Salmonella identification culture medium and preparation method thereof

Abstract

The invention provides a salmonella identification culture medium and a preparation method thereof. The salmonella identification culture medium comprises the following components: a basic substrate, a sugar agent, a chromogenic substrate and a selective inhibitor. The chromogenic substrate comprises sodium thiosulfate and ammonium ferric citrate. The selective inhibitor is prepared from sodium sulfite, lauryl sodium sulfate, 7-ethyl-2-methyl-4-undecyl sodium sulfate and vancomycin. According to the salmonella identification culture medium, the identification efficiency of salmonella can be effectively improved, and the identification accuracy of salmonella is improved.

Description

Technical Field

[0001] The invention relates to the technical field of microbial identification, in particular to a Salmonella identification culture medium and a preparation method thereof. Background Art

[0002] Salmonella is a group of Gram-negative rods that parasitize the intestines of humans and animals and have similar biochemical reactions and antigenic structures. There are more than 2,000 serotypes of Salmonella bacteria, which are mainly divided into typhoid and non-typhoid Salmonella infections. Typhoid is a manifestation of bloodstream infection, while non-typhoid Salmonella usually manifests as intestinal infection, usually causing diarrhea, fever and abdominal pain in patients; a few cause extraintestinal infections, which can cause bacteremia, urinary tract infections and otitis media. Therefore, the "Preventive Health Examination Management Measures" promulgated by the state stipulates that people engaged in food and drinking water production and operation, cosmetics production personnel, and public places directly serving customers must be checked for typhoid caused by Salmonella. There are generally two ways to identify and inspect Salmonella. One way is to use molecular biological methods for identification, such as PCR technology to detect specific gene fragments of bacteria, such as invention patent application with application number CN201410275816.5 and invention patent application with application number CN201410277037.9, both of which use PCR technology to detect and identify Salmonella. The other way is to collect stool samples from patients for selective culture and identification. Specifically, the stool samples collected from patients are transported to the laboratory through a transport medium, then cultured through a bacterial enrichment solution, and then transferred to an intestinal selective separation medium for separation and culture;

[0003] The commonly used transport medium is Cary-Blair medium, which mainly contains sodium, calcium and buffer components to ensure the vitality of microorganisms during transportation and has no bacteria-enhancing function;

[0004] Commonly used enrichment solutions are SC enrichment solution and GN enrichment solution, both of which are composed of nutrients and selective inhibitors, which can inhibit some bacteria and ensure the growth of target bacteria;

[0005] Commonly used intestinal selective separation culture media are mainly SS plate culture media or XLD plate culture media, both of which are composed of nutrients, selective inhibitors and hydrogen sulfide binding substrates. The colony center of the target bacteria will turn black, so as to identify the presence of the target bacteria. For example, the invention patent with application number CN201010550372.3 discloses a kit for simultaneous separation and identification of Salmonella and Shigella, which discloses the combination of non-selective enrichment liquid and selective enrichment liquid for selective culture, and further identification is performed by colorimetric agar plates, biochemical identification tubes, hydrogen sulfide filter paper and indole filter paper. The identification operation is cumbersome and lengthy, and when the selective culture is performed, non-target bacteria exist in the intestinal bacteria in the sample, such as Gram-negative bacteria such as Proteus mirabilis and Citrobacter freundii, which can also decompose sodium thiosulfate to produce hydrogen sulfide, thereby causing the colony center of the target bacteria to turn black, and the false positive rate is high. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a Salmonella identification culture medium and a preparation method thereof which can effectively improve the identification efficiency of Salmonella and improve the identification accuracy of Salmonella.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] A Salmonella identification culture medium, the Salmonella culture medium comprising the following components: a basic substrate, a sugar agent, a color developing substrate and a selection inhibitor;

[0009] The chromogenic substrate includes sodium thiosulfate and ammonium ferric citrate;

[0010] The selective inhibitors include sodium sulfite, sodium dodecyl sulfate, sodium 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin.

[0011] In one embodiment, each liter of the Salmonella culture medium comprises the following components: 8.5 g to 23.1 g of basic substrate, 15 g to 28 g of sugar, 6.2 g to 12.2 g of color substrate, 6 g to 16.6 g of selective inhibitor and the balance of water.

[0012] In one embodiment, the basic substrate includes yeast extract, tryptone, sodium chloride and agar.

[0013] In one embodiment, the sugar agent includes sucrose, lactose and xylose.

[0014] In one embodiment, the mass ratio of sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin in the selective inhibitor is (4-9): (1-5): (1-2.5): (0.004-0.009).

[0015] In one embodiment, the mass ratio of sodium thiosulfate to ammonium ferric citrate in the chromogenic substrate is (6-11): (0.2-1.2).

[0016] A method for preparing a Salmonella identification medium, used for preparing the Salmonella identification medium described in any one of the above embodiments, the method for preparing the Salmonella identification medium comprising the following steps:

[0017] Obtaining a basic substrate, a sugar, a chromogenic substrate and a selection inhibitor, wherein the selection inhibitor comprises sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin;

[0018] Adding water to the basic substrate to perform a dissolution operation to obtain a basic solution;

[0019] Adding water to the sugar, the chromogenic substrate and the selective inhibitor for secondary dissolution, and adding water to make up the balance to obtain a mixed solution;

[0020] The base liquid is added into the mixed liquid for uniform mixing to obtain a Salmonella identification medium.

[0021] In one embodiment, before the step of adding the base liquid to the mixed liquid for mixing, and after the step of adding water to the base substrate for dissolving, the method for preparing the Salmonella identification medium further comprises the following steps:

[0022] The base solution was sterilized by high pressure steam.

[0023] In one embodiment, before the step of adding the base liquid to the mixed liquid for mixing, and after the step of adding water for balance supplementation, the method for preparing the Salmonella identification medium further comprises the following steps:

[0024] The mixed solution is subjected to microfiltration.

[0025] In one embodiment, water is added to the sugar, the chromogenic substrate and the selective inhibitor for secondary dissolution, which specifically comprises the following steps:

[0026] Adding water to the sugar preparation to perform a dissolution-promoting operation to obtain a sugar solution;

[0027] Adding water to the chromogenic substrate to perform a secondary dissolution-promoting operation to obtain a chromogenic substrate solution;

[0028] Adding water to the selective inhibitor to perform dissolution-promoting operations three times to obtain a selective inhibitor solution;

[0029] The sugar solution, the color development substrate solution and the selection inhibition solution are mixed.

[0030] Compared with the prior art, the present invention has at least the following advantages:

[0031] The Salmonella identification culture medium of the present invention enables the basic substrate and the sugar agent to support the proliferation and growth of Salmonella, and the proliferation and growth of Salmonella can be achieved without inoculation in the transport culture medium and the growth promotion culture medium. When the sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin in the selective inhibitor are used in coordination, the inhibitory effect on Salmonella is weak, but it has a strong inhibitory effect on Proteus mirabilis and Citrobacter freundii, which are also Gram-negative rods, and the selective culture of Salmonella can be achieved without the need for concentrated inoculation of the growth promotion culture medium and the selective culture, that is, sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin. The synergistic use of sodium 2-methyl-4-undecyl sulfate and vancomycin has a good inhibitory effect on both Gram-positive and Gram-negative bacteria other than Salmonella, while the combination of sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin also has a certain inhibitory effect on Salmonella, but the inhibitory effect is weaker. The use of basic substrates and sugar agents can effectively achieve the proliferation and growth of Salmonella, and then the center of the colony of Salmonella will turn black under the action of the chromogenic substrate to achieve colorimetric identification of Salmonella, effectively improving the identification efficiency of Salmonella and improving the identification accuracy of Salmonella. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0033] Figure 1 The present invention is a flowchart of a method for preparing a Salmonella identification medium according to one embodiment of the present invention. DETAILED DESCRIPTION

[0034] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0035] It should be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may be a central element. When an element is considered to be "connected to" another element, it may be directly connected to the other element or there may be a central element at the same time. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only and do not represent the only implementation method.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0037] The present application provides a Salmonella identification medium. The above-mentioned Salmonella identification medium includes the following components: a basic substrate, a sugar, a chromogenic substrate and a selection inhibitor. The chromogenic substrate includes sodium thiosulfate and ammonium ferric citrate. The selection inhibitor includes sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin.

[0038] The above-mentioned Salmonella identification medium allows the basic substrate and sugar agent to be used to support the proliferation and growth of Salmonella, and the proliferation and growth of Salmonella can be achieved without inoculation in the transport medium and the growth-promoting medium. When used in conjunction with the selective inhibitors such as sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin, the inhibitory effect on Salmonella is weak, but it has a strong inhibitory effect on Proteus mirabilis and Citrobacter freundii, which are also Gram-negative rods. The selective culture of Salmonella can be achieved without the need for concentrated inoculation of the growth-promoting medium and the selective culture, that is, sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin. The synergistic use of sodium 2-methyl-4-undecyl sulfate and vancomycin has a good inhibitory effect on both Gram-positive and Gram-negative bacteria other than Salmonella, while the combination of sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin also has a certain inhibitory effect on Salmonella, but the inhibitory effect is weaker. The use of basic substrates and sugar agents can effectively achieve the proliferation and growth of Salmonella, and then the center of the Salmonella colony will turn black under the action of the chromogenic substrate to achieve colorimetric identification of Salmonella, effectively improving the identification efficiency of Salmonella and improving the identification accuracy of Salmonella.

[0039] In order to better understand the Salmonella identification medium of the present application, the Salmonella identification medium of the present application is further explained below:

[0040] The Salmonella identification medium of one embodiment comprises the following components: a basic substrate, a sugar, a chromogenic substrate and a selection inhibitor. The chromogenic substrate comprises sodium thiosulfate and ammonium ferric citrate. The selection inhibitor comprises sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin.

[0041] The above-mentioned Salmonella identification medium allows the basic substrate and sugar agent to be used to support the proliferation and growth of Salmonella, and the proliferation and growth of Salmonella can be achieved without inoculation in the transport medium and the growth-promoting medium. When used in conjunction with the selective inhibitors such as sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin, the inhibitory effect on Salmonella is weak, but it has a strong inhibitory effect on Proteus mirabilis and Citrobacter freundii, which are also Gram-negative rods. The selective culture of Salmonella can be achieved without the need for concentrated inoculation of the growth-promoting medium and the selective culture, that is, sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin. The synergistic use of sodium 2-methyl-4-undecyl sulfate and vancomycin has a good inhibitory effect on both Gram-positive and Gram-negative bacteria other than Salmonella, while the combination of sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin also has a certain inhibitory effect on Salmonella, but the inhibitory effect is weaker. The use of basic substrates and sugar agents can effectively achieve the proliferation and growth of Salmonella, and then the center of the Salmonella colony will turn black under the action of the chromogenic substrate to achieve colorimetric identification of Salmonella, effectively improving the identification efficiency of Salmonella and improving the identification accuracy of Salmonella.

[0042] In one embodiment, each liter of the Salmonella culture medium includes the following components: 8.5 g to 23.1 g of the basic substrate, 15 g to 28 g of the sugar agent, 6.2 g to 12.2 g of the color substrate, 6 g to 16.6 g of the selective inhibitor, and the balance of water. It can be understood that the basic substrate and the sugar agent provide sufficient water, carbon source, and nitrogen source for the proliferation and growth of Salmonella, effectively ensuring the effective proliferation and growth of Salmonella.

[0043] In one embodiment, the basic substrate includes yeast extract powder, tryptone, sodium chloride and agar. Further, the basic substrate also includes phenol red. It is understood that the culture medium is used for the culture and identification of Salmonella in the anal swab, and if the user does not collect a stool sample in the anal swab when using it, then in the presence of phenol red in the Salmonella culture medium, the color of the Salmonella culture medium does not change, that is, it remains red, and if the user collects a stool sample in the anal swab when using it, sucrose, lactose, and xylose are fermentable sugars that will produce acid, and in the presence of phenol red in the Salmonella culture medium, the color of the Salmonella culture medium will change, that is, from red to yellow, so that the missed detection of Salmonella can be effectively avoided. It can also be understood that sodium chloride maintains osmotic pressure.

[0044] In one embodiment, the basic substrate in each liter of Salmonella culture medium includes the following components by mass: 2g to 5g of yeast extract, 0.5g to 2g of tryptone, 3g to 8g of sodium chloride, and 3g to 8g of agar. Further, the basic substrate in each liter of Salmonella culture medium includes the following components by mass: 0.05g to 0.2g of phenol red. It can be understood that yeast extract and tryptone provide nitrogen sources, vitamins, and growth factors.

[0045] In one embodiment, the sugar agent includes sucrose, lactose and xylose. Further, the sugar agent in each liter of the Salmonella culture medium includes the following components in parts by weight: 6g-10g sucrose, 6g-10g lactose and 3g-8g xylose.

[0046] In one embodiment, the mass ratio of sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin in the inhibitor is (4-9): (1-5): (1-2.5): (0.004-0.009). It can be understood that when the ratio of sodium sulfite, sodium dodecyl sulfate and 7-ethyl-2-methyl-4-undecyl sulfate is adjusted and used in combination, both gram-negative bacteria and gram-positive bacteria are inhibited, but the inhibitory effect on Salmonella is weak, while it has a strong inhibitory effect on gram-negative bacteria other than Salmonella, especially for interfering bacteria, such as Proteus mirabilis and Citrobacter freundii. Further, combined with the enhancement of the effective inhibition of gram-positive bacteria by vancomycin, the effective inhibition of bacteria other than Salmonella is effectively achieved, and the effective proliferation and growth of Salmonella is ensured, thus effectively improving the identification accuracy of Salmonella. Furthermore, the selective inhibitor in each liter of Salmonella culture medium includes the following components: 4g-9g sodium sulfite, 1g-5g sodium dodecyl sulfate, 1g-2.5g sodium 7-ethyl-2-methyl-4-undecyl sulfate and 4mg-9mg vancomycin.

[0047] In one embodiment, the mass ratio of sodium thiosulfate to ammonium ferric citrate in the chromogenic substrate is (6-11): (0.2-1.2). Further, the chromogenic substrate in each liter of Salmonella culture medium includes the following components: 6g-11g of sodium thiosulfate and 0.2g-1.2g of ammonium ferric citrate.

[0048] The present application also provides a method for preparing a Salmonella identification culture medium, which is used to prepare the Salmonella identification culture medium of any of the above embodiments. The above-mentioned method for preparing the Salmonella identification culture medium includes the following steps: obtaining a basic substrate, a sugar agent, a chromogenic substrate and a selection inhibitor, wherein the selection inhibitor includes sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sodium sulfate and vancomycin; adding water to the basic substrate for a dissolution operation to obtain a basic liquid; adding water to the sugar agent, the chromogenic substrate and the selection inhibitor for a secondary dissolution operation, and adding water to make up the excess to obtain a mixed liquid; adding the basic liquid to the mixed liquid for a mixing operation to obtain a Salmonella identification culture medium.

[0049] The method for preparing the Salmonella identification culture medium can achieve rapid preparation of the Salmonella identification culture medium while ensuring the effectiveness of the components of each substance.

[0050] In order to better understand the preparation method of the Salmonella identification culture medium of the present application, the preparation method of the Salmonella identification culture medium of the present application is further explained below. The preparation method of the Salmonella identification culture medium of one embodiment comprises the following steps:

[0051] S100, obtaining a basic substrate, a sugar agent, a color developing substrate and a selection inhibitor, wherein the selection inhibitor comprises sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin.

[0052] S200, adding water to the basic substrate to perform a dissolution operation to obtain a basic solution.

[0053] S300, adding water to the sugar agent, the color developing substrate and the selection inhibitor for secondary dissolution operation, and adding water to make up the balance to obtain a mixed solution.

[0054] S400, adding the base liquid into the mixed liquid for uniform mixing to obtain a Salmonella identification medium.

[0055] The method for preparing the Salmonella identification culture medium can achieve rapid preparation of the Salmonella identification culture medium while ensuring the effectiveness of the components of each substance.

[0056] In one embodiment, water is added to the base substrate to perform a dissolution operation, and the specific steps are as follows:

[0057] Add water to yeast extract, tryptone and sodium chloride to completely dissolve, and then adjust the pH to 7.4-7.6 to obtain a semi-basic solution. Further, add water to yeast extract, tryptone, sodium chloride and phenol red to completely dissolve, and then adjust the pH to 7.4-7.6 to obtain a semi-basic solution. Further, add 10% sodium hydroxide and 10% hydrochloric acid to adjust the pH to 7.4-7.6.

[0058] Furthermore, water is added to the agar, heated and boiled until completely dissolved, and then added to the semi-basic liquid for mixing.

[0059] In one embodiment, before the step of adding the base liquid to the mixed liquid for mixing, and after the step of adding water to the base substrate for dissolving, the method for preparing the Salmonella identification medium further includes the following steps: sterilizing the base liquid with high pressure steam. Further, sterilizing the base liquid with high pressure steam at a temperature of 110°C to 121°C. Further, the high pressure steam sterilization treatment is performed for 15min to 30min. Further, the base liquid after high pressure steam sterilization is taken out and cooled to 55°C to 60°C.

[0060] In one embodiment, before the step of adding the base liquid to the mixed liquid for mixing, and after the step of adding water for the surplus supplementation, the preparation method of the Salmonella identification medium further comprises the following steps: microporous filtration of the mixed liquid. Further, the mixed liquid is microporous filtered using a microporous filter. Further, the pore size of the filter membrane of the microporous filter is 0.22um to 0.45um.

[0061] In one embodiment, water is added to the sugar, the chromogenic substrate and the selective inhibitor for a secondary dissolution operation, which specifically comprises the following steps:

[0062] Water is added to the sugar agent to perform a dissolution-promoting operation to obtain a sugar solution. Further, water is added to sucrose, lactose and xylose to completely dissolve them to obtain a sugar solution.

[0063] Further, water is added to the chromogenic substrate for a secondary dissolution operation to obtain a chromogenic substrate solution. Further, water is added to sodium thiosulfate and ammonium ferric citrate to completely dissolve, and then the pH is adjusted to 7.4-7.6 to obtain a chromogenic substrate solution. Further, the pH is adjusted to 7.4-7.6 by 10% sodium hydroxide and 10% hydrochloric acid.

[0064] Further, water is added to the selective inhibitor to perform a dissolution-promoting operation three times to obtain a selective inhibitor solution. Further, water is added to sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin to completely dissolve them to obtain a selective inhibitor solution.

[0065] Furthermore, the sugar solution, the chromogenic substrate solution and the selective inhibitor solution are mixed. It can be understood that since the sugar solution, the chromogenic substrate solution and the selective inhibitor solution are all solutions, they can be mixed quickly by simple stirring without a specific stirring speed or a specific stirring device.

[0066] Compared with the prior art, the present invention has at least the following advantages:

[0067] The Salmonella identification culture medium of the present invention enables the basic substrate and the sugar agent to support the proliferation and growth of Salmonella, and the proliferation and growth of Salmonella can be achieved without inoculation in the transport culture medium and the growth promotion culture medium. When the sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin in the selective inhibitor are used in coordination, the inhibitory effect on Salmonella is weak, but it has a strong inhibitory effect on Proteus mirabilis and Citrobacter freundii, which are also Gram-negative rods, and the selective culture of Salmonella can be achieved without the need for concentrated inoculation of the growth promotion culture medium and the selective culture, that is, sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin. The synergistic use of sodium 2-methyl-4-undecyl sulfate and vancomycin has a good inhibitory effect on both Gram-positive and Gram-negative bacteria other than Salmonella, while the combination of sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin also has a certain inhibitory effect on Salmonella, but the inhibitory effect is weaker. The use of basic substrates and sugar agents can effectively achieve the proliferation and growth of Salmonella, and then the center of the colony of Salmonella will turn black under the action of the chromogenic substrate to achieve colorimetric identification of Salmonella, effectively improving the identification efficiency of Salmonella and improving the identification accuracy of Salmonella.

[0068] Some specific examples are listed below, and if % is mentioned, it means percentage by weight. It should be noted that the following examples do not exhaust all possible situations, and the materials used in the following examples can be obtained from commercial sources unless otherwise specified.

[0069] Example 1

[0070] 1. Preparation of basic liquid: weigh 2g yeast extract powder, 0.5g tryptone, 3g sodium chloride and 0.05g phenol red, dissolve them in appropriate amount of water, and adjust the pH to 7.5; weigh 3g agar, heat and boil it in appropriate amount of water to dissolve, mix it with the previous liquid evenly, and sterilize it by high pressure at 115℃ for 15 minutes;

[0071] 2. Sugar solution preparation: weigh 6g sucrose, 6g lactose and 3g xylose, dissolve in appropriate amount of water and set aside;

[0072] 3. Preparation of reaction substrate solution: weigh 6 g sodium thiosulfate and 0.2 g ammonium ferric citrate, dissolve in appropriate amount of water, adjust pH to 7.5, and set aside;

[0073] 4. Preparation of inhibitors: weigh 4g sodium sulfite, 1g sodium dodecyl sulfate, 1g sodium 7-ethyl-2-methyl-4-undecyl sulfate and 4mg vancomycin, dissolve in appropriate amount of water and set aside;

[0074] 5. After mixing the sugar solution, reaction substrate solution and inhibitor, add enough water and filter with a sterilized microporous filter with a pore size of 0.45um; wait for the base liquid to cool to 55±0.5℃, add and mix well, use a dispensing device to quantitatively dispense into sterilized groove tubes, wait for it to solidify and then cover and package.

[0075] Example 2

[0076] 1. Preparation of basic liquid: weigh 3g yeast extract powder, 1g tryptone, 5g sodium chloride and 0.1g phenol red, dissolve them with appropriate amount of water, and adjust the pH to 7.5; weigh 5g agar, heat and boil it with appropriate amount of water to dissolve, mix it with the previous liquid evenly, and sterilize it by high pressure at 115℃ for 15 minutes;

[0077] 2. Sugar solution preparation: weigh 8g sucrose, 8g lactose and 5g xylose, dissolve in appropriate amount of water and set aside;

[0078] 3. Preparation of reaction substrate solution: weigh 8 g sodium thiosulfate and 0.6 g ammonium ferric citrate, dissolve in appropriate amount of water, adjust pH to 7.5, and set aside;

[0079] 4. Preparation of inhibitors: weigh 6 g of sodium sulfite, 3 g of sodium dodecyl sulfate, 1.8 g of 7-ethyl-2-methyl-4-undecyl sodium sulfate and 7 mg of vancomycin, dissolve in appropriate amount of water and set aside;

[0080] 5. After mixing the sugar solution, reaction substrate solution and inhibitor, add enough water and filter with a sterilized microporous filter with a pore size of 0.45um; wait for the base liquid to cool to 55±0.5℃, add and mix well, use a dispensing device to quantitatively dispense into sterilized groove tubes, wait for it to solidify and then cover and package.

[0081] Example 3

[0082] 1. Preparation of basic liquid: weigh 3g yeast extract powder, 1g tryptone, 5g sodium chloride and 0.1g phenol red, dissolve them with appropriate amount of water, and adjust the pH to 7.5; weigh 5g agar, heat and boil it with appropriate amount of water to dissolve, mix it with the previous liquid evenly, and sterilize it by high pressure at 115℃ for 15 minutes;

[0083] 2. Sugar solution preparation: weigh 8g sucrose, 8g lactose and 4g xylose, dissolve in appropriate amount of water and set aside;

[0084] 3. Preparation of reaction substrate solution: weigh 8.5 g sodium thiosulfate and 0.8 g ammonium ferric citrate, dissolve in appropriate amount of water, adjust pH to 7.5, and set aside;

[0085] 4. Preparation of inhibitors: weigh 6g of sodium sulfite, 2g of sodium dodecyl sulfate, 2g of 7-ethyl-2-methyl-4-undecyl sodium sulfate and 6mg of vancomycin, dissolve in appropriate amount of water and set aside;

[0086] 5. After mixing the sugar solution, reaction substrate solution and inhibitor, add enough water and filter with a sterilized microporous filter with a pore size of 0.45um; wait for the base liquid to cool to 55±0.5℃, add and mix well, use a dispensing device to quantitatively dispense into sterilized groove tubes, wait for it to solidify and then cover and package.

[0087] Example 4

[0088] 1. Preparation of basic liquid: weigh 4g yeast extract powder, 1.8g tryptone, 7g sodium chloride and 0.17g phenol red, dissolve them with appropriate amount of water, and adjust the pH to 7.5; weigh 6g agar, heat and boil it with appropriate amount of water to dissolve, mix it evenly with the previous liquid, and sterilize it by high pressure at 115℃ for 15 minutes.

[0089] 2. Sugar solution preparation: weigh 9g sucrose, 9g lactose and 7g xylose, dissolve in appropriate amount of water and set aside;

[0090] 3. Preparation of reaction substrate solution: weigh 10g sodium thiosulfate and 1g ammonium ferric citrate, dissolve in appropriate amount of water, adjust pH to 7.5, and set aside;

[0091] 4. Preparation of inhibitors: weigh 8g of sodium sulfite, 4g of sodium dodecyl sulfate, 2.2g of 7-ethyl-2-methyl-4-undecyl sodium sulfate and 8mg of vancomycin, dissolve in appropriate amount of water and set aside;

[0092] 5. After mixing the sugar solution, reaction substrate solution and inhibitor, add enough water and filter with a sterilized microporous filter with a pore size of 0.45um; wait for the base liquid to cool to 55±0.5℃, add and mix well, use a dispensing device to quantitatively dispense into sterilized groove tubes, wait for it to solidify and then cover and package.

[0093] Example 5

[0094] 1. Preparation of basic liquid: weigh 5g yeast extract powder, 2g tryptone, 8g sodium chloride and 0.2g phenol red, dissolve them with appropriate amount of water, and adjust the pH to 7.5; weigh 8g agar, heat and boil it with appropriate amount of water to dissolve, mix it with the previous liquid evenly, and sterilize it by high pressure at 115℃ for 15 minutes;

[0095] 2. Sugar solution preparation: weigh 10g sucrose, 10g lactose and 8g xylose, dissolve in appropriate amount of water and set aside;

[0096] 3. Preparation of reaction substrate solution: weigh 11g sodium thiosulfate and 1.2g ammonium ferric citrate, dissolve in appropriate amount of water, adjust pH to 7.5, and set aside;

[0097] 4. Preparation of inhibitors: weigh 9g sodium sulfite, 5g sodium dodecyl sulfate, 2.5g sodium 7-ethyl-2-methyl-4-undecyl sulfate and 9mg vancomycin, dissolve in appropriate amount of water and set aside;

[0098] 5. After mixing the sugar solution, reaction substrate solution and inhibitor, add enough water and filter with a sterilized microporous filter with a pore size of 0.45um; wait for the base liquid to cool to 55±0.5℃, add and mix well, use a dispensing device to quantitatively dispense into sterilized groove tubes, wait for it to solidify and then cover and package.

[0099] Comparative Example 1

[0100] 1. Preparation of basic liquid: weigh 3g yeast extract powder, 1g tryptone, 5g sodium chloride and 0.1g phenol red, dissolve them with appropriate amount of water, and adjust the pH to 7.5; weigh 5g agar, heat and boil it with appropriate amount of water to dissolve, mix it with the previous liquid evenly, and sterilize it by high pressure at 115℃ for 15 minutes;

[0101] 2. Sugar solution preparation: weigh 8g sucrose, 8g lactose and 4g xylose, dissolve in appropriate amount of water and set aside;

[0102] 3. Preparation of reaction substrate solution: weigh 8.5 g sodium thiosulfate and 0.8 g ammonium ferric citrate, dissolve in appropriate amount of water, adjust pH to 7.5, and set aside;

[0103] 4. Preparation of inhibitors: weigh 2g of sodium sulfite, 0.5g of sodium dodecyl sulfate, 0.5g of 7-ethyl-2-methyl-4-undecyl sodium sulfate and 2mg of vancomycin, dissolve in appropriate amount of water and set aside;

[0104] 5. After mixing the sugar solution, reaction substrate solution and inhibitor, add enough water and filter with a sterilized microporous filter with a pore size of 0.45um; wait for the base liquid to cool to 55±0.5℃, add and mix well, use a dispensing device to quantitatively dispense into sterilized groove tubes, wait for it to solidify and then cover and package.

[0105] Comparative Example 2

[0106] 1. Preparation of basic liquid: weigh 3g yeast extract powder, 1g tryptone, 5g sodium chloride and 0.1g phenol red, dissolve them with appropriate amount of water, and adjust the pH to 7.5; weigh 5g agar, heat and boil it with appropriate amount of water to dissolve, mix it with the previous liquid evenly, and sterilize it by high pressure at 115℃ for 15 minutes;

[0107] 2. Sugar solution preparation: weigh 8g sucrose, 8g lactose and 4g xylose, dissolve in appropriate amount of water and set aside;

[0108] 3. Preparation of reaction substrate solution: weigh 8.5 g sodium thiosulfate and 0.8 g ammonium ferric citrate, dissolve in appropriate amount of water, adjust pH to 7.5, and set aside;

[0109] 4. Preparation of inhibitors: weigh 12g sodium sulfite, 7g sodium dodecyl sulfate, 3g sodium 7-ethyl-2-methyl-4-undecyl sulfate and 12mg vancomycin, dissolve in appropriate amount of water and set aside;

[0110] 5. After mixing the sugar solution, reaction substrate solution and inhibitor, add enough water and filter with a sterilized microporous filter with a pore size of 0.45um; wait for the base liquid to cool to 55±0.5℃, add and mix well, use a dispensing device to quantitatively dispense into sterilized groove tubes, wait for it to solidify and then cover and package.

[0111] Comparative Example 3

[0112] 1. Preparation of basic liquid: weigh 3g yeast extract powder, 1g tryptone, 5g sodium chloride and 0.1g phenol red, dissolve them with appropriate amount of water, and adjust the pH to 7.5; weigh 5g agar, heat and boil it with appropriate amount of water to dissolve, mix it with the previous liquid evenly, and sterilize it by high pressure at 115℃ for 15 minutes;

[0113] 2. Sugar solution preparation: weigh 8g sucrose, 8g lactose and 4g xylose, dissolve in appropriate amount of water and set aside;

[0114] 3. Preparation of reaction substrate solution: weigh 8.5 g sodium thiosulfate and 0.8 g ammonium ferric citrate, dissolve in appropriate amount of water, adjust pH to 7.5, and set aside;

[0115] 4. Preparation of inhibitor: weigh 6g sodium sulfite, 2g 7-ethyl-2-methyl-4-undecyl sodium sulfate and 6mg vancomycin, dissolve in appropriate amount of water and set aside;

[0116] 5. After mixing the sugar solution, reaction substrate solution and inhibitor, add enough water and filter with a sterilized microporous filter with a pore size of 0.45um; wait for the base liquid to cool to 55±0.5℃, add and mix well, use a dispensing device to quantitatively dispense into sterilized groove tubes, wait for it to solidify and then cover and package.

[0117] Comparative Example 4

[0118] 1. Preparation of basic liquid: weigh 3g yeast extract powder, 1g tryptone, 5g sodium chloride and 0.1g phenol red, dissolve them with appropriate amount of water, and adjust the pH to 7.5; weigh 5g agar, heat and boil it with appropriate amount of water to dissolve, mix it with the previous liquid evenly, and sterilize it by high pressure at 115℃ for 15 minutes;

[0119] 2. Sugar solution preparation: weigh 8g sucrose, 8g lactose and 4g xylose, dissolve in appropriate amount of water and set aside;

[0120] 3. Preparation of reaction substrate solution: weigh 8.5 g sodium thiosulfate and 0.8 g ammonium ferric citrate, dissolve in appropriate amount of water, adjust pH to 7.5, and set aside;

[0121] 4. Preparation of inhibitors: weigh 6g of sodium sulfite, 2g of sodium dodecyl sulfate and 6mg of vancomycin, dissolve in appropriate amount of water and set aside;

[0122] 5. After mixing the sugar solution, reaction substrate solution and inhibitor, add enough water and filter with a sterilized microporous filter with a pore size of 0.45um; wait for the base liquid to cool to 55±0.5℃, add and mix well, use a dispensing device to quantitatively dispense into sterilized groove tubes, wait for it to solidify and then cover and package.

[0123] Comparative Example 5

[0124] 1. Preparation of basic liquid: weigh 3g yeast extract powder, 1g tryptone, 5g sodium chloride and 0.1g phenol red, dissolve them with appropriate amount of water, and adjust the pH to 7.5; weigh 5g agar, heat and boil it with appropriate amount of water to dissolve, mix it with the previous liquid evenly, and sterilize it by high pressure at 115℃ for 15 minutes;

[0125] 2. Sugar solution preparation: weigh 8g sucrose, 8g lactose and 4g xylose, dissolve in appropriate amount of water and set aside;

[0126] 3. Preparation of reaction substrate solution: weigh 8.5 g sodium thiosulfate and 0.8 g ammonium ferric citrate, dissolve in appropriate amount of water, adjust pH to 7.5, and set aside;

[0127] 4. Preparation of inhibitor: weigh 2g sodium dodecyl sulfate and 6mg vancomycin, dissolve in appropriate amount of water and set aside;

[0128] 5. After mixing the sugar solution, reaction substrate solution and inhibitor, add enough water and filter with a sterilized microporous filter with a pore size of 0.45um; wait for the base liquid to cool to 55±0.5℃, add and mix well, use a dispensing device to quantitatively dispense into sterilized groove tubes, wait for it to solidify and then cover and package.

[0129] The culture medium of Examples 1 to 5 and the culture medium of Comparative Examples 1 to 5 were used for the test, and the test verification process was as follows:

[0130] 1. Test verification plan

[0131] The performance of microbial culture medium mainly includes: sensitivity (the ability to detect true positives), specificity (the ability to exclude false positives) and anti-interference (the ability to simulate detection in real specimens).

[0132] This verification mainly verifies its performance through these three aspects.

[0133] 2. Experimental steps

[0134] 1) Quality control strains: Salmonella typhimurium (ATCC 14028), Proteus mirabilis (ATCC 7002), Citrobacter freundii (ATCC 43864), Escherichia coli (ATCC 25922)

[0135] 2) Preparation of bacterial solution: Fresh cultures of the F3 generation of Salmonella typhimurium were prepared into 0.5 McFarland units (about 1.5*10 8 cfu / mL) of bacterial solution, and then diluted it 10 times with sterile saline to prepare inoculum for sensitivity test; fresh culture of F3 generation of Escherichia coli was prepared into 0.5 McFarland unit (about 1.5*10 8 cfu / mL) of bacterial solution for interference test; fresh cultures of Proteus mirabilis and Citrobacter freundii F3 were prepared into 2.0 McFarland units of bacterial solution with sterile saline, and then diluted 10-fold with sterile saline to prepare inoculum for specificity test;

[0136] 3) Sensitivity test: 10 5 The inoculum of Salmonella typhimurium and Salmonella enteritidis with a concentration of 10 cfu / mL was inoculated into the five culture media of the examples, and cultured in a constant temperature incubator at 35-37° C. The culture results were observed every 2 hours starting from 18 hours.

[0137] 4) Interference test: In Salmonella typhimurium 10 8 Add the same volume of 10 8 The Escherichia coli liquid with a concentration of cfu / mL was mixed evenly and inoculated into the 5 culture media of the examples, and cultured in a constant temperature incubator at 35-37°C for 24 hours, and the culture results were observed.

[0138] 5) Specificity test: 2.0, 1.0, 0.5, 0.25, and 0.125 McFarland units of bacterial solution were inoculated into the best performing culture medium in the example and five comparative culture mediums, respectively, and cultured in a constant temperature incubator at 35-37° C. for 24 h, and the culture results were observed.

[0139] 3. Test results

[0140] 1) Sensitivity test results (minimum bacterial concentration for positive results)

[0141] Culture medium 18h Results 20h Results 22h Results 24h Results Final Result Example 1 <![CDATA[10 3 cfu / mL]]> <![CDATA[10 3 cfu / mL]]> <![CDATA[10 2 cfu / mL]]> <![CDATA[10 2 cfu / mL]]> <![CDATA[10 2 cfu / mL]]> Example 2 <![CDATA[10 3 cfu / mL]]> <![CDATA[10 2 cfu / mL]]> <![CDATA[10 2 cfu / mL]]> 10cfu / mL 10cfu / mL Example 3 10cfu / mL 10cfu / mL 10cfu / mL 10cfu / mL 10cfu / mL Example 4 <![CDATA[10 2 cfu / mL]]> 10cfu / mL 10cfu / mL 10cfu / mL 10cfu / mL Example 5 <![CDATA[10 2 cfu / mL]]> <![CDATA[10 2 cfu / mL]]> 10cfu / mL 10cfu / mL 10cfu / mL

[0142] Conclusion: Among the five culture media in the examples, the one with the highest sensitivity was Example 3, with a sensitivity of 10 cfu / mL after 18 h of culture, and the one with the lowest sensitivity was Example 1, with a sensitivity of 10 cfu / mL after 24 h of culture. 2 cfu / mL.

[0143] 2) Anti-interference test results (minimum bacterial concentration for positive results)

[0144] Culture medium 24h Results Example 1 <![CDATA[10 4 cfu / mL]]> Example 2 <![CDATA[10 3 cfu / mL]]> Example 3 <![CDATA[10 2 cfu / mL]]> Example 4 <![CDATA[10 5 cfu / mL]]> Example 5 <![CDATA[10 5 cfu / mL]]>

[0145] Conclusion: Among the five culture media in the examples, the culture medium in Example 3 has the best anti-interference property.

[0146] Based on the results of sensitivity and interference tests, different concentrations of inhibitors were formulated based on the culture medium formula of Example 3 to verify their specificity in order to obtain the best ratio of inhibitors.

[0147] 3) Specificity test results

[0148]

[0149] It can be seen from the above test results that Example 3 has the best specificity and can effectively inhibit Proteus mirabilis and Citrobacter freundii.

[0150] The above embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A Salmonella identification medium, characterized in that The Salmonella culture medium comprises the following components: a basic substrate, a sugar agent, a color developing substrate and a selection inhibitor; The chromogenic substrate includes sodium thiosulfate and ammonium ferric citrate; The selective inhibitors include sodium sulfite, sodium dodecyl sulfate, sodium 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin.

2. The Salmonella identification medium according to claim 1, characterized in that The salmonella culture medium comprises the following components per liter: 8.5 g to 23.1 g of basic substrate, 15 g to 28 g of sugar agent, 6.2 g to 12.2 g of color substrate, 6 g to 16.6 g of selective inhibitor and the balance of water.

3. The Salmonella identification medium according to claim 1 or 2, characterized in that The basic substrate includes yeast extract, tryptone, sodium chloride and agar.

4. The Salmonella identification medium according to claim 1 or 2, characterized in that The sugars include sucrose, lactose and xylose.

5. The Salmonella identification medium according to claim 1 or 2, characterized in that: The mass ratio of sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sulfate and vancomycin in the selective inhibitor is (4-9): (1-5): (1-2.5): (0.004-0.009).

6. The Salmonella identification medium according to claim 1 or 2, characterized in that: The mass ratio of sodium thiosulfate to ammonium ferric citrate in the color-developing substrate is (6-11): (0.2-1.2).

7. A method for preparing a Salmonella identification medium, for preparing the Salmonella identification medium according to any one of claims 1 to 6, characterized in that: The preparation method of the Salmonella identification culture medium comprises the following steps: Obtaining a basic substrate, a sugar, a chromogenic substrate and a selection inhibitor, wherein the selection inhibitor comprises sodium sulfite, sodium dodecyl sulfate, 7-ethyl-2-methyl-4-undecyl sodium sulfate and vancomycin; Adding water to the basic substrate to perform a dissolution operation to obtain a basic solution; Adding water to the sugar, the chromogenic substrate and the selective inhibitor for secondary dissolution, and adding water to make up the balance to obtain a mixed solution; The base liquid is added into the mixed liquid for uniform mixing to obtain a Salmonella identification medium.

8. The method for preparing the Salmonella identification medium according to claim 7, characterized in that: Before the step of adding the base liquid to the mixed liquid for mixing, and after the step of adding water to the base substrate for dissolving, the method for preparing the Salmonella identification medium further comprises the following steps: The base solution was sterilized by high pressure steam.

9. The method for preparing a Salmonella identification medium according to claim 7, characterized in that: Before the step of adding the base liquid to the mixed liquid for mixing, and after the step of adding water for making up the excess, the method for preparing the Salmonella identification medium further comprises the following steps: The mixed solution is subjected to microfiltration.

10. The method for preparing a Salmonella identification medium according to claim 7, characterized in that: Adding water to the sugar, the chromogenic substrate and the selective inhibitor for secondary dissolution operation specifically comprises the following steps: Adding water to the sugar preparation to perform a dissolution-promoting operation to obtain a sugar solution; Adding water to the chromogenic substrate to perform a secondary dissolution-promoting operation to obtain a chromogenic substrate solution; Adding water to the selective inhibitor to perform dissolution-promoting operations three times to obtain a selective inhibitor solution; The sugar solution, the color development substrate solution and the selection inhibition solution are mixed.

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