ISO 19045-2015 pdf free download

ISO 19045-2015 pdf free download.Ophthalmic optics Contact lens care products – Method for evaluating
Acanthamoeba encystment by contact lens care products
Optique ophtalmique — Produits d’entretien de lentilles de contact — Méthode d’évaluation de l’enkystement de Acanthamoeba au contact des produits d’entretien des lentilles de contact.
4.8.2.3 Mix contents of wells by gently pipetting up and down three times with a 1 000 tl pipettor set to deliver 500 tl or a 3 ml disposable pipette.
4.8.2.4 Immediately perform cell chamber counts using the hemocytometer on each of the three To cncystment negative control wells and record the individual and averaged total cell count/mi in TabIel.
4.8.2.5 Measure the background cyst count in the inoculum by adding 50 uI of the Sarkosyl-Calcofluor White solution (see Annex C) to each of the three To encystment negative control wells pipetting vigorously to mix and leaving at room temperature for S mm.
NOTE It has been observed that 5 mEn is critical for optimal assessment. Longer exposure times can cause the cysts to lyse and become difficult to recognize.
4.8.2.6 Again, mix vigorously by pipetting up and down using a 1 000 iii pipettor set to deliver 500 il or a 3 ml disposable pipette.
4.8.2.7 Immediately perform cell chamber counts using the hemocytometer on the Sarkosyl-Calcofluor White treated cells (under UV fluorescence with appropriate filter for Calcofluor white detection) to determine the background cyst level. Switch between UV fluorescence and white light to confirm observation of cysts. Count the number of refractile and fluorescent cells/mi and record in Tablel. These represent immature and mature cyst forms and give the background levels for the experiment (see Eigiue2 for representative images of encysted trophozoites).
4.8.2.8 The test is invalid if the background cyst count in the challenge inoculum is greater than 5,0 %. If greater than 5,0 %, prepare fresh inoculum and repeat the procedure.
4.8.3 Test samples
4.8.3.1 Mix all solutions vigorously immediately before dispensing. Dispense each test solution directly from the product bottles into three wells In the microtitre plates as in
4.8.3.2 Add 30 ul of 1,0 x 107/ml trophozoites in 1/4 strength Ringer’s solution to all wells. This is designed to yield a final concentration of 1 x 105 trophozoites/mI in the test wells. If the stock trophozoite concentration is found to be >1,0 x 107/ml. then add an appropriate volume to the wells to yield the required inoculum concentration of 1 x 105 trophozoites/mI (e.g. 20 uI if the stock suspension was counted as 1,5 107/ml).
4.8.3.3 Mix contents of wells by gently pipetting up and down three times with a 1 000 ul pipettor set to deliver 500 p1 or a 3 ml disposable pipette.ISO 19045-2015 pdf free download.