BS EN ISO 14730 pdf free download
BS EN ISO 14730 pdf free download. Ophthalmic optics – Contact lens care products – Antimicrobial preservative efficacy testing and guidance on determining discard date.
5.4 Inoculum challenge test procedure 5.4.1 Prepare one or more tubes (for each lot tested) containing a minimum of 1 0 ml of test solution per challenge organism. NOTE Sample tubes are used rather than lens cases to allow effective technical execution of the test. Since incompatibilities can exist between solution ingredients and tube materials, tubes of an appropriate material which is compatible with the ingredients should be considered. Inoculate the sample tube of the product to be tested with a suspension of test organisms sufficient to provide a final count of between 1 ,0 ? 1 0 5 cfu/ml and 1 ,0 ? 1 0 6 cfu/ml. Ensure that the volume of inoculum does not exceed 1 % of the sample volume. Ensure complete dispersion of the inoculum by adequate mixing. 5.4.2 Store the inoculated product at 20 °C to 25 °C. The temperature must be monitored using a calibrated device and the temperature documented. If the product is sensitive to light, it should be protected during the period of the test. 5.4.3 Take 1 ,0 ml aliquots of the inoculated product for determination of viable count at 7 d and 1 4 d. 5.4.4 After taking the 1 4 d sample, each sample is rechallenged as in 5.4.1 by using an inoculum level of 1 ,0 ? 1 0 4 cfu/ml to 1 ,0 ? 1 0 5 cfu/ml. 5.4.5 Take 1 ,0 ml aliquots of the inoculated product for determination of the viable count at 21 d and 28 d. 5.4.6 Subject each of the 1,0 ml aliquots, removed at the specified time intervals, to a suitable series of decimal dilutions in validated neutralizing media. Mix the suspension well by vortexing vigorously and let stand to allow neutralization to be completed. Neutralization conditions shall be based on recovery-medium control testing (see 5.5.2).If an antimicrobial agent in the formulation cannot be adequately inactivated or neutralized, eliminate it using a validated membrane filtration procedure (see annex A). 5.4.7 Determine the viable count of organisms in appropriate dilutions by preparation of triplicate plates (unless otherwise justified) of a suitable recovery medium (e.g. TSA for bacteria and SDA for mould and yeast). If membrane filtration has been employed to remove or neutralize antimicrobial agents, culture the membranes on these media as appropriate. If the pour-plate method is utilized, keep the agar for pour plates below 50 °C prior to pouring. NOTE The agar media used for determination of viable counts may also contain antimicrobial inactivators or neutralizers, if required. 5.4.8 Incubate bacterial recovery plates at 30 °C to 35 °C. Incubate yeast recovery plates at 20 °C to 25 °C or 30 °C to 35 °C. Incubate mould recovery plates at 20 °C to 25 °C. Incubation times for optimal recovery of bacteria, yeast and moulds shall be determined. Minimum incubation times shall be based on recovery medium control testing (see 5.5.2). Record the number of cfu observed on countable plates. Plates should be observed periodically during incubation to prevent the occurrence of uncountable plates due to overgrowth. 5.4.9 Determine the average number of colony-forming units on countable plates. Calculate the microbial reduction at the specified time points. NOTE Countable plates refer to 30 cfu to 300 cfu per plate for bacteria and yeast, and 8 cfu to 80 cfu per plate for moulds, except when colonies are observed only for the 1 0 0 or 10 –1 dilution plates. 5.4.10 The absence of microorganisms shall be documented, e.g., by recording a “0” or “NR” (no recovery), when plates for all dilutions of a sample at a single time point have zero colonies. 5.4.11 The concentration of survivors is calculated at each point of time. The concentration of viable organisms following the 1 4 d rechallenge is the sum of the rechallenge inoculum concentration and the 1 4 d survivor concentration. 5.5 Controls 5.5.1 Inoculum controls The initial and rechallenge inoculum concentrations are calculated by dispersing an identical aliquot of the inoculum into the same volume as used in 5.4.1 of a suitable diluent to achieve a final concentration not less than 1 ,0 ? 1 0 5 cfu/ml to 1 ,0 ? 1 0 6 cfu/ml for the initial inoculum or 1 ,0 ? 1 0 4 to 1 ,0 ? 1 0 5 cfu/ml for the rechallenge. The volume of inoculum does not exceed 1 % of the sample volume. Ensure dispersion of the inoculum by adequate mixing. Evaluate this control sample for cfu/ml at the beginning of the test in order to demonstrate the suitability of the medium used for growth of the test organism and provide an estimate of the initial inoculum concentration. Plate the appropriate aliquot from each tube onto the recovery agar plates in triplicate (unless otherwise justified).BS EN ISO 14730 pdf download.
