Microbiological examination of non-sterile products. Screening tests and microbiological counts (Eur. Pharm. 2.6.12 and Eur. Pharm. 2.6.13. Eur Pharmacopoeia 11).
These tests describe the quantitative count of mesophilic bacteria and fungi that can develop under aerobic conditions, to determine if a substance or preparation meets a microbiological quality specification, indicating the number of samples to be studied and how to interpret the results obtained (Eur. Pharm. 2.6.12) and to determine the presence of some specific types of microorganisms (Eur. Pharm. 2.6.13). These methods are not applicable to products that contain viable microorganisms as active ingredients.
As a general rule, these tests must be performed with methods that guarantee the absence of external microbial contamination with any microorganism in the product under test. In addition, the antimicrobial activity that the products may contain must be eliminated or neutralized and any toxic effect for microorganisms must be avoided during the test performace. The methods used for counting can be membrane filtration or plate count methods. The Most Probable Number (MPN) method is the least accurate method for microbiological enumeration and should only be used for products with a very low microbial load, in which case it may be the most appropriate method. The method chosen depends on the nature of the product and the limit of microorganisms required. A negative control with the diluent used instead of the pharmaceutical preparation under test is included in the tests, in which no growth of microorganisms should be obtained.
In the tests 10 g or 10 mL of the product must be used with the following exceptions: for liquids or solids in liquid form or solids addressed to be used in aerosol form, 10 containers should be used. For products in the form of transdermal patches, 10 patches samples. The amounts can be reduced for active substances that are formulated under the following conditions: amounts per unit dose (e.g. tablets, capsules, injection) that are equal to or less than 1 mg or the amount per gram or milliliter (for products presented in unit dose) is less than 1 mg. In thoses cases the amount tested is not less than the amount present in 10 unit doses or 10 g or 10 mL of product. For materials such as active substances where the amounts in the sample quantities are limited or the batch sizes are too small (less than 1,000 mL or 1,000 g), the amount tested may be 1 percent of the batch, unless otherwise specified, prescribed a less or justified and authorized amount. For products where the total number of units in a batch is less than 200 (e.g, samples used in a clinical trial), the sample size may be reduced to 2 units, or 1 unit if the size is less than 100.
Samples should be selected at random from the batch or containers available for preparation. To obtain the required sample, mix the contents of a sufficient number of containers to provide the sample.
The Eur. Pharm 2.6.12, describes the determination of the TAMC (Total Aerobic Microbial Count) and the TYMC (Total combined Yeast/Molds Counts), whereas the Eur. Pharm 2.6.13, specifies detection and count of some microorganisms, to determine its absence or limited presence:
- Bile-tolerant gram-negative bacteria.
- Candida albicans.
- Clostridia.
- Escherichia coli.
- Pseudomonas aeruginosa.
- Salmonella spp.
- Staphylococcus aureus.