BS EN ISO 16954 pdf free download
BS EN ISO 16954:2015 pdf free download.Dentistry一Test methods for dental unit waterline biofilm treatment.
4 Treatment methods
Depending upon the specific technical approach ot a treatment method and its intended benents, the performance objectives ofa dental unit procedural water delivery system treatment method can include one or both ofthe following:
— prevention or inhibition of biofilm formation on surfaces within the dental unit procedural water delivery system;
— removal of biofilm from surfaces within the dental unit procedural water delivery system.
This International Standard specifies separate test methods for each of the above performance objectives. These requirements can be expanded upon, for example to include additional replicates or test scenarios. Additions to the test method shall follow the general principles of this International Standard and be fully described in the test report.
5 Test water and bacterial challenge suspensions
5.1 Test water
This subclause specifies the preparation oftest water prior to inoculation.
5.1.1 Reagents
5.1.1.1 Water, in accordance with ISO 3696:1987, grade 3.
5.1.1.2 Calcium chloride (CaC12), or an equivalent molar quantity ofa calcium chloride hydrate.
5.1.1.3 Magnesium chloride (MgC12), or an equivalent molar quantity ofa magnesium chloride hydrate.
5.1.1.4 Sodium bicarbonate (NaHCO3).
5.1.1.5 Tryptic soy broth (TSB), 1/3-strength, 10,0 g tryptic soy medium per litre broth.
5.1.1.6 Sodium hydroxide (NaOH), 1 mol/l.
5.1.1.7 Hydrochloric acid (HCI), 1 mol/l.
5.1.2 Preparation ofhardness stock solution 1
Dissolve 74,0 g of calcium chloride (5.1.1.2) and 31,7 g of magnesium chloride (5.1.1.3) in 1,00 1 water (5.1.1.1). Hardness stock solution 1 shall be sterilized by heat or filter-sterilized using a 0,2 im microfilter and used within 24 h or stored at (5 ± 3) °C for up to 6 months.
5.1.3 PreparatIon ofhardness stock solution 2
Dissolve 56,0 g ofsodium bicarbonate (5.1.1.4) in 1,00 1 water (5.1.1.1). Hardness stock solution 2 shall be filter-sterilized using 0,2 im microfilter and used within 24 h or stored at (5 ± 3) °C for up to 6 months. Hardness stock solution 2 is not to be heat sterilized.
5.1.4 Preparation oftest water prior to inoculation
For each litre of test water to be prepared, add 1,00 ml of 1/3-strength TSB (5d,15) and 1,80 ml of hardness stock solution 1 (5.1.2) to 1,00 I water (5.1.1.1) and steam sterilize. After the sterilized solution has cooled, for each litre of test water add 4,00 ml of hardness stock solution 2 which has been filtersterilized using a 0,2 pm microfilter. Adjust the pH to 7,0 to 8,0, measured according to ISO 10523, by adding sodium hydroxide (S.1.t6) or hydrochloric acid (5.1.1.7). The test water shall be used within 24 h or stored at (5 ± 3) °C for up to one week.
NOTE 1 The hardness ofthe prepared test water is approximately 1,8 mmol ofcalcium ions per litre (equivalent to approximately 180 mg/I as CaCO3). This corresponds to the upper limit of the generally accepted range for hard waterjill
NOTE 2 The test water has a concentration of approximately 10 mg TSB per litre, which yields a total organic carbon (TOC) level ofapproximately 4 mg/I, although the exact TOC level might vary somewhat. This approximate TOC level is consistent with the recommended upper limit of4 mg/I for TOC in chlorinated drinking waterli] and is included in the test water to reduce the time for biofilm formation.
5.2 Bacterial challenge
Bacterial challenge suspensions used to inoculate the test water shall be prepared with the following bacteria from the American Type Culture Collection (ATCC) or an international authorized ATCC distributor:
a) Pseudomonas aeruginosa (ATCC #700888);
b) Kiebsiella pneurnoniae (ATCC #13882).
Alternate strains ofP. aeruginosa and K. pneumoniae (i.e. different ATCC numbers for the same bacterium species) can be substituted ifthe specified strains are not available, provided that the isolation source indicated by ATCC is water or a water system.
The bacterium species shall be separately reconstituted in sterilized dilute TSB, having a concentration of0,3 g TSB per litre. The reconstituted cultures shall be used within eight transfer passages. Cultivation of the bacteria for inoculating test water shall be performed one day before preparing the inoculated test water in accordance with 53.
Compliance with appropriate laboratory safety practices is critical when working with these bacteria, including the handling of waste that can be contaminated with these bacteria.
5.3 Inoculated test water
On days of test apparatus operation inoculated test water shall be prepared within two hours before the commencement of the daily flow program specified in 6.2.2 by inoculating sterilized test water (Si) with both of the reconstituted bacterial cultures (5.2), to achieve a concentration of 5 x 101 CFU/ml to 5 x 102 CFU/ml of each of the bacteria and a total bacterial concentration of 102 CFU/mI to 10 CFU/ml in the inoculated test water. The temperature of the sterilized test water at the time of inoculation shall be (23 ± 3) °C.
To ensure accurate concentrations of each bacterium species in the inoculated test water, it can be useful to centrifuge and re-suspend each of the reconstituted bacterial cultures in sterile phosphate buffer and determine the approximate bacterial concentration using turbidimetric measurements. Using these results, the volume of each of the single-species bacterial suspensions to be added to the test water can be calculated. Alternatively, other methods for achieving the specified inoculation range can be used.
6 Test apparatus
The test apparatuses shall consist of a specified number of either dental units or surrogate dental unit water systems which closely replicate the dental unit procedural water system.BS EN ISO 16954 pdf download.
