Counting, microbial methods

Hospital steriliza tion is more limited in the availabiHty of steriliza tion methods and of packaging materials. Microbial invasion can occur particularly when articles are wrapped in traditional fabrics such as muslin (140-thread-count cotton). The expected shelf life of hospital-wrapped and sterilized articles is considered to be ca 21—30 days when a double-wrapping technique is used. Double-wrapping requires two successive wraps, each having a layer or layers of an approved packaging material. 

Historically, measurement of the microbial biomass has been a tedious, time-consuming occupation involving staining and direct counting or use of culture media and enumeration of individual microbial communities. However, in the last 20 years, a suite of methods have been developed for more rapid assessment of the microbial biomass. These include the substrate-induced respiration method (Anderson and Domsch 1978), the chloroform fumigation-incubation method (Jenkinson and... 

A similar approach to that employed in aerobic microbial count validation is employed but quantification is not possible. A low level (<100 cells) of specified organism is added to various product and broth mixtures and recovery viewed on the resultant selective plates. For the method to be considered valid, growth on plates must be comparable to that derived from parallel control cultures containing no product. Parallel controls not only must be run at validation stage but also as a matter of routine to indicate acceptable preparation and performance of media. 

Determine the number of colony-forming units per liter in each suspension by plate count method, using the conditions of media and microbial recovery incubation times listed in Table 3 to confirm the initial CPUs per ml estimate. 

Microscopic counting is still the most direct method of estimating the amount of microbial biomass in soil but is technically difficult and completely unsuitable for routine use. Thin films are prepared from an agar-soil suspension, are mounted on microscope slides, and then are treated with an appropriate stain. Phenolic aniline blue is often used as it stains protein and is thus considered to give an estimate of the entire population. The numbers... 

Back [46] describes a method to check the microbial reduction capability of an installed filtration line. This method employs the use of five different types of microorganisms, which are suspended in water, and consists of inoculating a suspension of about 10 microorganisms the non-filtered beverage with a batch size of 100-500 hL. The ratio of the microbiological count found between the inlet to the first main filtration equipment and the outlet of the final filtration equipment should be higher than 10, typically higher than 10. ... 

The microbial count of air may be reduced by filtration, chemical disinfection and to a limited extent by ultraviolet (UV) light. Filtration is the most commonly used method and filters may be made of a variety of materials such as cellulose, glass wool, fibreglass mixtures or polytetrafluorethylene (PTFE) with resin or acrylic binders. There are standards in both the UK and USA for the quality of moving air, in the UK there is a grading system from... 

After sampling, the products are prepared in the laboratory. The sample preparation is illustrated below with two simple methods for determining the total microbial count ... 

The term bioburden refers to the amount of microbial flora that can be detected on an item or surface or in a solution. The microbial recovery method used depends on the type of material being evaluated. Aerobic bioburden counts in parenteral solutions are obtained by conducting the total aerobic count and total yeast and molds count as specified in the USP microbial limits test (<61>) or a equivalent compendial test. Alternatively, a modified membrane filtration technique can be used to allow filtration of larger volumes of solution to assess bioburden recoveries where sample results are expected to contain a negligible number of microbial flora from the overall solution. 

Microbial limit tests, as described in USP 24-NF 19 (<61>) provide the information necessary to conduct bioburden counts by quantitative estimations of viable aerobic microorganisms in pharmaceutical articles, raw materials, and finished dosage forms. Consult the EP or JP for any differences in these methods from those of the USP. Note that USP (<61>) is currently under revision and that the next version will be fully harmonized between the USP, EP, and JP. The microbial limit test comprises the total aerobic bacterial (microbial) count (TAG), the total combined yeasts and molds count (TYMC), and tests for indicator organisms. Bioburden tests are also referred to as total viable counts. This test estimates the total number of viable (culturable) aerobic microorganisms in various items including parenteral UBPS. The total viable count is obtained by adding the counts from the TAC and the TYMC. 

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