BS ISO 19045 pdf free download

BS ISO 19045-2015 pdf free download. Ophthalmic optics — Contact lens care products — Method for evaluating Acanthamoeba encystment by contact lens care products.
3 Principle
3.1 General The assay tests the capability for a solution to induce Acanthamoeba trophozoite encystment as this physiological event can afford the organism protection from disinfection.
3.2 Encystment test The encystment test is used to measure a disinfecting solution’s potential for inducing trophozoite encystment to either the immature or mature cyst form. Assessment of this phenomenon is considered important as Acanthamoeba cysts can be resistant to many disinfecting systems at operating conditions. In the encystment test, contact lens disinfecting solutions are exposed to Acanthamoeba trophozoites. Following detergent treatment and calcofluor white staining to lyse remaining trophozoites and stain the inner cell wall, the organisms are observed microscopically for the production of immature and mature cysts.
4.4.4 3 ml sterile disposable plastic Pasteur pipettes.
4.4.5 Fluorescence microscope with ×10, ×20, and ×40 phase contrast and fluorescence objectives with
a UV-2A filter, excitation 330 nm–380 nm, and emission greater than 420 nm.
4.4.6 An inverted microscope with ×10, ×20, and ×40 objectives.
4.4.7 28 °C ± 2 °C and 32,5 °C ± 2,5 °C incubators.
4.4.8 Centrifuge.
4.4.9 Vortex mixer.
4.4.10 Cell counting chamber (e.g. Modified Fuchs Rosenthal INCYTO disposable hemocytometer).
4.4.11 Optional: Pivoting blade cell scraper.
4.4.12 Sterile 75 cm 2 and 150 cm 2 /180 cm 2 flat polystyrene tissue culture flasks.
4.5 Test samples
Aliquots of the product to be tested shall be representative of the product to be marketed. The product should be taken directly from the final product container immediately prior to testing. Three lots of product shall be tested. Each lot of product shall be tested with a separate inoculum preparation.
4.6 Culture maintenance
4.6.1 The strain should not be subcultured more than five passes as per American Type Culture Collection (ATCC) protocols.
4.6.2 Maintenance of stock cultures (see E.1).
4.6.3 Scaling up cultures for testing (24 h prior to test) (see E.2).
4.7 Preparation of microbial challenge
4.7.1 Grow trophozoites as described in 4.6.2 and 4.6.3.
NOTE Prepare a sufficient number of flasks based on the size of the experiment and the number of trophozoites required.
4.7.2 Vigorously shake flasks to dislodge adherent trophozoites (rinse with a pipette if necessary).
NOTE Scrape the bottom of the flask with a cell scraper if necessary.
4.7.3 Decant trophozoites into 50 ml polypropylene centrifuge tubes and centrifuge at 500 × g for 5 min at room temperature.
4.7.4 Resuspend one tube pellet in 10 ml of 1/4 strength Ringer’s solution (see Annex B) and use to resuspend the other pellets if additional inoculum is required.
4.7.5 Wash ×3 with 10 ml of 1/4 strength Ringer’s solution by centrifugation at 500 × g for 2 min at room temperature.
4.7.6 Resuspend pellet by vortexing in 1 ml to 2 ml of 1/4 strength Ringer’s solution.
4.7.7 Enumerate trophozoite numbers using a cell counting chamber (make a 1:10 to 1:100 dilution in 1/4 strength Ringer’s solution to assist) and record number/ml. A volume of 20 µl is used for cell counting using the hemocytometer.
NOTE A 1:100 dilution can be prepared by two 1:10 serial dilutions of 100 μl into 900 μl.
4.7.8 Adjust the stock concentration to 1,0 × 10 7 /ml to 1,5 × 10 7 /ml in 1/4 strength Ringer’s solution and use immediately for testing.
4.8 Encystment procedure
4.8.1 General
The encystment procedure consists of pipetting 3,0 ml ± 0,1 ml or weighing 3,0 g ± 0,1 g of control and/or test solutions into a well of a 12 well microtitre plate.
4.8.2 Control plate
4.8.2.1 Dispense the encystment positive control solution into three wells and dispense the negative control solution (see Annex D) into six wells each of a 12 well microtitre plate as shown in Figure 1.BS ISO 19045 pdf download.