The ability of the procedure to detect challenge microorganisms in the presence of a suitably neutralized product to be tested must be established. The suitability of the procedure must be reconfirmed if a change is made in materials or methods or if a change is made in the product or direct product contact materials that may affect the outcome of the test.

The growth-promoting capabilities of media used in this procedure must be established.

Use standardized suspensions of test strains or prepare as stated below. Seed-lot culture maintenance techniques (seed-lot systems) are used so that the viable microorganisms used for inoculation are NMT five passages removed from the original master seed lot. Grow each of the bacterial and fungal test strains separately (see Table 2).

Use cultures of the following microorganisms:1 Candida albicans (ATCC No. 10231), Aspergillus brasiliensis (ATCC No. 16404), Escherichia coli (ATCC No. 8739), Pseudomonas aeruginosa (ATCC No. 9027), and Staphylococcus aureus (ATCC No. 6538). The viable microorganisms used in the procedure should be part of a freshly growing culture (e.g., in logarithmic growth phase) with the exception of A. brasiliensis spores. The culture conditions for the inoculum culture are described (see Table 2) in which the suitable media are Soybean–Casein Digest or Sabouraud Dextrose Agar Medium.

To harvest the bacterial and C. albicans cultures, use sterile saline TS to wash the surface growth, and collect it in a suitable vessel. To harvest the spores of A. brasiliensis, use sterile saline TS containing 0.05% of polysorbate 80. The spore suspension should be aseptically treated (e.g., filtration through sterile glass wool) to remove hyphae. All microbial suspensions should be prepared to ensure that there is no carry over of residual growth medium from the inoculum (e.g., centrifugation followed by resuspension in appropriate sterile suspending fluid.)

Alternatively, the stock culture organisms may be grown in a suitable liquid medium (i.e., Soybean–Casein Digest Broth or Sabouraud Dextrose Broth) and the cells harvested by centrifugation, then washed and resuspended in appropriate sterile suspending fluid. The microbial suspensions used for inoculations should be adjusted to obtain a microbial count of about 1 × 108 cfu/mL. Use the bacterial and yeast suspensions within 2 h, or within 24 h if stored between 2 and 8. A stable spore suspension can be prepared and then may be maintained at 2–8 for up to 7 days. [Note—The estimate of inoculum concentration may be obtained by turbidimetric procedures for the challenge microorganisms and later confirmed by plate count. ]

Media used in this procedure must be capable of supporting microbial growth. Test each batch of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from the ingredients described.

For solid media, counts obtained must be at least 50% of the calculated value for a standardized inoculum. For a freshly prepared inoculum, growth of the microorganisms occurs comparable to that previously obtained with a previously tested and approved batch of medium.

For the purpose of testing, compendial articles have been divided into four categories (see Table 1). The criteria of antimicrobial effectiveness for these products are a function of the route of administration. It is expected that formulations containing preservatives will meet minimal efficacy standards, whether packaged as multidoses or unit doses.

The procedure can be conducted either in five original containers if a sufficient volume of product is available in each container and if the product container can be entered aseptically (i.e., needle and syringe through an elastomeric rubber stopper), or in five sterile, capped bacteriological containers [inert relative to the antimicrobial agent(s)] of suitable size into which a sufficient volume of product has been transferred. Inoculate each container with one of the prepared and standardized inocula, and mix. The volume of the suspension inoculum used is between 0.5% and 1.0% of the volume of the product to minimize potential effects on the product. The concentration of test microorganisms that is added to the product (Category 1, 2, or 3) is such that the final concentration of the test preparation after inoculation is between 1 × 105 and 1 × 106 cfu/mL of the product. For Category 4 products (antacids), the final concentration of the test preparation after inoculation is between 1 × 103 and 1 × 104 cfu/mL of the product.

The initial concentration of viable microorganisms in each test preparation is estimated based on the concentration of microorganisms in each of the standardized inocula as determined by the plate-count method. Incubate the inoculated containers at 22.5 ± 2.5. Sample each container at the appropriate intervals (specified in Table 3). Record any changes observed in appearance at these intervals. Determine, by the plate-count procedure, the number of cfu present in each test preparation for the applicable intervals (see General Procedures in Microbial Enumeration Tests 61). Plate counts will be conducted using a minimum of duplicate plates, with the cfu averaged before determination of deduced cfu/mL. If membrane filtration is used, duplicate membrane filters will be used for each estimate. Using the calculated concentrations of cfu/mL present at the start of the test, calculate the change in log10 values of the concentration of cfu/mL for each microorganism at the applicable test intervals, and express the changes in concentration in terms of log reductions. The log reduction is defined as the difference between the log10 unit value of the starting concentration of cfu/mL in the suspension and the log10 unit value of cfu/mL of the survivors at that time point.