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Bacteria quantitative killing test

2021-04-30 13:46:45

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2.1.1.7.1 Purpose

Measure the required dose of disinfectant to kill the bacterial propagule and bacterial spores in the suspension or on the carrier in the laboratory to verify the practical disinfectant dose.

2.1.1.7.2 Test equipment

(1) Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis black variant spore suspensions or bacterial tablets prepared according to the method shown in 2.1.1.2. In addition, according to the specific purpose of the disinfectant or the special needs of the test, prepare suspensions or bacterial tablets of other bacterial species.

(2) The disinfectant solution should be prepared with sterile hard water unless there are special regulations. The concentration of the disinfectant solution should be based on the effective ingredients contained. For example, chlorine-containing disinfectants are based on the concentration of available chlorine, iodophors are based on the available iodine, peracetic acid is based on the amount of peracetic acid, and the concentration of compound disinfectants is based on the content of the main bactericidal active ingredients. . The calculation of the effective ingredient concentration of each group of disinfectant solution should be based on the final concentration of the effective ingredient in the mixture of the test bacteria and the disinfectant.

(3) Neutralizers or equipment to remove residual disinfectants (neutralizers or related equipment that have been certified qualified by the appraisal test).

(4) Hard water for dilution of disinfectant: see Appendix A.

(5) Organic interference substances. The suspension test uses 3% BSA solution, and the carrier test directly uses TSB (see Appendix A).

(6) TSA medium: see Appendix A.

(7) Tryptone soybean broth medium containing neutralizer (neutralizer TSB), and the neutralizer is qualified.

(8) Graduated straws (1.0ml, 5.0ml).

(9) Constant temperature water bath.

(10) Sprayer (used for spray disinfection test).

(11) Electric mixer.

(12) Stopwatch.

2.1.1.7.3 Test grouping

The test should be divided into the following groups:

(1) Test group. There are two options for testing purposes.

The first is suitable for the identification of disinfection products. According to Table 1-1 in this specification and instructions for use, select the test bacteria and a disinfectant concentration (that is, the lowest concentration specified in the product instructions) and 3 action times (the instructions specify the shortest action time, 0.5 of the specified shortest action time Times, 1.5 times of the specified minimum action time. If the minimum action time is specified as 20min in the manual, the test should be carried out for 10min, 20min, and 30min.

The second type is suitable for daily monitoring by disinfection product supervision agencies. According to the tested bacteria and the disinfectant's ability to kill the bacteria, select a more resistant bacteria and a disinfectant concentration (that is, the lowest concentration specified in the product instruction manual) and an action time (the instructions specify the shortest effect) Time) to experiment.

(2) Positive control group. According to the regulations of various tests, use the diluent instead of the disinfectant solution, and carry out the test according to the same procedure as above. The result obtained represents the original concentration of the bacterial solution, which is used as the initial concentration for calculating the kill logarithm.

2.1.1.7.4 Suspension quantitative sterilization test operation procedure

(1) First, prepare the disinfectant according to the product specification. If there is no special instructions, use sterile hard water to prepare, and the prepared concentration is 1.25 times the concentration to be tested (for example, if the concentration of the disinfectant to be evaluated is 200mg/L, 250mg/L should be prepared), and place in a 20℃±1℃ water bath spare.

(2) Prepare experimental bacterial suspension according to 2.1.1.2, with a concentration of 1×108cfu/ml~5×108cfu/ml.

(3) Aseptic test tube for detoxification test, first add 0.5ml test bacteria suspension, then add 0.5ml organic interfering substance, mix well, put in a water bath at 20℃±1℃ for 5min, then suck the above with a sterile pipette The concentration of disinfectant solution 4.0 ml is injected into it, mixed quickly and the time is counted immediately.

(4) After the test bacteria interact with the disinfectant to each predetermined time, respectively add 0.5ml of the test bacteria and disinfectant mixture to 4.5ml of the sterilized neutralizer, and mix well.

(5) After each tube of the test bacteria and disinfectant mixture is added with neutralizer for 10 minutes, draw 1.0ml of the sample solution respectively, and determine the number of viable bacteria according to the method of viable bacteria culture and counting. Each tube of sample solution can be inoculated with 2 plates. If there are a large number of colonies growing on the plate, a series of 10-fold dilutions can be carried out, and then the live bacteria culture can be counted.

(6) At the same time, use the diluent instead of the disinfectant, and conduct a parallel test as a positive control.

(7) All test samples were cultured in a 37°C incubator, and the bacterial propagules were cultured for 48 hours to observe the final results; bacterial spores need to be cultured for 72 hours to observe the final results.

(8) The experiment was repeated 3 times, the concentration of viable bacteria (cfu/ml) of each group was calculated and converted to a logarithmic value (N), and then the logarithmic killing value was calculated as follows:

Killing logarithmic value (KL) = Logarithmic value of the average viable bacteria concentration of the control group (No)-Logarithmic value of the viable bacteria concentration of the test group (Nx)

When calculating the logarithm of killing, take two decimal places to round off the numbers. If the average number of colonies produced in the disinfection test group after disinfection treatment is less than or equal to 1, its killing log value is greater than or equal to the log value of the average viable bacteria concentration of the control group before the test.


Author: Zhejiang Oujie Youhu Technology Co., Ltd.
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Bacteria quantitative killing test
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