CN110819691A - Method for identifying and evaluating sterilization effect of disinfectant - Google Patents
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- 239000000645 desinfectant Substances 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 19
- 230000001954 sterilising effect Effects 0.000 title claims abstract description 15
- 238000004659 sterilization and disinfection Methods 0.000 title claims abstract description 12
- 230000000694 effects Effects 0.000 title claims abstract description 6
- 238000012360 testing method Methods 0.000 claims abstract description 134
- 230000001580 bacterial effect Effects 0.000 claims abstract description 37
- 238000002156 mixing Methods 0.000 claims abstract description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 12
- 238000012258 culturing Methods 0.000 claims abstract description 9
- 239000013068 control sample Substances 0.000 claims abstract description 6
- 239000002504 physiological saline solution Substances 0.000 claims abstract 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 17
- 241000894006 Bacteria Species 0.000 claims description 16
- 235000015097 nutrients Nutrition 0.000 claims description 13
- 229920001817 Agar Polymers 0.000 claims description 12
- 239000008272 agar Substances 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000012137 tryptone Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 3
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 239000001888 Peptone Substances 0.000 claims description 2
- 108010080698 Peptones Proteins 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims description 2
- 229940041514 candida albicans extract Drugs 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims description 2
- 238000002844 melting Methods 0.000 claims description 2
- 230000008018 melting Effects 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 239000003755 preservative agent Substances 0.000 claims description 2
- 230000002335 preservative effect Effects 0.000 claims description 2
- 239000000523 sample Substances 0.000 claims description 2
- 239000012138 yeast extract Substances 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims 8
- 238000004364 calculation method Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 12
- 230000003385 bacteriostatic effect Effects 0.000 abstract description 10
- 244000005700 microbiome Species 0.000 abstract description 3
- 239000003242 anti bacterial agent Substances 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 4
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 229920001661 Chitosan Polymers 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000001223 reverse osmosis Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
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Abstract
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.
Description
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.
Example 1
(1) Arranging 8 sterilized test tubes on a test tube rack, marking the numbers of the test tubes, (2) adding 0.9% sterilized normal saline to each test tube, wherein 9mL is added to the test tube No. 1-5, 10mL is added to the test tube No. 6-8, placing 8 test tubes into a water bath at 20 +/-2 ℃, (3) adding 1mL of bacterial solution to the test tube No. 1, mixing the mixture, adding 1mL of bacterial solution to the test tube No. 2, mixing the mixture, adding 1mL of bacterial solution to the test tube No. 3, test tube No. 4, test tube No. 5, mixing the solutions of the test tubes No. 6-8, adding the solutions of the test tubes No. 6-8 to the test tube No. 3-5, mixing the solutions, adding 10mL of the solutions to the test tube No. 6-8, (4) taking 30. mu.l of normal saline from the test tube No. 3, adding 30. mu.l of PHMG (namely, the concentration of disinfectant is 3ppm) to the test tube No. 3, adding the disinfectant to the test tube No. 4, adding the disinfectant to the test tube, mixing the broth, adding the broth to the test tube No. 6-5, adding the broth to the broth, mixing the broth, adding the broth to the broth, stirring, adding the broth, stirring, adding the broth, stirring, adding the broth, stirring, adding the broth, stirring:
| concentration of disinfectant | 0ppm | 3ppm | 5ppm | 10ppm | 30ppm | 50ppm |
| Rate of inhibition of bacteria | 0％ | 96.75％ | 98.78％ | 99.84％ | 99.99％ | 99.99％ |
| Positive/negative | + | + | - | - | - | - |
Example 2
(1) Arranging 6 sterilized test tubes on a test tube rack, marking the number of the test tubes, (2) adding 0.9% of sterilized normal saline into each test tube, wherein 9mL is added into the test tube 1-4, 10mL is added into the test tube 5-6, placing 6 test tubes into a water bath at 20 +/-2 ℃, (3) adding 1mL of bacterial liquid into the test tube 1, uniformly mixing, taking three 1mL of bacterial liquid from the test tube 2, respectively, transferring into the test tube 3 and the test tube 4, respectively, uniformly mixing, sequentially pouring the solution of the test tube 6-8 into the test tube 3-5, uniformly mixing, respectively transferring 10mL of solution from the test tube 3-5 into the test tube 6-8, (4) taking 30 μ l of normal saline from the test tube 3, taking 10000 μ l of nano silver ion antibacterial agent (namely, the concentration of the antibacterial agent is 30ppm), uniformly mixing, taking 60 μ l of normal saline from the test tube 4, taking 10 μ l of nano silver ion antibacterial agent from the test tube 3, taking 10 μ l of the nano silver ion antibacterial agent (namely, the concentration of the antibacterial agent is 30 μ l, namely, the concentration is 30ppm, the concentration is taken from the test tube 3 to the test tube 4, the test tube 4. the test tube, taking the test tube, the test tube 10 μ l of the test tube, the test tube is taken, the test tube is taken, the test tube is taken, the test tube, the:
| concentration of disinfectant | 0ppm | 30ppm | 60ppm | 100ppm |
| Rate of inhibition of bacteria | 0％ | 64.76％ | 86.45％ | 97.65％ |
| Positive/negative | + | + | + | - |
Example 3
(1) Arranging 8 sterilized test tubes on a test tube rack, marking the number of the test tubes, (2) adding 0.9% of sterilized normal saline into each test tube, wherein 9mL of the 1-5 test tube is added, 10mL of the 6-8 test tube is added, placing 8 test tubes into a water bath at 20 +/-2 ℃, (3) adding 1mL of bacterial liquid into the 1 st test tube, mixing uniformly, taking three 1mL of bacterial liquid from the 2 nd test tube, respectively, transferring into the 3 rd test tube, the 4 th test tube and the 5 th test tube, mixing uniformly the 3-5 test tubes, sequentially pouring the 6-8 test tube into the 3-5 test tube, mixing uniformly, transferring 10mL of bacterial liquid from the 3-5 test tube into the 6-8 test tube, (4) taking 50 μ l of normal saline from the 3 test tube, taking 50 μ l of chitosan disinfectant (namely, the concentration of 50ppm of disinfectant is 50ppm) into the 3 rd test tube, taking 100 μ l of disinfectant from the 4 th test tube, taking out of the normal saline from the 3 rd test tube, taking out of the 10 μ l of disinfectant, taking the broth from the 1 st test tube, taking the broth from the broth, mixing uniformly, taking the broth from the broth, taking the broth, mixing, taking the broth, taking the broth, taking the broth:
| concentration of disinfectant | 0ppm | 50ppm | 100ppm | 300ppm | 500ppm | 1000ppm |
| Rate of inhibition of bacteria | 0％ | 72.65％ | 87.64％ | 95.84％ | 98.29％ | 99.99％ |
| Positive/negative | + | + | + | + | - | - |
The above description is only presented as an enabling solution for the present invention and should not be taken as a sole limitation on the solution itself.
Claims (6)
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.
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| CN112378902A (en) * | 2020-11-03 | 2021-02-19 | 华中农业大学 | Method for rapidly evaluating disinfection effect of antibacterial disinfectant |
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| CN111398260A (en) * | 2020-03-31 | 2020-07-10 | 广州立白企业集团有限公司 | Method for rapidly evaluating antibacterial efficacy and method for displaying antibacterial efficacy on surface of carrier |
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