluent before filtration
- Antibiotics: Composite sample prepared and dissolved in fluid A
- Devices: Collect eluates by flushing the lumen with fluid B
Method B: Direct Inoculation
This method is used when membrane filtration is not suitable.
Procedure
The sample is directly inoculated into FTM and SCDM. The volume of sample should not exceed 10% of the volume of the medium unless specified.
- Aqueous solutions: Transfer sample directly
- Oils: Use emulsifying agents (polysorbate 80, etc.)
- Ointments/creams: Dilute 1:10 before inoculation
- Solids: Add directly to medium
- Dressings: Cut into pieces and immerse
- Devices: Immerse entirely or flush lumen with medium
Incubate inoculated media for 14 days under standard temperatures.
Interpretation of Results
After incubation:
- No turbidity: Sample passes sterility test
- Turbidity/growth: Sample fails, unless test is proven invalid
Test is Invalid If:
- Growth appears in negative controls
- Environmental monitoring shows contamination
- Procedure errors are identified
- Isolated microorganism is confirmed to be due to procedural fault
Key Points
- Sterility testing ensures pharmaceutical products are free from viable microorganisms.
- FTM, ATM and SCDM are the primary media used.
- Membrane filtration is the preferred method for most products.
- Direct inoculation is used for products unsuitable for filtration.
- Media must be tested for sterility, growth promotion and neutralization capacity.
- Results must be interpreted carefully after 14 days of incubation.
Detailed Notes:
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