CN111471619A - Culture medium for isolated culture of avibacterium paragallinarum in laboratory and preparation method and application thereof - Google Patents
Culture medium for isolated culture of avibacterium paragallinarum in laboratory and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of isolated culture of avibacterium paragallinarum, and discloses a culture medium for isolated culture of avibacterium paragallinarum in a laboratory, and a preparation method and application thereof, wherein the culture medium is a chocolate agar culture medium which is prepared from the following raw materials in parts by volume: 100 parts of nutrient agar culture medium; 10 parts of healthy chicken whole blood; the preparation method of the culture medium comprises the following steps: A1. sterilizing the prepared nutrient agar culture medium at high temperature, and keeping the temperature at 70-85 ℃; A2. adding the healthy chicken whole blood into the nutrient agar culture medium prepared in the step A1 according to the volume parts, heating at 70-85 ℃ for 5-10 min, and shaking up while heating to obtain a culture medium added with the healthy chicken whole blood; A3. and (4) preparing a sterile plate by using the culture medium which is obtained in the step A2 and is added with the healthy chicken whole blood, namely a chocolate agar culture medium for later use. The invention brings great convenience for the preparation of the culture medium for the isolated culture of the avibacterium paragallinarum and simultaneously reduces the cost investment of the poultry breeding industry.
Description
Technical Field
The invention relates to the technical field of isolated culture of avibacterium paragallinarum, and in particular relates to a culture medium for isolated culture of avibacterium paragallinarum in a laboratory, and a preparation method and application thereof.
Background
Avian paragallinarum (avibacterium paragallinarum) is a pathogen of avian infectious rhinitis and is one of the main pathogens causing acute respiratory diseases of poultry, and chickens are the main hosts of the avian paragallinarum and are mainly characterized by inflammation of nasal cavities and sinuses, sneezing and face swelling. The avian infectious rhinitis can be infected by chickens of all ages, and is particularly most susceptible to bred chickens and laying hens. The infection of the disease can cause the increase of the number of rejected chickens and the obvious reduction of the laying rate of the chickens, so the disease can cause great economic loss to the poultry breeding industry, and further has great influence on the poultry breeding industry.
Therefore, poultry farming usually monitors whether the farmed poultry carries avibacterium paragallinarum in real time in the farming process, the monitoring method is to collect samples from the poultry bodies, separate and culture the collected samples, detect whether the samples contain the avibacterium paragallinarum by using a series of detection methods, and perform corresponding administration treatment on the farmed poultry according to whether the samples contain the avibacterium paragallinarum and the content of the avibacterium paragallinarum in the samples, so as to reduce the infection of avian infectious rhinitis in the poultry, and reduce the economic loss and other influences of the poultry farming industry as much as possible. The culture medium is often needed in the process of isolated culture of the avibacterium paragallinarum.
At present, a culture medium for separating and culturing the avibacterium paragallinarum is a sheep blood culture medium, namely sheep blood is added into a commercially available nutrient agar culture medium, so that necessary nutrient substances are provided for the separation and culture of the avibacterium paragallinarum. For the acquisition of sheep blood, two acquisition ways are generally available, one is that the sheep blood sold in the market is adopted, and because the sheep blood in the market has longer storage time, when the sheep blood is used for separating and culturing avibacterium paragallinarum, the nutritional effect of a plurality of substances in the sheep blood is reduced, so the separation and culture effect on the avibacterium paragallinarum in the actual use is poorer, the misjudgment on the detection of the avian infectious rhinitis infection in poultry can be caused finally, and the price of the sheep blood sold in the market is high; the other way is to collect sheep blood by self, and the process of collecting sheep blood is complicated and inconvenient to collect sheep blood. Therefore, in view of the above-mentioned drawbacks caused by the culture medium for avibacterium paragallinarum, there is no method for overcoming the above-mentioned drawbacks.
Disclosure of Invention
Aiming at the defects of the existing method, the first purpose of the invention is to provide a culture medium for isolated culture of avibacterium paragallinarum in a laboratory, healthy chicken whole blood is used for replacing sheep blood to prepare the culture medium for isolated culture of avibacterium paragallinarum, and the healthy chicken whole blood has low cost, is easy to obtain, is not limited by purchasing time and can be prepared for use on site.
The second purpose of the invention is to provide a preparation method of a culture medium for isolated culture of avibacterium paragallinarum in a laboratory, which is characterized in that the culture medium for isolated culture of avibacterium paragallinarum is prepared by utilizing healthy chicken whole blood and is prepared by a specific preparation method, so that great convenience is brought to isolated culture of avibacterium paragallinarum.
The third purpose of the invention is to provide a new method for the laboratory separation culture of avibacterium paragallinarum, the chocolate agar culture medium prepared by the preparation method is used for the separation culture of avibacterium paragallinarum, and the whole blood of healthy chickens is low in cost, easy to obtain, free from the limitation of purchasing time and ready to use, and in addition, the whole blood collection process of healthy chickens is simple and convenient, so that great convenience is brought to the laboratory separation culture of avibacterium paragallinarum, and the cost investment of the laboratory separation culture of avibacterium paragallinarum is reduced. The culture method provided by the invention can separate and culture the avibacterium paragallinarum more efficiently compared with the culture method of inoculating the avibacterium paragallinarum into a culture medium which uses sheep blood as a coenzyme I donor.
The invention also provides a method for detecting the poultry infectious rhinitis infection, which comprises the steps of detecting whether a satellite phenomenon exists on a blood agar culture medium by matching the chocolate agar culture medium and the blood agar culture medium, preliminarily judging whether the poultry is infected by the poultry infectious rhinitis, and further formulating a corresponding treatment strategy according to the poultry infectious rhinitis infection condition.
In order to achieve the first object, the invention provides the following method scheme: the culture medium for the laboratory isolated culture of the avibacterium paragallinarum is characterized by being a chocolate agar culture medium which is prepared from the following raw materials in parts by volume: 100 parts of nutrient agar culture medium; 10 parts of healthy chicken whole blood;
the preparation method of the chocolate agar culture medium comprises the following steps:
A1. sterilizing the prepared nutrient agar culture medium at high temperature, and keeping the temperature at 70-85 ℃;
A2. adding the healthy chicken whole blood into the nutrient agar culture medium prepared in the step A1 according to the volume parts, heating at 70-85 ℃ for 5-10 min, and shaking up while heating to obtain a culture medium added with the healthy chicken whole blood;
A3. and (4) preparing a sterile plate by using the culture medium which is obtained in the step A2 and is added with the healthy chicken whole blood, namely a chocolate agar culture medium for later use.
Coenzyme I is required in the growth process of avibacterium paragallinarum, is an important coenzyme in redox reaction and has important roles in substance metabolism and energy metabolism, so that the supply of the coenzyme I required for the growth of the avibacterium paragallinarum in the growth process of the avibacterium paragallinarum is very important. In the prior art, sheep blood is added to a culture medium for separating and culturing avibacterium paragallinarum on the basis of a basic nutrient agar culture medium, and in the process of culturing avibacterium paragallinarum by using the culture medium, the sheep blood provides coenzyme I for the growth of avibacterium paragallinarum in the process, but the whole blood of healthy chickens, namely the blood of chickens, comprises blood cells and blood plasma, and the blood cells contain abundant coenzyme I because the sheep blood introduced in the background technology has the defects of difficult preparation, short storage time and high price. Therefore, the healthy chicken whole blood can provide coenzyme I for the growth of the avibacterium paragallinarum, the whole blood collection process of the fresh healthy chicken is simple and convenient, the cost is low, the laboratory operation is convenient, the existing collection can be realized, the limitation of the purchase time is avoided, the preparation of the culture medium for the isolated culture of the avibacterium paragallinarum is greatly facilitated, the isolated culture efficiency of the avibacterium paragallinarum is further improved, and the cost input of the poultry breeding industry is reduced. According to experimental analysis, the healthy chicken whole blood is suitable for being used as a nutrient component for providing coenzyme I in a culture medium for culturing the avibacterium paragallinarum.
The invention prepares the chocolate agar culture medium in the culture medium for separating and culturing the avibacterium paragallinarum by utilizing the basic nutrient agar culture medium and the healthy chicken whole blood. Through the research of the laboratory and the combination with the test conditions of the avibacterium paragallinarum, the volume parts of the nutrient agar culture medium and the healthy chicken whole blood in the chocolate agar culture medium provided by the invention are finally determined to be 100 parts and 10 parts respectively. The poultry bacillus paragallinarum is inoculated on a chocolate agar culture medium, and the culture medium can better separate and culture the poultry bacillus paragallinarum.
The chocolate agar culture medium is prepared by the method, heat preservation is carried out at 70-85 ℃ after high-temperature sterilization, on one hand, the sterilized nutrient agar culture medium cannot be solidified in the temperature range, and the healthy chicken whole blood and the nutrient agar culture medium are favorably mixed uniformly, on the other hand, the healthy chicken whole blood is added into the culture medium and heated for 5-10 min, blood cells in the healthy chicken whole blood can be cracked, coenzyme I in the blood cells can enter the culture medium, and the coenzyme I can be directly and uniformly mixed on a prepared chocolate agar culture medium sterile flat plate, so that the utilization of coenzyme I by avibacterium paragallinarum is favorably realized, and the separation culture of avibacterium paragallinarum is favorably realized.
Further, the whole blood of the healthy chicken is obtained by artificially collecting sterile blood under the wings of the healthy chicken.
By adopting the method scheme, the whole blood of the healthy chickens is obtained by monitoring the chickens to be sampled in real time from a poultry breeding center monitored in a laboratory, and collecting the whole blood of the healthy chickens to be sampled in a healthy state; the blood sampling mode is aseptic wing-down blood sampling. Therefore, the whole blood of the healthy chicken is simple in acquisition mode, convenient to prepare and low in cost. The chicken without the avibacterium paragallinarum antibody and the infectious disease can be used as the healthy chicken in the invention through detection.
Further, the whole blood of the healthy chicken is used as it is.
By adopting the method scheme, in order to ensure that the coenzyme I in the whole blood of the healthy chickens is in the best action period, the whole blood of the collected healthy chickens is collected and used at the moment.
Further, the nutrient agar culture medium comprises the following raw material components:
peptone 9.0 g/L-11.0 g/L;
2.5 g/L-3.5 g/L of beef extract powder;
4.0 g/L-5.5 g/L of sodium chloride;
agar 11.0 g/L-17.0 g/L.
Further, the preparation method of the nutrient agar culture medium comprises the steps of mixing the components according to the raw material component ratio and sterilizing at 121 ℃ for 15-20 min.
By adopting the scheme of the method, the nutrient agar culture medium prepared by the preparation method is used as a basic culture medium for preparing the chocolate agar culture medium, the nutrient agar culture medium prepared by the method and the components and proportion is added with fresh healthy chicken whole blood, and the chocolate agar culture medium is obtained by a specific preparation method and is used for separating and culturing the avibacterium paragallinarum. Through experimental analysis, the components are more beneficial to the separation and culture of the subsequently prepared chocolate agar culture medium to the avibacterium paragallinarum compared with the nutrient agar culture medium prepared within the range.
In order to achieve the second object, the invention provides the following method scheme: a preparation method of a culture medium for isolated culture of avibacterium paragallinarum in a laboratory comprises the following specific steps:
(1) obtaining whole blood of healthy chicken: artificially collecting sterile blood under wings of healthy chickens, wherein the whole blood of the healthy chickens needs to be collected at present;
(2) preparation of chocolate agar medium:
A1. sterilizing the prepared nutrient agar culture medium at high temperature, and keeping the temperature at 70-85 ℃;
A2. 100 parts of nutrient agar culture medium; adding the healthy chicken whole blood into the nutrient agar culture medium prepared in the step A1, heating at 70-85 ℃ for 5-10 min, and shaking up while heating to obtain a culture medium added with the healthy chicken whole blood;
A3. and (4) preparing a sterile plate by using the culture medium which is obtained in the step A2 and is added with the healthy chicken whole blood, namely a chocolate agar culture medium for later use.
By adopting the method scheme, the preparation method of the culture medium for separating and culturing the avibacterium paragallinarum is elaborated in detail; the preparation of fresh healthy chicken whole blood and chocolate agar culture medium respectively needs special attention to the adding time of the healthy chicken whole blood in the chocolate agar culture medium. The chocolate agar culture medium is prepared by the method, heat preservation is carried out at 70-85 ℃ after high-temperature sterilization, on one hand, the sterilized nutrient agar culture medium cannot be solidified in the temperature range, and the healthy chicken whole blood and the nutrient agar culture medium are favorably mixed uniformly, on the other hand, the healthy chicken whole blood is added into the culture medium and heated for 5-10 min, blood cells in the healthy chicken whole blood can be cracked, coenzyme I in the blood cells can enter the culture medium, and the coenzyme I can be directly and uniformly mixed on a prepared chocolate agar culture medium sterile flat plate, so that the utilization of coenzyme I by avibacterium paragallinarum is favorably realized, and the separation culture of avibacterium paragallinarum is favorably realized.
In order to achieve the third object, the present invention provides the following method: a novel method for the isolated culture of avibacterium paragallinarum in a laboratory is characterized in that the culture medium for the isolated culture of avibacterium paragallinarum in the laboratory is applied to the culture method of avibacterium paragallinarum.
Further, the novel method for the laboratory isolated culture of avibacterium paragallinarum comprises the following specific steps:
inoculating the avibacterium paragallinarum on a chocolate agar culture medium plate, culturing for 18-24h in a 5% CO2 carbon dioxide incubator at 37 ℃, and culturing to obtain the avibacterium paragallinarum strain.
By adopting the technical scheme, the invention also provides a culture method for separating and culturing the avibacterium paragallinarum by utilizing the chocolate agar culture medium for culturing the avibacterium paragallinarum, the avibacterium paragallinarum is inoculated to the chocolate agar culture medium for culturing, the separation of the avibacterium paragallinarum is ensured, and the avibacterium paragallinarum is inoculated to a culture medium which utilizes sheep blood as a coenzyme I donor.
To achieve the fourth object, the present invention provides the following method: the application of the culture medium for the isolated culture of the avibacterium paragallinarum in the laboratory is to apply the culture medium for the isolated culture of the avibacterium paragallinarum in the detection of the avian infectious rhinitis infection in poultry.
Further, the application of the culture medium for the isolated culture of the avibacterium paragallinarum in the laboratory specifically comprises the following steps:
C1. sampling: taking at least one of infraorbital sinus secretion and nasal sinus secretion of a chicken suspected to be sick as a collected sample by aseptic operation;
C2. coating and culturing: inoculating one loop of the sample collected in the step C1 on a chocolate agar culture medium plate, culturing for 18-24h in a 5% CO2 carbon dioxide incubator at 37 ℃ to obtain a strain A;
C3. and (3) detection: simultaneously cross-streaking the strain A obtained by the culture in the step C2 and the pre-cultured staphylococcus onto a blood agar culture medium plate, culturing for 18-24h in a 5% CO2 carbon dioxide incubator at 37 ℃, and detecting whether a satellite phenomenon exists around a staphylococcus colony on the blood agar culture medium plate; if the satellite phenomenon appears on the blood agar culture medium flat plate, the strain A cultured in the step C2 is avibacterium paragallinarum, and the infection phenomenon of avian infectious rhinitis appears in poultry; if the satellite phenomenon does not appear on the blood agar culture medium plate, the strain A cultured in the step C2 is not the avibacterium paragallinarum, and the poultry is proved not to have the infection phenomenon of the avian infectious rhinitis.
By adopting the method scheme, the characteristic that the avibacterium paragallinarum can grow on the chocolate agar culture medium is utilized, the sample collected from the poultry is inoculated on the chocolate agar culture medium for culture, and then the cultured strain and the staphylococcus are simultaneously and crossly inoculated on the blood agar culture medium; when the chocolate agar culture medium is prepared, the nutrient agar culture medium is subjected to heat preservation at 70-85 ℃ after high-temperature sterilization, and the healthy chicken whole blood is added and heated for 5-10 min, so that blood cells in the healthy chicken whole blood can be ruptured, and coenzyme I in the blood can enter the culture medium, so that nutrient substances are provided for the culture of the avibacterium paragallinarum, and the isolated culture of the avibacterium paragallinarum is ensured; the preparation method of the blood agar culture medium is different from the preparation method of the chocolate agar culture medium, only the healthy chicken whole blood is kept and added at 50 ℃, blood cells in the healthy chicken whole blood cannot be broken at the temperature, coenzyme I in the healthy chicken whole blood cannot enter the culture medium, so that less nutrient substances can be provided for the culture of the avibacterium paragallinarum in the culture of the avibacterium paragallinarum, and corresponding nutrient substances can be provided for the growth of the avibacterium paragallinarum in the growth process of staphylococcus, so that the avibacterium paragallinarum only grows near the staphylococcus on the blood agar culture medium, but does not grow at other streaked positions near the staphylococcus, and the phenomenon that the avibacterium paragallinarum grows near the staphylococcus is called as a satellite phenomenon. Under the cooperation of a chocolate agar culture medium and a blood agar culture medium, a collected sample is firstly inoculated on the chocolate agar culture medium, then a strain growing on the chocolate agar culture medium is inoculated on the blood agar culture medium, if the strain grows on the chocolate agar culture medium and a satellite phenomenon appears on the blood agar culture medium, the collected sample can be preliminarily judged to contain the avibacterium paragallinarum, further, the poultry is preliminarily judged to be infected with the avian infectious rhinitis, otherwise, the collected sample can be preliminarily judged to contain no the avibacterium paragallinarum, and further, the poultry is preliminarily judged to be not infected with the avian infectious rhinitis. The strain subjected to separation culture is subjected to corresponding biochemical detection, and the strain subjected to separation culture is further identified to be the avibacterium paragallinarum, so that the detection result of the detection method is reliable, and the detection method is simple and convenient to operate, short in period and sensitive in detection degree, so that the culture medium for the separation culture in the avibacterium paragallinarum laboratory has better application in detection of avian infectious rhinitis infection in poultry.
In summary, in one aspect, the invention provides a culture medium for isolated culture of avibacterium paragallinarum in a laboratory and a preparation method thereof, healthy chicken whole blood is used for replacing sheep blood to prepare the culture medium for isolated culture of avibacterium paragallinarum, and the culture medium is prepared by a specific preparation method, and as the healthy chicken whole blood is low in cost and easy to obtain, is not limited by purchasing time and can be prepared on site, in addition, the collection process of the healthy chicken whole blood is simple and convenient, great convenience is brought to the preparation of the culture medium for isolated culture of avibacterium paragallinarum, the isolated culture efficiency of the avibacterium paragallinarum is improved, and the cost input of poultry breeding industry is reduced; on the other hand, the invention also provides a novel method for isolated culture of the avibacterium paragallinarum in the culture medium laboratory, and the avibacterium paragallinarum is inoculated on the provided chocolate agar culture medium in the method, so that the isolated culture of the avibacterium paragallinarum is ensured; in addition, the invention also provides a method for detecting the avian infectious rhinitis infection in poultry by applying the culture medium for laboratory isolated culture of the avibacterium paragallinarum to detection of the avian infectious rhinitis infection in poultry, wherein the detection method has the advantages of reliable detection result, simple and convenient operation, short period and sensitive detection degree, and has good application prospect.
Drawings
FIG. 1 is a PCR electrophoresis of avian bacterium paragallinarum in the present invention.
In the figure, lane I: separating the product from the bacteria; lane N: negative control; lane P: a positive control; lane M: DNASMarker.
Detailed Description
Instruments and medicines used in the invention are all from common market, so detailed description is not provided herein; the glass instrument and the culture medium used in the invention are all subjected to aseptic treatment before use; the used healthy chicken is cultivated in a health care center laboratory of Beijing poultry breeding Limited company; the staphylococcus used in the invention is staphylococcus epidermidis, is preserved in China center for culture Collection of microorganisms, and has a strain preservation number of CICC 24067.
The invention provides a culture medium for isolated culture of avibacterium paragallinarum in a laboratory, which is a chocolate agar culture medium and is prepared from the following raw materials, by volume, 100 parts of a nutrient agar culture medium and 10 parts of healthy chicken whole blood, wherein the nutrient agar culture medium comprises the following raw material components of peptone 9.0 g/L-11.0 g/L, beef extract powder 2.5 g/L-3.5 g/L, sodium chloride 4.0 g/L-5.5 g/L, agar 11.0 g/L-17.0 g/L, and the final pH is 7.2 +/-0.2, and the components are uniformly mixed according to the distribution ratio of the raw materials and are sterilized at the high temperature of 121 ℃ for 15-20 min.
The invention also provides a preparation method of the culture medium for the isolated culture of the avibacterium paragallinarum in the laboratory, which comprises the following specific steps:
(1) obtaining whole blood of healthy chicken: artificially collecting sterile blood under wings of healthy chickens, wherein the whole blood of the healthy chickens needs to be collected at present;
(2) preparation of chocolate agar medium:
A1. sterilizing the prepared nutrient agar culture medium at high temperature, and keeping the temperature at 70-85 ℃;
A2. 100 parts of nutrient agar culture medium; adding the healthy chicken whole blood into the nutrient agar culture medium prepared in the step A1, heating at 70-85 ℃ for 5-10 min, and shaking up while heating to obtain a culture medium added with the healthy chicken whole blood;
A3. and (4) preparing a sterile plate by using the culture medium which is obtained in the step A2 and is added with the healthy chicken whole blood, namely a chocolate agar culture medium for later use.
The invention also provides a novel method for the isolated culture of the avibacterium paragallinarum in the laboratory, the culture medium for the isolated culture of the avibacterium paragallinarum in the laboratory can be applied to the culture method of the avibacterium paragallinarum, and the novel method for the isolated culture of the avibacterium paragallinarum in the laboratory comprises the following specific steps: inoculating the avibacterium paragallinarum on a chocolate agar culture medium plate, culturing for 18-24h in a 5% CO2 carbon dioxide incubator at 37 ℃, and culturing to obtain the avibacterium paragallinarum strain.
The invention further provides an application of the culture medium for the isolated culture of the avibacterium paragallinarum in the laboratory, which is applied to the detection of the avian infectious rhinitis infection in poultry by utilizing the culture medium for the isolated culture of the avibacterium paragallinarum in the laboratory and specifically comprises the following steps:
C1. sampling: taking at least one of infraorbital sinus secretion and nasal sinus secretion of a chicken suspected to be sick as a collected sample by aseptic operation;
C2. coating and culturing: inoculating one loop of the sample collected in the step C1 on a chocolate agar culture medium plate, culturing for 18-24h in a 5% CO2 carbon dioxide incubator at 37 ℃ to obtain a strain A;
C3. and (3) detection: simultaneously cross-streaking the strain A obtained by the culture in the step C2 and the pre-cultured staphylococcus onto a blood agar culture medium plate, culturing for 18-24h in a 5% CO2 carbon dioxide incubator at 37 ℃, and detecting whether a satellite phenomenon exists around a staphylococcus colony on the blood agar culture medium plate; if the satellite phenomenon appears on the blood agar culture medium flat plate, the strain A cultured in the step C2 is avibacterium paragallinarum, and the infection phenomenon of avian infectious rhinitis appears in poultry; if the satellite phenomenon does not appear on the blood agar culture medium plate, the strain A cultured in the step C2 is not the avibacterium paragallinarum, and the poultry is proved not to have the infection phenomenon of the avian infectious rhinitis.
The present invention will be described in further detail below with reference to the accompanying drawings, preparation examples, application examples, and detection of Acinetobacter paragallinarum.
Preparation example
Preparation example 1
The preparation method of the nutrient agar culture medium comprises the steps of weighing 1.0g of peptone, 0.3g of beef extract powder and 0.5g of sodium chloride respectively, mixing the peptone, the beef extract powder and the sodium chloride in a beaker, dissolving the peptone with a proper amount of deionized water, weighing 1.4g of agar, adding the agar into the solution, heating the agar, stirring the agar while heating until the agar is dissolved, continuously adding the deionized water into the solution until the total volume of the solution is 100m L, adjusting the final pH value of the solution to 7.2 +/-0.2, and sterilizing the solution at 121 ℃ for 15min to obtain the nutrient agar culture medium.
Preparation example 2
The preparation method of the nutrient agar culture medium comprises the steps of weighing 0.9g of peptone, 0.25g of beef extract powder and 0.4g of sodium chloride respectively, mixing the peptone, the beef extract powder and the sodium chloride in a beaker, dissolving the peptone with a proper amount of deionized water, weighing 1.1g of agar, adding the agar into the solution, heating the agar, stirring the agar until the agar is dissolved, continuously adding the deionized water into the solution until the total volume of the solution is 100m L, adjusting the final pH value of the solution to be 7.2 +/-0.2, and sterilizing the solution at 121 ℃ for 15min to obtain the nutrient agar culture medium.
Preparation example 3
The preparation method of the nutrient agar culture medium comprises the steps of weighing 1.10g of peptone, 0.35g of beef extract powder and 0.55g of sodium chloride respectively, mixing the peptone, the beef extract powder and the sodium chloride in a beaker, dissolving the peptone with a proper amount of deionized water, weighing 1.7g of agar, adding the agar into the solution, heating the agar, stirring the agar until the agar is dissolved, continuously adding the deionized water into the solution until the total volume of the solution is 100m L, adjusting the final pH value of the solution to be 7.2 +/-0.2, and sterilizing the solution at 121 ℃ for 20min to obtain the nutrient agar culture medium.
Preparation example 4
In this preparation example, the chocolate agar medium was prepared using the nutrient agar medium prepared in preparation example 1, and since healthy chicken whole blood was added to the medium, it was necessary to obtain healthy chicken whole blood first. The formula of the chocolate agar culture medium is as follows: 100ml of nutrient agar culture medium; healthy chicken whole blood 10 ml.
The preparation method of the chocolate agar culture medium comprises the following steps:
(1) obtaining whole blood of healthy chicken: the method comprises the following steps of (1) artificially collecting sterile blood under wings of healthy chickens, wherein the whole blood of the healthy chickens needs to be collected at present, the blood collection amount is based on actual needs, and in order to ensure that the volume of the whole blood of the healthy chickens for preparing a chocolate agar culture medium is enough and the blood consumption in the actual operation process is considered, the blood collection amount of the whole blood of the healthy chickens in the embodiment is 12 ml;
(2) preparation of chocolate agar medium:
A1. preparing 100m L nutrient agar culture medium according to the method of preparation example 1, sterilizing at high temperature, cooling to 70-85 deg.C, maintaining the temperature in water bath, cooling to 80 deg.C, and maintaining the temperature in water bath;
A2. adding 10ml of the collected healthy chicken whole blood into 100ml of the nutrient agar culture medium prepared in the step A1, and continuously heating in a water bath kettle at 80 ℃ for 5-10 min, wherein the continuous heating time in the preparation example is 8min, and the culture medium added with the healthy chicken whole blood is obtained by uniformly shaking while heating;
A3. and (4) pouring the culture medium added with the healthy chicken whole blood obtained in the step A2 into an aseptic plate to prepare an aseptic plate, namely a chocolate agar culture medium, and after the aseptic examination, storing the aseptic plate in a refrigerator at 4 ℃ for later use.
Preparation example 5
In this preparation example, the blood agar medium was prepared using the nutrient agar medium prepared in preparation example 1, and since healthy chicken whole blood was added to the medium, it was necessary to obtain healthy chicken whole blood first. The formula of the blood agar culture medium is as follows: 100ml of nutrient agar culture medium; healthy chicken whole blood 10 ml.
The preparation method of the blood agar culture medium comprises the following steps:
(1) obtaining whole blood of healthy chicken: the method comprises the following steps of (1) artificially collecting sterile blood under wings of healthy chickens, wherein the whole blood of the healthy chickens is collected at present, the blood collection amount is based on actual needs, and in order to ensure that the volume of the whole blood of the healthy chickens for preparing a blood agar culture medium is enough and the loss of the blood amount in the actual operation process is considered, the blood collection amount of the whole blood of the healthy chickens in the embodiment is 12 ml;
(2) preparation of blood agar medium:
B1. preparing 100m L nutrient agar culture medium according to the method of preparation example 1, sterilizing at high temperature, cooling to 50 deg.C, and maintaining the temperature in water bath;
B2. adding 10ml of the collected healthy chicken whole blood into the 100ml of nutrient agar culture medium prepared in the step B1, and shaking up to obtain a culture medium added with the healthy chicken whole blood;
B3. and (4) pouring the culture medium added with the healthy chicken whole blood obtained in the step B2 into an aseptic plate to prepare an aseptic plate, namely a blood agar culture medium, and after aseptic examination, storing the aseptic plate in a refrigerator at 4 ℃ for later use.
Examples
Example 1
The embodiment mainly provides a new method for isolated culture of avibacterium paragallinarum in a laboratory, and the culture medium for isolated culture of avibacterium paragallinarum in the laboratory is applied to the culture method of avibacterium paragallinarum. The chocolate agar culture medium prepared in preparation example 4 is used in the present example, and the specific steps of the new method for laboratory isolated culture of avibacterium paragallinarum are as follows: the avian pullobacter paragallinarum strain was streaked and inoculated on a chocolate agar medium plate prepared in preparation example 4 at 37 ℃ with 5% CO2Culturing in carbon dioxide incubator for 18-24h, inoculating Acinetobacter paragallinarum strain chocolate agar medium plate at 37 deg.C and 5% CO2Culturing in a carbon dioxide incubator for 18h to obtain the avibacterium paragallinarum strain.
Example 2
The embodiment mainly provides a detection method for isolated culture of avibacterium paragallinarum in a laboratory, and a culture medium for isolated culture of avibacterium paragallinarum in the laboratory is applied to the detection method for avibacterium paragallinarum. This example used the blood agar medium prepared in preparation example 5, avian pullus paragallinarum laboratoryThe detection method of the separation culture comprises the following specific steps: the same blood agar medium plate prepared in preparation example 5 was cross-streaked with avian paragallinarum strain and staphylococcus strain simultaneously at 37 ℃ with 5% CO2Culturing in carbon dioxide incubator for 18-24 hr, inoculating avian Paragallinarum strain in blood agar culture medium plate at 37 deg.C and 5% CO2After being cultured in a carbon dioxide incubator for 20 hours, the avibacterium paragallinarum grows around staphylococcus colonies and shows a satellite phenomenon.
Example 3
The embodiment provides a new method for isolated culture of avibacterium paragallinarum in a laboratory and a detection method thereof, and the method comprises the following specific steps:
(1) culturing avibacterium paragallinarum: the avian pullobacter paragallinarum strain was streaked and inoculated on a chocolate agar medium plate prepared in preparation example 4 at 37 ℃ with 5% CO2Culturing in carbon dioxide incubator for 18-24h, inoculating Acinetobacter paragallinarum strain chocolate agar medium plate at 37 deg.C and 5% CO2Culturing for 24h in a carbon dioxide incubator to obtain the avibacterium paragallinarum strain.
(2) Detection of avibacterium paragallinarum: streaking the avibacterium paragallinarum obtained by culturing in the step (1) on a blood agar medium plate prepared in preparation example 5, and simultaneously streaking staphylococcus on the blood agar medium plate inoculated with the avibacterium paragallinarum, wherein the streaked path of the staphylococcus is crossed with the streaked path of the avibacterium paragallinarum; placing the streaked blood agar medium plate at 37 deg.C and 5% CO2Culturing in carbon dioxide incubator for 18-24 hr, inoculating avian Paragallinarum strain in blood agar culture medium plate at 37 deg.C and 5% CO2After 24 hours of culture in the carbon dioxide incubator, the avibacterium paragallinarum grows around staphylococcus colonies and shows a satellite phenomenon.
Application example
Application example 1
In the present application example, the chocolate agar medium provided in preparation example 4 and the blood agar medium provided in preparation example 5 were applied to detection of infectious rhinitis infection of poultry in poultry, and suspected sick chickens at the healthcare center of Beijing poultry Breeding Co., Ltd were used as detection targets.
The method for detecting the infectious rhinitis infection of the poultry specifically comprises the following steps:
C1. sampling: taking at least one of infraorbital sinus secretion and nasal sinus secretion of a suspected sick chicken as an acquired sample through aseptic operation, and selecting a mixture of the infraorbital sinus secretion and the nasal sinus secretion as the suspected sick chicken secretes the infraorbital sinus secretion and the nasal sinus secretion at the same time for the sampling as the acquired sample;
C2. coating and culturing: inoculating one loop of the sample collected in the step C1 on a chocolate agar culture medium plate prepared in the preparation example 4, culturing for 24h in a 5% CO2 carbon dioxide incubator at 37 ℃ to obtain a strain A;
C3. and (3) detection: simultaneously cross-streaking the strain A and the staphylococcus obtained by the culture in the step C2 on a blood agar culture medium plate, placing the streaked blood agar culture medium plate in a 5% CO2 carbon dioxide incubator at 37 ℃ for 24 hours, and observing whether a satellite phenomenon exists around the staphylococcus colony on the blood agar culture medium plate;
C4. and (4) analyzing results: bacterial colony A grows around staphylococcus on the blood agar culture medium plate in the step C3, a satellite phenomenon appears on the blood agar culture medium plate, the strain A cultured in the step C2 is preliminarily presumed to be avibacterium paragallinarum, a sample collected in the step C1 contains the avibacterium paragallinarum, and the infection phenomenon of avian infectious rhinitis in poultry is preliminarily proved.
Detection test
The colony A obtained by isolation and culture in application example 1 was subjected to the following detection.
1. Microscopic morphology identification
Colony A isolated and cultured in application example 1 was subjected to a conventional gram stain, and the bacterial morphology was observed under an oil mirror (100-fold). The observation shows that the thallus is polymorphic, shows gram-negative coccobacillus during primary separation, is dyed by two poles, does not form spores, is not capsulated, has no flagellum and can not move; after 24h of culture, the thalli are rod-shaped or ball-rod-shaped and tend to be filamentation; after 48h of culture, the cells were degenerated, fragmented and represented irregular morphology. The microscopic morphology of the strain is the same as that of the paragallibacterium gallinarum, and the colony A isolated and cultured in application example 1 can be further judged to be the paragallibacterium gallinarum.
2. Identification by biochemical test
The colony A isolated and cultured in the application example 1 is taken for biochemical identification, the items of the biochemical identification comprise the production conditions of catalase, ONPG (β -galactoside), L-arabinose, D-galactose, maltose, D-mannitol, D-sorbitol, trehalose and a-glucosidase and the growth conditions in the air, and the detection of each item is carried out according to the detection standard of national standard (NY/T538-2015). through the detection, the strain can reduce nitrate into nitrite, ferment glucose but not produce gas, have oxidase activity, alkaline phosphatase, produce no indole, hydrolyze no urea and liquefy no gelatin, and other biochemical identification results are shown in the following table:
TABLE 1 Biochemical identification test for haemophilus paragallinarum
According to the detection result, the biochemical test result of the strain is the same as the biochemical characteristic of the avibacterium paragallinarum, and the colony A isolated and cultured in the application example 1 can be further judged to be the avibacterium paragallinarum.
3. Serotype identification
A plate was prepared from the monovalent positive serum of avian paragallinarum, and colony A isolated and cultured in application example 1 was inoculated onto a plate containing the monovalent positive serum of avian paragallinarum to perform a plate agglutination test. The univalent positive serum of the avibacterium paragallinarum contains an antibody of the avibacterium paragallinarum, the cultured strain is used as an antigen, the test result is positive, the strain inoculated on the univalent positive serum plate containing the avibacterium paragallinarum is the antigen of the univalent positive serum of the avibacterium paragallinarum, and the colony A which is isolated and cultured in application example 1 can be continuously further judged to be the avibacterium paragallinarum.
PCR identification
(1) PCR reagent
6 × DNA L loading Buffer, 2 × Master Mix, TAE, agarose, DNA Marker D L5000, GoldView, negative control, positive control.
(2) Primer and method for producing the same
TABLE 2 Gene amplification primers
(3) DNA extraction
Adding the pure culture of the bacteria obtained by culture into 100ul of sterile ultrapure water by the inoculation amount of one loop, uniformly mixing, carrying out boiling water bath for 10min, carrying out ice bath for 5min, and centrifuging at 12000r/min for 1 min, wherein the supernatant is used as a template for gene amplification.
(4) PCR reaction system
TABLE 3 identification of PCR reaction System for avibacterium paragallinarum
| Components | Volume (ul) |
| ddH2O | 16 |
| 2×MasterMix | 25 |
| Upstream primer (10 um/L) | 2 |
| Downstream primer (10 um/L) | 2 |
| DNA template | 5 |
And simultaneously, setting yin-yang contrast. The PCR reaction conditions are as follows:
1)94℃，3min；
2)94℃，30s，55℃，30s，72℃，30s，30circles：
3)72℃，5min。
(5) detection of PCR products
The PCR product was subjected to 1% agarose gel electrophoresis by adding 6 × DNA L loading Buffer 0.25ul, and the band size of the amplified product was observed.
(6) Determination of results
As shown in FIG. 1, the positive control shows the amplified fragment, the negative control shows the unamplified fragment, and the gel imaging system or the UV-light analysis of the PCR amplified product can determine that the fragment product of about 412bp amplified in lane 1 is positive, so the test is established.
As can be seen from the above PCR results, the colony A isolated and cultured in application example 1 was identified and judged as the pathogen of avian paragallibacterium by PCR test.
According to the detection result, the strain A obtained by separation and culture in the application example 1 is avibacterium paragallinarum, so that the sample collected in the step C1 in the application example 1 can be judged to contain avibacterium paragallinarum, and the infection phenomenon of avian infectious rhinitis in detected poultry is proved. Aiming at the results, a corresponding dosing scheme is formulated for the detection object, and the avibacterium paragallinarum carried in the poultry is regularly detected and monitored so as to relieve the infection phenomenon of the avian infectious rhinitis in the poultry.
Application example 2
In the present application example, the chocolate agar medium provided in preparation example 4 and the blood agar medium provided in preparation example 5 were applied to detection of infectious rhinitis infection of poultry in poultry, and suspected sick chickens at the healthcare center of Beijing poultry Breeding Co., Ltd were used as detection targets.
The method for detecting the infectious rhinitis infection of the poultry specifically comprises the following steps:
C1. sampling: taking at least one of infraorbital sinus secretion and nasal sinus secretion of a suspected sick chicken as an acquired sample through aseptic operation, and selecting the infraorbital sinus secretion of the suspected sick chicken as the acquired sample in the sampling;
C2. coating and culturing: inoculating one loop of the sample collected in the step C1 on a chocolate agar culture medium plate prepared in the preparation example 4, culturing for 24h in a 5% CO2 carbon dioxide incubator at 37 ℃ to obtain a strain A;
C3. and (3) detection: simultaneously and crossly streaking the strain A obtained by the culture in the step C2 and the pre-cultured staphylococcus onto a blood agar culture medium plate, placing the streaked blood agar culture medium plate in a 5% CO2 carbon dioxide incubator at 37 ℃ for 24 hours, and observing whether a satellite phenomenon exists around a staphylococcus colony on the blood agar culture medium plate;
C4. and (4) analyzing results: on the blood agar culture medium plate in the step C3, bacterial colony A and staphylococcus both grow on the blood agar culture medium plate, but the growth range of the bacterial colony A is not limited to the periphery of the staphylococcus, and the phenomenon of satellite does not appear on the blood agar culture medium plate, so the bacterial strain A cultured in the step C2 is not the avibacterium paragallinarum, the sample collected in the step C1 does not contain the avibacterium paragallinarum, and the infection phenomenon of avian infectious rhinitis does not appear in poultry is preliminarily judged.
In conclusion, by utilizing the results of microscopic morphological identification, biochemical test identification, serotype identification and PCR identification, the culture medium for laboratory isolated culture of avibacterium paragallinarum provided by the invention can be used for isolated culture of avibacterium paragallinarum, the preparation method of the culture medium and the novel method for laboratory isolated culture of avibacterium paragallinarum can be applied to isolated culture of avibacterium paragallinarum, and the blood agar culture medium and the chocolate agar culture medium provided by the invention can be applied to detection of avian infectious rhinitis infection in poultry.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and the person skilled in the art can make modifications without inventive contribution to the present embodiment as needed after reading the present specification, but is protected by patent law within the scope of the claims of the present invention.
Claims (10)
1. The culture medium for the laboratory isolated culture of the avibacterium paragallinarum is characterized by being a chocolate agar culture medium which is prepared from the following raw materials in parts by volume: 100 parts of nutrient agar culture medium; 10 parts of healthy chicken whole blood;
the preparation method of the chocolate agar culture medium comprises the following steps:
A1. sterilizing the prepared nutrient agar culture medium at high temperature, and keeping the temperature at 70-85 ℃;
A2. adding the healthy chicken whole blood into the nutrient agar culture medium prepared in the step A1 according to the volume parts, heating at 70-85 ℃ for 5-10 min, and shaking up while heating to obtain a culture medium added with the healthy chicken whole blood;
A3. and (4) preparing a sterile plate by using the culture medium which is obtained in the step A2 and is added with the healthy chicken whole blood, namely a chocolate agar culture medium for later use.
2. The culture medium for the isolated culture of avibacterium paragallinarum in the laboratory according to claim 1, wherein the culture medium comprises: the whole blood of the healthy chicken is obtained by artificially collecting sterile blood under wings of the healthy chicken.
3. The culture medium for the isolated culture of avibacterium paragallinarum in the laboratory according to claim 1, wherein the culture medium comprises: the use mode of the healthy chicken whole blood is adopted at present.
4. The culture medium for the isolated culture of the avibacterium paragallinarum in the laboratory according to claim 1, wherein the nutrient agar culture medium comprises the following raw material components:
peptone 9.0 g/L-11.0 g/L;
2.5 g/L-3.5 g/L of beef extract powder;
4.0 g/L-5.5 g/L of sodium chloride;
agar 11.0 g/L-17.0 g/L.
5. The culture medium for the isolated culture of avibacterium paragallinarum in the laboratory according to claim 4, wherein the culture medium comprises: the preparation method of the nutrient agar culture medium comprises mixing the above raw materials at a ratio, and sterilizing at 121 deg.C for 15-20 min.
6. The preparation method of the culture medium for the isolated culture of the avibacterium paragallinarum in the laboratory according to any one of claims 1 to 5, which is characterized by comprising the following specific steps:
(1) obtaining whole blood of healthy chicken: artificially collecting sterile blood under wings of healthy chickens, wherein the whole blood of the healthy chickens needs to be collected at present;
(2) preparation of chocolate agar medium:
A1. sterilizing the prepared nutrient agar culture medium at high temperature, and keeping the temperature at 70-85 ℃;
A2. 100 parts of nutrient agar culture medium; adding the healthy chicken whole blood into the nutrient agar culture medium prepared in the step A1, heating at 70-85 ℃ for 5-10 min, and shaking up while heating to obtain a culture medium added with the healthy chicken whole blood;
A3. and (4) preparing a sterile plate by using the culture medium which is obtained in the step A2 and is added with the healthy chicken whole blood, namely a chocolate agar culture medium for later use.
7. A new method for laboratory isolated culture of avibacterium paragallinarum, characterized in that the culture medium for laboratory isolated culture of avibacterium paragallinarum according to any one of claims 1 to 5 is applied to the culture method of avibacterium paragallinarum.
8. The new method for the isolated culture of avibacterium paragallinarum in the laboratory according to claim 7, wherein the new method for the isolated culture of avibacterium paragallinarum in the laboratory comprises the following specific steps:
inoculating avian paragallibacterium to chocolate agar culture medium plate, and culturing at 37 deg.C and 5% CO2Culturing in a carbon dioxide incubator for 18-24h to obtain the avibacterium paragallinarum strain.
9. The application of the culture medium for the isolated culture of the avibacterium paragallinarum in the laboratory is characterized in that the culture medium for the isolated culture of the avibacterium paragallinarum in the laboratory is applied to the detection of the infectious rhinitis infection of poultry.
10. The application of the culture medium for the isolated culture of the avibacterium paragallinarum in the laboratory according to claim 9 is characterized by comprising the following steps:
C1. sampling: taking at least one of infraorbital sinus secretion and nasal sinus secretion of a chicken suspected to be sick as a collected sample by aseptic operation;
C2. coating and culturing: inoculating one loop of the sample collected in step C1 on chocolate agar medium plate, and culturing at 37 deg.C with 5% CO2Culturing in a carbon dioxide incubator for 18-24h to obtain a strain A;
C3. and (3) detection: crossing the strain A cultured in step C2 and the pre-cultured staphylococcus simultaneously onto blood agar culture medium plate, and culturing at 37 deg.C and 5% CO2Culturing in a carbon dioxide incubator for 18-24h, and detecting whether a satellite phenomenon exists around a staphylococcus colony on a blood agar culture medium plate; if the satellite phenomenon appears on the blood agar culture medium flat plate, the strain A cultured in the step C2 is avibacterium paragallinarum, and the infection phenomenon of avian infectious rhinitis appears in poultry; if the satellite phenomenon does not appear on the blood agar culture medium plate, the strain A cultured in the step C2 is not the avibacterium paragallinarum, and the poultry is proved not to have the infection phenomenon of the avian infectious rhinitis.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5240705A (en) * | 1990-09-05 | 1993-08-31 | Akzo Nv | Haemophilus paragallinarum vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5240705A (en) * | 1990-09-05 | 1993-08-31 | Akzo Nv | Haemophilus paragallinarum vaccine |
Non-Patent Citations (3)
| Title |
|---|
| D.V.UMALI等: "Characterization of Ornithobacterium rhinotracheale from commercial layer chickens in eastern Japan", 《POULTRY SCIENCE》 * |
| 程东庆: "《病原生物学检验试验指导》", 30 June 2014, 杭州：浙江工商大学出版社 * |
| 项大实: "《实用兽医手册》", 30 June 1990, 北京：北京出版社 * |
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