Microbiological examination of non-sterile products. Screening tests and microbiological counts (Eur Pharmacopoeia 11, chapters 2.6.12 and 2.6.13.) 

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 performance. 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. For the detection of specific microorganisms, the pharmacopoeia describes a detection and identification procedure for each microorganism. 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.

Chapter 2.6.12 describes the determination of TAMC (total aerobic microorganism count) and TYMC (total combined yeast and mould count). In these 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. For orodispersible films, a sample of 10 films should be analysed. 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.

The total aerobic microbial count (TAMC) is considered to be the number of CFU found on TSA medium, while the total yeast/mold count (TYMC) is considered to be equal to the number of CFU found on Saboraud-dextrose agar.

Chapter 2.6.13 of the Pharmacopoeia specifies the detection and enumeration of some microorganisms, to determine their absence or limited presence:

-        Bile-tolerant gram-negative bacteria.

-        Candida albicans.

-        Clostridia.

-        Escherichia coli.

-        Pseudomonas aeruginosa.

-        Salmonella spp.

-        Staphylococcus aureus. 

For each determination, 1 g or 1 mL of the product to be examined is required, except for the determination of Salmonella spp., which requires at least 10 g or 10 mL and for the determination of Clostridia, which requires not less than 2 g or 2 mL of product.

Samples for any of the determinations must be selected at random from the batch or from the available containers. If necessary, to obtain the required sample, the contents of a sufficient number of containers must be mixed.

General Chapter 5.1.4 of the European Pharmacopoeia describes the acceptance criteria for the microbiological quality of non-sterile dosage forms and non-sterile substances for pharmaceutical use.