阅读说明：本技术 一种消毒剂灭菌效果的鉴定和评价方法 (Method for identifying and evaluating sterilization effect of disinfectant ) 是由 王娟 张文英 南紫萱 梁芳慧 岳琳 张伟亮 贾鹏飞 葛凤燕 张星辰 刘占旗 邢云 于 2019-12-06 设计创作，主要内容包括：本发明公开了一种消毒剂灭菌效果的鉴定和评价方法,属于微生物的测定或检测技术领域。包括以下步骤(1)标记8支试管(2)加入生理盐水,1-5号加9mL,6-8号加10mL,20±2℃水浴(3)1试管加1mL菌液混匀后,取1mL移入第2试管,混匀,从第2试管内取1mL移入第3、4和5,将3-5试管分别混匀后,将6-8试管依次倒入3-5试管混匀后,再分别移取10mL于6-8试管内(4)加入消毒剂于3-7作为实验样；第8管内作为对照样(5)定性和定量培养(5)观察结果计算抑菌率。本发明检测方法可以检测任意浓度消毒剂,可以同时检测两批及以上的消毒剂抑菌效果,检测浓度范围广,节省时间。(The invention discloses a method for identifying and evaluating the sterilization effect of a disinfectant, belonging to the technical field of determination or detection of microorganisms. Marking 8 test tubes (2), adding physiological saline, adding 9mL into No. 1-5 test tubes, adding 10mL into No. 6-8 test tubes, adding 1mL of bacterial liquid into 1 test tube in a water bath (3) at 20 +/-2 ℃, uniformly mixing, transferring 1mL into the No. 2 test tube, uniformly mixing, transferring 1mL from the No. 2 test tube into No. 3, 4 and 5 test tubes, respectively uniformly mixing 3-5 test tubes, sequentially pouring 6-8 test tubes into 3-5 test tubes, uniformly mixing, then respectively transferring 10mL into 6-8 test tubes, adding a disinfectant into 3-7 test tubes (4) to serve as experimental samples; and (4) in the 8 th tube, qualitatively and quantitatively culturing as a control sample (5) and observing results to calculate the bacteriostasis rate. The detection method can detect the disinfectant with any concentration, can simultaneously detect the bacteriostatic effect of two or more batches of the disinfectant, has wide detection concentration range and saves time.)

1. A method for identifying and evaluating the sterilization effect of a disinfectant is completed in a sterile table, and is characterized by comprising the following steps of (1) arranging 8 sterilized test tubes on a test tube rack, marking test tube numbers, (2) adding 0.9% sterilized physiological saline into each test tube, adding 9mL of the test tube 1-5 and 10mL of the test tube 6-8, placing 8 test tubes into a water bath at 20 +/-2 ℃, adding 1mL of bacterial liquid into the test tube 1, uniformly mixing, adding 1mL of the solution into the test tube 2, uniformly mixing, taking 3 mL of the solution from the test tube 2 into the test tube 3, the test tube 4 and the test tube 5, uniformly mixing the solutions from the test tube 3 to the test tube 5, pouring the solutions from the test tube 6 to the test tube 5 in turn, uniformly mixing, transferring 10mL of the solution from the test tube 3 to the test tube 5 into the test tube 6 to 8, taking a supernatant of the solution from the test tube 3 to the test tube 5 as a sterile culture broth, and taking a supernatant of a culture broth from the test broth 6 to a broth 7 th test broth, and observing the growth of the culture broth, wherein the culture broth, the culture broth is obtained by adding the disinfectant into the culture broth, the:

y is the bacteriostasis rate;

w-number of control bacteria;

q is the number of bacteria in the experimental sample.

2. The method for identifying and evaluating the sterilizing effect of a disinfectant according to claim 1, wherein: the preparation method of the bacterial liquid used in the step (3) comprises the following steps:

(1) activation of lyophilized bacteria

Melting and dispersing freeze-dried bacteria in 5mL of nutrient broth to form a suspension, culturing for 18h-24h at 37 +/-2 ℃ to prepare a bacterial suspension, taking the bacterial suspension by using an inoculating loop, inoculating the bacterial suspension on an agar culture medium plate by a scribing method, culturing for 18h-24h at 37 +/-2 ℃, taking a typical bacterial colony from a culture dish, inoculating the typical bacterial colony in an agar culture medium inclined tube, culturing for 18h-24h at 37 +/-2 ℃, storing the inclined tube in a refrigerator at 5-10 ℃ as a preservative, wherein the preservation period is not more than one month, the passage is carried out once per month, and the number of passage is not more than 10 generations;

(2) preparation of test bacterial solution

1) Inoculating the preserved strain to an agar culture medium plate by a streaking method, and culturing at 37 +/-2 ℃ for 24 h; the plate is stored at 5-10 ℃ and used within 1 week;

2) taking 20mL of nutrient broth, putting the nutrient broth into a 100mL Erlenmeyer flask, and inoculating a typical bacterial colony on the plate in the step 1) into the broth for culture; the culture conditions are that the temperature is 37 +/-2 ℃, the rotation speed is adjusted to 130r/min, and the time is 18-24 h;

3) the nutrient broth was diluted 20-fold with distilled water, and the concentration of the cultured bacteria was adjusted to 1X108CFU/mL-5X108CFU/mL to prepare a test bacterial solution.

3. The method for identifying and evaluating the sterilizing effect of a disinfectant according to claim 1 or 2, wherein: the bacteria used were gram-negative in Escherichia coli (8099 or ATCC 11229).

4. The method for identifying and evaluating the sterilizing effect of a disinfectant according to claim 1 or 2, wherein: the agar medium had the composition: each 1000mL of agar culture medium contains 10g of tryptone, 10g of sodium chloride, 17.5g of agar powder, 5g of yeast extract powder and the balance of distilled water; adjusting the pH value to 7.2 +/-0.2 after sterilization.

5. The method for identifying and evaluating the sterilizing effect of a disinfectant according to claim 1 or 2, wherein: the nutrient broth comprises the following components: every 1000mL of nutrient broth contains 15g of tryptone, 5g of plant peptone, 5g of sodium chloride and the balance of distilled water; adjusting the pH value to 7.2 +/-0.2 after sterilization.

6. The method for identifying and evaluating the sterilizing effect of a disinfectant according to claim 1, wherein: the calculation method of the number of the bacteria in the control sample comprises the following steps: and (4) diluting the control sample bacterial liquid to the number of the clear colonies on a plate after the plate can be coated, and calculating the number of the control sample colonies according to the number.

Technical Field

The invention relates to the technical field of determination or detection of microorganisms.

Background

Along with the increasing living standard, the requirement of people on sanitation is also increased. Along with the generation of various disinfectants, people have higher and higher requirements on the bacteriostatic effect of the disinfectants. The detection method implemented at present is GB15981-1995, a disinfectant is diluted by distilled water to a series of concentration gradients, then diluted bacterial suspension is added and mixed uniformly for a period of time, a small amount of neutralizer is taken and added, after neutralization is carried out for 10 minutes, a small amount of nutrient broth is taken out and added, and whether the broth is turbid or not is observed to judge the bacteriostatic effect of the broth. Although the detection method has the advantages of simple required instruments and equipment, obvious phenomenon and easy observation, the method can accurately evaluate the killing effect of the disinfectant on the microorganisms. However, the method can only dilute the disinfectant according to a certain concentration gradient, and the detection process takes a long time, so that a detection method which is suitable for disinfectants with various concentrations and has a short detection period is urgently needed.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for identifying and evaluating the sterilization effect of a disinfectant.

A method for identifying and evaluating the sterilization effect of a disinfectant is completed in a sterile table and comprises the following steps of (1) arranging 8 sterilized test tubes on a test tube rack and marking test tube numbers, (2) adding 0.9% sterilized normal saline to each test tube, adding 9mL of the test tubes from 1 to 5 and 10mL of the test tubes from 6 to 8, putting 8 test tubes into a water bath at 20 +/-2 ℃, after adding 1mL of bacterial liquid into the test tube 1 and mixing uniformly, adding 1mL of the solution into the test tube 2 and mixing uniformly, adding 3 mL of the solution into the test tubes from 2 and transferring into test tubes 3 to 5 respectively, after mixing the solution in the test tubes 3 to 5 respectively, pouring the solution in the test tubes from 6 to 8 in sequence, mixing uniformly, transferring 10mL of the solution into the test tubes from 3 to 5 respectively, taking the solution from the test tubes 3 to 5 as a test tube 6 to 8, and taking a supernatant of a broth from the test tube 3 to 7 as a sterile broth, and taking a broth as a sterile culture broth, and observing the growth of a bacterial colony, if the growth of the bacterial colony is observed, the bacterial colony is observed after adding the disinfectant into the culture broth, the broth:

y is the bacteriostasis rate;

w is the number of bacteria in the control sample (the number of colonies of the control sample is calculated by diluting the bacterial liquid to a certain concentration and counting the number of clear colonies on a plate after the plate coating can be carried out);

q is the number of bacteria in the experimental sample.

Judging the bacteriostatic effect according to the calculated bacteriostatic rate, wherein the bacteriostatic rate is qualified if more than or equal to 99.9%.

The preparation method of the bacterial liquid used in the step (3) comprises the following steps:

(1) activation of lyophilized bacteria

Melting and dispersing freeze-dried bacteria in 5mL of nutrient broth to form a suspension, culturing for 18h-24h at 37 +/-2 ℃ to prepare a bacterial suspension, taking the bacterial suspension by using an inoculating loop, inoculating the bacterial suspension on an agar culture medium plate by a scribing method, culturing for 18h-24h at 37 +/-2 ℃, taking a typical bacterial colony from a culture dish, inoculating the typical bacterial colony in an agar culture medium inclined tube, culturing for 18h-24h at 37 +/-2 ℃, storing the inclined tube in a refrigerator at 5-10 ℃ as a preservative, wherein the preservation period is not more than one month, the passage is carried out once per month, and the number of passage is not more than 10 generations;

(2) preparation of test bacterial solution

1) Inoculating the preserved strain to an agar culture medium plate by a streaking method, and culturing at 37 +/-2 ℃ for 24 h; the plate is stored at 5-10 ℃ and used within 1 week;

2) taking 20mL of nutrient broth, putting the nutrient broth into a 100mL Erlenmeyer flask, and inoculating a typical bacterial colony on the plate in the step 1) into the broth for culture; the culture conditions are that the temperature is 37 +/-2 ℃, the rotation speed is adjusted to 130r/min, and the time is 18-24 h;

3) the nutrient broth was diluted 20-fold with distilled water, and the concentration of the cultured bacteria was adjusted to 1X108CFU/mL-5X108CFU/mL to prepare a test bacterial solution.

The bacteria used were gram-negative in Escherichia coli (8099 or ATCC 11229).

The agar medium had the composition: each 1000mL of agar culture medium contains 10g of tryptone, 10g of sodium chloride, 17.5g of agar powder, 5g of yeast extract powder and the balance of distilled water; adjusting the pH value to 7.2 +/-0.2 after sterilization.

The nutrient broth comprises the following components: every 1000mL of nutrient broth contains 15g of tryptone, 5g of plant peptone, 5g of sodium chloride and the balance of distilled water; adjusting the pH value to 7.2 +/-0.2 after sterilization.

The equipment used in the invention comprises the following equipment: common laboratory instruments such as a desktop clean bench, a constant-temperature incubator, a micropipette, a plate, a test tube, a triangular flask, an alcohol lamp and a triangular coating rod; an autoclave: the temperature is maintained at 121 ℃ and the pressure can be maintained at 103 kPa.

Reagent: the reagents used in the assay should be analytically pure or suitable for microbiological testing. The test water is analytically pure water, i.e., the above-mentioned distilled water, for preparing the microbial culture medium, and can be prepared by ion exchange, distillation or filtration with reverse osmosis device, and has no toxicity and bacteriostatic substances.

Adopt the produced beneficial effect of above-mentioned technical scheme to lie in:

compared with GB 15981-1995: the detection method adopted by the invention can be used for detecting the antibacterial performance of the disinfectant with any concentration and in any batch by uniformly mixing the diluted bacterial suspension and the disinfectant, and the detection concentration range is wide.

The national standard method is used for detecting the antibacterial performance of the disinfectant by uniformly mixing the disinfectant with a certain diluted concentration gradient and the bacterial suspension, and the process for detecting the disinfectant with different concentrations and different batches is complicated, time-consuming and complex to operate. The detection method of the invention can simultaneously detect disinfectants with different concentrations and different batches, thereby saving a great deal of time.

The invention can simultaneously carry out the disinfectant qualitative disinfection test and the disinfectant quantitative disinfection test, thereby greatly saving time.

Drawings

FIG. 1 is a bacteriostatic effect diagram of disinfectant concentrations of 5ppm, 10ppm and 15ppm

FIG. 2 is a diagram of the bacteriostatic effect of 3ppm, 10ppm, 30ppm and 300ppm disinfectant concentration

FIG. 3 is a diagram of the bacteriostatic effect of 10ppm, 30ppm, 300ppm and 3000ppm disinfectant concentration

FIG. 4 is a diagram of the bacteriostatic effect of 3ppm, 15ppm and 30ppm disinfectant concentration

Detailed Description

The following examples illustrate the invention in detail. The raw materials and various devices used in the invention are conventional commercially available products, and can be directly obtained by market purchase.