USP 61 Nonsterile Product Microbial Enumeration

How many microbes are in your drug product?  USP <61> will  determine the bioburden per gram of substance, by use of various culture methods.  The end goal is “colony forming units” or cfu per gram.  Each organism or “organism cluster” in a product that is properly diluted and cultured, with appropriate  media and temperature conditions, will eventually grow to a ”cfu” that can be visibly counted with the naked eye.  A notoriously inaccurate test, it is nevertheless the standard in determining cleanliness of drug products.

  • Aerobic and Yeast and Mold Plate Count
  • 10 gm of sample is diluted with a pH balanced diluent, usually supplemented with  neutralizers for antimicrobials present, and processed by shaking, stomaching, vortexing or other means.  Aliquots are placed in petri plates with nutrient agar.

  • Membrane Filtration
  • As above but rather than plating, diluent with product and microbes are passed through a 0.45 micron filter, capturing microbes while allowing diluent and product to pass through.  Filter is placed on nutrient agar plates, and microbes growth to visible colony forming units.

  • Microbial Suitability
  • Any microbial enumeration test must be validated to show that any organisms in the product can actually be cultured.  It is always possible that antimicrobial properties of the product will inhibit the growth of a microbe to a visible colony forming unit.  Validation or “suitability” is accomplished by inoculating plated product with low levels (<100 cfu) of test species and demonstrating equivalence to recovery of that organism in the absence of the product.  Microbial enumeration methods are considered suitable if product results are 50 – 200% of control results.