It is relatively straightforward to conduct a TVC and perform tests to detect the common objectionable organisms of pharmaceutical importance if the sample is a pure culture of healthy organisms suspended in a simple aqueous solution. Unfortunately, the reality is that samples frequently pose problems, because the organisms to be enumerated or detected are present in low concentrations as part of a mixture of organisms, and they may be starved and slow growing, dormant in the form of spores that are slow or difficult to germinate, or sublethally damaged. Furthermore, the sample might pose solubility problems, contain suspended solid particles to which the organisms are adsorbed and from which they are difficult to remove, or it may possess intrinsic antimicrobial activity that reduces the growth rate and ease of detection of organisms of interest. It is important to recognise the problems that might be posed by particular dosage forms, and the need to ensure that the procedure adopted does not present an opportunity for the bioburden organisms to reproduce simply by virtue of the long time period required for sample preparation. Delayed-release or enteric-coated tablets, for example, may not dissolve quickly in peptone water, so it may be necessary to aseptically triturate them using a sterile pestle and mortar. Bioburden testing of medical devices can often represent the greatest challenge of all because of the problems posed by sheer size and inaccessibility of internal surfaces to liquid culture medium.
The EP and USP give directions on the preparation of specimens for analysis. Both pharmacopoeias consider water-soluble products, nonfatty insoluble materials and fatty products. In addition, the EP covers transdermal patches and the USP considers aerosols. Useful information on sample preparation is provided by Millar,9 the Parenteral Drug Association,10 and, for medical devices, by ISO 11737.11
Where possible, samples should be dissolved in the culture medium or diluting fluid recommended in the pharmacopoeias. If the sample is insoluble in water it must be suspended, or, for fatty products, emulsified using surfactants and heat (< 40°C). In the rare cases where emulsification is not feasible, use of nonaqueous solvents is a possibility, but very few have acceptably low toxicity. Isopropyl myristate, for example, is recommended for solubilizing or diluting water-insoluble materials for compendial sterility tests (though not currently mentioned for bioburden determinations). Thorough validation, particularly with respect to toxicity to microorganisms, would be necessary if a case were to be made for using such a solvent. Insoluble solids are also suspended in an aqueous medium, and neither pharmacopoeia clarifies that the intention is normally to keep the sample in uniform suspension throughout the dilution and plating process, rather than to remove microorganisms from the suspended particles and count or detect them in the supernatant after the solid has sedimented. The latter strategy may occasionally be necessary if the density of suspended material is so great that colonies growing in, or on, the culture medium could not clearly be distinguished, but in this case validation to confirm the extent of removal of organisms from solid particles would be essential. Viscosity of the solubilized, suspended or emulsified sample should be considered, both from the perspective of volumetric errors resulting from incomplete drainage from pipettes, and, in the case of samples enumerated by membrane filtration, the problems of low flow rates. Measuring by weight overcomes the first, and raising both the temperature (to a value not exceeding 40°C) and the transmembrane pressure should increase flow rates.
Antimicrobial activity may be exhibited by components of the formulation; these may be either the active (e.g., antibiotics) or, more commonly, preservatives. While preservatives usually effect some degree of microbial killing, they will not render the product sterile, so bioburden determinations are still required. To avoid the problem of antimicrobial ingredients carrying over into diluting fluids and Petri dishes and retarding the growth and detection of any organisms that survived their presence in the undiluted product, the ingredient in question must be removed or inactivated.
Medical devices can rarely be handled in the ways commonly used for medicines. They are often large, always insoluble and possess many surfaces to which liquids cannot readily gain access. It is necessary either to immerse the device totally in culture medium or diluting fluid or subject it to surface sampling. Total immersion is preferred and may necessitate a supply of large, strong, sterile and sealable bags that can be filled with medium. The device is dismantled as far as practicable and any valves or taps must be opened to facilitate liquid entry. Surface sampling using contact plates or swabs is less desirable and requires extensive validation. It is necessary to confirm that the area sampled is representative of the whole, and to quantify the efficiency of removal of attached microorganisms. Ultrasonics, shaking with or without glass beads and flushing are other techniques that may be employed to facilitate removal, and these are considered in detail elsewhere.10,11
5 QUANTITATIVE METHODS
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