Culture Media for Compendial Methods

Compendial pharmacopeial tests are standardized methods for many pharmaceutical raw materials and finished products. Testing and compliance with these standards detailed within compendial methods is a basic requirement for the global product development, release, and distribution of pharmaceutical ingredients and drug products. Compendial tests are performed to determine drug compatibility, and safety or for routine quality control.

MICROBIOLOGICAL QUALITY CONTROL OF NON-STERILE PRODUCTS

The three internationally most important Pharmacopoeias (Ph Eur/JP/USP) published harmonized versions of their chapters on microbial examination of non-sterile products in 2009. In addition, the 2016 edition of the Chinese Pharmacopoeia is, for the most part, harmonized in terms of media composition and performance criteria, although growth promotion testing needs to be performed with strains from the Chinese strain collection. These methods address the total aerobic count (TAMC and TYMC) and the test for specified microorganisms.

Our culture media portfolio for microbial examination of non-sterile products complies with the harmonized Pharmacopoeia. As compendial methods change, be assured that we monitor this thoroughly, and any changes to composition or performance testing will be implemented quickly so that you won’t need to worry about the regulatory compliance of our media.

We provide a choice of products for all culture media and substances required for:

• Sample Preparation
• Microbial Enumeration Tests
• Tests for Specified Microorganisms

Browse from our range of products to support your compendial pharmacopeial tests:

• Dilution media
• Dehydrated culture media (DCM)
• Culture media - Ready-to-use plate
• Culture media - Ready-to-use liquid
• Neutralizing agents

MICROBIOLOGICAL QUALITY CONTROL OF NON-STERILE PRODUCTS

The three internationally most important Pharmacopoeias (Ph Eur/JP/USP) published harmonized versions of their chapters on microbial examination of non-sterile products in 2009. In addition, the 2016 edition of the Chinese Pharmacopoeia is, for the most part, harmonized in terms of media composition and performance criteria, although growth promotion testing needs to be performed with strains from the Chinese strain collection. These methods address the total aerobic count (TAMC and TYMC) and the test for specified microorganisms.

Our culture media portfolio for microbial examination of non-sterile products complies with the harmonized Pharmacopoeia. As compendial methods change, be assured that we monitor this thoroughly, and any changes to composition or performance testing will be implemented quickly so that you won’t need to worry about the regulatory compliance of our media.

PHARMACOPEIA COMPENDIAL TESTING

Regulatory agencies must implement pharmaceutical drug testing and follow the respective pharmacopeia methods or validated methods.

European Directorate for the Quality of Medicines and Healthcare (2017)

The European Pharmacopoeia. 9th Ed. Chapter 2.6.12 Microbiological examination of non-sterile products: Microbial enumeration tests and Chapter 2.6.13 Microbiological examination of non-sterile products: Test for specified products. Strasbourg, France.

Japanese Ministry of Health, Labour and Welfare (2016)

The Japanese Pharmacopoeia. 17th Ed. Chapter 4.05 Microbial Limit Test I. Microbiological examination of non-sterile products: Total viable aerobic count and II. Microbiological examination of non-sterile products: Test for specified products. Japanese Ministry of Health, Labour, and Welfare. Tokyo, Japan.

United States Pharmacopeial Convention (2014)

The United States Pharmacopeia 38/National Formulation 33, Supp. 2. Chapter <61> Microbiological examination of non-sterile products: Microbial enumeration tests and Chapter <62> Microbiological examination of non-sterile products: Test for specified products. Rockville, Md., USA.

SAMPLE PREPARATION FOR COMPENDIAL TESTING

The choice of the dilution media and additives used depends on the properties of the test sample. The products with antimicrobial properties can be countered by using neutralizing agents.

Table 1. Products used as diluents and neutralizing agents during sample preparation.

Medium/ Substance	Intended Use
Buffered Sodium Chloride Peptone Solution pH 7.0 Phosphate Buffer Solution pH 7.2 Soybean Casein Digest Broth	Diluents for all non-fatty products, soluble or insoluble in water
Polysorbate 80	Products insoluble in water; Added to assist the suspension of substances with poor wettability fatty products. Neutralizing QACs, iodine, and parabens
Isopropyl Myristate	Diluents for fatty products
Lecithin	Transdermal patches. Neutralizing quaternary ammonium compounds (QACs), parabens and bis-biguanides
Sodium Hydrogen Sulfite	Neutralizing glutaraldehyde and mercurials
Glycine	Neutralizing aldehydes
Thioglycollate	Neutralizing mercurials
Thiosulfate	Neutralizing mercurials, halogens, and aldehydes

COMPENDIAL METHODS FOR MICROBIAL ENUMERATION

Microbial Enumeration Tests are quantitative methods to determine the contamination level of mesophilic bacteria and fungi that grow under aerobic conditions in a pharmaceutical product. The tests include detecting the total aerobic microbial count (TAMC) and total yeast and mold count (TYMC). Membrane Filtration, Plate-Count, or the Most-Probable-Number (MPN) is the most common enumeration method, and the choice of method depends on the test product.

Before a method is chosen, the suitability should be proven in the presence of the product. This involves the preparation of suitable test strains, growth promotion tests of the culture media, and suitability tests for the counting method in the presence of the product.

Table 2. Media suitability for test strains

Test Strains	Preparation of Test Strains	Growth Promotion	Suitability of Counting Method in the Presence of Product
Staphylococcus aureus	Soybean Casein Digest Agar or Broth	Soybean Casein Digest Agar or Broth	Soybean Casein Digest Agar (MPN) or Broth
Pseudomonas aeruginosa	Soybean Casein Digest Agar or Broth	Soybean Casein Digest Agar or Broth	Soybean Casein Digest Agar (MPN) or Broth
Bacillus subtilis	Soybean Casein Digest Agar or Broth	Soybean Casein Digest Agar or Broth	Soybean Casein Digest Agar (MPN) or Broth
Candida albicans	Sabouraud Dextrose Agar or Broth	Soybean Casein Digest Agar (TAMC) Sabouraud Dextrose Agar (TYMC)	Soybean Casein Digest Agar (TAMC) Sabouraud Dextrose Agar (TYMC)
Aspergillus brasiliensis	Sabouraud Dextrose Agar or Potato Dextrose Agar and Polysorbate 80 (optional)	Soybean Casein Digest Agar (TAMC) Sabouraud Dextrose Agar (TYMC)	Soybean Casein Digest Agar (TAMC) Sabouraud Dextrose Agar (TYMC)

Figure 1. Diagram for microbial enumeration testing between 1. TAMC: Mixed microbial growth on Soybean Casein Digest Agar, and 2. TYMC: Mixed Yeast and Mold growth on Sabourated Dextrose Agar. A) Soybean Casein Digest Agar B) Soybean Casein Digest Broth C) Sabouraud Dextrose Agar D) Sabouraud Dextrose Agar with Chloramphenicol*

*Can be used to suppress bacterial growth threatening to exceed TYMC acceptance criteria.

Tests for Specified Microorganisms

Depending on the microbiological quality specification of each non-sterile pharmaceutical product, the manufacturer is required to determine the absence or limited occurrence of the following seven organisms:

1. Bile-Tolerant Gram-Negative Bacteria
2. Escherichia coli
3. Salmonella
4. Pseudomonas aeruginosa
5. Staphylococcus aureus
6. Clostridia
7. Candida albicans

These organisms serve as general indicator organisms, but depending on the nature of the product, its designated use, the recipient group, and the method of application, other pathogenic microorganisms may be relevant. The following overview of required media is limited to explicit Pharmacopoeia requirements.

Organism:

Bile-Tolerant Gram-Negative Bacteria

Dilution / Enrichment:
Soybean Casein Digest Broth
Enterobacteria Enrichment
Broth Mossel

Subcultivation:
Violet RedBile Glucose Agar

Organism:

Escherichia coli

Dilution / Enrichment:
Soybean Casein Digest Broth
MacConkey Broth

Subcultivation:
MacConkey Agar

Organism:

Salmonella

Dilution / Enrichment:
Soybean Casein Digest Broth
Rappaport-Vassiliadis
Salmonella Enrichment Broth

Subcultivation:
Xylose Lysine Deoxycholate Agar

Organism:

Pseudomonas aeruginosa

Dilution / Enrichment:
Soybean Casein Digest Broth

Subcultivation:
Cetrimide Agar

Organism:

Staphylococcus aureus

Dilution / Enrichment:
Soybean Casein Digest Broth

Subcultivation:
Mannitol Salt Agar

Organism:

Candida albicans

Dilution / Enrichment:
Sabouraud Dextrose Broth

Subcultivation:
Sabouraud Dextrose Agar

Organism:

Clostridia

Dilution / Enrichment:
Reinforced Medium for Clostridia

Subcultivation:
Columbia Agar