CN110923167A - Application of escherichia coli freeze-dried powder in testing fumigation disinfection effect - Google Patents
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Abstract
The invention provides application of escherichia coli freeze-dried powder in testing fumigation and disinfection effects, and relates to the technical field of detection methods. The escherichia coli freeze-dried powder is prepared by the following method: pre-freezing: uniformly mixing the escherichia coli bacterial suspension and a freeze-drying stabilizer, and pre-freezing; and (3) drying: and (3) carrying out vacuum drying on the pre-frozen escherichia coli to obtain the escherichia coli freeze-dried powder. The escherichia coli freeze-dried powder is applied to the inspection of the fumigation disinfection effect, and the disinfection effect of the disinfectant can be quickly, simply and accurately inspected.
Description
Technical Field
The invention relates to the technical field of detection methods, in particular to application of escherichia coli freeze-dried powder in testing fumigation disinfection effects.
Background
The fumigation method is a rapid, effective and simple disinfection method. The fumigant adopted in the fumigation method is a molecular gas disinfectant, has the effect of killing viruses, germs or other harmful organisms, can be diffused to the whole closed space, including small corners, gaps and the like, and can kill all corners in the closed space, so that the complete disinfection is really realized. After the fumigation and disinfection are finished, the door and the window are opened to ventilate and disperse air, so that no residue is left. The fumigation method is a disinfection mode very suitable for breeding places, and is more and more accepted by most farmers at present.
At present, more and more disinfectants which can be used for fumigation in the market have different quality and different disinfection effects. The fumigation effect cannot be directly observed by naked eyes, and the market problem of how to distinguish the quality of the disinfectant is difficult. The conventional method for testing the fumigation effect at present comprises the following steps: the field is coated and sampled before and after disinfection, bacteria culture is carried out after fumigation, and the bacteria killing rate is calculated according to the number of growing colonies. The method has high requirements on experimenters and experimental sites, most of the farms are difficult to meet relevant requirements, and multi-point collection is needed to detect as accurately as possible, so that the labor components are increased. Therefore, it is necessary to develop a simple and effective method for testing the fumigation effect.
Disclosure of Invention
Therefore, in order to solve the above problems, it is necessary to provide an application of escherichia coli lyophilized powder in testing fumigation and disinfection effects, and the escherichia coli lyophilized powder is used to test the fumigation and disinfection effects conveniently, quickly and accurately, so as to reduce the economic burden of farmers.
The application of the escherichia coli freeze-dried powder in the inspection of the fumigation disinfection effect is characterized in that the escherichia coli freeze-dried powder is prepared by the following method:
pre-freezing: uniformly mixing the escherichia coli bacterial suspension and a freeze-drying stabilizer, and pre-freezing;
and (3) drying: and (3) carrying out vacuum drying on the pre-frozen escherichia coli to obtain the escherichia coli freeze-dried powder.
The escherichia coli freeze-dried powder has the following characteristics: 1) the microorganism can not lose activity after being dried in a low-temperature state; 2) during the low-temperature drying process, the growth of microorganisms and the action of enzymes can not be basically carried out, and the original properties and quantity of the freeze-dried substances can be best maintained; 3) the freeze-dried powder is in a sponge shape, cannot be dried and shrunk, has large contact area when being dissolved by water, and can quickly recover the original properties; 4) can remove 95-99% of water in the thalli and greatly prolongs the storage time of the strains.
In one embodiment, the freeze-drying stabilizer is a mixture of sucrose, gelatin and skimmed milk powder, wherein the concentration of the sucrose is 35-40 g/L, the concentration of the skimmed milk powder is 6-7 g/L, and the concentration of the gelatin is 12-13 g/L. The freeze-drying stabilizer comprises a saccharide stabilizer, a protein stabilizer and the like. Preferably, the saccharide stabilizer is a disaccharide such as trehalose, sucrose, lactose, and the like. Preferably, the protein-based stabilizer is milk protein, collagen or soy protein. The inventor finds that the mixture of sucrose, gelatin and skimmed milk powder is adopted as the freeze-drying stabilizer, and has obvious advantages: the skimmed milk powder has good dispersibility and homogeneity, not only can play a role of a protective layer on the cell surface, but also can enlarge the distance between cells, and is easy to obtain a homogeneous product; gelatin is used as a filler to prevent the effective components from sublimating and dissipating with water vapor; the sucrose solution can form a glass state in the freeze drying process, has extremely high viscosity, is not easy to form crystals, has very low diffusion coefficient of molecules, is surrounded around protein molecules to form a carbohydrate glass body similar to ice in structure, prevents the movement of macromolecular substances, prevents the stretching and precipitation of the molecular structure of the protein, maintains the three-dimensional stable structure of the protein molecules, and plays a role in protecting the activity of cells.
In one embodiment, the escherichia coli in the escherichia coli bacterial suspension is 3 rd generation to 14 th generation. The 3 rd to 14 th generation escherichia coli have good biological activity and high stability, and the test result is more convincing.
In one embodiment, the pre-freezing step specifically comprises: diluting the Escherichia coli bacterial suspension to 3-5 multiplied by 10 by using a diluent4cfu/mL, adding lyophilized stabilizer, and mixingAfter being mixed evenly, the mixture is placed in an environment with the temperature of minus 45 to minus 35 ℃ and is pre-frozen for 22 to 26 hours. The sterilization rate of the disinfectant can not reach 100% basically, in the actual use process, due to the difference between the sterilization place and the type of the disinfectant, the sterilization rate is generally lower than 99.9%, if the bacterial content in the freeze-dried powder is too high, the number of bacterial colonies growing on the sterilized culture medium can not be counted, and therefore the sterilization rate cannot be calculated. The inventor finds that the bacterium content of the escherichia coli freeze-dried powder is 10 orders of magnitude in the long-term research and exploration process4cfu/g, preferably 3 to 5X 104And cfu/g, the bacterial colony can be counted conveniently, and the sterilization rate can be calculated accurately.
In one embodiment, the pre-freezing step specifically comprises: the diluent is tryptone physiological saline, and the pH value is 7.0-7.2.
In one embodiment, the drying step specifically comprises: and (3) putting the pre-frozen escherichia coli into a dryer, and carrying out vacuum drying at the temperature of-85 to-75 ℃ for 7-9 hours.
The invention also provides a method for testing the fumigation effect, which comprises the following steps:
and (3) resuscitation: dissolving the lyophilized powder of Escherichia coli in water, and uniformly coating on nutrient agar culture medium to obtain experimental group and control group;
and (3) disinfection: placing the experimental group in a field to be fumigated and disinfected, and simultaneously placing the control group in a sterile environment;
culturing: after the fumigation is finished, placing the experimental group and the control group in the same environment for culturing;
and (3) calculating: after the culture, the colony numbers of the experimental group and the control group are calculated, and the sterilization rate is calculated.
The method for testing the fumigation disinfection effect can realize the purpose of quickly, simply and conveniently testing the fumigation disinfection effect of the disinfectant by utilizing the characteristics of the escherichia coli freeze-dried powder.
In one embodiment, the water adding amount in the escherichia coli freeze-dried powder is as follows: and adding 0.8-1.2 mL of water into each gram of escherichia coli freeze-dried powder.
In one embodiment, in the culturing step, the culturing temperature is 37 +/-1 ℃, and the culturing time is 48-50 h.
In one embodiment, the calculated sterilization rate is calculated using the following formula I:
η ═ 100% · (B-a)/B (formula I);
wherein η represents the bactericidal activity, A represents the average number of colonies in the experimental group, and B represents the average number of colonies in the control group.
Compared with the prior art, the invention has the following beneficial effects:
the method for testing the fumigation effect applies the escherichia coli freeze-dried powder to the method for testing the fumigation effect, the number of thalli in the escherichia coli freeze-dried powder is stable, the activity after recovery is high, the preservation is convenient, the escherichia coli freeze-dried powder can be recovered and used at any time according to needs, the testing operation is simple and rapid, the method does not depend on precise instruments and high-operation-level personnel excessively, the fumigation effect can be tested rapidly and accurately, and the economic burden of a breeding plant is greatly reduced.
Drawings
FIG. 1 is a sample of E.coli lyophilized powder of the examples;
FIG. 2 shows the growth of E.coli after dilution of lyophilized powder for different times;
wherein: a is diluted by 1 time, B is diluted by 10 times, C is diluted by 100 times, D is diluted by 1000 times, E is diluted by 10000 times, and F is blank control;
FIG. 3 is a graph showing the test results of Experimental example 3;
wherein a and b are experimental groups, and c and d are control groups.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. The following is a description of preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Example 1
1. Reagent and apparatus
(1) 3 rd to 14 th generation of escherichia coli by using a nutrient agar culture medium slant culture;
(2) culture medium: nutrient agar culture medium
(3) Reagent: distilled water, 0.96% physiological saline, skimmed milk powder, sucrose, and gelatin
(4) Diluting liquid: tryptone physiological saline (1.0 g of tryptone and 8.5g of sodium chloride were dissolved in 900mL or more of distilled water, pH was adjusted to 7.0, volume was adjusted to 1000mL with distilled water, and autoclaving at 121 ℃ C.).
2. Preparation of lyophilized powder of Escherichia coli
(1) Preparing an escherichia coli bacterial suspension: taking a slant culture of a nutrient agar culture medium of 3 rd generation to 14 th generation of escherichia coli, sucking 3.0mL of diluent by using a 5.0mL suction tube, adding the diluent into a slant test tube, repeatedly blowing and sucking, then transferring a washing solution into another sterile test tube by using the 5.0mL suction tube, adding 100mL of diluent, uniformly knocking by 50 times to obtain a primary bacterial suspension, roughly detecting the concentration of bacteria by using a bacteria concentration turbidimetry method, and after the detection is finished, taking the bacterial suspension and using a nutrient agar culture dish to respectively dilute by 10 times and 100 times for culture so as to ensure that about 40000 bacterial colonies are contained in 1mL of bacterial suspension; the suspension was then diluted with diluent to about 40000 colonies per 1mL of E.coli suspension according to the results of the assay.
(2) Preparation of freeze-drying stabilizer:
an agent A: weighing 11g of skimmed milk powder, adding 40mL of water, heating in a water bath at 60 ℃ to dissolve uniformly, adding 60g of sucrose, dissolving completely, diluting to 100mL, performing damp-heat sterilization at 110 ℃ for 15min, and sterilizing for later use;
and (2) agent B: weighing 20g of gelatin, heating to dissolve to 100mL, and sterilizing at 121 deg.C for 30min for use.
And uniformly mixing the agent A and the agent B according to the volume ratio of 1:1 to obtain the freeze-drying stabilizer.
(3) Preparing freeze-dried powder:
mixing the prepared bacterial suspension with a freeze-drying stabilizer according to the volume ratio of 7:1, and fully and uniformly shaking by using an oscillator to prepare freeze-dried bacterial suspension for later use;
precisely sucking 2mL of the freeze-dried bacterial suspension by using a 2mL sterile suction pipe, adding the freeze-dried bacterial suspension into a 5mL penicillin bottle, taking care of preventing the upper part of a bottle mouth from being splashed, preventing pollution, covering a rubber plug, not tightly covering, leaving a gap, freezing in a freezer at-40 ℃ for 24 hours, placing a pre-frozen freeze-dried powder sample in a drying box of a freeze dryer, carrying out freeze drying at-80 ℃ for 8 hours, and then sealing by a gland to obtain the final escherichia coli freeze-dried powder (the product is shown in figure 1), wherein the escherichia coli freeze-dried powder needs to be placed in the refrigerator for freeze storage at.
The escherichia coli freeze-dried powder obtained in the embodiment is revived at different times, and the survival rate of escherichia coli is tested, and the results are shown in table 1:
TABLE 1 survival rate of lyophilized Escherichia coli powder after preservation
| Days of storage | 0 | 30 | 60 | 90 days |
| Survival rate | 89.67％ | 89.22％ | 88.53％ | 87.24％ |
Example 2
A method for testing the fumigation effect comprises the following steps:
(1) and (3) resuscitation: 4g of escherichia coli freeze-dried powder in example 1 is added with 4mL of sterilized water to prepare a bacterial solution, 4 culture dishes filled with nutrient agar culture media are taken, 1mL of bacterial solution is added into each culture dish, two culture dishes serve as experimental groups, and the other two culture dishes serve as control groups;
(2) and (3) disinfection: placing the experimental group in a field to be fumigated and disinfected, and simultaneously placing the control in an aseptic environment for fumigation;
(3) culturing: after the fumigation is finished, placing the experimental group and the control group in an incubator at 37 ℃ for culturing for 48 hours;
(4) and (3) calculating: after the culture, calculating the colony numbers of the experimental group and the control group, and calculating the sterilization rate, wherein the sterilization rate is calculated by adopting the following formula I:
η ═ 100% · (B-a)/B (formula I);
wherein η represents the bactericidal activity, A represents the average number of colonies in the experimental group, and B represents the average number of colonies in the control group.
Experimental example 1
The escherichia coli freeze-dried powder in the example 1 is diluted by adding water, wherein the dilution times are respectively 1, 10, 100, 1000 and 10000 times, the standard of the dilution of 1 time is that 1mL of sterilized water is added into each gram of escherichia coli freeze-dried powder, the dilution of 10 times is that a diluent diluted by 1 time is diluted by 10 times, the dilution of 100 times is that a diluent diluted by 10 times is diluted by 10 times, and the like. 1ml of the above bacterial liquid was added to nutrient agar medium and cultured in an incubator at 37 ℃ for 48 hours, and the growth of colonies was as shown in FIG. 2.
As can be seen from the colony growth situation in FIG. 2, the number of colonies diluted 1 time is too large to count, the number of colonies diluted 10 times is too large to count, the number of colonies diluted 100 times is about 300-400 colonies, the number of colonies diluted 1000 times is about 45 colonies, the number of colonies diluted 10000 times is 5 colonies, and the number of colonies not grown by the blank control is not counted.
Experimental example 2
The influence of different freeze-drying stabilizers on the survival rate of thalli in the freeze-dried powder is tested, the escherichia coli freeze-dried powder prepared by adopting the different freeze-drying stabilizers is respectively revived, the survival rate of escherichia coli is tested, and the results are shown in table 2:
TABLE 2 protective Effect of different freeze-drying stabilizers on E.coli
| Classes of protective Agents | Survival rate |
| 6.8g/L milk powder | 58.25％ |
| 12.5g/L gelatin | 52.19％ |
| 37.5g/L sucrose | 55.87％ |
| Lyophilized stabilizer of example 1 | 89.67％ |
Experimental example 3
A method for testing the fumigation effect comprises the following steps:
(1) testing and selecting materials: the fumigant is a 'Zhengzheng' fumigant produced by Guangzhou Migao chemical company Limited with the batch number of 20190901, the product 'Zhengzheng' fumigant is a binary package, the main components of the fumigant are sodium chlorite (liquid) and anhydrous sodium bisulfate (solid), the liquid is poured into the solid when the fumigant is used, and the sodium chlorite and the sodium bisulfate react chemically to produce chlorine dioxide gas to generate a fumigating effect;
(2) and (3) resuscitation: taking two bottles (4g) of the escherichia coli freeze-dried powder in example 1, adding 2mL of sterile water into the escherichia coli freeze-dried powder respectively to prepare a bacterial solution, taking 4 culture dishes filled with nutrient agar culture media, adding 1mL of bacterial solution into each culture dish, wherein two culture dishes are used as an experimental group, and the other two culture dishes are used as a control group;
(3) and (3) disinfection: placing the experimental group in a field to be fumigated and disinfected, fumigating for 60 minutes by using a positive seeking fumigant, and simultaneously placing a control in an aseptic environment;
(4) culturing: after the fumigation is finished, placing the experimental group and the control group in an incubator at 37 ℃ for culturing for 48 hours; the colony growth is shown in FIG. 3;
(5) as a result: as shown in FIG. 3, the average number of colonies counted in the fumigation group was about 140, the average number of colonies counted in the control group was close to A in FIG. 2, and the average number of colonies counted in the control group was about 4X 10 as determined by the dilution results of Experimental example 14Therefore, the fumigation disinfection and killing rate is calculated to be 99.65%.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. The application of the escherichia coli freeze-dried powder in the inspection of the fumigation disinfection effect is characterized in that the escherichia coli freeze-dried powder is prepared by the following method:
pre-freezing: uniformly mixing the escherichia coli bacterial suspension and a freeze-drying stabilizer, and pre-freezing;
and (3) drying: and (3) carrying out vacuum drying on the pre-frozen escherichia coli to obtain the escherichia coli freeze-dried powder.
2. The use of claim 1, wherein the freeze-drying stabilizer is a mixture of sucrose, gelatin and skimmed milk powder, wherein the concentration of sucrose is 35-40 g/L, the concentration of skimmed milk powder is 6-7 g/L, and the concentration of gelatin is 12-13 g/L.
3. The use of claim 1, wherein the escherichia coli in the escherichia coli bacterial suspension is from generation 3 to generation 14.
4. Use according to claim 1, wherein the pre-freezing step is in particular: diluting the Escherichia coli bacterial suspension to 3-5 multiplied by 10 by using a diluent4cfu/mL, adding a freeze-drying stabilizer, uniformly mixing, and pre-freezing for 22-26 h at-45 to-35 ℃.
5. The use according to claim 4, wherein the pre-freezing step is in particular: the diluent is tryptone physiological saline, and the pH value is 7.0-7.2.
6. The use according to any one of claims 1 to 5, wherein the drying step is in particular: and (3) putting the pre-frozen escherichia coli into a dryer, and carrying out vacuum drying at the temperature of-85 to-75 ℃ for 7-9 hours.
7. A method for testing the fumigation effect comprises the following steps:
and (3) resuscitation: dissolving the escherichia coli freeze-dried powder of any one of claims 1 to 6 in water, and uniformly coating the solution on a nutrient agar culture medium to divide the solution into an experimental group and a control group;
and (3) disinfection: placing the experimental group in a field to be fumigated and disinfected, and simultaneously placing the control group in a sterile environment;
culturing: after the fumigation is finished, placing the experimental group and the control group in the same environment for culturing;
and (3) calculating: after the culture, the colony numbers of the experimental group and the control group are calculated, and the sterilization rate is calculated.
8. The method according to claim 7, wherein in the resuscitation step, the amount of water added into the escherichia coli freeze-dried powder is as follows: and adding 0.8-1.2 mL of water into each gram of escherichia coli freeze-dried powder.
9. The method according to claim 7, wherein in the culturing step, the culturing temperature is 37 +/-1 ℃ and the culturing time is 48-72 hours.
10. The method of claim 7 or 8, wherein the calculated sterilization rate is calculated using formula I, η ═ 100% · (B-a)/B (formula I);
wherein η represents the bactericidal activity, A represents the average number of colonies in the experimental group, and B represents the average number of colonies in the control group.
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| CN103444797A (en) * | 2013-08-15 | 2013-12-18 | 五河县畜牧兽医技术推广中心 | Disinfecting fumigant for animal building and preparation method of disinfecting fumigant |
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| CN103444797A (en) * | 2013-08-15 | 2013-12-18 | 五河县畜牧兽医技术推广中心 | Disinfecting fumigant for animal building and preparation method of disinfecting fumigant |
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| 方振荣: "过氧乙酸熏蒸消毒试验研究", 《中国动物检疫》 * |
| 梁山县诚达二手化工设备购销部: "细菌冻干机：冻干保存细菌方法及步骤", 《梁山县诚达二手化工设备购销部》 * |
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