CN111206067B - Method for evaluating wet tissue anticorrosion effect - Google Patents
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Abstract
The invention belongs to the technical field of wet tissue anticorrosion effect evaluation, and relates to a wet tissue anticorrosion effect evaluation method. The evaluation method comprises the following steps: s1 bacterial contamination of the cloth: mixing various bacterial liquids and fungal suspensions in equal proportion to obtain a mixed bacterial liquid; dividing 10 pieces of wet tissues into 5 pieces of bacteria samples and 5 pieces of fungi samples, flatly spreading the samples, dripping mixed bacteria liquid, and putting the samples into a sterile bottle, wherein the samples are marked as bacteria 0d, bacteria 7d, bacteria 14d, bacteria 21d, bacteria 28d, fungi 0d, fungi 7d, fungi 14d, fungi 21d and fungi 28 d; adding eluent into the mixture at 0d, 7d, 14d, 21d and 28d respectively to wash down the microorganisms on the sample, absorbing the sample solution or the diluent, placing the sample solution or the diluent in a sterilization flat dish, pouring the culture medium, turning the flat dish after agar is solidified, and counting viable bacteria colonies after the culture; calculating the result of S2; and S3 evaluation method. The evaluation method is simple and easy to operate, and is suitable for large-batch screening.
Description
Technical Field
The invention belongs to the technical field of wet tissue anticorrosion effect evaluation, and particularly relates to a wet tissue anticorrosion effect evaluation method.
Background
Wet wipes are moist paper towels used to wipe the skin; the cleaning agent is prepared by taking non-woven fabrics, dust-free paper or other materials as carriers, taking purified water as production water and adding a proper amount of auxiliary materials such as humectant, essence, preservative and the like; a product has cleaning effect on human body surface or object surface. With the continuous improvement of living standard of people, the consumption of various wet tissues is also continuously increased, the growth of the global sanitary product industry is turning from a developed market to an emerging market, and the wet tissues and the wet tissue products still have potential in the developed market. The production technology of the wiping towel products is continuously improved, and the market application is also continuously expanded. The facial mask is various in variety from various facial masks, car wiping wet tissues, makeup removing wet tissues and disinfection wet tissues to sanitary care products for children and the like. Because the wet tissue is rich in water and nutrient substances and is easy to cause microbial pollution and deterioration, manufacturers of the wet tissue need to strictly disinfect the wet tissue in the manufacturing process of the wet tissue and also need to have quite good anti-corrosion and anti-mildew effects in the shelf life of the wet tissue.
Factors affecting the effectiveness of antimicrobial agents in wet wipes include: 1. a wet tissue liquid formula; 2. the type of nonwoven fabric; 3. production processes, etc., and thus the performance of the same antimicrobial agent in different products may vary greatly. The method of evaluating the effectiveness of an antimicrobial agent in a product is important. Due to the characteristics of the cloth piece, the cloth piece cannot be evaluated as a whole by decontaminating bacteria like liquid, and USP42<51> Antimicrobial effective Testing is not suitable for wet paper towels. Therefore, it is necessary to provide a method for evaluating the preservative effect that can be used for wet tissues.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a method for evaluating the antiseptic effect of a wet tissue. The method for evaluating the anticorrosion effect of the wet tissue is simple and easy to operate, is suitable for large-batch screening, and is suitable for evaluating the anticorrosion effect of the wet tissue.
The technical scheme of the invention is as follows:
the method for evaluating the antiseptic effect of the wet tissue comprises the following steps:
s1 bacterial contamination of the cloth: respectively mixing various bacterial liquids in equal proportion, and mixing various fungus suspensions in equal proportion to obtain mixed bacterial liquids;
dividing 10 pieces of wet tissue samples with the size of 15cm multiplied by 20cm into 5 pieces of bacteria samples and 5 pieces of fungi samples, after the samples are flattened, respectively dripping 0.2mL of mixed bacterial liquid at four corners and the middle to ensure that the bacterial colony number of each piece of bacteria sample reaches 105cfu/tablet-3X 106cfu/piece, the number of fungal colonies per piece of fungal sample is 105cfu/tablet-3X 106cfu/piece, after drop-dyeing, putting into a sterile bottle respectively, and marking as bacterium 0d, bacterium 7d, bacterium 14d, bacterium 21d, bacterium 28d, fungus 0d, fungus 7d, fungus 14d, fungus 21d, and fungus 28 d;
adding 100mL of diluent as eluent to wash down microorganisms on a wet tissue sample at 0d, 7d, 14d, 21d and 28d respectively, absorbing sample liquid, or taking 1mL of diluent with 2-3 dilutions after dilution, placing the diluted sample liquid or the diluted diluent in sterilization flat dishes, inoculating two sterilization flat dishes to each sample liquid or the diluted diluent, pouring bacteria by using 15mL of nutrient agar culture medium cooled to 40-45 ℃, pouring fungi by using 15mL of Sabouraud's dextrose agar culture medium cooled to 40-45 ℃, rotating the flat dishes to fully and uniformly turn the flat dishes after agar is solidified, culturing the bacteria at 33-37 ℃ for 48 hours, culturing the fungi at 26-30 ℃ for 3-5 d, and counting viable bacteria colonies;
s2 calculation of results: m ═ rxzx 100, where M is the number of bacteria or fungi per time point and R is the dilution factor; z is the average of the number of colonies on the two plates, and is the unit cfu; 100 is the dosage of the eluent, and the unit is mL;
evaluation method of S3: the reduction of the total number of bacteria from the initial to 14 days on a logarithmic scale cannot be less than 2.0, and the number of bacteria from 14 to 28 days cannot be increased; the total number of fungi could not increase from the initial to 14 days and 28 days, which indicates that the wet tissue has antiseptic effect.
Further, the bacterial liquid in step S1 includes staphylococcus aureus liquid, escherichia coli liquid, and pseudomonas aeruginosa liquid.
Further, the preparation method of the bacterial liquid in the step S1 includes:
(1) opening a freeze-dried strain tube under aseptic operation, adding a nutrient broth culture medium, and gently blowing and sucking to melt and disperse strains to obtain a strain suspension; dripping strain suspension into a test tube containing 5.0-10.0 mL of nutrient broth culture medium, culturing at 37 ℃ for 18-24 h, taking the strain suspension cultured for 1 generation by using an inoculating loop, streaking and inoculating the strain suspension onto a nutrient agar culture medium flat plate, culturing at 37 ℃ for 18-24 h, selecting typical bacterial colonies in 2 generations, inoculating on a nutrient agar inclined plane, and culturing at 37 ℃ for 18-24 h to obtain a 3 rd generation culture;
(2) taking a fresh culture of a 3 rd-14 th generation nutrient agar culture medium slant, sucking 3.0-5.0 mL of diluent, adding the diluent into a slant test tube, repeatedly blowing and sucking, washing off bacterial lawn, transferring a washing liquid into another sterile test tube, and uniformly suspending bacteria to obtain an original bacterial suspension;
(3) firstly, the bacteria-containing concentration of the original bacteria suspension obtained in the step (2) is measured by a bacteria concentration turbidimetry method, and then the original bacteria suspension is diluted to 1 x 10 by a diluent8cfu/mL～5×108cfu/mL, and obtaining the product.
Further, the fungus suspension of step S1 includes candida albicans suspension and aspergillus niger spore suspension.
Further, the preparation method of the candida albicans suspension comprises the following steps:
(a) taking a freeze-dried strain tube, opening the freeze-dried strain tube in an aseptic operation, adding the freeze-dried strain tube into a sandcastle liquid culture medium tube, and gently blowing and sucking to melt and disperse strains to obtain a strain suspension; dripping strain suspension into a test tube containing 5.0-10.0 mL of sandcastle liquid culture medium, culturing at 37 ℃ for 18-24 h, taking a typical colony in a 1 st generation culture by using an inoculating loop, streaking and inoculating the typical colony on a sandcastle agar culture medium flat plate, culturing at 37 ℃ for 18-24 h, selecting a typical colony in a second generation culture, inoculating on a sandcastle agar slant, and culturing at 37 ℃ for 18-24 h to obtain a 3 rd generation slant culture;
(b) during test, continuously passaging the 3 rd generation slant culture obtained in the step (a) on a sandcastle agar slant by the same method as the 3 rd generation, taking a 5 th generation or 6 th generation sandcastle agar culture medium slant fresh culture, sucking 3.0-5.0 mL of diluent, adding the diluent into a slant test tube, repeatedly blowing and sucking, washing off thalli, transferring a washing solution into another sterile test tube, and enabling candida albicans suspension to be uniform to obtain an original bacterial suspension;
(c) measuring the bacteria-containing concentration of the original bacterial suspension obtained in step (b) by using a bacteria concentration turbidimetry method, and then diluting the original bacterial suspension to 1 x 10 by using a diluent7cfu/mL～5×107cfu/mL, and obtaining the product.
Further, the preparation method of the aspergillus niger spore suspension comprises the following steps:
taking a freeze-dried strain tube, opening the freeze-dried strain tube in a sterile operation, sucking normal saline, adding the normal saline into the strain tube, and slightly blowing and sucking to melt and disperse strain precipitates to obtain a precipitate suspension; adding the precipitate suspension into a test tube containing 5.0mL of a Sabouraud's dextrose agar culture medium, culturing for 42-48 h at 30 +/-1 ℃, inoculating the 1 st generation culture into a plate of the Sabouraud's dextrose agar culture medium by using an inoculating loop streak, culturing for 42-48 h in an incubator at 30 +/-1 ℃, taking a typical bacterial colony in the plate culture, inoculating the typical bacterial colony in the culture medium, and culturing for 42-48 h in the incubator at 30 +/-1 ℃, thus obtaining a 3 rd generation slant culture;
(II) during the test, inoculating the 3 rd generation slant culture obtained in the step (I) into a culture medium, and culturing at 30 +/-1 ℃ for 7-9 days to obtain Aspergillus niger conidia;
(III) taking 5.0-10.0 mL of Tween 80 physiological saline solution with the volume concentration of 0.05%, scraping and washing the Aspergillus niger conidia obtained in the step (II) in the solution, transferring the spore suspension into an aseptic test tube, filtering by sterilized absorbent cotton to remove hyphae, observing whether the hyphae exists under a microscope after filtering, centrifuging for 20min at 5000-6000 r/min if the hyphae exists in the suspension, observing under the microscope again, and centrifuging again until the aseptic silk exists if the hyphae still exists in the suspension until the bacterial content of the bacterial suspension is 1 x 107cfu/mL～5×107cfu/mL。
Further, the preparation method of the Aspergillus niger spore suspension comprises the step (I) and the step (II), wherein the culture medium is a culture medium bevel of Saccharum sinensis Roxb.
Compared with the prior art, the invention has the following advantages:
(1) improvement of the Aspergillus niger spore culture method: the aspergillus niger is cultured by adopting the slant culture medium, and the operation is simpler compared with the culture by using a Roche flask.
(2) Due to the characteristics of the cloth piece, the cloth piece cannot be evaluated as a whole by decontaminating bacteria like liquid, and USP42<51> Antimicrobial effective Testing is not suitable for wet paper towels. According to the evaluation method for the antiseptic effect of the wet tissue, each time point is provided with the cloth piece with the same initial bacterial infection amount, the number of microorganisms on the corresponding cloth piece is measured after the cloth piece is placed for a certain time, and the antiseptic effect of the wet tissue is evaluated according to the change of the microorganisms at different time points.
(3) The method for evaluating the anticorrosion effect of the wet tissue is simple and easy to operate, and is suitable for large-batch screening.
(4) The evaluation method for the wet tissue anticorrosion effect quantizes the evaluation result, so that the result is objective, direct and accurate.
Detailed Description
The present invention is further described in the following description of the specific embodiments, which is not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art according to the basic idea of the invention, within the scope of the invention, as long as they do not depart from the basic idea of the invention.
In the present invention, the starting materials are commercially available unless otherwise specified. For example, the Staphylococcus aureus is Staphylococcus aureus (Staphylococcus aureus) ATCC 6538, the Escherichia coli is Escherichia coli (Escherichia coli)8099, the Pseudomonas aeruginosa is Pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC 15442, the Candida albicans is Candida albicans ATCC 10231, and the Aspergillus niger is Aspergillus niger ATCC 16404, which are all purchased from Guangdong province microorganism culture collection.
Nutrient broth medium (cat # 022010), nutrient agar medium (cat # 022020), Sabouraud dextrose agar medium (cat # 021099), and Sabouraud broth medium (cat # 021091) were all purchased from Kyoto Kay Microscience, Inc.
Example 1 preparation of bacterial suspension:
the bacterial liquid comprises staphylococcus aureus liquid, escherichia coli liquid and pseudomonas aeruginosa liquid.
The preparation method of the staphylococcus aureus liquid comprises the following steps:
(1) taking a freeze-dried staphylococcus aureus tube, wherein the amount of the freeze-dried strain in the freeze-dried staphylococcus aureus tube is 1.5 multiplied by 107Opening the cfu/bottle under aseptic operation, adding 3mL of nutrient broth culture medium, and gently blowing and sucking to melt and disperse the strains to obtain strain suspension; dripping 2mL of strain suspension into a test tube containing 8.0mL of nutrient broth culture medium, culturing at 37 ℃ for 22h, taking the strain suspension cultured for 1 generation by using an inoculating loop, streaking and inoculating the strain suspension on a nutrient agar culture medium plate, culturing at 37 ℃ for 22h, selecting typical colonies in 2 generations, inoculating the typical colonies on a nutrient agar slant, and culturing at 37 ℃ for 22h to obtain a 3 rd generation culture;
(2) taking a fresh culture of a slant of a nutrient agar culture medium of the 3 rd generation of the strain, sucking 4.0mL of diluent, adding the diluent into a slant test tube, repeatedly blowing and sucking, washing off lawn, transferring the washing liquid into another sterile test tube, and uniformly suspending bacteria to obtain an original bacterial suspension;
(3) firstly, the original material obtained in the step (2) is measured by a bacteria concentration turbidimetry methodThe bacteria concentration of the bacterial suspension is diluted to 3 x 10 by using a diluent8cfu/mL, and obtaining the product.
The preparation methods of the escherichia coli bacterial liquid and the pseudomonas aeruginosa bacterial liquid are similar to the preparation method of the staphylococcus aureus bacterial liquid.
The diluent is PBS diluent, and the preparation method of the PBS diluent comprises the following steps: weighing 2.83g of anhydrous disodium hydrogen phosphate and 1.36g of potassium dihydrogen phosphate, adding 1000mL of distilled water, adjusting the pH value to 7.2 after completely dissolving, and sterilizing for 20min by using pressure steam at 121 ℃ for later use.
Example 2 preparation of fungal suspension:
the fungus suspension comprises candida albicans suspension and aspergillus niger spore suspension.
The preparation method of the candida albicans suspension comprises the following steps:
(a) taking a candida albicans freeze-dried strain tube, wherein the amount of freeze-dried strains in the freeze-dried strain tube is 1.2 multiplied by 106Opening the cfu/bottle in a sterile operation, sucking the cfu/bottle by using a capillary pipette, adding 3mL of a sandcastle liquid culture medium into the cfu/bottle, and gently blowing and sucking the cfu/bottle to melt and disperse the strains to obtain a strain suspension; dripping strain suspension into a test tube containing 6.0mL of sandcastle liquid culture medium, culturing at 37 ℃ for 20h, taking a typical colony in a 1 st generation culture by using an inoculating loop, streaking and inoculating the typical colony on a sandcastle agar culture medium plate, culturing at 37 ℃ for 20h, selecting a typical colony in a second generation culture, inoculating on a sandcastle agar slant, and culturing at 37 ℃ for 20h to obtain a 3 rd generation slant culture;
(b) during test, continuously passaging the 3 rd generation slant culture obtained in the step (a) on a Sabouraud agar slant by the same method as the 3 rd generation, taking a fresh culture of the 5 th generation slant culture medium of the Sabouraud agar, sucking 4.0mL of diluent, adding the diluent into a slant test tube, repeatedly blowing and sucking, washing bacterial lawn, transferring washing liquor into another sterile test tube, and enabling the Candida albicans suspension to be uniform to obtain an original bacterial suspension;
(c) measuring the bacteria-containing concentration of the original bacterial suspension obtained in step (b) by using a bacteria concentration turbidimetry method, and then diluting the original bacterial suspension to 4 x 10 by using a diluent7cfu/mL, and obtaining the product.
The preparation method of the Aspergillus niger spore suspension comprises the following steps:
taking Aspergillus niger freeze-dried strain tube, wherein the amount of freeze-dried strain in the freeze-dried strain tube is 1.3 multiplied by 106The cfu/bottle is opened in a sterile operation, 3mL of physiological saline is absorbed by a capillary pipette and added into the strain tube, and the strain is slightly sucked by blowing, so that the strain precipitate is melted and dispersed to obtain precipitate suspension; adding the precipitate suspension into a test tube containing 5.0mL of a Sabouraud's dextrose agar culture medium, culturing for 45h at 30 ℃, inoculating the 1 st generation culture to a plate of the Sabouraud's dextrose agar culture medium by using an inoculating loop streak, culturing for 45h in a 30 ℃ incubator, taking a typical colony in the plate culture, inoculating to a slope of the Sabouraud's dextrose agar culture medium, and culturing for 45h in the 30 ℃ incubator to obtain a 3 rd generation slope culture;
(II) during the test, inoculating the 3 rd generation slant culture obtained in the step (I) to a slant of a Sabouraud's dextrose agar culture medium, and culturing for 8 days at 30 ℃ to obtain aspergillus niger conidia;
(III) taking 6.0mL of Tween 80 physiological saline solution with the volume concentration of 0.05%, scraping and washing the Aspergillus niger conidia obtained in the step (II) in the solution, transferring the spore suspension into a sterile test tube, filtering by using sterilized absorbent cotton to remove hyphae, observing whether hyphae exist under a microscope after filtering, centrifuging for 20min at 5600r/min if hyphae exist in the suspension, observing under the microscope again, and centrifuging again until the sterile hyphae exist if the hyphae still exist in the suspension until the bacterial hyphae exist, wherein the bacterial content of the bacterial suspension is 2 x 10 when the bacterial suspension is used7cfu/mL。
The diluent is PBS diluent, and the preparation method of the PBS diluent comprises the following steps: weighing 2.83g of anhydrous disodium hydrogen phosphate and 1.36g of potassium dihydrogen phosphate, adding 1000mL of distilled water, adjusting the pH value to 7.2 after completely dissolving, and sterilizing for 20min by using pressure steam at 121 ℃ for later use.
Example 3 evaluation method of Wet tissue antiseptic Effect
The method for evaluating the antiseptic effect of the wet tissue comprises the following steps:
s1 bacterial contamination of the cloth: respectively mixing the bacterial liquids prepared in the embodiment 1 in equal proportion and the fungal suspensions prepared in the embodiment 2 in equal proportion to obtain mixed bacterial liquids;
10 pieces of wet paper with the size of 15cm multiplied by 20cm are put in a vacuumThe towel samples are divided into 5 pieces of bacteria samples and 5 pieces of fungi samples, after the samples are flattened, 0.2mL of mixed bacterial liquid is respectively dripped at four corners and the middle of the samples, so that the bacterial colony number of each piece of bacteria sample reaches 2.5 multiplied by 106cfu/piece, the number of fungal colonies per piece of fungal sample was 1.5X 106cfu/piece, after drop-dyeing, putting into a sterile bottle respectively, and marking as bacterium 0d, bacterium 7d, bacterium 14d, bacterium 21d, bacterium 28d, fungus 0d, fungus 7d, fungus 14d, fungus 21d, and fungus 28 d;
respectively adding 100mL of diluent as eluent to 0d, 7d, 14d, 21d and 28d to wash down microorganisms on a wet tissue sample, taking 1mL of diluent with 2 dilutions after dilution, placing the diluent in sterilization plates, inoculating two sterilization plates to each diluent, pouring 15mL of nutrient agar culture medium cooled to 43 ℃, pouring 15mL of fungus with Sabouraud's glucose agar culture medium cooled to 43 ℃, rotating the plates to be fully uniform, turning the plates after agar solidification, culturing the bacteria at 35 ℃ for 48h, culturing the fungus at 28 ℃ for 4d, and counting viable bacteria colonies;
s2 calculation of results: m ═ rxzx 100, where M is the number of bacteria or fungi per time point and R is the dilution factor; z is the average of the number of colonies on the two plates, and is the unit cfu; 100 is the dosage of the eluent, and the unit is mL;
evaluation method of S3: the reduction of the total number of bacteria from the initial to 14 days on a logarithmic scale cannot be less than 2.0, and the number of bacteria from 14 to 28 days cannot be increased; the total number of fungi could not increase from the initial to 14 days and 28 days, which indicates that the wet tissue has antiseptic effect.
The diluent used as the eluent in the step S1 is a PBS diluent, and the preparation method of the PBS diluent is as follows: weighing 2.83g of anhydrous disodium hydrogen phosphate and 1.36g of potassium dihydrogen phosphate, adding 1000mL of distilled water, adjusting the pH value to 7.2 after completely dissolving, and sterilizing for 20min by using pressure steam at 121 ℃ for later use.
Test example I, evaluation results of wet tissue antiseptic effect
Cloth pieces from Nbond nowcovers were taken and water, glycerol and preservative sodium benzoate were mixed in 99: 0.5: 0.5 to obtain feed liquid, and spraying the feed liquid on a cloth piece to obtain the wet tissue. When the wet tissue preservative effect evaluation method of example 3 was used to evaluate the preservative effect of the wet tissue, the log reduction value of the total bacteria count from the initial to 14 days could not be less than 2.0, and the bacteria count could not increase from 14 to 28 days; the total number of fungi can not increase from the initial day to 14 days and 28 days, namely, the wet tissue has the antiseptic effect, and the result is judged to be passed. The evaluation results are shown in table 1.
Table 1: evaluation results of Wet tissue antiseptic Effect
As can be seen from table 1, the evaluation method for the wet tissue antiseptic effect provided by the invention quantifies the evaluation result, can objectively, directly and accurately judge the antiseptic effect of the wet tissue, is suitable for large-batch screening, and is suitable for evaluating the antiseptic effect of the wet tissue.
Claims (1)
1. The method for evaluating the anticorrosion effect of the wet tissue is characterized by comprising the following steps of:
s1 bacterial contamination of the cloth: respectively mixing the prepared bacterial liquids in equal proportion, and mixing the prepared fungal suspensions in equal proportion to obtain mixed bacterial liquids;
dividing 10 pieces of wet tissue samples with the size of 15cm multiplied by 20cm into 5 pieces of bacteria samples and 5 pieces of fungi samples, after the samples are flattened, respectively dripping 0.2mL of mixed bacterial liquid at four corners and in the middle to ensure that the bacterial colony number of each piece of bacteria sample reaches 2.5 multiplied by 106cfu/piece, the number of fungal colonies per piece of fungal sample was 1.5X 106cfu/piece, after drop-dyeing, putting into a sterile bottle respectively, and marking as bacterium 0d, bacterium 7d, bacterium 14d, bacterium 21d, bacterium 28d, fungus 0d, fungus 7d, fungus 14d, fungus 21d, and fungus 28 d;
respectively adding 100mL of diluent as eluent to 0d, 7d, 14d, 21d and 28d to wash down microorganisms on a wet tissue sample, taking 1mL of diluent with 2 dilutions after dilution, placing the diluent in sterilization plates, inoculating two sterilization plates to each diluent, pouring 15mL of nutrient agar culture medium cooled to 43 ℃, pouring 15mL of fungus with Sabouraud's glucose agar culture medium cooled to 43 ℃, rotating the plates to be fully uniform, turning the plates after agar solidification, culturing the bacteria at 35 ℃ for 48h, culturing the fungus at 28 ℃ for 4d, and counting viable bacteria colonies;
s2 calculation of results: m ═ rxzx 100, where M is the number of bacteria or fungi per time point and R is the dilution factor; z is the average of the number of colonies on the two plates, and is the unit cfu; 100 is the dosage of the eluent, and the unit is mL;
evaluation method of S3: the reduction of the total number of bacteria from the initial to 14 days on a logarithmic scale cannot be less than 2.0, and the number of bacteria from 14 to 28 days cannot be increased; the total number of fungi cannot be increased from the initial time to 14 days and 28 days, namely, the wet tissue has the preservative effect;
the diluent used as the eluent in the step S1 is a PBS diluent, and the preparation method of the PBS diluent is as follows: weighing 2.83g of anhydrous disodium hydrogen phosphate and 1.36g of potassium dihydrogen phosphate, adding 1000mL of distilled water, adjusting the pH value to 7.2 after completely dissolving, and sterilizing for 20min by using pressure steam at 121 ℃ for later use;
the bacterial liquid comprises staphylococcus aureus liquid, escherichia coli liquid and pseudomonas aeruginosa liquid;
the preparation method of the staphylococcus aureus liquid comprises the following steps:
(1) taking a freeze-dried staphylococcus aureus tube, wherein the amount of the freeze-dried strain in the freeze-dried staphylococcus aureus tube is 1.5 multiplied by 107Opening the cfu/bottle under aseptic operation, adding 3mL of nutrient broth culture medium, and gently blowing and sucking to melt and disperse the strains to obtain strain suspension; dripping 2mL of strain suspension into a test tube containing 8.0mL of nutrient broth culture medium, culturing at 37 ℃ for 22h, taking the strain suspension cultured for 1 generation by using an inoculating loop, streaking and inoculating the strain suspension on a nutrient agar culture medium plate, culturing at 37 ℃ for 22h, selecting typical colonies in 2 generations, inoculating the typical colonies on a nutrient agar slant, and culturing at 37 ℃ for 22h to obtain a 3 rd generation culture;
(2) taking a fresh culture of a slant of a nutrient agar culture medium of the 3 rd generation of the strain, sucking 4.0mL of diluent, adding the diluent into a slant test tube, repeatedly blowing and sucking, washing off lawn, transferring the washing liquid into another sterile test tube, and uniformly suspending bacteria to obtain an original bacterial suspension;
(3) firstly, the bacteria-containing concentration of the original bacteria suspension obtained in the step (2) is measured by a bacteria concentration turbidimetry method, and then the original bacteria suspension is diluted to 3 x 10 by a diluent8cfu/mL, namely obtaining;
the preparation methods of the escherichia coli liquid and the pseudomonas aeruginosa liquid are similar to the preparation method of the staphylococcus aureus liquid;
the diluent is PBS diluent, and the preparation method of the PBS diluent comprises the following steps: weighing 2.83g of anhydrous disodium hydrogen phosphate and 1.36g of potassium dihydrogen phosphate, adding 1000mL of distilled water, adjusting the pH value to 7.2 after completely dissolving, and sterilizing for 20min by using pressure steam at 121 ℃ for later use;
the fungus suspension comprises candida albicans suspension and aspergillus niger spore suspension;
the preparation method of the candida albicans suspension comprises the following steps:
(a) taking a candida albicans freeze-dried strain tube, wherein the amount of freeze-dried strains in the freeze-dried strain tube is 1.2 multiplied by 106Opening the cfu/bottle in a sterile operation, sucking the cfu/bottle by using a capillary pipette, adding 3mL of a sandcastle liquid culture medium into the cfu/bottle, and gently blowing and sucking the cfu/bottle to melt and disperse the strains to obtain a strain suspension; dripping strain suspension into a test tube containing 6.0mL of sandcastle liquid culture medium, culturing at 37 ℃ for 20h, taking a typical colony in a 1 st generation culture by using an inoculating loop, streaking and inoculating the typical colony on a sandcastle agar culture medium plate, culturing at 37 ℃ for 20h, selecting a typical colony in a second generation culture, inoculating on a sandcastle agar slant, and culturing at 37 ℃ for 20h to obtain a 3 rd generation slant culture;
(b) during test, continuously passaging the 3 rd generation slant culture obtained in the step (a) on a Sabouraud agar slant by the same method as the 3 rd generation, taking a fresh culture of the 5 th generation slant culture medium of the Sabouraud agar, sucking 4.0mL of diluent, adding the diluent into a slant test tube, repeatedly blowing and sucking, washing bacterial lawn, transferring washing liquor into another sterile test tube, and enabling the Candida albicans suspension to be uniform to obtain an original bacterial suspension;
(c) firstly, the bacteria concentration is measured by a turbidimetry methodThe original bacterial suspension obtained in the step (b) contains bacterial concentration, and then the bacterial concentration is diluted to 4 multiplied by 10 by using diluent7cfu/mL, namely obtaining;
the preparation method of the Aspergillus niger spore suspension comprises the following steps:
taking Aspergillus niger freeze-dried strain tube, wherein the amount of freeze-dried strain in the freeze-dried strain tube is 1.3 multiplied by 106The cfu/bottle is opened in a sterile operation, 3mL of physiological saline is absorbed by a capillary pipette and added into the strain tube, and the strain is slightly sucked by blowing, so that the strain precipitate is melted and dispersed to obtain precipitate suspension; adding the precipitate suspension into a test tube containing 5.0mL of a Sabouraud's dextrose agar culture medium, culturing for 45h at 30 ℃, inoculating the 1 st generation culture to a plate of the Sabouraud's dextrose agar culture medium by using an inoculating loop streak, culturing for 45h in a 30 ℃ incubator, taking a typical colony in the plate culture, inoculating to a slope of the Sabouraud's dextrose agar culture medium, and culturing for 45h in the 30 ℃ incubator to obtain a 3 rd generation slope culture;
(II) during the test, inoculating the 3 rd generation slant culture obtained in the step (I) to a slant of a Sabouraud's dextrose agar culture medium, and culturing for 8 days at 30 ℃ to obtain aspergillus niger conidia;
(III) taking 6.0mL of Tween 80 physiological saline solution with the volume concentration of 0.05%, scraping and washing the Aspergillus niger conidia obtained in the step (II) in the solution, transferring the spore suspension into a sterile test tube, filtering by using sterilized absorbent cotton to remove hyphae, observing whether hyphae exist under a microscope after filtering, centrifuging for 20min at 5600r/min if hyphae exist in the suspension, observing under the microscope again, and centrifuging again until the sterile hyphae exist if the hyphae still exist in the suspension until the bacterial hyphae exist, wherein the bacterial content of the bacterial suspension is 2 x 10 when the bacterial suspension is used7cfu/mL；
The diluent is PBS diluent, and the preparation method of the PBS diluent comprises the following steps: weighing 2.83g of anhydrous disodium hydrogen phosphate and 1.36g of potassium dihydrogen phosphate, adding 1000mL of distilled water, adjusting the pH value to 7.2 after completely dissolving, and sterilizing for 20min by using pressure steam at 121 ℃ for later use.
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