CN117757893A - Simulated water body for detecting bacteriostasis and sterilization effects of bactericide in aquaculture water body and application method - Google Patents
Simulated water body for detecting bacteriostasis and sterilization effects of bactericide in aquaculture water body and application method Download PDFInfo
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Abstract
The application relates to the technical field of antibacterial and bactericidal effect testing of aquaculture bactericides, in particular to a simulated water body for detecting antibacterial and bactericidal effects of bactericides in aquaculture water bodies and a using method. The simulated water body is used for detecting the bacteriostatic and bactericidal effect of the bactericide in the aquaculture water body and is characterized by comprising a simulated water body, single bacteria and the bactericide, wherein the simulated water body is one of a simulated seawater conventional water body, a simulated seawater seedling pool water body, a simulated fresh water conventional water body, a simulated fresh water seedling pool water body and a simulated fresh water seedling pool water body. The simulated water body and the using method have the advantages of low cost, short detection period, high efficiency, high accuracy, wide application range, simple using method and the like, and the gap of the high-efficiency method for testing the antibacterial effect of the bactericide in the aquaculture water body at present is overcome.
Description
Technical Field
The application relates to the technical field of antibacterial and bactericidal effect testing of aquaculture bactericides, in particular to a simulated water body for detecting antibacterial and bactericidal effects of bactericides in aquaculture water bodies and a using method.
Background
Bactericides are one of the most commonly used medicines in aquaculture processes and are used for eliminating or killing pathogenic microorganisms and other harmful microorganisms in the external environment. In aquaculture, in order to control various infectious diseases, it is generally necessary to sterilize aquaculture water, animal seedlings, feeds, etc. to destroy various harmful microorganisms, thereby creating a sanitary and safe living environment for the aquaculture animals.
The laboratory general test methods for testing the bactericidal and bacteriostatic effects of the bactericides generally comprise three methods, namely 1, oxford cup or drug sensitive test paper, 2, mic test 3 and killing index, wherein the three methods have higher nutrient contents, the test indexes are not suitable for aquaculture water, and the use concentration data of the obtained bactericides are far higher than the actual use concentration. Meanwhile, the content of pathogenic bacteria in the culture water body is reduced, some pathogenic bacteria such as vibrio are symbiotic bacteria in practice, and most pathogenic causes are opportunistic. After the vibrio is completely killed, secondary problems are easy to occur, especially shrimp metamorphosis is limited in growth, and death is caused. Therefore, the test method has too high sterilization degree and is not suitable for the test method of the bactericide in aquaculture.
Oxford cups or drug sensitive test papers are mostly used for antibiotics, and the test strains are sensitive or resistant to the antibiotics, and the test for aquatic preparations has the following problems: 1. the macromolecular bacteriostatic agent is slowly diffused, needs to be refrigerated for diffusion or coated after diffusion, the data is easy to depend on the diffusion rate of the bacteriostatic agent, and the data error is large, the experiment is tedious and the repeatability is poor; 2. the bacteriostasis environment is a solid interface, the agar nutrient is higher than the culture water body, and the solid interface is different from the growth state of microorganisms in the water body, so that the data are different, and 3, the oxford cup is completely bacteriostasis and is different from the bacteriostasis requirement of aquaculture, so that the data obtained by the oxford cup test are difficult to be used in actual use.
In the Mic method, bacteria are inoculated after a bactericide is added to a liquid, and turbidity is observed, so that the following problems are caused: 1. when not clouded (when the concentration of bacteria is less than 1X 10) 8 cfu/mL, the aquaculture is unacceptable, and the aquaculture is generally controlled at 1X10 3-4 cfu/mL or less); 2. the nutrient concentration of the liquid culture is higher, and the corresponding antibacterial concentration is also improved, so that the data cannot represent the actual use concentration; 3. growth was judged by haze (visual inspection of haze required to a concentration of bacteria greater than 1X 10) 8 cfu/mL), cannot determine the level of inhibition, whether it is completely or mildly (1X 10) 5-7 cfu/mL), or powerful bacteriostasis (1X 10) 2-4 cfu/mL)。
The killing index is a method for testing the sterilizing effect of the bactericide, is very suitable for production and living scenes, but has certain defects for aquaculture, 1, the killing index is mainly the short-term killing effect, the long-term bacteriostasis level is not considered, and the aquaculture is more careful about the long-term bacteriostasis effect. 2. The laboratory sterilizing effect inspection method of the national standard disinfectant uses a culture medium with high nutrition level, and simultaneously adds an organic interference object, and the measured sterilizing data is not matched with the environment in which the aquatic product is used.
At present, the use concentration of the aquatic bactericide mostly adopts the experience concentration, a temporary culture pond or an actual test is used, the cost for developing a new bactericide product is high, and the use concentrations of different pathogenic bacteria cannot be accurately distinguished. Therefore, it is very valuable to develop a simulated water body and a using method for detecting the bacteriostasis and sterilization effects of the bactericide in the culture water body.
Disclosure of Invention
Aiming at the defects of the prior art, the application aims to provide the simulated water body for detecting the bacteriostasis and sterilization effect of the bactericide in the culture water body and the application method thereof.
In a first aspect, the present application provides a simulated water body for detecting bacteriostatic and bactericidal effects of a bactericide in a culture water body, which adopts the following technical scheme:
the simulated water body comprises a simulated seawater conventional water body, a simulated seawater seedling pool water body, a simulated fresh water conventional water body and a simulated fresh water seedling pool water body, and a simulated fresh water seedling pool water body.
Preferably, the simulated seawater conventional water body comprises the following components in percentage by mass: 1000g of primary pure water, 30g of sea salt, 20mg of peptone and 10mg of yeast powder; the simulated seawater seedling pool water body or the simulated seawater seedling pool water body comprises the following components in percentage by mass: 1000g of primary pure water, 30g of sea salt, 70mg of peptone and 30mg of yeast powder.
Preferably, the simulated fresh water conventional water body comprises the following components in percentage by mass: 1000g of primary pure water, 5g of sea salt, 20mg of peptone and 10mg of yeast powder; the simulated fresh water seedling pool water body or the simulated fresh water seedling pool water body comprises the following components in percentage by mass: 1000g of primary pure water, 5g of sea salt, 70mg of peptone and 30mg of yeast powder.
Preferably, the synthetic method of the simulated seawater conventional water body comprises the following steps: sequentially adding 30g of sea salt, 20mg of peptone and 10mg of yeast powder into 1000g of primary pure water according to the mass ratio, uniformly mixing to obtain a simulated seawater conventional water body, respectively taking 50mL of the simulated seawater conventional water body, subpackaging into 250mL shake flasks, correspondingly marking 50/250mL shake flasks A1-A20, and sterilizing at 121 ℃ for 20min for later use; the synthesis method of the simulated seawater seedling pool water body or the simulated seawater seedling pool water body comprises the following steps: sequentially adding 30g of sea salt, 70mg of peptone and 30mg of yeast powder into 1000g of primary pure water according to the mass ratio, uniformly mixing to obtain a simulated seawater seedling pool water body or a simulated seawater seedling pool water body respectively, respectively taking 50mL of the simulated seawater seedling pool water body or the simulated seawater seedling pool water body, subpackaging the simulated seawater seedling pool water body or the simulated seawater seedling pool water body into 250mL shake flasks, correspondingly marking 50/250mL shake flasks B1-B20 and C1-C20, and sterilizing at 121 ℃ for 20min for later use.
Preferably, the synthetic method of the simulated fresh water conventional water body comprises the following steps: sequentially adding 5g of sea salt, 20mg of peptone and 10mg of yeast powder into 1000g of primary pure water according to the mass ratio, uniformly mixing to obtain a simulated fresh water conventional water body, respectively taking 50mL of the simulated fresh water conventional water body, subpackaging the simulated fresh water conventional water body into 250mL shake flasks, correspondingly marking the simulated fresh water conventional water body as 50/250mL shake flasks D1-D20, and sterilizing at 121 ℃ for 20min for later use; the synthesis method of the simulated fresh water seedling pool water body or the simulated fresh water seedling pool water body comprises the following steps: according to the mass ratio, adding 5g of sea salt, 70mg of peptone and 30mg of yeast powder into 1000g of primary pure water in sequence, uniformly mixing to obtain a simulated fresh water seedling pool water body or a simulated fresh water seedling pool water body respectively, respectively taking 50mL of the simulated fresh water seedling pool water body or the simulated fresh water seedling pool water body, subpackaging the simulated fresh water seedling pool water body or the simulated fresh water seedling pool water body into 250mL shake flasks, correspondingly marking 50/250mL shake flasks E1-E20 and F1-F20, and sterilizing at 121 ℃ for 20min for later use.
Preferably, the single bacteria are one of ATCC17802 vibrio parahaemolyticus, vibrio parahaemolyticus selected from transparent shrimp seedlings of Xiamen, vibrio parahaemolyticus selected from fish gill, algicidal, parahaemolyticus, havickers, vibrio vulnificus, vibrio lautus, aeromonas hydrophila and escherichia coli.
Preferably, the bactericide is one of povidone iodine bactericide, quaternary ammonium salt bactericide and polyhexamethylene monoguanidine crystal bactericide with purity of more than 98%.
In a second aspect, the application provides a use method of a simulated water body for detecting the bacteriostatic and bactericidal effects of a bactericide in a culture water body, which adopts the following technical scheme:
the application method of the simulated water body for detecting the bacteriostasis and sterilization effect of the bactericide in the culture water body adopts the water body to prepare initial seed liquid, and the preparation method of the initial seed liquid comprises the following steps: inoculating 1uL of single bacteria by using a 1uL inoculating loop, putting the inoculated single bacteria into a corresponding 50/250mL shaking bottle, and culturing for 18-24 hours at 37 ℃ to obtain corresponding initial seed liquid; adding the bactericide to be detected into a corresponding 50/250mL shaking bottle, adding 0.5mL of corresponding initial seed liquid, measuring the single-fungus solubility in 0.5-2h, and converting into a logarithmic value, wherein the logarithmic value is a short-term sterilization index; after 24 hours or more, the single bacteria solubility is measured and converted into logarithmic value, and the logarithmic value is the long-term bacteriostasis index.
Preferably, the concentration of the single bacteria in the corresponding initial seed solution is as follows, namely the concentration of the single bacteria in the simulated seawater conventional water body or the simulated freshwater conventional water body is 1 multiplied by 10 6 cfu/mL-1×10 7 cfu/mL; the concentration of single bacteria in the simulated seawater seedling pool water body, the simulated fresh water seedling pool water body or the simulated fresh water seedling pool water body is 1 multiplied by 10 7 cfu/mL-1×10 8 cfu/mL。
In summary, the beneficial technical effects of the present application are: the simulated water body and the using method have the advantages of low cost, short detection period, high efficiency, high accuracy, wide application range, simple using method and the like, and the gap of the high-efficiency method for testing the bacteriostasis and sterilization effects of the bactericide in the aquaculture water body at present is overcome.
Detailed Description
Embodiments of the present application will be described in detail below with reference to examples and preparations, but it will be understood by those skilled in the art that the following examples and preparations are only for illustration of the present application and should not be construed as limiting the scope of the present application. The specific conditions in the examples and preparations were not specified, and the procedures were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The application provides a simulation water body for detecting bacteriostasis and sterilization effect of bactericide in aquaculture water body, including simulation water body, single fungus and bactericide, wherein, the simulation water body is including simulation sea water conventional water body, simulation sea water seedling pool water body, simulation sea water washing seedling pool water body, simulation fresh water conventional water body, simulation fresh water seedling pool water body, simulation fresh water washing seedling pool water body.
In one embodiment, the simulated seawater normal body of water comprises the following components in mass ratio: 1000g of primary pure water, 30g of sea salt, 20mg of peptone and 10mg of yeast powder; the simulated seawater seedling pool water body comprises the following components in percentage by mass: 1000g of primary pure water, 30g of sea salt, 70mg of peptone and 30mg of yeast powder.
In the application, the simulated seawater seedling pool water bodies comprise the following components in percentage by mass: 1000g of primary pure water, 30g of sea salt, 70mg of peptone and 30mg of yeast powder; the simulated fresh water conventional water body comprises the following components in percentage by mass: 1000g of primary pure water, 5g of sea salt, 20mg of peptone and 10mg of yeast powder; the simulated fresh water seedling pool water body or the simulated fresh water seedling pool water body comprises the following components in percentage by mass: 1000g of primary pure water, 5g of sea salt, 70mg of peptone and 30mg of yeast powder; .
In one embodiment, the synthetic method of the simulated seawater conventional water body comprises the following steps: sequentially adding 30g of sea salt, 20mg of peptone and 10mg of yeast powder into 1000g of primary pure water, uniformly mixing to obtain a simulated seawater conventional water body, respectively taking 50mL of the simulated seawater conventional water body, subpackaging into 250mL shake flasks, correspondingly marking as 50/250mL shake flasks A1-A20, and sterilizing at 121 ℃ for 20min for later use; the synthesis method of the simulated seawater seedling pool water body comprises the following steps: sequentially adding 30g of sea salt, 70mg of peptone and 30mg of yeast powder into 1000g of primary pure water, uniformly mixing to respectively obtain a simulated sea water seedling pool water body or a simulated sea water seedling pool water body, respectively taking 50mL of the simulated sea water seedling pool water body or the simulated sea water seedling pool water body, subpackaging the simulated sea water seedling pool water body or the simulated sea water seedling pool water body into 250mL shake flasks, correspondingly marking 50/250mL shake flasks B1-B20, and sterilizing at 121 ℃ for 20min for later use.
In the application, the synthesis method of the simulated seawater seedling pool water body further comprises the following steps: sequentially adding 30g of sea salt, 70mg of peptone and 30mg of yeast powder into 1000g of primary pure water, uniformly mixing to obtain a simulated sea water seedling pool water body or a simulated sea water seedling pool water body respectively, respectively taking 50mL of the simulated sea water seedling pool water body or the simulated sea water seedling pool water body, subpackaging the simulated sea water seedling pool water body or the simulated sea water seedling pool water body into 250mL shake flasks, correspondingly marking the shake flasks as 50/250mL C1-C20, and sterilizing at 121 ℃ for 20min for later use. The synthetic method of the simulated fresh water conventional water body comprises the following steps: sequentially adding 5g of sea salt, 20mg of peptone and 10mg of yeast powder into 1000g of primary pure water, uniformly mixing to obtain a simulated fresh water conventional water body, respectively taking 50mL of the simulated fresh water conventional water body, subpackaging into 250mL shake flasks, correspondingly marking as 50/250mL shake flasks D1-D20, and sterilizing at 121 ℃ for 20min for later use; the synthesis method of the simulated fresh water seedling pool water body or the simulated fresh water seedling pool water body comprises the following steps: sequentially adding 5g of sea salt, 70mg of peptone and 30mg of yeast powder into 1000g of primary pure water, uniformly mixing to obtain a simulated fresh water seedling pool water body or a simulated fresh water seedling pool water body respectively, respectively taking 50mL of the simulated fresh water seedling pool water body or the simulated fresh water seedling pool water body, subpackaging the simulated fresh water seedling pool water body or the simulated fresh water seedling pool water body into 250mL shake flasks, correspondingly marking 50/250mL shake flasks as E1-E20 and F1-F20, and sterilizing at 121 ℃ for 20min for later use.
In one embodiment, the single bacterium is one of ATCC17802 vibrio parahaemolyticus and Vibrio parahaemolyticus selected from transparent shrimp larvae of Xiamen.
In the application, the single bacterium is one of algicidal, parahaemolytic, havickers, vibrio vulnificus, vibrio splendidus, aeromonas hydrophila and escherichia coli.
In one embodiment, the biocide is a polyhexamethylene monoguanidine crystal biocide having a purity greater than 98%.
In the application, the bactericide is also one of povidone iodine bactericide and quaternary ammonium salt bactericide.
The application also provides a using method of the simulated water body for detecting the bacteriostasis and sterilization effect of the bactericide in the culture water body, the water body is used for preparing initial seed liquid, and the preparing method of the initial seed liquid is as follows: inoculating 1uL of single bacteria by using a 1uL inoculating loop, putting the inoculated single bacteria into a corresponding 50/250mL shaking bottle, and culturing for 18-24 hours at 37 ℃ to obtain corresponding initial seed liquid; adding the bactericide to be detected into a corresponding 50/250mL shaking bottle, adding 0.5mL of corresponding initial seed liquid, measuring the single-fungus solubility in 0.5-2h, and converting into a logarithmic value, wherein the logarithmic value is a short-term sterilization index; after 24 hours or more, the single bacteria solubility is measured and converted into logarithmic value, and the logarithmic value is the long-term bacteriostasis index.
In one embodiment, the concentration of the corresponding single bacteria in the initial seed solution is as follows:the concentration of single bacteria in the conventional water body of the simulated seawater is 1 multiplied by 10 6 cfu/mL-1×10 7 cfu/mL; the concentration of single bacteria in the simulated seawater seedling pool water body and the simulated seawater seedling pool water body is 1 multiplied by 10 7 cfu/mL-1×10 8 cfu/mL。
The application also comprises the corresponding concentration of the single bacteria in the initial seed liquid, wherein the concentration of the single bacteria in the conventional water body simulating fresh water is 1 multiplied by 10 6 cfu/mL-1×10 7 cfu/mL; the concentration of single bacteria in the water body of the simulated fresh water seedling pool or the water body of the simulated fresh water seedling pool is 1 multiplied by 10 7 cfu/mL-1×10 8 cfu/mL。
Example 1
A simulated water body for detecting bacteriostasis and sterilization effects of a bactericide in a culture water body comprises a simulated seawater conventional water body, ATCC17802 vibrio parahaemolyticus and polyhexamethylene guanidine crystal bactericide with purity of more than 98 percent, and the synthetic method of the simulated seawater conventional water body comprises the following steps: sequentially adding 30g of sea salt, 20mg of peptone and 10mg of yeast powder into 1000g of primary pure water, uniformly mixing to obtain a simulated seawater conventional water body, respectively taking 50mL of the simulated seawater conventional water body, subpackaging into 250mL shake flasks, correspondingly marking as 50/250mL shake flasks A1-A20, and sterilizing at 121 ℃ for 20min for later use;
the application method of the simulated water body for detecting the bacteriostasis and sterilization effect of the bactericide in the culture water body adopts the water body to prepare initial seed liquid, and the preparation method of the initial seed liquid comprises the following steps: inoculating 1uL of single bacteria by using a 1uL inoculating loop, putting the inoculated single bacteria into a corresponding 50/250mL shaking bottle, and culturing for 24 hours at 37 ℃ to obtain corresponding initial seed liquid; adding polyhexamethylene monoguanidine crystal bactericide into a corresponding 50/250mL shaking bottle, adding 0.5mL of corresponding initial seed liquid, measuring the single bacteria solubility in 0.5-2h, and converting into logarithmic value, wherein the logarithmic value is a short-term sterilization index; after more than or equal to 24 hours, measuring the single bacteria solubility, and converting the single bacteria solubility into logarithmic value, wherein the logarithmic value is a long-term bacteriostasis index; the concentration of single bacteria in the conventional water body of the simulated seawater is 5 multiplied by 10 6 cfu/mL。
Example 2
A simulated water body for detecting bacteriostasis and sterilization effects of a bactericide in a culture water body comprises simulated seawater conventional water body, vibrio parahaemolyticus screened by Xiamen transparent shrimp larvae and polyhexamethylene monoguanidine crystal bactericide with purity of more than 98%, wherein the synthetic method of the simulated seawater conventional water body comprises the following steps: sequentially adding 30g of sea salt, 20mg of peptone and 10mg of yeast powder into 1000g of primary pure water, uniformly mixing to obtain a simulated seawater conventional water body, respectively taking 50mL of the simulated seawater conventional water body, subpackaging into 250mL shake flasks, correspondingly marking as 50/250mL shake flasks A1-A20, and sterilizing at 121 ℃ for 20min for later use;
the application method of the simulated water body for detecting the bacteriostasis and sterilization effect of the bactericide in the culture water body adopts the water body to prepare initial seed liquid, and the preparation method of the initial seed liquid comprises the following steps: inoculating 1uL of single bacteria by using a 1uL inoculating loop, putting the inoculated single bacteria into a corresponding 50/250mL shaking bottle, and culturing for 24 hours at 37 ℃ to obtain corresponding initial seed liquid; adding polyhexamethylene monoguanidine crystal bactericide into a corresponding 50/250mL shaking bottle, adding 0.5mL of corresponding initial seed liquid, measuring the single bacteria solubility in 0.5-2h, and converting into logarithmic value, wherein the logarithmic value is a short-term sterilization index; after more than or equal to 24 hours, measuring the single bacteria solubility, and converting the single bacteria solubility into logarithmic value, wherein the logarithmic value is a long-term bacteriostasis index; the concentration of single bacteria in the conventional water body of the simulated seawater is 7 multiplied by 10 6 cfu/mL。
Example 3
The application provides a simulated water body for detecting bacteriostasis and sterilization effects of a bactericide in a culture water body, which comprises a simulated seawater conventional water body, vibrio parahaemolyticus screened at a fish gill position and a polyhexamethylene monoguanidine crystal bactericide with purity of more than 98%, wherein the synthetic method of the simulated seawater conventional water body is as follows: sequentially adding 30g of sea salt, 20mg of peptone and 10mg of yeast powder into 1000g of primary pure water, uniformly mixing to obtain a simulated seawater conventional water body, respectively taking 50mL of the simulated seawater conventional water body, subpackaging into 250mL shake flasks, correspondingly marking as 50/250mL shake flasks A1-A20, and sterilizing at 121 ℃ for 20min for later use;
the application method of the simulated water body for detecting the bacteriostasis and sterilization effect of the bactericide in the culture water body adopts the water body to prepare initial seed liquid, and the preparation method of the initial seed liquid comprises the following steps: 1uL of single bacteria were inoculated using a 1uL inoculating loop into corresponding 50/250mL shake flasks,culturing at 37deg.C for 24 hr to obtain corresponding initial seed solution; adding polyhexamethylene monoguanidine crystal bactericide into a corresponding 50/250mL shaking bottle, adding 0.5mL of corresponding initial seed liquid, measuring the single bacteria solubility in 0.5-2h, and converting into logarithmic value, wherein the logarithmic value is a short-term sterilization index; after more than or equal to 24 hours, measuring the single bacteria solubility, and converting the single bacteria solubility into logarithmic value, wherein the logarithmic value is a long-term bacteriostasis index; the concentration of single bacteria in the conventional water body of the simulated seawater is 8 multiplied by 10 6 cfu/mL。
Example 4
A simulated water body for detecting bacteriostasis and sterilization effects of a bactericide in a culture water body comprises a simulated seawater seedling pool water body, vibrio parahaemolyticus screened by Xiamen transparent shrimp seedlings and 0.5ppm polyhexamethylene monoguanidine crystal bactericide with purity of more than 98%, wherein the synthetic method of the simulated seawater seedling pool water body comprises the following steps: sequentially adding 30g of sea salt, 70mg of peptone and 30mg of yeast powder into 1000g of primary pure water, uniformly mixing to respectively obtain a simulated sea water seedling pool water body or a simulated sea water seedling pool water body, respectively taking 50mL of the simulated sea water seedling pool water body or the simulated sea water seedling pool water body, subpackaging the simulated sea water seedling pool water body or the simulated sea water seedling pool water body into 250mL shake flasks, correspondingly marking 50/250mL shake flasks B1-B20, and sterilizing at 121 ℃ for 20min for later use.
The application method of the simulated water body for detecting the bacteriostasis and sterilization effect of the bactericide in the culture water body adopts the water body to prepare initial seed liquid, and the preparation method of the initial seed liquid comprises the following steps: inoculating 1uL of single bacteria by using a 1uL inoculating loop, putting the single bacteria into a corresponding 50/250mL shaking flask, and culturing for 124 hours at 37 ℃ to obtain corresponding initial seed liquid; adding polyhexamethylene monoguanidine crystal bactericide into a corresponding 50/250mL shaking bottle, adding 0.5mL of corresponding initial seed liquid, measuring the single bacteria solubility in 0.5-2h, and converting into logarithmic value, wherein the logarithmic value is a short-term sterilization index; after more than or equal to 24 hours, measuring the single bacteria solubility, and converting the single bacteria solubility into logarithmic value, wherein the logarithmic value is a long-term bacteriostasis index; the concentration of single bacteria in the water body of the simulated seawater seedling pool water body is 1 multiplied by 10 8 cfu/mL。
Property testing
1. Conventional water body testing
The water body of a transparent shrimp seed pond of Xiamen is adopted.
The concentration of the vibrio in the initial fermentation liquid is 1 multiplied by 10 6 cfu/mL, three types of ATCC17802 vibrio parahaemolyticus marked as Vibrio A, vibrio parahaemolyticus marked as Vibrio B screened by transparent shrimp larvae of Xiamen and Vibrio parahaemolyticus marked as Vibrio C screened at fish gill are adopted for testing;
the bactericide is as follows: polyhexamethylene monoguanidine crystal bactericide with purity of more than 98%;
determining the logarithmic value of cfu/L of three vibrios under the conditions that different purity is more than 98% and the concentration of polyhexamethylene guanidine crystal bactericides is detected; the test method comprises the following steps: oxford cup test (diameter of inhibition zone mm), test results are shown in table 1; mic test, test results are shown in Table 2; the kill index method was tested and the test results are shown in table 3.
Table 1 oxford cup test
| Bactericide concentration/ppm | Vibrio A | Vibrio B | Vibrio C |
| 2 | Without any means for | Without any means for | Without any means for |
| 4 | Without any means for | Without any means for | Without any means for |
| 8 | Without any means for | 11 | Without any means for |
| 16 | 10 | 23 | 9 |
| 32 | 17 | 25 | 10 |
| 64 | 18 | 27 | 10 |
From Table 1, it can be seen that the use of oxford cup test suggests a concentration of at least 16ppm of bactericide.
TABLE 2Mic test
From Table 2, it can be seen that the use of MIC tests suggests a concentration of at least 8ppm of biocide.
Table 3 kill index test
| Bactericide concentration/ppm | Vibrio A | Vibrio B | Vibrio C |
| 2 | 1.4 | 1.0 | 0.4 |
| 4 | 1.9 | 1.8 | 0.8 |
| 8 | 2.8 | 2.4 | 1.5 |
| 16 | 3.7 | 3.9 | 2.7 |
| 32 | 4.8 | 5.1 | 4.6 |
| 64 | 5.5 | Greater than 6 | 5.4 |
From Table 3, the concentration of the bactericide was at least 8ppm when Vibrio was reduced to the 4 th order using the killing index test.
As can be seen from tables 1-3, the above data are far higher than the effective values in practical use, and generally, the concentration of the bactericide is generally 1ppm for light penaeus vannamei, the concentration of the bactericide is generally 2-3ppm for emergency or later stage of penaeus vannamei, and the daily prevention and control can be as low as 0.3-0.5 ppm.
2. Simulated seawater conventional water body test
2.1 50mL of the test solution of the conventional seawater-simulated water body in example 1 was added to a concentration of 5X 10 of Vibrio 0.5mLA 6 After cfu/mL are uniformly mixed, the sterilizing effect of the polyhexamethylene guanidine crystal bactericide with different concentrations is tested at the time of 0.5h,24h and 7d respectively, the concentration of vibrio at the moment is converted into a logarithmic value representation, and the result is shown in Table 4.
TABLE 5 sterilizing Effect of example 1
2.2 50mL of the test solution from the conventional seawater-simulated water body of example 2 was added to a concentration of 7X 10 of 0.5 mVibrio paradisi 6 After cfu/mL are uniformly mixed, the sterilizing effect of the polyhexamethylene guanidine crystal bactericide with different concentrations is tested at the time of 0.5h,24h and 7d respectively, the concentration of vibrio at the moment is converted into a logarithmic value representation, and the result is shown in Table 5.
TABLE 5 sterilizing Effect of example 2
2.3 50mL of the test solution from the conventional seawater-simulated body of water of example 3 was added to a concentration of 8X 10 of Vibrio 0.5mLC 6 After cfu/mL are uniformly mixed, the sterilizing effect of the polyhexamethylene guanidine crystal bactericide with different concentrations is tested at the time of 0.5h,24h and 7d respectively, the concentration of vibrio at the moment is converted into a logarithmic value representation, and the result is shown in Table 6.
TABLE 6 example 3 sterilizing Effect
From tables 4 to 6, it is understood that when the concentration of the polyhexamethylene guanidine crystal bactericide used in a general culture water body or a conventional seawater-like water body is 0.5ppm, excessive propagation of Vibrio parahaemolyticus can be controlled by conventional control.
2. Growth test for rice bran-shaped larva of white shrimp in conventional seawater simulation water body
The method in the embodiment 2 is adopted to prepare the simulated seawater conventional water body, 18 litopenaeus vannamei ponds are configured, each pond is 2m long, 2m wide and 1.5m high, 200 bran-shaped larvae of litopenaeus vannamei ponds are arranged in each pond, the repetition number of each group is 3, 6 groups are set in the test, and 21 ponds are totally arranged, wherein, the vibrio B is added in 12 ponds, and the concentration is 1x10 5 cfu/L, the average survival number of 1d,3d and 7d prawns was calculated, and the test results are shown in Table 7.
The test method comprises the following steps: the concentrations of polyhexamethylene monoguanidine crystal bactericide are respectively added to 9 corresponding shrimp ponds at the same time of adding the B vibrio, namely 0.25ppm+B vibrio, 0.5ppm+B vibrio and 1ppm+B vibrio, and the other 3 shrimp ponds are respectively marked as the B vibrio, and the polyhexamethylene monoguanidine crystal bactericide is not added at the same time of adding the B vibrio; in addition, 3 shrimp ponds, the concentration of the crystal bactericide added with the polyhexamethylene guanidine is 0.25ppm, the vibrio B is not added, and the mark is 1ppm; 3 shrimp ponds were left as blank controls (no ATCC17802 bacteria and bactericide added).
Table 7 simulates the survival number of Litopenaeus vannamei in the conventional water chaff larvae of seawater
As can be seen from Table 7, the correlation degree between the simulated seawater conventional water body and the actual culture data is better, and the addition amount of the bactericide can be selected for the specific discovery of pathogenic bacteria at the seawater pond mouth so as to control the culture of the disease bacteria.
3. Simulated seawater seedling pool water body test
Example 450mL of test solution of the simulated seawater seedling pool water body is added with 1X10 of the concentration of 0.5mLB vibrio 8 After cfu/mL are uniformly mixed, the sterilizing effect of the polyhexamethylene guanidine crystal bactericide with different concentrations is tested at the time of 0.5h,24h and 7d respectively, the concentration of vibrio at the moment is converted into a logarithmic value representation, and the result is shown in Table 8.
TABLE 8 sterilizing Effect of example 4
As can be seen from Table 8, the apparent infection of Vibrio parahaemolyticus can be controlled when the concentration of the polyhexamethylene guanidine crystal bactericide is 1-2ppm, which is rich in nutrition and has high pollution.
4. Test for simulating growth of chaff larva of white shrimp in seawater seedling pool water body
The method in example 4 is adopted to prepare a simulated seawater seedling pool water body, 18 Penaeus vannamei Boone pools are configured, each pool is 2m long, 2m wide and 1.5m high, 200 chaff larvae of Penaeus vannamei Boone are arranged in each pool, the repetition number of each group is 3, 6 groups are set in the test, 21 pools are total, wherein, vibrio B is added in 12 pools, and the concentration is 1x10 5 cfu/L, the average survival number of 1d,3d and 7d prawns was calculated, and the test results are shown in Table 8.
The test method comprises the following steps: the concentrations of polyhexamethylene monoguanidine crystal bactericide are respectively added to 9 corresponding shrimp ponds at the same time of adding the B vibrio, namely 0.25ppm+B vibrio, 0.5ppm+B vibrio and 1ppm+B vibrio, and the other 3 shrimp ponds are respectively marked as the B vibrio, and the polyhexamethylene monoguanidine crystal bactericide is not added at the same time of adding the B vibrio; in addition, 3 shrimp ponds, the concentration of the crystal bactericide added with the polyhexamethylene guanidine is 0.25ppm, the vibrio B is not added, and the mark is 1ppm; 3 shrimp ponds were left as blank controls (no ATCC17802 bacteria and bactericide added).
Table 8 simulates the survival number of Litopenaeus vannamei in bran-like larvae in a seawater pond water body
From table 8, it can be seen that the correlation degree between the simulated seawater seedling pool water body and the actual culture data is better, and for finding diseases such as glass seedlings, the bactericide with corresponding concentration is added immediately, so that corresponding symptoms can be treated, the survival rate is improved, and the death of the pond is prevented.
The foregoing embodiments are merely for illustrating the technical solution of the present application and are not intended to be limiting, and although the foregoing embodiments specifically illustrate the present application, it should be understood by those skilled in the relevant art that modifications and equivalents can be made to the specific embodiments of the present application without departing from the spirit and scope of the present application.
Claims (9)
1. The simulated water body is used for detecting the bacteriostatic and bactericidal effect of the bactericide in the aquaculture water body and is characterized by comprising a simulated water body, single bacteria and the bactericide, wherein the simulated water body is one of a simulated seawater conventional water body, a simulated seawater seedling pool water body, a simulated fresh water conventional water body, a simulated fresh water seedling pool water body and a simulated fresh water seedling pool water body.
2. A simulated water body for detecting the bacteriostatic and bactericidal effect of a bactericide in a body of aquaculture water according to claim 1, wherein said simulated seawater regular water body comprises the following components in mass ratio: 1000g of primary pure water, 30g of sea salt, 20mg of peptone and 10mg of yeast powder; the simulated seawater seedling pool water body or the simulated seawater seedling pool water body comprises the following components in percentage by mass: 1000g of primary pure water, 30g of sea salt, 70mg of peptone and 30mg of yeast powder.
3. A simulated water body for detecting the bacteriostatic and bactericidal effect of a bactericide in a body of aquaculture water according to claim 1, wherein said simulated fresh water body comprises the following components in mass ratio: 1000g of primary pure water, 5g of sea salt, 20mg of peptone and 10mg of yeast powder; the simulated fresh water seedling pool water body or the simulated fresh water seedling pool water body comprises the following components in percentage by mass: 1000g of primary pure water, 5g of sea salt, 70mg of peptone and 30mg of yeast powder.
4. The simulated water body for detecting the bacteriostatic and bactericidal effect of a bactericide in a culture water body according to claim 2, wherein the synthetic method of the simulated seawater conventional water body is as follows: sequentially adding 30g of sea salt, 20mg of peptone and 10mg of yeast powder into 1000g of primary pure water according to the mass ratio, uniformly mixing to obtain a simulated seawater conventional water body, respectively taking 50mL of the simulated seawater conventional water body, subpackaging into 250mL shake flasks, correspondingly marking 50/250mL shake flasks A1-A20, and sterilizing at 121 ℃ for 20min for later use; the synthesis method of the simulated seawater seedling pool water body or the simulated seawater seedling pool water body comprises the following steps: sequentially adding 30g of sea salt, 70mg of peptone and 30mg of yeast powder into 1000g of primary pure water according to the mass ratio, uniformly mixing to obtain a simulated seawater seedling pool water body or a simulated seawater seedling pool water body respectively, respectively taking 50mL of the simulated seawater seedling pool water body or the simulated seawater seedling pool water body, subpackaging the simulated seawater seedling pool water body or the simulated seawater seedling pool water body into 250mL shake flasks, correspondingly marking 50/250mL shake flasks B1-B20 and C1-C20, and sterilizing at 121 ℃ for 20min for later use.
5. A simulated water body for detecting the bacteriostatic and bactericidal effect of a bactericide in a aquaculture water body according to claim 3, wherein the synthetic method of the simulated fresh water conventional water body is as follows: sequentially adding 5g of sea salt, 20mg of peptone and 10mg of yeast powder into 1000g of primary pure water according to the mass ratio, uniformly mixing to obtain a simulated fresh water conventional water body, respectively taking 50mL of the simulated fresh water conventional water body, subpackaging the simulated fresh water conventional water body into 250mL shake flasks, correspondingly marking the simulated fresh water conventional water body as 50/250mL shake flasks D1-D20, and sterilizing at 121 ℃ for 20min for later use; the synthesis method of the simulated fresh water seedling pool water body or the simulated fresh water seedling pool water body comprises the following steps: according to the mass ratio, adding 5g of sea salt, 70mg of peptone and 30mg of yeast powder into 1000g of primary pure water in sequence, uniformly mixing to obtain a simulated fresh water seedling pool water body or a simulated fresh water seedling pool water body respectively, respectively taking 50mL of the simulated fresh water seedling pool water body or the simulated fresh water seedling pool water body, subpackaging the simulated fresh water seedling pool water body or the simulated fresh water seedling pool water body into 250mL shake flasks, correspondingly marking 50/250mL shake flasks E1-E20 and F1-F20, and sterilizing at 121 ℃ for 20min for later use.
6. The simulated water body for detecting the bacteriostatic and bactericidal effect of a bactericide in a culture water body according to claim 1, wherein the single bacterium is one of ATCC17802 vibrio parahaemolyticus, vibrio parahaemolyticus selected from transparent shrimp larvae of Xiamen, vibrio parahaemolyticus selected from fish gill, algicidal, parahaemolyticus, haven, vibrio vulnificus, vibrio lautus, aeromonas hydrophila and escherichia coli.
7. The simulated water body for detecting the bacteriostatic and bactericidal effect of a bactericide in a culture water body according to claim 1, wherein the bactericide is one of povidone iodine bactericide, quaternary ammonium salt bactericide and polyhexamethylene monoguanidine crystal bactericide with purity of more than 98%.
8. A method of using a simulated body of water for detecting the bacteriostatic and bactericidal effects of a bactericide in a body of aquaculture water, characterized by employing the body of water of any one of claims 4 or 5 for the preparation of an initial seed solution, the method of preparing the initial seed solution comprising: inoculating 1uL of single bacteria by using a 1uL inoculating loop, putting the inoculated single bacteria into a corresponding 50/250mL shaking bottle, and culturing for 18-24 hours at 37 ℃ to obtain corresponding initial seed liquid; adding the bactericide to be detected into a corresponding 50/250mL shaking bottle, adding 0.5mL of corresponding initial seed liquid, measuring the single-fungus solubility in 0.5-2h, and converting into a logarithmic value, wherein the logarithmic value is a short-term sterilization index; after 24 hours or more, the single bacteria solubility is measured and converted into logarithmic value, and the logarithmic value is the long-term bacteriostasis index.
9. The method for detecting a simulated water body having an antibacterial effect as claimed in claim 8, wherein the concentration of the single bacteria in the initial seed solution is 1×10 in the simulated seawater normal water body or the simulated freshwater normal water body 6 cfu/mL-1×10 7 cfu/mL; simulated seawater seedling pool water body and simulated seawater washingThe concentration of single bacteria in the seedling pool water body, the simulated fresh water seedling pool water body or the simulated fresh water seedling pool water body is 1 multiplied by 10 7 cfu/mL-1×10 8 cfu/mL。
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