阅读说明：本技术 一种评估消毒剂效果的菌片及其制备方法与应用 (Bacterial tablet for evaluating effect of disinfectant, and preparation method and application thereof ) 是由 陈潇 刘辉 李忠 程琳越 刘岚铮 胡光春 钟源霞 时玉雯 崔雯 于 2021-09-03 设计创作，主要内容包括：本发明属于消毒剂技术领域,具体涉及一种评估消毒剂效果的菌片及其制备方法与应用。所述制备方法具体为：将菌液以冷冻干燥的方式加载于载体上,形成菌片。本发明所提供的菌片能够有效将高浓度的菌株加载于载体上,进而能够对大多数消毒剂进行评估体系,消毒效果检测时间由现有技术中的几天甚至半个月的时间缩短为48～72h,具有高效、保存时间长、方便、省时省力等优点。(The invention belongs to the technical field of disinfectants, and particularly relates to a bacterial tablet for evaluating the effect of a disinfectant, and a preparation method and application thereof. The preparation method specifically comprises the following steps: and (3) loading the bacterial liquid on the carrier in a freeze drying mode to form bacterial tablets. The bacterial tablet provided by the invention can effectively load high-concentration bacterial strains on a carrier, and further can evaluate most disinfectants, the disinfection effect detection time is shortened to 48-72 h from several days or even half a month in the prior art, and the bacterial tablet has the advantages of high efficiency, long preservation time, convenience, time and labor saving and the like.)

1. A preparation method of a fungus tablet is characterized by comprising the following steps: loading the bacterial liquid on a carrier in a freeze drying mode to form bacterial tablets;

the carrier is one of filter paper, metal, glass, cotton cloth or plastic.

2. The method of claim 1, wherein: the method comprises the following specific steps:

(1) sterilizing the carrier;

(2) performing generation recovery on the preserved strain on a culture medium;

(3) diluting the recovered strain with a preservation solution prepared by freeze-drying and preserving components of the strain to obtain a strain suspension;

(4) loading the bacterial suspension on a carrier, and then carrying out freeze drying on the obtained carrier to obtain freeze-dried bacterial tablets;

(5) and (5) freezing and storing the freeze-dried bacterial tablets.

3. The method of claim 2, wherein: when the disinfectant such as aerosol or ultra-low volume spray is evaluated, the carrier is a metal sheet or a glass sheet;

or, the carriers except the filter paper are subjected to degreasing treatment before being infected with bacteria;

the degreasing method comprises the following steps:

(1) boiling the carrier in water containing detergent for 30 min;

(2) washing with tap water;

(3) boiling with distilled water for 10 min;

(4) rinsing with distilled water until pH is neutral;

(5) airing and ironing for later use;

or the sterilization condition of the carrier is that the carrier is treated for 10-20min at the temperature of 120-130 ℃.

4. The method of claim 2, wherein: the strain is propagated from 3 rd generation to 8 th generation, and the culture time is 18h-24 h.

5. The method of claim 2, wherein: the preservation solution comprises nutrient components and a freeze-drying protective agent, wherein the freeze-drying protective agent is glycerol, and the nutrient components are one or more of proteins, sugar alcohols and amino acids;

further, the protein includes one or more of bovine serum albumin, brain heart infusion or casein;

further, the sugar alcohol comprises one or more of sucrose, trehalose and glucose.

6. The method of claim 2, wherein: for the disinfectant used for the disinfection objects which are not cleaned or dirty, the bacteria content of the bacterial suspension after the preservation solution is diluted is 1 multiplied by 10⁹～5×10⁹CFU/mL, then adding 3.0% bovine serum albumin equal to the bacterial suspension to make the bacterial suspension concentration 1X 10⁸～5×10⁸CFU/mL, and then loading; for the disinfectant used for cleaned or cleaner disinfection objects, the bacterium content of the bacterium suspension after the preservation solution is diluted is 1 multiplied by 10⁹～5×10⁹CFU/mL, then adding 0.3% bovine serum albumin equal to the bacterial suspension to make the bacterial suspension concentration 1X 10⁸～5×10⁸CFU/mL, and then loading; the disinfectant for strictly cleaned or extremely cleaned disinfection objects does not use bovine serum albumin, and the concentration of the bacterial suspension after the preservation solution is diluted is 1 multiplied by 10⁸～5×10⁸CFU/mL, and then loading;

further, when bovine serum albumin with the same amount as the bacterial suspension is not added, the preserving fluid comprises 1% of sucrose, 0.25% of sodium glutamate, 20% of glycerol and 3.7% of brain heart infusion culture medium; when bovine serum albumin with the same quantity as the bacterial suspension is added, the preserving fluid comprises 2% of sucrose, 0.5% of sodium glutamate, 40% of glycerol and 7.4% of brain heart infusion culture medium;

further, when the bacterial tablet is used for evaluating the low-temperature on-site disinfection effect, bovine serum albumin is replaced by trypsin, preferably tryptone soybean broth culture medium.

7. The method of claim 2, wherein: when the bacterial suspension is loaded on the carrier, 10 mu L of bacterial suspension needs to be dripped on each square carrier with 100 square millimeters or each round carrier with 113.04 square millimeters;

or, the freeze drying condition is freezing for 24 hours at minus 30 to minus 60 ℃;

or the freeze-dried bacterium slices are stored at the temperature of-20 ℃ to-40 ℃.

8. The method according to any one of claims 1 to 7, wherein the number of recovered bacteria in the mushroom pieces is 5X 10⁵cfu/tablet-5X 10⁶cfu/tablet.

9. A method for evaluating the disinfection effect of a disinfectant, comprising: the method specifically comprises the following steps: a sterilization simulation test using the disc of claim 8 to evaluate the sterilization effect of the disinfectant.

10. The method of claim 9, wherein: the disinfection simulation test is a carrier soaking sterilization test;

the method specifically comprises the following steps: disinfectant with different concentrations and 5.0 mL/piece are respectively injected into different sterile plates to simulate different disinfection conditions;

placing the plate containing disinfectant in 20 + -1 deg.C water bath for 5-8min, and soaking the bacteria tablet in the disinfectant;

taking out the sterilized bacterial tablet with sterile forceps, adding into 5.0-8mL neutralizer, mixing for 20-30s electrically or beating with palm for 80-90 times for neutralization for 10-15min, inoculating into a plate, and determining the number of viable bacteria;

furthermore, the disinfection simulation test needs to replace a disinfectant with a diluent or standard hard water to make a positive control test.

Technical Field

The invention belongs to the technical field of disinfectants, and particularly relates to a bacterial tablet for evaluating the effect of a disinfectant, and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

At present, methods for evaluating the effect of a disinfectant on killing microorganisms mainly comprise the steps of preparing bacterial suspension, preparing bacterial tablets and counting and culturing viable bacteria, wherein the preparation of the bacterial tablets is a key step in the process of detecting the disinfection effect of the disinfectant. According to technical specification of disinfection (2002 edition), the preparation process of the bacterial tablet comprises the steps of recovery culture of related standard bacterial strains, preparation of bacterial propagules, spore or fungal suspensions, low-staining of carriers and the like. After the carrier of the bacterial tablet is pretreated by bacterial contamination, the bacterial tablet is made by bacterial contamination with a low-staining method, and the bacterial tablet is dried in a 37 ℃ incubator or is used for a disinfectant sterilization test after being dried conventionally. In the process of preparing the bacterial tablet, the preparation process of the bacterial suspension for the bacterial infection is complex and long in time. The preparation process of the bacterial propagule suspension takes about 54-72 hours, the bacterial suspension is stored in a refrigerator at 4 ℃ after being prepared, and the bacterial suspension cannot be used overnight on the same day; the preparation process of the bacterial spore suspension is relatively complex relative to a propagule, the whole process takes 9-11 days, the steps are complicated and long, and the prepared spore suspension can be stored in a refrigerator at 4 ℃ for half a year; due to the long culture time of some fungi such as aspergillus niger, the preparation time of the fungal suspension is the longest compared with the bacterial suspension, about 13d to 15d is consumed, and the fungal conidium suspension can not be stored for more than two days under the condition of 2 ℃ to 8 ℃. In addition, bacteria, spores or fungi on the carrier can cause partial death in the baking process in the bacterial contamination process by the drop dyeing method, and even if the carrier is immediately placed in a refrigerator at 4 ℃ after preparation, the natural death rate of the strains is high and the storage time is short because no strain protective agent exists. Therefore, when the disinfection effect of the disinfectant is detected every time, experimenters need to repeat the tedious experimental steps to ensure the freshness of the bacterial tablets and the accuracy of experimental results, and a great deal of time, manpower, material resources and financial resources are consumed.

With the popularization of freeze drying equipment and the gradual maturity of the technology, at present, a freeze drying method is used as a method for storing bacteria for a long time in domestic and foreign conditional laboratories. The vacuum freeze drying technology is that the microbe is frozen into solid state in low temperature environment with added protecting agent, and the water is sublimated directly into gas state in vacuum to make the microbe in dry, oxygen-deficient and physiological activity inhibited state while avoiding direct damage. The inventor finds that the strain source based on the microporous filter membrane has limited bacterial load, can only carry out counting comparison of limited bacterial load, and often needs a high-concentration strain in the actual disinfectant evaluation process, so the use of the strain source is very limited, and the effective evaluation of the disinfectant cannot be realized.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides the bacterial tablet for evaluating the effect of the disinfectant, and the preparation method and the application thereof, the bacterial tablet provided by the invention can effectively load high-concentration bacterial strains on a carrier, so that the disinfectant can be effectively evaluated, the detection time of the disinfection effect is shortened to 48-72 h from the time of several days or even half a month in the prior art, and the bacterial tablet has the advantages of high efficiency, long preservation time, convenience, time saving, labor saving and the like.

The specific scheme is as follows:

the invention provides a preparation method of a bacterial tablet for evaluating the effect of a disinfectant, which comprises the following steps: loading the bacterial liquid on a carrier in a freeze drying mode to form bacterial tablets;

the carrier is one of filter paper, metal, glass, cotton cloth or plastic.

The method comprises the following specific steps:

(1) sterilizing the carrier;

(2) performing generation recovery on the preserved strain on a culture medium;

(3) diluting the recovered strain with a preservation solution prepared by freeze-drying and preserving components of the strain to obtain a strain suspension;

(4) loading the bacterial suspension on a carrier, and then carrying out freeze drying on the obtained carrier to obtain freeze-dried bacterial tablets;

(5) and (5) freezing and storing the freeze-dried bacterial tablets.

The invention provides a bacterial tablet for evaluating the effect of the disinfectant, which is obtained by the preparation method.

The third aspect of the invention provides a method for evaluating the disinfection effect of a disinfectant, which comprises the following steps: the bacterial tablet is adopted to carry out disinfection simulation test to evaluate the disinfection effect of the disinfectant.

One or more embodiments of the present invention have at least the following advantageous effects:

(1) the bacterial tablet provided by the invention can effectively load high-concentration bacterial strains on the carrier through the structural characteristics of the carrier and the freeze drying process, so that most of disinfectants can be evaluated, and the bacterial tablet has practical application value.

(2) When the disinfectant disinfection effect of the disinfectant is detected, the detection time is greatly shortened, the detection can be completed within 48-72 hours, and the disinfectant evaluation efficiency can be remarkably improved. Moreover, the fungus piece provided by the invention is a disposable and ready-to-use product, and can be directly purchased and used in a laboratory, so that the fungus piece preparation work before a disinfection effect detection experiment is not needed, and the problem of the preservation time of the fungus piece is solved, so that corresponding inspectors and resources are liberated, and the detection efficiency is further improved.

(3) The preparation work of the fungus pieces can be delivered to manufacturers, and the manufacturers replace a large amount of long, time-consuming, complex and repetitive fungus piece preparation work in laboratories through batch production, so that the total social cost is saved.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a photograph of a mushroom chip prepared in example 1 of the present invention;

FIG. 2 is a graph showing the number of recovered cells in a cell patch versus time in example 1 of the present invention;

FIG. 3 is a graph showing the time required for the method of example 3 of the present invention to detect the disinfecting effect of a disinfectant in comparison with the time required for the original method.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background art, the prepared bacterial tablets can be used for evaluating the effect of the disinfectant by using the microporous filter membrane as a carrier and loading the bacterial strains on the microporous filter membrane in a freeze-drying mode, but the bacterial tablets based on the microporous filter membrane have limited bacterial carrying capacity, can only count and compare the bacterial carrying capacity of a limited number of bacteria, are very limited in the actual use process, and cannot effectively evaluate the disinfectant.

In order to solve the above technical problems, a first aspect of the present invention provides a method for preparing a bacterial tablet for evaluating the effect of a disinfectant, wherein a bacterial solution is applied to a carrier in a freeze-drying manner to form a bacterial tablet;

the carrier is one of filter paper, metal, glass, cotton cloth or plastic.

The problems specifically solved are as follows:

(1) the invention relates to a problem of thallus protection, which uses a vacuum freeze-drying method, but in the freeze-drying process of thallus, a large amount of water in the thallus can be frozen to form ice crystals, and the ice crystals can damage the integrity of cell membranes, thereby influencing the functions of the thallus. The invention selects a proper freeze-drying protective agent, can reduce cell damage to the maximum extent and keep the activity and performance of thalli.

(2) The preparation process of the bacterial suspension is long in time consumption, the bacterial propagule suspension and the fungus suspension are limited by environmental conditions, the bacterial propagule suspension and the fungus suspension need to be prepared in situ before each preparation of the bacterial tablet, the process is complex, the time consumption is long, the storage time is short, and the preparation process is complex and long although the storage time of the bacterial spore suspension is relatively long. The invention hands over the preparation procedure of the bacterial suspension to manufacturers, and the prepared bacterial suspension is made into disposable freeze-dried bacterial tablets, which can be stored for a long time, save the experimental time, improve the experimental efficiency and be used for a long time in laboratories.

(3) The method has the advantages that the freeze-drying preservation solution is added with nutrient components such as saccharides and proteins, so that the long-term survival of the thalli can be ensured, and a good preservation effect is achieved.

It is particularly emphasized that the strain source described in patent CN 112980729 a previously published by the present inventors is mainly used for evaluating the culture medium, and although it is described that the strain source can also be used for evaluating the disinfection effect of the disinfectant, it is very limited in the using process because the strain-carrying body in the strain source is a microporous filter membrane and can only carry a recovery strain at a low concentration, as described in the patent: ' the concentration of the recovered strain after being diluted by the freeze-dried preserved components of the thallus is not higher than 10³CFU/ml ", and in practice in the testing of the disinfecting efficacy of disinfectants, general disinfectant evaluationThe process requires the introduction of a large number of strains, generally up to 10⁸CFU/ml, therefore, the source of the strain in this patent is not well suited for most disinfectant efficacy testing procedures.

Based on the problems existing when the strain source in the patent is used for evaluating the disinfection effect of the disinfectant, the scheme provided by the invention is provided for specifically evaluating the effect of the disinfectant.

The preparation method specifically comprises the following steps:

(1) sterilizing the carrier;

(2) performing generation recovery on the preserved strain on a culture medium;

(3) diluting the recovered strain with a preservation solution prepared by freeze-drying and preserving components of the strain to obtain a strain suspension;

(4) loading the bacterial suspension on a carrier, and then carrying out freeze drying to obtain freeze-dried bacterial tablets;

(5) and (5) freezing and storing the freeze-dried bacterial tablets.

Further, in the evaluation of a disinfectant such as an aerosol or an ultra-low volume spray, a carrier having an adsorption ability such as a cloth sheet or a filter paper sheet is not used, and a metal sheet or a glass sheet is selected.

Furthermore, the carriers except the filter paper need to be degreased before being infected with bacteria.

The degreasing method comprises the following steps:

(1) boiling the carrier in water containing detergent for 30 min;

(2) washing with tap water;

(3) boiling with distilled water for 10 min;

(4) rinsing with distilled water until pH is neutral;

(5) air drying and ironing for later use.

Further, the sterilization condition of the carrier is 120-130 ℃ for 10-20 min.

Further, the carrier is 10mm × 10mm square;

preferably, the metal sheet is a circular shape having a diameter of 12mm and a thickness of 0.5mm when used as a carrier.

In one or more embodiments of the invention, the strain is a thallus obtained by passaging a standard strain from a traceable strain preservation institution, or a strain which is verified by a laboratory to have stable representative characteristics of the strain and is passaged for 3 rd generation to 8 th generation, and the culture time is 18h to 24 h.

Further, the culture medium of the strain is a solid culture medium.

In one or more embodiments of the invention, the preservation solution comprises a nutrient component and a freeze-drying protective agent, wherein the freeze-drying protective agent is glycerol, and the nutrient component is a compound of one or more of proteins, sugar alcohols and amino acids.

Further, the protein includes one or more of bovine serum albumin, brain heart infusion or casein;

further, the sugar alcohol comprises one or more of sucrose, trehalose and glucose.

In one or more embodiments of the invention, for the disinfectant used for disinfection objects without cleaning or dirtying, the bacteria content of the bacterial suspension after the preservation solution is diluted is 1 x 10⁹～5×10⁹CFU/mL, then adding 3.0% bovine serum albumin equal to the bacterial suspension to make the bacterial suspension concentration 1X 10⁸～5×10⁸CFU/mL, and then loading; for the disinfectant used for cleaned or cleaner disinfection objects, the bacterium content of the bacterium suspension after the preservation solution is diluted is 1 multiplied by 10⁹～5×10⁹CFU/mL, then adding 0.3% bovine serum albumin equal to the bacterial suspension to make the bacterial suspension concentration 1X 10⁸～5×10⁸CFU/mL, and then loading; the disinfectant for strictly cleaned or extremely cleaned disinfection objects does not use bovine serum albumin, and the concentration of the bacterial suspension after the preservation solution is diluted is 1 multiplied by 10⁸～5×10⁸CFU/mL, and then loading；

Further, when bovine serum albumin with the same amount as the bacterial suspension is not added, the preserving fluid comprises 1% of sucrose, 0.25% of sodium glutamate, 20% of glycerol and 3.7% of brain heart infusion culture medium; when bovine serum albumin with the same quantity as the bacterial suspension is added, the preservation solution comprises 2% of sucrose, 0.5% of sodium glutamate, 40% of glycerol and 7.4% of brain heart infusion culture medium.

Further, when the bacterial tablet is used for evaluating the low-temperature on-site disinfection effect, bovine serum albumin is replaced by trypsin, preferably tryptone soybean broth culture medium. Wherein, bovine serum albumin and trypsin are used as organic interferents.

In one or more embodiments of the invention, the bacterial suspension is loaded onto the carrier by dripping 10. mu.L of bacterial suspension per 100 mm square carrier or 113.04 mm square carrier.

Further, the freeze-drying condition is vacuum freezing for 24 hours at-30 to-60 ℃.

Furthermore, the freeze-dried bacterium tablets are stored at the temperature of-20 ℃ to-40 ℃.

In a second aspect of the present invention, there is provided a pellet obtained by the above-mentioned method, wherein the number of recovered fungi in the pellet is 5X 10⁵cfu/tablet-5X 10⁶cfu/tablet. The bacterial tablet can be used for sterilizing the surface of an object in a epidemic area at normal temperature or sterilizing the surface of an object in a preventive and epidemic area at low temperature.

The third aspect of the invention provides a method for evaluating the disinfection effect of a disinfectant, which comprises the following steps: the bacterial tablet is adopted to carry out disinfection simulation test to evaluate the disinfection effect of the disinfectant.

The disinfection simulation test is a carrier soaking sterilization test;

the method specifically comprises the following steps: disinfectant with different concentrations and 5.0 mL/piece are respectively injected into different sterile plates to simulate different disinfection conditions;

placing the plate containing disinfectant in 20 + -1 deg.C water bath for 5-8min, and soaking the strain pieces in the disinfectant.

Taking out the sterilized bacterial tablet with sterile forceps, adding into 5.0-8mL neutralizer, mixing for 20-30s electrically or beating with palm for 80-90 times, neutralizing for 10-15min, inoculating into plate, and determining viable bacteria number.

Furthermore, the disinfection simulation test needs to replace a disinfectant with a diluent or standard hard water to make a positive control test.

Further, the sterilization simulation test calculates the amount of live bacteria (CFU/tablet), and converts the amount of live bacteria into a logarithmic value (N), and the method for calculating the killing logarithmic value comprises the following steps:

KL＝N_d-N_s

in the formula:

KL-log kill;

N_d-log of the mean viable bacterial count of the control group;

N_slog of the amount of live bacteria in the experimental group.

In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the technical solution of the present invention will be described in detail below with reference to specific examples and comparative examples.

Example 1

A preparation method of a fungus tablet comprises the following steps:

sterilizing a filter paper sheet cut into 10mm multiplied by 10mm, and performing wet heat sterilization at 121 ℃ for 15 min;

the preserved escherichia coli 8099 is subjected to generation recovery on a nutrient agar test tube inclined plane and is passaged to the 5 th generation, and the culture time is 18 hours;

preparing a freeze-drying protective agent which comprises 2% of sucrose, 0.5% of sodium glutamate, 40% of glycerol and 7.4% of brain heart infusion culture medium, and sterilizing with high-pressure steam at 115 ℃ for 20 min;

eluting Escherichia coli 8099 on the inclined plane with lyophilized protectant with equilibrium value at room temperature to obtain product with bacteria content of about 1 × 10⁹CFU/mL～5×10⁹CFU/mL, and then adding 3.0% bovine serum albumin equal to the bacterial suspension to make the bacterial suspension concentration 5X 10⁸CFU/mL；

Putting the sterile filter paper sheet into a sterile flat plate, wherein each plate is provided with 10 sheets, loading bacterial suspension onto the filter paper sheet by a drop-dyeing method, and dripping 10 mu L of bacterial suspension into each sheet;

putting the flat plate containing the fungus slices into a vacuum freeze dryer, and freeze-drying for 24 hours at the temperature of-30 ℃ to obtain freeze-dried filter paper sheets, namely the fungus slices;

placing the freeze-dried filter paper sheet into a temperature of-20 ℃ for freezing and storing, and respectively detecting the recovery rate of the filter paper sheet after 24h, one week and one month, wherein the result is shown in figure 2;

example 2

The carrier is selected from white muslin cloth and is degreased. The degreasing method comprises the following steps:

(1) boiling cotton cloth in water containing detergent for 30 min;

(2) washing with tap water;

(3) boiling with distilled water for 10 min;

(4) rinsing with distilled water until pH is neutral;

(5) air drying and ironing for later use.

Before cutting, the degreased cloth block is cut off according to the size of 10mm multiplied by 10mm, warp and weft yarns on the periphery of the edge are respectively removed, and then the cloth block is cut off according to a yarn drawing mark, so that the cloth piece with the consistent size and no rough selvedge, which is 10mm multiplied by 10mm, is obtained.

Sterilizing the cloth sheet at 121 deg.C for 15 min;

the preserved black variety ATCC9372 of the bacillus subtilis is subjected to generation recovery on a nutrient agar test tube inclined plane to passage 8, and the culture time is 24 hours;

preparing a freeze-drying protective agent which comprises 1% of sucrose, 0.25% of sodium glutamate, 20% of glycerol and 3.7% of brain heart infusion culture medium, and sterilizing by high-pressure steam at 115 ℃ for 20 min;

eluting the Bacillus subtilis var niger ATCC9372 from the slant with a lyoprotectant at equilibrium room temperature to give a suspension concentration of 5X 10⁸CFU/mL。

Placing 10 pieces of sterile cloth pieces into a sterile flat plate, loading bacterial suspension onto the cloth pieces by a drop-dyeing method, and dripping 10 mu L of bacterial suspension into each piece;

putting the flat plate containing the fungus slices into a vacuum freeze dryer, and freeze-drying for 24h at the temperature of minus 60 ℃ to obtain freeze-dried cloth slices, namely the fungus slices;

comparative example 1

Sterilizing a microporous filter membrane cut into 10mm multiplied by 10mm, and sterilizing by using ethylene oxide or irradiation;

the preserved escherichia coli 8099 is subjected to generation recovery on a nutrient agar test tube inclined plane and is passaged to the 5 th generation, and the culture time is 18 hours;

preparing a freeze-drying protective agent which comprises 2% of sucrose, 0.5% of sodium glutamate, 40% of glycerol and 7.4% of brain heart infusion culture medium, and sterilizing with high-pressure steam at 115 ℃ for 20 min;

eluting Escherichia coli 8099 on the inclined plane with lyophilized protectant with equilibrium value at room temperature to obtain product with bacteria content of about 1 × 10⁹CFU/mL～5×10⁹CFU/mL, then adding 3.0% bovine serum albumin equal to the bacterial suspension to make the concentration of the bacterial suspension about 5X 10⁸CFU/mL；

Loading bacteria on a filter membrane, and loading bacteria liquid on a microporous filter membrane in a filtering mode; the filtration mode in the step of loading the thalli on the filter membrane is that a specific amount of bacteria liquid passes through a Kjeldahl filter and liquid is filtered in a negative pressure suction filtration mode, so that the thalli are uniformly distributed on the filter membrane and are prevented from being distributed at the edge of the filter membrane; and (3) dropwise adding a certain amount of preservation solution prepared by freeze-drying and preserving the thallus after filtering. And (3) putting the filter membrane loaded with the bacterial liquid in the sterile container into a vacuum freeze dryer, and freeze-drying for 24 hours at the temperature of minus 30 ℃.

Example 3

A method for evaluating the disinfection effect of a disinfectant comprises the following steps:

a sterilization simulation test was performed using the bacterial pellet prepared in example 1 to evaluate the sterilization effect of the disinfectant:

preparing 60mL of 84 disinfectant with the effective chlorine content of 50 mg/L;

taking 4 sterile plates, and injecting disinfectant solution into the plates according to the amount of 5mL (total 15mL) of each plate;

placing the dish containing disinfectant in a water bath kettle at 20 deg.C for 5min, placing the prepared fungus pieces with sterile tweezers, and soaking in the disinfectant;

after 4 plates act for 1min, 3min, 5min and 8min respectively, taking out the bacterial slices, putting the bacterial slices into a 10mL D/E broth neutralizer test tube, shaking for 20s, standing for 5min, fully mixing, diluting according to a certain proportion, sucking 1mL, inoculating into a nutrient agar plate by using a pouring method, and measuring the number of viable bacteria; i.e. the number of viable bacteria of the experimental group.

Another plate is taken, 10mL of standard hard water is injected to replace the disinfectant, 2 bacterial tablets are put in the plate, and the steps are repeated to be used as a positive control group;

and (5) putting the counting plate into an incubator at 37 ℃, culturing for 48h, and recording the number of viable bacteria.

The sterilization simulation test calculates the live bacterial quantity (CFU/tablet), and converts the live bacterial quantity into a logarithmic value (N), and the method for calculating the killing logarithmic value comprises the following steps:

KL＝N_d-N_s

in the formula:

KL-log kill;

N_d-log of the mean viable bacterial count of the control group;

N_slog of the amount of live bacteria in the experimental group.

Example 4

A method for evaluating the disinfection effect of a disinfectant comprises the following steps:

the bacterial sheets prepared in comparative example 1 were used for a disinfection simulation test to evaluate the disinfectant effect of the disinfectant:

preparing 60mL of 84 disinfectant with the effective chlorine content of 50 mg/L;

treating microporous filter membrane with disinfectant for 1min, 3min, 5min, and 8 min;

placing the microporous filter membrane in a nutrient agar plate, culturing and counting;

evaluation of disinfectant effect requires that the concentration of initial bacterial suspension reaches 10⁸CFU/mL, compared with example 3, this example uses microporous membrane as carrier, when the disinfectant concentration is low, the action time is short, because the bacterial load is too large, each thallus can not distribute on the membrane evenly to form single colony, thallus links into one piece after culturing, can not realize the counting function.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.