Sterility testing of pharmaceutical or medical products helps assess whether they are free from contaminating microorganisms. These products should pass sterility testing because unsterile medical products can be hazardous to health.
Sterile means completely free from microorganisms, including highly resistant bacterial endospores. Either the part or the whole product is incubated in a suitable culture medium. Different pharmacopeia like USP (United State Pharmacopeia), British Pharmacopeia (BP), and Indian Pharmacopeia (IP) recommends fluid thioglycollate medium (FTM), alternative thioglycollate medium (ATM), and soybean casein digest medium (SCDM) as a suitable culture medium for sterility testing.
Table of Contents
Media for Sterility Testing
The three media which is used for sterility testing are:
Fluid Thioglycollate Medium (FTM)
Fluid thioglycollate medium supports the growth of the aerobes, anaerobes, and microphiles. It is used for the sterility testing of pharmaceutical products. It is used to examine clear liquid or water-soluble materials and store blood in the blood bank. Incubation is done at 30-35 ℃ aerobically.
| Ingredients | Gms/liter | Use |
| Agar | 0.75 g | Supports the growth of aerobes and anaerobes |
| L-cystine | 0.5 g | Lowers the oxidation-reduction potential |
| Sodium chloride | 2.5 g | Osmotic equilibrium |
| Dextrose monohydrate | 5.5 g | Growth factor for bacterial multiplication |
| Yeast | 5 g | Growth factor for bacterial multiplication |
| Pancreatic digest of casein | 15 g | Source of nitrogen and carbon |
| Sodium thioglycollate | 0.5 g | Reducing agent and neutralizes the toxic effects of mercurial |
| Resazurin sodium | 1 ml | Color indicator |
| Distilled water | 1000 ml | As a solvent |
Final pH (at 25 °C): 7.1±0.2
Alternative Thioglycollate Medium (ATM)
Alternative thioglycollate medium is used for the sterility testing of turbid and viscous products. It is also used for devices for determining the anaerobes in narrow tubes. It has a similar composition as the FTM except for the agar and the resazurin sodium. Incubation is done at 30-35 ℃, anaerobically.
| Ingredients | Gms/liter | Use |
| L-cystine | 0.5 g | Lower the oxidation-reduction potential |
| Sodium chloride | 2.50 g | Osmotic equilibrium |
| Dextrose monohydrate | 5.50 g | Growth factor for bacterial multiplication |
| Yeast extract | 5 g | Growth factor for bacterial multiplication |
| Pancreatic digest of casein | 15 g | Source of nitrogen and carbon |
| Sodium thioglycollate | 0.5 g | Reducing agent and neutralizes the toxic effects of mercurial |
| Distilled water | 1000 ml | As a solvent |
Final pH (at 25 °C): 7.1±0.2
Soybean Casein Digest Medium (SCDM)
Soybean casein digest medium (SCDM) is used for sterility testing of molds and the lower bacteria. Incubation is done at 20-25 ℃ aerobically.
| Ingredients | Gms/liter | Use |
| Dextrose | 2.5 g | Growth factor for bacterial multiplication |
| Dipotassium hydrogen phosphate | 2.5 g | Lower the oxidation-reduction potential |
| Pancreatic digest of casein | 17.0 | Source of amino acids and peptides |
| Papaic digest of soybean meal | 3.0 | Reducing agent and neutralizes the toxic effects of mercurial |
| Sodium chloride | 5.0 | Osmotic equilibrium |
| Distilled water | 1000 ml | As a solvent |
Final pH (at 25 °C) 7.3 ± 0.2
Methods of Sterility Testing
Sterility testing is done by membrane filtration and direct inoculation method.
Membrane Filtration
- Membrane filtration sterility testing for pharmaceutical products is recommended by United States Pharmacopeia (USP), European Pharmacopoeia (Ph. Eur), and Japanese (JP) Pharmacopoeia.
- Membrane filtration is the method of the filtration of fluids through the sterile membrane filter having a pore size ≤ 0.45 μm and a diameter of approximately 50mm.
- A sample is filtered through the membrane filters having a flow rate of 55-75ml/min at a pressure of 70mm Hg. Membrane filters use cellulose nitrate for aqueous, oily, and weak alcoholic solutions and cellulose acetate filters for strong alcoholic solutions.
- If there is the presence of any microorganisms, then it is retained in the filter. Under the aseptic condition, filter through the membrane. Then aseptically remove the membrane and cut it into two halves.
- Then immerse one-half of the membrane in 100 ml of soybean casein digest medium and incubate at 20-25 ℃ for 14 days. Then immerse the other half in 100 ml of fluid thioglycollate medium and incubate at 30-35 ℃ for 14 days.
The samples whose quality needs to be checked can be any of the forms. It needs to be dissolved in a suitable diluent if it’s water-soluble. If it is oil soluble, then it is dissolved in a suitable solvent. Membrane filtration is used to test the following substances:
- Oil or oily preparation
- Ointments in solutions
- Soluble powder or bacteriostatic, fungistatic liquid
- Solid without antimicrobial properties and not soluble in the medium
- Nonbacteriostatic solids are not readily soluble in culture medium
Direct Inoculation
In the direct inoculation method, a test sample is directly inoculated in the culture media using a sterile pipette or syringe. If the test sample is an antimicrobial agent, it is neutralized by adding suitable inactivating agents to the medium. Then its incubated at 20-25 ℃ (soybean casein digest) and 30-35 ℃ (fluid thioglycollate) for 14 days.
Inactivation of Antimicrobial Agents in Sterility Testing
If any preservative is present in the product or the test sample is bacteriostatic or fungistatic, use the suitable sterile neutralizing agent. Then its action will be nullified so that it won’t prevent the growth of the contaminating microorganisms. To inactivate the antimicrobial agent, dilution can also be done. When the antimicrobial agent is diluted in the culture medium, it reaches the level at which it ceases to have any activity. An appropriate neutralizing or inactivating agent is incorporated into the culture media.
| Antimicrobial agent | Method of Inactivation/Inactivating agent |
| Penicillin | Penicillinase |
| Cephalosporins | Cephalosporinase |
| Streptomycin | Streptomycin adenyltransferase, Streptomycin phosphotransferase |
| Aminoglycosides | Acetyl-coenzyme A |
| Barbiturates | Dilute to 0.2% in culture medium with a pH 7.0 |
| Sulfonamides | P-aminobenzoic acid |
| Sorbic acid | Dilution and polysorbate-80 |
| Azide | Azolectin |
| Chloramphenicol | Chloramphenicol acetyltransferase |
| Organic acids | Polysorbate-80 |
| Phenol disinfectants | Dilution |
| Halogens | Sodium thiosulphate |
| Quaternary ammonium compounds | Lecithin+Lubrol or Tween 80 |
| Dyes | Membrane filtration |
| Heavy metals | Thioglycolic acid |
| Ethyl alcohol | Due to less than 1 % |
| Other antibiotics | Membrane filtration |
Results and Interpretation
- The sample passes the sterility test if there is no growth of microbes after the incubation.
- If growth is observed and turbidity is seen, then a re-test is done.
- If growth is observed again in the second test and cannot be distinguished from the second test, it fails.
- But if it can be distinguished from the first test, then the second re-test is done using twice the number of samples.
- If growth is not observed in the second re-test, it passes the test. If growth is observed, then it fails the test.
References
- Denyer, S. P., Hodges, N., Gorman, S. P., & Gilmore, B. F. (2011). Hugo and Russell’s Pharmaceutical Microbiology (Eighth). Wiley-Blackwell.
- Sandle, T. (2020). Sterility Testing – Overcoming Difficult Products. December.
- Bugno, A., Saes, D. P. S., Almodovar, A. A. B., Dua, K., Awasthi, R., Ghisleni, D. D. M., Hirota, M. T., de Oliveira, W. A., & de Jesus Andreoli Pinto, T. (2018). Performance Survey and Comparison Between Rapid Sterility Testing Method and Pharmacopoeia Sterility Test. Journal of Pharmaceutical Innovation, 13(1), 27–35. https://doi.org/10.1007/s12247-017-9303-z
- Bowman, F. W. (1969). The sterility testing of pharmaceuticals. Journal of Pharmaceutical Sciences, 58(11), 1301–1308. https://doi.org/10.1002/jps.2600581102