CN111118104A - Culture medium and preparation method thereof, kit, detection device and detection method - Google Patents
Culture medium and preparation method thereof, kit, detection device and detection method Download PDFInfo
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Abstract
The invention discloses a culture medium and a preparation method thereof, a kit, a detection device and a detection method, wherein the culture medium comprises: a target carbon source and/or a target nitrogen source, and triphenyltetrazolium chloride. Through the mode, the method can simplify the steps of microorganism screening and improve the screening efficiency.
Description
Technical Field
The invention relates to the technical field of microbial detection, in particular to a culture medium and a preparation method thereof, a kit, a detection device and a detection method.
Background
The identification and screening of the microorganism are important research subjects in clinical medicine, and have important significance for efficiently and accurately identifying and screening the microorganism for monitoring and preventing microbial infection.
In the prior art, identification and screening of microorganisms are generally performed by using phenotypic biochemical reactions, such as observing color change or state change of culture medium before and after inoculation of microorganisms. In the process of screening and identifying microorganisms by observing color change of the culture medium, the culture medium needs to be adjusted below a color-changing site, and the operation is complex; meanwhile, in the process of identifying the microorganisms, the color change of the indicator is not obvious or the color development time is long, so that the judgment of the detection result is not facilitated, and the screening efficiency of the microorganisms is low.
In a long-term research and development process, the inventor finds that the existing microorganism screening method is complicated in operation process and low in screening efficiency.
Disclosure of Invention
The invention mainly solves the technical problem of providing a culture medium, a preparation method thereof, a kit, a detection device and a detection method, which can simplify the steps of microorganism screening and improve the screening efficiency.
In order to solve the technical problems, the invention adopts a technical scheme that: a culture medium for screening microorganisms is provided.
Wherein the culture medium comprises:
a target carbon source and/or a target nitrogen source, and triphenyltetrazolium chloride.
In order to solve the technical problem, the invention adopts another technical scheme that: a kit for screening microorganisms is provided.
Wherein, the kit comprises:
a cassette comprising at least one media containment chamber;
a culture medium disposed in the medium holding chamber, the culture medium comprising any one of the culture media.
In order to solve the technical problem, the invention adopts another technical scheme that: provides a preparation method of a culture medium for screening microorganisms.
Wherein the method comprises the following steps:
providing a starting material for preparing any of said media to prepare a media mixture;
and (3) sterilizing the culture medium mixture to obtain the culture medium.
In order to solve the technical problems, the invention adopts a technical scheme that: a sample analysis instrument is provided.
Wherein the sample analyzer comprises:
and the coded disc is used for accommodating a detection card, and the detection card comprises any culture medium.
In order to solve the technical problem, the invention adopts another technical scheme that: a method for detecting a sample is provided.
Wherein the method comprises the following steps:
providing any of said culture media;
inoculating the microorganism to be detected to the culture medium and culturing under a preset condition;
when the microorganism to be detected is a target microorganism and the target carbon source and/or the target nitrogen source is a carbon source or a nitrogen source which can be directly utilized by the target microorganism, the target microorganism directly utilizes the target carbon source and/or the target nitrogen source to grow, so that the growth indicator triphenyltetrazolium chloride develops color.
In order to solve the technical problem, the invention adopts another technical scheme that: a method for detecting a sample is provided.
Wherein the method comprises the following steps:
providing any of said culture media;
inoculating the microorganism to be detected to the culture medium and culturing under a preset condition;
when the microorganism to be detected is a target microorganism and the target carbon source and/or the target nitrogen source is a carbon source or a nitrogen source which cannot be directly utilized by the target microorganism, the target microorganism generates a decomposition enzyme to decompose the target carbon source and/or the target nitrogen source to obtain the carbon source or the nitrogen source which can be directly utilized by the target microorganism, and the carbon source and/or the nitrogen source is directly utilized for growth, so that the growth indicator triphenyltetrazolium chloride is developed.
The invention has the beneficial effects that: different from the situation of the prior art, the culture medium utilizes the specificity of a target microorganism on certain nutrient substances, when the culture medium for screening the microorganisms is prepared, the added carbon source and/or nitrogen source can be utilized only by the target microorganism, namely only the target microorganism can grow and propagate on the culture medium, and the added triphenyltetrazolium chloride and dehydrogenase generated in the metabolic process of the target microorganism are used for carrying out electron transfer, so that the culture medium is subjected to obvious color change to judge the target microorganism, and the culture medium is simple to operate and high in identification efficiency.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts. Wherein:
FIG. 1 is a schematic structural view of one embodiment of a kit for screening microorganisms according to the present invention;
FIG. 2 is a schematic flow chart showing one embodiment of a method for screening microorganisms according to the present invention;
FIG. 3 is a schematic flow chart of one embodiment of a method for preparing a medium for screening microorganisms according to the present invention;
FIG. 4 is a schematic diagram of the structure of one embodiment of a sample analyzer of the present invention;
FIG. 5 is a schematic flow chart of a first embodiment of a method for detecting a sample according to the present invention;
FIG. 6 is a flow chart of a second embodiment of a method for detecting a sample according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The present embodiment discloses a medium for screening microorganisms, the medium comprising: a target carbon source and/or a target nitrogen source, and triphenyltetrazolium chloride.
The culture medium for screening the microorganisms comprises carbon sources, nitrogen sources, inorganic salts and other conventional components, the specificity of the target microorganisms for utilizing certain nutrient substances is utilized, when the culture medium for screening the microorganisms is prepared, the added target nutrient substances (the target carbon sources and/or the target nitrogen sources) are only capable of being utilized by the target microorganisms, namely only the target microorganisms can grow and propagate on the culture medium, and electron transfer is carried out between the added triphenyltetrazolium chloride and dehydrogenase generated in the metabolic process of the target microorganisms, so that the culture medium undergoes obvious color change to judge the target microorganisms, and the culture medium is simple to operate and high in identification efficiency.
In this embodiment, the target carbon source is a carbon source that cannot be utilized by the non-target microorganism and the target nitrogen source is a nitrogen source that cannot be utilized by the non-target microorganism, that is, only a carbon source and/or a nitrogen source that can be utilized by the target microorganism is present in the medium. In the growth and propagation process of the microorganism, both the carbon source and the nitrogen source are necessary, and the microorganism cannot grow and propagate due to the lack of the available carbon source or nitrogen source, so that the target carbon source or target nitrogen source added in the embodiment can prevent the non-target microorganism from surviving, thereby realizing the screening of the target microorganism.
The triphenyltetrazolium chloride is a microorganism growth indicator for dyeing living cells, and the indicator can perform electron transfer with intermediate dehydrogenase in a microorganism metabolic process so as to enable the culture medium to have obvious color change. And further, the target microorganism is judged, so that the operation is simple and the identification efficiency is high.
Of course, other target nutrients indispensable in the growth and reproduction process of the target microorganism and the microorganism growth indicator can be combined, the target microorganism is screened out through the indispensable target nutrients in the growth and reproduction process of the target microorganism, the target microorganism is indicated to be in a growth state through the microorganism growth indicator, and the target microorganism can be efficiently and accurately identified and screened through the cooperation of the target microorganism and the microorganism growth indicator.
Optionally, the target carbon source includes an organic acid salt, and the type of the target carbon source may be determined according to the target microorganism. In order to confirm whether the microorganisms to be identified can utilize acetate or citrate, the target carbon source comprises acetate or citrate, and optionally, culture media respectively taking acetate and citrate as unique carbon sources are adopted for identifying the microorganisms to be identified. Further, the acetate comprises potassium acetate, magnesium acetate, zinc acetate and the like; the citrate comprises potassium citrate, magnesium citrate or calcium citrate and the like.
In this embodiment, the organic acid can be directly utilized by the microorganism, and therefore, the target microorganism (capable of utilizing the target carbon source and/or the target nitrogen source) can directly utilize the organic acid as a carbon source for fermentation in the process of screening the microorganism. Meanwhile, triphenyltetrazolium chloride is an indicator for dyeing living cells, namely, dehydrogenase generated by the target microorganism in a survival state in a metabolic process can reduce the triphenyltetrazolium chloride, so that the target microorganism is dyed, the culture medium presents corresponding colors, observation is facilitated, biochemical reaction paths of different microorganisms using carbon sources are determined, and the genus of the microorganism is determined. Specifically, in the process of identifying the microorganisms through citrate, the escherichia, shigella, edwardsiella and yersinia are all negative, the salmonella and klebsiella are usually positive, and the slime and liquefaction serratia and some proteus and citrobacter are positive. In the process of identifying the sanitary strains through acetate, the Escherichia coli is positive, and the Shigella is negative.
Alternatively, the target carbon source may also be a polysaccharide, which may be a starch, in particular a hydrolysed starch, and when the target carbon source is a starch, the target microorganism is a microorganism capable of producing an amylase. This is because the microorganism cannot directly utilize starch, and the microorganism capable of producing amylase can hydrolyze starch in the medium and utilize it as a carbon source, and therefore, when preparing a microorganism for screening for amylase-producing microorganisms, the carbon source to be added can only hydrolyze starch, thereby enabling the screening of a target microorganism capable of producing amylase. Meanwhile, triphenyltetrazolium chloride is an indicator for dyeing living cells, namely, dehydrogenase generated by the target microorganism in a survival state in a metabolic process can reduce the triphenyltetrazolium chloride, so that the target microorganism is dyed, the culture medium presents corresponding colors, observation is facilitated, biochemical reaction ways of different microorganisms using carbon sources are determined, and further the genus of the microorganism is determined. Specifically, in the process of identifying the microorganisms by starch, diphtheria corynebacteria are negative and escherichia coli are positive for light and medium starch hydrolysis reactions.
Optionally, the triphenyltetrazolium chloride can be solid powder or a triphenyltetrazolium chloride solution with a certain concentration. In one embodiment, to avoid insufficient dissolution of the solid powder and improve the preparation efficiency of the culture medium, triphenyltetrazolium chloride is added into the culture medium through 0.5% by mass of triphenyltetrazolium chloride aqueous solution.
Further, the medium may further include a nitrogen source (when the target nitrogen source is included in the medium, the nitrogen source is not required to be added to the medium; when the target nitrogen source is not included in the medium, the nitrogen source is required to be added to the medium), a phosphorus source, a potassium source, an inorganic salt, distilled water, and the like. The nitrogen source can be ammonium salt, such as ammonium sulfate, ammonium chloride, ammonium nitrate or ammonium dihydrogen phosphate and other ammonium salts or the combination of more than one of the ammonium salts; the phosphorus source may be a phosphate salt, such as ammonium phosphate, ammonium dihydrogen phosphate, and the like; the inorganic salt may be provided by sodium chloride or the like, and the buffer may be provided by a hydrogen salt of a polybasic acid, such as dibasic hydrogen phosphate or monobasic hydrogen phosphate, or the like. The potassium source may include potassium sulfate, potassium chloride, or the like. Specifically, the culture medium comprises ammonium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride and distilled water. The ammonium dihydrogen phosphate can play the roles of a nitrogen source, a phosphorus source and a buffering agent in the growth and metabolism process of microorganisms; the dipotassium phosphate can play the roles of inorganic salt, a phosphorus source and a buffering agent; the sodium chloride is used to provide inorganic salt ions. In addition, when it is required to provide the culture medium with a sulfur source, the culture medium can also include ammonium sulfate, or the ammonium dihydrogen phosphate is replaced by ammonium sulfate.
Optionally, in order to obtain better screening and detection effects, the culture medium may further include a reaction auxiliary agent, where the reaction auxiliary agent includes one or more of a reaction coenzyme, an enzyme activator, or an inhibitor. Further, the reaction coenzyme comprises one or more of nicotinic acid, riboflavin or pyridoxal phosphate in combination; the enzyme activation auxiliary agent comprises one or more of magnesium salt, calcium salt and folic acid; the inhibitor comprises iodoacetic acid. Wherein, the magnesium salt can comprise one or more of magnesium sulfate, magnesium chloride or magnesium nitrate; the calcium salt comprises calcium chloride. Correspondingly, the types of the selected reaction auxiliary agents are different according to different target microorganisms and different target nutrient substance (such as a target carbon source or a target nitrogen source) systems.
When the target microorganism is screened by the target carbon source, the reaction auxiliary agent comprises one or more of riboflavin, nicotinic acid, magnesium salt or iodoacetic acid. In the embodiment, triphenyltetrazolium chloride and dehydrogenase generated in the metabolic process of the target microorganism carry out electron transfer, so that the culture medium is changed from colorless to red. The magnesium salt is an enzyme activator, and as the metabolism and growth process of the microorganism comprises a plurality of enzymatic reactions, the provision of the enzyme activator is beneficial to improving the metabolism efficiency of the microorganism, thereby improving the detection efficiency. The riboflavin and the nicotinic acid can promote the electron transfer between the dehydrogenase and the triphenyltetrazolium chloride, so that the color development of a culture medium is facilitated. The iodoacetic acid is an inhibitor of triphenyltetrazolium chloride, microorganisms can be killed due to a certain sterilization effect of the triphenyltetrazolium chloride, and a proper amount of inhibitors such as the iodoacetic acid and the like can be added into the culture medium to improve the color development effect. When the culture medium comprises one or more of riboflavin, nicotinic acid or iodoacetic acid, a proper dosage can be selected to obtain a better color development effect.
In one embodiment, the target nutrient is a target carbon source, and specifically, the target carbon source can be sodium acetate, sodium citrate or hydrolyzed starch; in the culture medium, the mass of the target carbon source such as the sodium acetate, the sodium citrate or the hydrolyzed starch is 2-8g, such as 2g, 6g or 8 g; the ammonium dihydrogen phosphate has a mass of 1-5g, e.g., 1g, 3g, or 5 g; the dipotassium hydrogen phosphate has a mass of 1-5g, such as 1g, 3g or 5 g; the sodium chloride has a mass of 3-5g, e.g., 3g, 4g, or 5 g; the volume of the distilled water is 800-; the volume of the 0.5% aqueous solution of triphenyltetrazolium chloride is 12.5-50ml, such as 12.5ml, 30ml or 50 ml.
In this embodiment, the mass of riboflavin is optionally between 0.005 and 0.01g, e.g., 0.005g, 0.008g, or 0.01 g; the weight of nicotinic acid is 0.005-0.01g, such as 0.005g, 0.008g or 0.01 g; the mass of the iodoacetic acid is 0.1-0.2g, such as 0.1g, 0.15g or 0.2 g. The magnesium salt is magnesium sulfate heptahydrate, and the magnesium sulfate heptahydrate has a mass of 0.2-0.5g, such as 0.2g, 0.4g, or 0.5 g.
When the target microorganism is screened by the target nitrogen source, the reaction auxiliary agent comprises one or more of calcium salt, folic acid, magnesium salt and pyridoxal phosphate. In this embodiment, magnesium salt, folic acid and the calcium salt are enzyme activation auxiliary agent, because the metabolism and the growth process of microorganism include multiple enzymatic reaction, provide the enzyme activator and be favorable to improving the metabolic efficiency of microorganism, and then improve detection efficiency, it is specific, folic acid is the protein synthesis promoter, pyridoxal phosphate is amino acid conversion coenzyme, adds reaction auxiliary agent can improve the growth and the metabolic rate of microorganism, can shorten the time of arriving the color development focus (the moment that the culture medium colour no longer deepens), and then improve target microorganism's screening efficiency.
In another embodiment, the target nutrient may be a target nitrogen source, the target nitrogen source is gelatin, and the mass of the gelatin in the medium is 120g, such as 120g, 120g or 120 g; the mass of the ammonium sulfate is 1-3g, e.g., 1g, 2g, or 3 g; the dipotassium hydrogen phosphate has a mass of 1-5g, such as 1g, 3g or 5 g; the sodium chloride has a mass of 0.5-2g, e.g., 0.2g, 2g, or 5 g; the volume of the distilled water is 800-; the volume of the 0.5% aqueous solution of triphenyltetrazolium chloride is 12.5-50ml, such as 12.5ml, 30ml or 50 ml. In addition, the culture medium may further include a target nutrient and the reaction auxiliary.
In this embodiment, optionally, the calcium chloride has a mass of 0.1 to 0.5g, e.g., 0.1g, 0.3g, or 0.5 g; the magnesium sulfate heptahydrate has a mass of 0.1-0.5g, e.g., 0.1g, 0.3g, or 0.5 g; the folic acid has a mass of 0.1-0.5g, e.g., 0.1g, 0.3g, or 0.5 g; the pyridoxal phosphate has a mass of 0.1-0.5g, such as 0.1g, 0.3g or 0.5 g.
In the present embodiment, when gelatin is used as a target nitrogen source, it cannot be directly used by a microorganism. Only microorganisms capable of producing gelatinase are able to hydrolyze gelatin by gelatinase into small amino acids or polypeptides and use the resulting amino acids or polypeptides as a nitrogen source for fermentation. Meanwhile, triphenyltetrazolium chloride is an indicator for dyeing living cells, namely, dehydrogenase generated by the target microorganism in a survival state in a metabolic process can reduce the triphenyltetrazolium chloride, so that the target microorganism is dyed, the culture medium presents corresponding colors, observation is facilitated, biochemical reaction ways of different microorganisms using a nitrogen source are determined, and further the genus of the microorganism is determined. Specifically, in the process of identifying microorganisms by gelatin, proteus mirabilis, proteus vulgaris, serratia, negative enterobacter and the like can liquefy the gelatin, namely the result is positive, and the escherichia coli is negative.
Meanwhile, compared with the method for observing the state change of the gelatin, the method of the embodiment has short detection time, is convenient for observing the detection result, and is beneficial to improving the detection efficiency.
In order to solve the technical problem, the invention adopts another technical scheme that: a kit for screening microorganisms is provided.
Referring to fig. 1, fig. 1 is a schematic structural diagram of an embodiment of a kit for screening microorganisms according to the present invention, wherein the kit 1 includes: a cartridge 10, said cartridge 10 comprising at least one medium receiving chamber 20; a culture medium 30 disposed in the culture medium receiving chamber 20, the culture medium 30 including any one of the culture media. In this embodiment, the culture medium 30 is used for screening microorganisms, and at least includes a target carbon source and/or a target nitrogen source, and triphenyltetrazolium chloride. Further, the kit 10 may include a plurality of medium accommodating chambers 20, the medium 30 accommodated in each medium accommodating chamber 20 may include the same or different kinds of target nutrients, and when the medium 30 accommodated in each medium accommodating chamber 20 includes the same kind of target nutrients, the kit 10 may perform parallel experiments on the same kind of microorganism to be identified; when the kinds of target nutrients contained in the culture medium 30 contained in each of the culture medium accommodating chambers 20 are different, the kit 10 can further narrow the kinds of microorganisms to be identified in a plurality of different ways, such as a carbon source, a nitrogen source, and the like, and when the number of the culture medium accommodating chambers 20 is sufficiently large, the kinds of the microorganisms can be accurately identified.
In order to solve the technical problem, the invention adopts another technical scheme that: provides a preparation method for screening microorganisms.
Referring to fig. 2, fig. 2 is a schematic flow chart of an embodiment of a method for screening microorganisms according to the present invention, wherein the method comprises:
s100, providing a culture medium for screening microorganisms, wherein the culture medium comprises any culture medium.
In step S100, the culture medium may be located in a test tube or in a culture medium accommodating chamber of the kit. The culture medium may further include other nutrients necessary for the growth of microorganisms, such as inorganic salts, buffers, etc., in addition to the target carbon source and/or target nitrogen source, and triphenyltetrazolium chloride.
Further, the culture medium is a sterilized culture medium, and the sterilization method comprises filtration sterilization and/or high-temperature sterilization.
S200, inoculating the microorganism to be detected to the culture medium, and culturing under a preset culture condition.
In the step S200, the culture is performed under a determined culture condition according to the kind and property of the microorganism to be detected inoculated onto the culture medium, and the predetermined culture condition includes a culture time, a culture temperature, a rotation speed, and the like. When the microorganism to be identified is Escherichia coli, the culture temperature is 36-38 deg.C, the culture time is 6-8 hours (e.g., 6 hours, 7 hours, or 8 hours), and the rotation speed is 100-150 rpm. In addition, the strain is a rejuvenated bacterial liquid of the microorganism to be detected, and the inoculation amount is 1% of the mass of the culture medium.
S300, detecting the change of the color of the culture medium after the culture time is up.
In step S300, the culture medium is observed after the culture time is reached, and when the color of the culture medium is not deepened, the detection end point is determined, if the culture medium is red, the detection result is positive, that is, the inoculated microorganisms include the target microorganism, and if the culture medium is colorless, the detection result is negative, that is, the inoculated microorganisms do not include the target microorganism.
Further, the color change of the medium can be observed visually or by a colorimeter. To improve the efficiency of identification of microorganisms, particularly for a kit comprising a plurality of medium-containing chambers, the change in color of the medium is automatically observed by a colorimeter.
In order to solve the technical problem, the invention adopts another technical scheme that: a method for screening microorganisms is provided.
Referring to fig. 3, fig. 3 is a schematic flow chart illustrating a method for preparing a culture medium for screening microorganisms according to an embodiment of the present invention, wherein the method comprises:
s10, providing raw materials for preparing any one of the culture media to prepare a culture medium mixture.
In step S10, the culture medium may further include other nutrients necessary for the growth of microorganisms, such as inorganic salts, buffers, etc., in addition to the target nutrients (target carbon source and/or target nitrogen source) and tritolyltetrazole chloride.
S20, sterilizing the culture medium mixture to obtain the culture medium.
In the step S20, the culture medium mixture is sterilized by filtration, specifically, the culture medium mixture obtained by mixing all the components constituting the culture medium is sucked by a syringe, and the culture medium mixture is injected into a microbial filtration membrane to filter out bacteria, so as to obtain the culture medium.
Optionally, in the process of sterilizing the culture medium by adopting a high-temperature sterilization mode, a culture medium mixture obtained by mixing all components (except for triphenyltetrazolium chloride) forming the culture medium is sterilized at the temperature of 110-130 ℃ (e.g., 110 ℃, 120 ℃ or 130 ℃) for 15-30 minutes (e.g., 15 minutes, 20 minutes or 30 minutes), cooled to room temperature, added with triphenyltetrazolium chloride, and uniformly mixed to obtain the culture medium.
In order to solve the technical problems, the invention adopts a technical scheme that: a sample analyzer is provided.
Referring to fig. 4, fig. 4 is a schematic structural diagram of an embodiment of a sample analyzer of the present invention, wherein the sample analyzer 100 includes: a code wheel 120 for receiving a test card (not shown) comprising any of the above mentioned culture media. Specifically, the sample analyzer 100 includes an analyzer body 110, the code wheel 120 is disposed on the analyzer body 110 and includes a plurality of detection card receiving locations 130, and the detection card receiving locations 130 are used for receiving the detection card. In use, the sample analyzer 100 is used to inoculate a strain to be detected onto the culture medium of the corresponding test card and detect a color change in the test card.
Furthermore, the technical details and technical advantages of the culture medium have been explained in detail above and will not be described further here.
In order to solve the technical problem, the invention adopts another technical scheme that: a method for detecting a sample is provided.
Referring to fig. 5, fig. 5 is a schematic flow chart of a first embodiment of a sample detection method according to the present invention, wherein the method includes:
s110, providing any culture medium.
S120, inoculating the microorganism to be detected to the culture medium and culturing under a preset condition.
S130, when the microorganism to be detected is a target microorganism and the target carbon source and/or the target nitrogen source is a carbon source or a nitrogen source which can be directly utilized by the target microorganism, the target microorganism directly utilizes the target carbon source and/or the target nitrogen source to grow, so that the growth indicator, namely the triphenyltetrazolium chloride, develops color.
In the present embodiment, the target carbon source and/or the target nitrogen source is a carbon source or a nitrogen source that can be directly utilized by the microorganism, such as an organic acid, peptone, or the like. Therefore, in the process of sample detection, microorganisms can directly utilize the carbon source and/or the nitrogen source, and the color of the greening tritetrazol is changed through dehydrogenase generated in the growth process, so that the culture medium is changed. Accordingly, the biochemical pathway of the microorganism to utilize the target carbon source and/or the target nitrogen source can be determined by observing the change of the color of the culture medium, and the genus of the microorganism can be determined.
In order to solve the technical problem, the invention adopts another technical scheme that: a method for detecting a sample is provided.
Referring to fig. 6, fig. 6 is a schematic flow chart of a first embodiment of a sample detection method according to the present invention, wherein the method includes:
s11, providing any culture medium.
S12, inoculating the microorganism to be detected on the culture medium and culturing under preset conditions.
S13, when the microorganism to be detected is a target microorganism and the target carbon source and/or the target nitrogen source is a carbon source or a nitrogen source which cannot be directly utilized by the target microorganism, the target microorganism generates a decomposition enzyme to decompose the target carbon source and/or the target nitrogen source to obtain the carbon source or the nitrogen source which can be directly utilized by the target microorganism, and the carbon source and/or the nitrogen source are directly utilized to grow, so that the growth indicator, namely the triphenyltetrazolium chloride, is developed.
In the present embodiment, the target carbon source and/or the target nitrogen source is a carbon source or a nitrogen source that cannot be directly utilized by the microorganism, such as gelatin, starch, or the like. Therefore, during the process of sample detection, the microorganism needs to generate corresponding decomposition enzyme to decompose the carbon source or the nitrogen source which cannot be directly utilized into nutrient substances (such as the carbon source and/or the nitrogen source) which can be directly utilized by the microorganism, and the dehydrogenase generated in the growth process enables the triphenyltetrazolium chloride to change color, thereby changing the color of the culture medium. Correspondingly, whether the microorganism can produce corresponding hydrolase can be determined by observing the change of the color of the culture medium, namely, the biochemical pathway of the microorganism for utilizing a target carbon source and/or a target nitrogen source is judged, and the genus of the microorganism is further determined.
In addition, the embodiment can screen out microorganisms capable of producing amylase, compared with the existing process that iodine solution is dripped outside a bacterial colony growing on a starch culture dish and whether a transparent ring is generated or not is observed or the identification is carried out by using 3, 5-dinitrosalicylic acid for color development, the detection time is shortened to 24h from 48h of the existing scheme, the requirement of adding a detection reagent and completing observation within 10min is omitted, the operation is simpler, and the detection efficiency is higher.
Meanwhile, the embodiment can screen out microorganisms capable of producing gelatinase, in the conventional method for detecting the gelatinase produced by the microorganisms by observing the state of the culture medium, the detection time is 2-7 days, the culture medium needs to be taken out daily for freezing and observation, the embodiment only needs to observe the change of the color of the culture medium, the detection time is shortened to 1 day, the operation is simple, and the detection efficiency is high.
The technical solution of the present invention is explained in detail by the following specific examples:
note: microorganism 1 is dysentery bacillus; microorganism 2 is Escherichia coli. Microorganism 3 is corynebacterium diphtheriae; microorganism 4 is Bacillus subtilis. Microorganism 5 is Proteus mirabilis.
Example 1
1g of ammonium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride, 0.5g of magnesium sulfate, 2g of sodium acetate, 1000ml of distilled water and 12.5ml of 0.5% aqueous solution of triphenyltetrazolium chloride are mixed to obtain a culture medium mixture, and the culture medium mixture is sterilized by adopting a filtration sterilization mode to obtain the culture medium 1.
The culture medium 1 was inoculated with the microorganism to be detected 1, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
Inoculating the culture medium 1 with the microorganism to be detected 2, culturing at 37 ℃ for 7 hours at the rotating speed of 120rpm, and observing color change.
Example 2
3g of ammonium dihydrogen phosphate, 3g of dipotassium hydrogen phosphate, 4g of sodium chloride, 0.35g of magnesium sulfate, 6g of sodium acetate, 900ml of distilled water and 30ml of 0.5% aqueous solution of triphenyltetrazolium chloride are mixed to obtain a culture medium mixture, and the culture medium mixture is sterilized by adopting a filtration sterilization mode to obtain a culture medium 2.
The culture medium 2 is inoculated with the microorganism to be detected 1, cultured for 8 hours at 36 ℃, the rotating speed is 130rpm, and the color change is observed.
The culture medium 2 is inoculated with the microorganism to be detected 2, the culture is carried out for 8 hours at 36 ℃, the rotating speed is 130rpm, and the color change is observed.
Example 3
5g of ammonium dihydrogen phosphate, 1g of dipotassium hydrogen phosphate, 5g of sodium chloride, 0.2g of magnesium sulfate, 8g of sodium acetate and 800ml of distilled water are mixed to obtain a culture medium mixture, the culture medium mixture is sterilized at 120 ℃ for 20 minutes in a high-temperature extinction mode, 50ml of 0.5% triphenyltetrazolium chloride aqueous solution is added after the culture medium mixture is cooled to the room temperature, and the culture medium 3 is obtained after the mixture is uniformly stirred.
Inoculating the culture medium 3 to the microorganism to be detected, culturing at 38 ℃ for 6 hours at the rotation speed of 150rpm, and observing color change.
Inoculating the culture medium 3 to the microorganism to be detected for 2, culturing at 38 ℃ for 6 hours at the rotating speed of 150rpm, and observing color change.
Example 4
1g of ammonium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride, 0.5g of magnesium sulfate, 2g of sodium citrate, 1000ml of distilled water and 12.5ml of 0.5% aqueous solution of triphenyltetrazolium chloride are mixed to obtain a culture medium mixture, and the culture medium mixture is sterilized by adopting a filtration sterilization mode to obtain a culture medium 4.
The culture medium 4 was inoculated with the microorganism to be detected 1, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
The culture medium 4 is inoculated with the microorganism to be detected for 2, the culture is carried out for 7 hours at 37 ℃, the rotating speed is 120rpm, and the color change is observed.
Example 5
1g of ammonium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride, 0.5g of magnesium sulfate, 2g of hydrolyzed starch, 1000ml of distilled water and 12.5ml of 0.5% aqueous solution of triphenyltetrazolium chloride are mixed to obtain a culture medium mixture, and the culture medium mixture is sterilized by adopting a filtration sterilization mode to obtain a culture medium 5.
The culture medium 5 is inoculated with the microorganism to be detected for 3, the microorganism is cultured for 7 hours at 37 ℃, the rotating speed is 120rpm, and the color change is observed.
The culture medium 5 was inoculated with the microorganism to be tested 4, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
Example 6
1g of ammonium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride, 0.5g of magnesium sulfate, 0.3g of calcium chloride, 110g of gelatin, 1000ml of distilled water and 12.5ml of 0.5% aqueous solution of triphenyltetrazolium chloride are mixed to obtain a culture medium mixture, and the culture medium mixture is sterilized by filtration sterilization to obtain a culture medium 6.
The culture medium 6 was inoculated with the microorganism 5 to be detected, cultured at 37 ℃ for 7 hours at 120rpm, and the color change was observed.
The culture medium 6 was inoculated with the microorganism to be tested 2, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
Example 7
1g of ammonium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride, 0.5g of magnesium sulfate, 2g of sodium acetate, 0.008g of nicotinic acid, 1000ml of distilled water and 12.5ml of 0.5% aqueous solution of triphenyltetrazolium chloride are mixed to obtain a culture medium mixture, and the culture medium mixture is sterilized by filtration sterilization to obtain a culture medium 7.
The culture medium 7 was inoculated with the microorganism to be detected 1, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
The culture medium 7 is inoculated with the microorganism to be detected for 2, the culture is carried out for 7 hours at 37 ℃, the rotating speed is 120rpm, and the color change is observed.
Example 8
1g of ammonium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride, 0.5g of magnesium sulfate, 2g of sodium acetate, 0.008g of riboflavin, 1000ml of distilled water and 12.5ml of 0.5% aqueous solution of triphenyltetrazolium chloride are mixed to obtain a culture medium mixture, and the culture medium mixture is sterilized by filtration sterilization to obtain a culture medium 8.
The culture medium 8 was inoculated with the microorganism to be detected 1, cultured at 37 ℃ for 7 hours at 120rpm, and the color change was observed.
The culture medium 8 is inoculated with the microorganism to be detected for 2, the culture is carried out for 7 hours at 37 ℃, the rotating speed is 120rpm, and the color change is observed.
Example 9
1g of ammonium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride, 0.5g of magnesium sulfate, 2g of sodium acetate, 0.008g of nicotinic acid, 0.008g of riboflavin, 1000ml of distilled water and 12.5ml of 0.5% aqueous solution of triphenyltetrazolium chloride are mixed to obtain a culture medium mixture, and the culture medium mixture is sterilized by filtration sterilization to obtain a culture medium 9.
The culture medium 9 was inoculated with the microorganism to be detected 1, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
The culture medium 9 was inoculated with the microorganism to be tested 2, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
Example 10
1g of ammonium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride, 0.5g of magnesium sulfate, 2g of sodium acetate, 0.008g of nicotinic acid, 0.15g of iodoacetic acid, 1000ml of distilled water and 12.5ml of 0.5% aqueous solution of triphenyltetrazolium chloride are mixed to obtain a culture medium mixture, and the culture medium mixture is sterilized by filtration sterilization to obtain the culture medium 10.
The culture medium 10 was inoculated with the microorganism to be detected 1, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
The culture medium 10 was inoculated with the microorganism to be tested 2, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
Example 11
1g of ammonium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride, 0.5g of magnesium sulfate, 2g of sodium acetate, 0.008g of riboflavin, 0.15g of iodoacetic acid, 1000ml of distilled water and 12.5ml of 0.5% aqueous solution of triphenyltetrazolium chloride are mixed to obtain a culture medium mixture, and the culture medium mixture is sterilized by filtration sterilization to obtain a culture medium 11.
The culture medium 11 was inoculated with the microorganism to be detected 1, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
The culture medium 11 was inoculated with the microorganism to be detected 2, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
Example 12
1g of ammonium dihydrogen phosphate, 5g of the dipotassium hydrogen phosphate, 3g of sodium chloride, 0.5g of magnesium sulfate, 2g of sodium acetate, 0.008g of nicotinic acid, 0.008g of riboflavin, 0.15g of iodoacetic acid, 1000ml of distilled water and 12.5ml of 0.5% aqueous solution of triphenyltetrazolium chloride are mixed to obtain a culture medium mixture, and the culture medium mixture is sterilized by filtration sterilization to obtain a culture medium 12.
The culture medium 12 was inoculated with the microorganism to be detected 1, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
The culture medium 12 was inoculated with the microorganism to be detected 2, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
Example 13
1g of ammonium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride, 0.5g of magnesium sulfate, 0.3g of calcium chloride, 110g of gelatin, 0.3g of folic acid, 1000ml of distilled water and 12.5ml of 0.5% aqueous solution of triphenyltetrazolium chloride are mixed to obtain a culture medium mixture, and the culture medium mixture is sterilized by filtration sterilization to obtain a culture medium 13.
The culture medium 13 was inoculated with the microorganism to be detected 5, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
The culture medium 13 was inoculated with the microorganism to be detected 2, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
Example 14
1g of ammonium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride, 0.5g of magnesium sulfate, 0.3g of calcium chloride, 110g of gelatin, 0.3g of pyridoxal phosphate, 1000ml of distilled water and 12.5ml of 0.5% aqueous solution of triphenyltetrazolium chloride are mixed to obtain a culture medium mixture, and the culture medium mixture is sterilized by filtration sterilization to obtain a culture medium 14.
The culture medium 14 was inoculated with the microorganism to be detected 5, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
The culture medium 14 was inoculated with the microorganism to be detected 2, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
Example 15
1g of ammonium dihydrogen phosphate, 5g of the dipotassium hydrogen phosphate, 3g of sodium chloride, 0.5g of magnesium sulfate, 0.3g of calcium chloride, 110g of gelatin, 0.3g of folic acid, 0.3g of pyridoxal phosphate, 1000ml of distilled water and 12.5ml of a 0.5% aqueous solution of triphenyltetrazolium chloride are mixed to obtain a culture medium mixture, and the culture medium mixture is sterilized by filtration sterilization to obtain a culture medium 15.
The culture medium 15 was inoculated with the microorganism to be detected 5, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
The culture medium 15 was inoculated with the microorganism to be tested 2, cultured at 37 ℃ for 7 hours at 120rpm, and observed for color change.
TABLE 1 identification of target microorganisms in media containing different target carbon sources
| Color change (inoculation of microorganism to be tested 1) | Color change (inoculation of microorganism to be tested 2) | |
| |
Colorless to colorless | Colorless-red |
| Medium 2 | Colorless to colorless | Colorless-red |
| Medium 3 | Colorless to colorless | Colorless-red |
| Medium 3 | Colorless to colorless | Colorless-red |
Note: the development time is the time from the beginning of the color change to the point where the color no longer darkens.
As can be seen from table 1, the microorganism to be tested 1 does not contain a target microorganism that utilizes acetate or citrate as a carbon source, and the microorganism to be tested 2 contains a target microorganism that utilizes acetate or citrate as a carbon source. In the embodiment, the carbon source added in the culture medium for screening the microorganisms is only the carbon source utilized by the target microorganisms, so that the biochemical way of the microorganisms to be tested for utilizing the carbon source is determined; meanwhile, triphenyltetrazolium chloride is added to carry out the identification and screening process of the microorganisms, so that the complex operation of adjusting the alkalinity of the cultured amino acid can be avoided, the color change of the target microorganisms is obvious, the identification efficiency is high, and the operation is simple.
TABLE 2 identification of target microorganisms in media containing different target nutrients
Note: the development time is the time from the beginning of the color change to the point where the color no longer darkens.
As can be seen from table 2, the microorganism to be tested 1 does not contain a target microorganism that utilizes acetate as a carbon source, the microorganism to be tested 3 does not contain a target microorganism that utilizes hydrolyzed starch as a carbon source, and the microorganism to be tested 5 does not contain a target microorganism that utilizes gelatin as a nitrogen source; the microorganism to be tested 2 contains a target microorganism using acetate as a carbon source, the microorganism to be tested 4 contains a target microorganism using hydrolyzed starch as a carbon source, and the microorganism to be tested 2 is capable of producing gelatinase and contains a target microorganism using gelatin as a nitrogen source. In the embodiment, when the culture medium for screening the microorganisms is prepared, the added target nutrient substance (target carbon source or target nitrogen source) carbon source or nitrogen source is only available for the target microorganisms, and the added triphenyltetrazolium chloride and dehydrogenase generated in the metabolic process of the target microorganisms are subjected to electron transfer, so that the culture medium undergoes obvious color change to judge the target microorganisms, and the method is simple to operate and high in identification efficiency.
TABLE 3 identification results of target microorganisms in the medium containing the reaction auxiliary and the target carbon source
Note: the development time is the time from the beginning of the color change to the point where the color no longer darkens.
Comparing the culture medium 1, the culture medium 7, the culture medium 8 and the culture medium 9 in table 3, it can be seen that the addition of the reaction coenzyme can promote the growth and the propagation of the target microorganism, thereby shortening the color development time and improving the detection efficiency, and the addition of the reaction coenzyme niacin and the reaction coenzyme riboflavin can cooperate with each other to further shorten the color development time. Comparing the culture medium 7 and the culture medium 10, the culture medium 8 and the culture medium 11, and the culture medium 9 and the culture medium 12 in table 3, it can be seen that the added iodoacetic acid plays a role of an inhibitor, and the detection efficiency is further improved by shortening the color development time by preventing triphenyltetrazolium chloride from killing excessive microorganisms.
TABLE 4 identification results of target microorganisms in the medium containing the reaction auxiliary and the target nitrogen source
Note: the development time is the time from the beginning of the color change to the point where the color no longer darkens.
As is clear from Table 4, the microorganism 2 to be tested does not contain a target microorganism that utilizes gelatin as a nitrogen source, and the microorganism 5 to be tested contains a target microorganism that utilizes gelatin as a nitrogen source. Whereas, since gelatin is a nitrogen source that cannot be directly utilized by the microorganism, discoloration of the medium by microorganism 5 indicates that microorganism 5 is capable of producing gelatinase that degrades gelatin. In the embodiment, the nitrogen source added into the culture medium for screening the microorganisms is only the nitrogen source utilized by the target microorganisms, and the triphenyltetrazolium chloride is added for identifying and screening the microorganisms, so that compared with a mode of observing the state change of the culture medium, the method does not need constant-temperature incubation, the observation time is shortened from 2-7 days to less than 2 days, and the method is high in identification efficiency and simple to operate. In addition, the reaction auxiliary agent is added to shorten the color development time, and the added auxiliary agents folic acid and pyridoxal phosphate can perform synergistic action to further shorten the color development time to 24 hours.
In summary, the invention discloses a culture medium for microorganism screening, a preparation method thereof and a kit, wherein the culture medium comprises: a target carbon source and/or a target nitrogen source, and triphenyltetrazolium chloride. Through the mode, the method can simplify the steps of the microorganism screening process and improve the detection efficiency.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (13)
1. A medium for screening microorganisms, the medium comprising:
a target carbon source and/or a target nitrogen source, and triphenyltetrazolium chloride.
2. The medium according to claim 1, wherein the target carbon source comprises an organic acid salt or a saccharide; the target nitrogen source comprises gelatin.
3. The culture medium of claim 2, wherein the organic acid salt comprises an acetate salt or a citrate salt; the saccharide comprises starch.
4. The culture medium of claim 1, wherein the triphenyltetrazolium chloride is 0.5% aqueous triphenyltetrazolium chloride solution in parts by weight.
5. The culture medium of claim 1, further comprising a source of phosphorus, a source of potassium, and an inorganic salt.
6. The culture medium of claim 5, wherein the source of phosphorus comprises a phosphate salt, the source of potassium comprises a potassium salt, and the inorganic salt comprises sodium chloride.
7. The culture medium of claim 1, further comprising a reaction auxiliary comprising one or a combination of a reaction coenzyme, an enzyme activator, or an inhibitor.
8. The culture medium of claim 7, wherein the reactive coenzyme comprises one or a combination of more of niacin, riboflavin, or pyridoxal phosphate; the enzyme activation auxiliary agent comprises one or more of magnesium salt, calcium salt and folic acid; the inhibitor comprises iodoacetic acid.
9. A kit for screening microorganisms, the kit comprising:
a cassette comprising at least one media containment chamber;
a culture medium disposed in the medium containment chamber, the culture medium comprising the culture medium of any one of claims 1-8.
10. A method of preparing a culture medium for screening microorganisms, the method comprising:
providing a starting material for preparing a culture medium according to any one of claims 1 to 8 to prepare a culture medium mixture;
and (3) sterilizing the culture medium mixture to obtain the culture medium.
11. A sample analyzer comprising a code wheel for receiving a test card comprising the medium of any of claims 1-8.
12. A method for detecting a sample, the method comprising:
providing a culture medium according to any one of claims 1 to 8;
inoculating the microorganism to be detected to the culture medium and culturing under a preset condition;
when the microorganism to be detected is a target microorganism and the target carbon source and/or the target nitrogen source is a carbon source or a nitrogen source which can be directly utilized by the target microorganism, the target microorganism directly utilizes the target carbon source and/or the target nitrogen source to grow, so that the growth indicator triphenyltetrazolium chloride develops color.
13. A method for detecting a sample, the method comprising:
providing a culture medium according to any one of claims 1 to 8;
inoculating the microorganism to be detected to the culture medium and culturing under a preset condition;
when the microorganism to be detected is a target microorganism and the target carbon source and/or the target nitrogen source is a carbon source or a nitrogen source which cannot be directly utilized by the target microorganism, the target microorganism generates a decomposition enzyme to decompose the target carbon source and/or the target nitrogen source to obtain the carbon source or the nitrogen source which can be directly utilized by the target microorganism, and the carbon source and/or the nitrogen source is directly utilized for growth, so that the growth indicator triphenyltetrazolium chloride is developed.
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