BS EN 14561:2006 pdf download – Chemical disinfectants and antiseptics — Quantitative carrier test for the evaluation of bactericidal activity for instruments used in the medical area — Test method and requirements (phase 2, step 2)
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test. The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g. mineral substances, protein, carbohydrates, lipids, detergents) shall be defined.
NOTE In the following, the term “interfering substance” is used even if it contains more than one substance.
220.127.116.11.2 Clean conditions (bovine albumin solution – low concentration)
Dissolve 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of diluent (18.104.22.168).
Sterilize by membrane filtration (22.214.171.124), keep in a refrigerator (126.96.36.199) and use within 1 month.
The final concentration of the bovine albumin in the test procedure (5.5) is 0,3 g/l.
188.8.131.52.3 Dirty conditions (Mixture of bovine albumin solutions – high concentration with sheep erythrocytes (see 184.108.40.206)) Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of diluent (220.127.116.11).
Sterilize by membrane filtration (18.104.22.168). Prepare at least 8,0 ml fresh sterile defibrinated sheep blood (22.214.171.124). Centrifuge the sheep blood at 800 gN for 10 min. After discarding the supernatant, resuspend erythrocytes in diluent (126.96.36.199). Repeat this procedure at least 3 times, until the supernatant is colourless. Resuspend 3 ml of the packed sheep erythrocytes in the 97 ml of sterilized bovine albumin solution (see above). To avoid contamination this mixture should be split in portions probably needed per day and kept in separate containers for a maximum of 7 days in a refrigerator at 2° C to 8° C. The final concentration of bovine albumin and sheep erythrocytes in the test procedure (5.5) shall be 3 g/l and 3 ml/l respectively.
5.3 Apparatus and glassware
5.3.1 General Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and reagents or the sample, except those which are supplied sterile, by one of the following methods: a) by moist heat, in the autoclave [188.8.131.52 a)] ;
b) by dry heat, in the hot air oven [184.108.40.206 b)].
5.3.2 Usual microbiological laboratory equipment 2) and in particular, the following:
220.127.116.11 Apparatus for sterilization: a) for moist heat sterilization, an autoclave capable of being maintained at ( ) C 121 3 0 ° + for a minimum holding time of 15 min;
b) for dry heat sterilization, a hot air oven capable of being maintained at (180 5 0 + ) °C for a minimum holding time of 30 min, at (170 5 0 + ) °C for a minimum holding time of 1 h or at (160 5 0 + ) °C for a minimum holding time of 2 h.
18.104.22.168 Water baths, capable of being controlled at 20° C ± 1 °C, at 45 °C ± 1 °C (if pour plate technique is used – see 22.214.171.124) and at additional test temperatures ± 1 °C (5.5.1).
126.96.36.199 Incubator, capable of being controlled at either 36 °C ± 1 °C or at 37 °C ± 1 °C. The same temperature shall be used for all incubations performed during a test and its controls and validation.
188.8.131.52 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1)°C NOTE For measuring the pH of the agar-media (184.108.40.206) a puncture electrode or a flat membrane electrode should be used.
a) Electromechanical agitator, e.g. Vortex ® mixer 3) .
b) Mechanical shaker
220.127.116.11 Membrane filtration apparatus, constructed of a material compatible with the substances to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter 47 mm to 50 mm and 0,45 µm pore size for sterilization of hard water (18.104.22.168) and bovine albumin (22.214.171.124). The vacuum source used shall give an even filtration flow rate. In order to obtain an uniform distribution of the microorganisms over the membrane and in order to prevent overlong filtration, the device shall be set so as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
126.96.36.199 Refrigerator, capable of being controlled at 2 °C to 8 °C. 188.8.131.52 Graduated pipettes of nominal capacities 10 ml and 1 ml and 0,1 ml. Calibrated automatic pipettes may be used.