Sterilization experiment procedure
(1)According to the test requirements, the spore suspension and tablet of Bacillus subtilis were prepared. Put the bacterial pieces into double-layer polyethylene plastic bags for sealing and packaging, 2 pieces for each bag. 20 bags for each test.
(2) There are three layers of sterilizer: upper layer, middle layer and lower layer. Each layer has one point inside, middle layer and outer layer respectively, 9 points in total. One bag shall be placed in the middle of each item, and a total of 18 bags of test bacterial tablets are required. Another bag (2 pieces) of bacterial tablet was placed at room temperature as a positive control.
(3) The ethylene oxide shall be sterilized under full load according to the ethylene oxide concentration, action time, temperature and relative humidity in the cabinet specified in the operation manual. Full load items, depending on the use of the sterilizer. After the treatment, take out the bacterial pieces, and transfer them into 5 ml nutrient broth culture medium respectively, put them into 37 ℃ incubator for culture, and observe the final results (qualitative culture detection) after 7 days.
Take 0.2ml suspension of the nutrient broth tube which is difficult to judge the result and inoculate it into the nutrient agar plate, smear it evenly with the sterile L-bar, and culture it in the 37 ℃ incubator. 48 hours later, smear staining, observation of colony morphology under the microscope, or further test to determine whether there is growth or whether the growth is the test bacteria. In case of non test bacteria, the test shall be carried out again.
(4) During the test, both qualitative and quantitative positive control group and negative control group should be set up.
(5) In the quantitative positive control group, the same batch of test tablets were placed at room temperature. After the disinfection or sterilization test group reached the specified action time, the two tablets were immediately put into PBS tubes containing 5.0ml, and each tablet was vibrated 80 times. Take the lotion and count the living bacteria according to the method shown in 18.104.22.168.
(6) In the qualitative positive control group, the same batch of test bacterial tablet was placed at room temperature, and after the sterilization test group reached the specified action time, two pieces of the bacterial tablet were immediately inoculated in 5.0ml nutrient broth medium, respectively, and put into the incubator for qualitative culture to observe the bacterial growth.
(7) In the negative control group, two samples of the same batch of test were inoculated with 5.0ml of nutrient broth medium, and the uninoculated nutrient broth medium was put into the incubator for qualitative culture to observe whether there was bacterial growth.
(8) The test was repeated 5 times.
(9) In the five tests, the recovery amount of the positive control tablet in each test should be between 5 × 10 CFU / tablet and 5 × 10 CFU / tablet; in the qualitative positive control group, the bacterial growth was good; in the negative control group, the bacterial growth was sterile. If the results of the positive or negative control do not meet the above requirements, the test shall be cancelled and carried out again.