CN110846375B

CN110846375B - Culture medium composition for bacterial resistance counting, preparation method and application thereof

Info

Publication number: CN110846375B
Application number: CN201911012056.8A
Authority: CN
Inventors: 崔璟; 唐明忠
Original assignee: Shenzhen Aier Biotechnology Co ltd
Current assignee: Beijing Xingyuanhui Technology Co ltd; Tang Mingzhong
Priority date: 2019-10-23
Filing date: 2019-10-23
Publication date: 2023-05-05
Anticipated expiration: 2039-10-23
Also published as: CN110846375A

Abstract

The present invention provides a medium comprising: MH broth at a concentration of 0.5 g/l to 40g/l, brain heart infusion at a concentration of 0.5 g/l to 60 g/l, and water. The technical scheme of the invention has the characteristics of accelerating the growth of microorganisms, reducing the background and reducing the occurrence of bacterial agglomeration growth, and the culture medium is applied to the counting of bacteria by a resistance method, so that the function of rapidly culturing the bacteria is achieved on one hand, and the requirements of reducing the background, namely reducing particulate matters, required by the resistance counting are met on the other hand.

Description

Culture medium composition for bacterial resistance counting, preparation method and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a culture medium, especially a bacterial culture medium.

Background

The culture medium is an in-vitro enrichment culture reagent, provides proper nutrition components and growth environment for the growth and propagation of microorganisms and accelerates the growth speed of the microorganisms. At present, the culture medium in domestic market has tens and various types, and can meet the requirements of culturing common clinical microorganisms. However, if the medium is used as a medium for counting bacteria by a resistance method, on one hand, the function of rapidly culturing bacteria is achieved, the background reduction required by resistance counting, namely the requirement of reducing particulate matters is also met, the medium on the market at present only meets the requirement of culturing common bacteria, and the requirement of the medium for counting bacteria by the resistance method cannot be met, so the medium for meeting the requirement of resistance counting is required.

Disclosure of Invention

The embodiment of the invention provides a bacterial culture medium used in a bacterial counting process by a resistance method, which at least solves the technical problems of slower bacterial culture and higher cost in the prior art.

According to an embodiment of the present invention, there is provided a medium composition for bacterial resistance counting, the medium composition comprising: MH broth at a concentration of 0.5 g/l to 40g/l, brain heart infusion at a concentration of 0.5 g/l to 60 g/l, and water.

Optionally, the above-described culture medium composition comprises: MH broth at a concentration of 5g/l to 27g/l, brain heart infusion at a concentration of 5g/l to 40g/l, and water.

Alternatively, the MH broth has a concentration of 21g/L, the brain heart infusion has a concentration of 12g/L, and the water has a volume of 1L.

Alternatively, the MH broth has a concentration of 5g/l, the brain heart infusion has a concentration of 40g/l, and the water has a volume of 1 l.

Optionally, the MH broth has a concentration of 27g/L, the brain heart infusion has a concentration of 5g/L, and the water has a volume of 1L.

The embodiment of the invention provides a preparation method of a culture medium composition, which comprises the following steps:

step one, cleaning an instrument used for weighing and dissolving the culture medium composition;

weighing the MH broth, the brain heart infusion and the water;

step three, dissolving the MH broth, the brain heart infusion and the water obtained by weighing to obtain a dissolved substance;

step four, filtering the dissolved substances to obtain a culture medium composition;

and fifthly, performing high-pressure damp-heat sterilization on the culture medium composition.

Optionally, the sterilized culture medium is subjected to sterility testing.

Optionally, filtering the dissolved material, including: the dissolved matter is subjected to primary filtration and then is subjected to secondary filtration, wherein the secondary filtration is carried out once, twice or three times.

Optionally, the first filtration uses a filter with a pore size of 0.3 m to 1 micron and the second filtration uses a filter with a pore size of 0.1 micron to 0.3 micron.

Optionally, the primary filtration uses a filter with a pore size of 0.4 microns and the secondary filtration uses a filter with a pore size of 0.22 microns.

Optionally, the sterilized culture medium is cultured at 35 ℃ for 48 hours, and then the sterility test is carried out.

Alternatively, a sterile-inspected acceptable medium is inoculated with clinically common bacteria to verify the culture capacity of the medium.

The use of the above-described culture medium in bacterial resistance counting.

The Chinese meaning of MH (Mueller-Hinton Broth, MH) Broth is hydrolyzed casein medium.

The technical scheme of the invention has the characteristics of accelerating the growth of microorganisms, reducing the background and reducing the occurrence of bacterial agglomeration growth, and the culture medium is applied to the counting of bacteria by a resistance method, so that the function of rapidly culturing the bacteria is achieved on one hand, and the requirements of reducing the background, namely reducing particulate matters, required by the resistance counting are met on the other hand. During the growth process of bacteria, the cocci have the phenomenon of agglomeration and aggregation growth, and the phenomenon achieves the aim of dispersing and growing the bacteria as much as possible through the brain heart immersion liquid in the technical scheme of the invention.

The technical scheme of the invention has good stability, can keep the stability of the granular materials within one year after the preparation is finished, and can ensure the culture capacity of bacteria; the content of the particulate matters is low, and the requirement of resistance counting can be met; the process is mature and the cost is low.

Description of{ Medium }

The present application discloses a medium that can be used for bacterial culture for bacterial enumeration. For example, the medium may be used for bacterial cultivation prior to counting results of a method and/or apparatus for bacterial counting to better achieve a method and/or apparatus for bacterial counting. For another example, the culture medium can be used as an in vitro enrichment culture reagent to provide suitable nutrients and growth environments for microbial growth and to accelerate the growth rate of microorganisms.

In some embodiments, the culture medium applied to bacterial counting may be applied in a variety of bacterial counting methods, such as counter assays (also may be referred to as microscopic counting methods), electronic counter counting methods (also may be referred to as resistance counting methods), living cell counting methods, gravimetric methods, and the like.

In some embodiments, a solvent may refer to a liquid used to dissolve and/or dilute other components (e.g., bacteria, fixatives, additives, etc.) in the composition. Exemplary solvents may include, but are not limited to, water, dimethyl sulfoxide, trifluoroacetic acid, methanol, ethanol, dimethylformamide, acetonitrile, acetic acid, acetone, pyridine, dioxane, chloroform, isopropanol, tetramethyl ethylenediamine, triethylamine, methyl formate, tributylamine, methyl ethyl ketone, ethyl acetate, trioctylamine, dimethyl carbonate, tetrahydrofuran, propanol, n-butanol, methylene chloride, benzene, diethyl ether, isopropyl ether, n-butyl ether, trichloroethylene, diphenyl ether, methylene chloride, toluene, carbon tetrachloride, carbon disulfide, cyclohexane, hexane, petroleum ether, and the like, or any combination thereof. Preferably, the solvent may be pure water. In some embodiments, the solvent may comprise 40% -75% of the total volume of the composition. More preferably, the solvent may comprise 41% to 70% of the total volume of the composition. More preferably, the solvent may comprise 42% to 65% of the total volume of the composition. More preferably, the solvent may comprise 43% to 60% of the total volume of the composition. More preferably, the solvent may comprise 44% to 59% of the total volume of the composition. More preferably, the solvent may comprise 45% -58% of the total volume of the composition. More preferably, the solvent may comprise from 46% to 57% of the total volume of the composition. More preferably, the solvent may comprise 47% to 56% of the total volume of the composition. More preferably, the solvent may comprise 48% to 54% of the total volume of the composition. More preferably, the solvent may comprise 54% of the total volume of the composition. More preferably, the solvent may comprise 53% of the total volume of the composition. More preferably, the solvent may comprise 52% of the total volume of the composition. More preferably, the solvent may comprise 51% of the total volume of the composition. More preferably, the solvent may comprise 50% of the total volume of the composition. More preferably, the solvent may comprise 49% of the total volume of the composition. Particularly preferably, the solvent may comprise 48% of the total volume of the composition.

Bacteria may be the final component of the composition that is counted. When the composition is used to verify a bacteria counting device or method, the value detected is the number of bacteria contained in the composition. In some embodiments, the bacteria may be selected from one or any combination of bacillus, coccus, helicobacter, and the like. Preferably, the bacteria may be selected from one or any combination of bacillus or coccus. Exemplary bacilli may include, but are not limited to, enterobacter aerogenes, enterobacter cloacae, enterobacter agglomerans, escherichia coli, escherichia friesen, escherichia helman, serratia marcescens, serratia rubra, klebsiella pneumoniae, proteus, providencia, salmonella, enterobacter trifoliatus, algobacter americanus, butje's bacteria, sildencia, shigella, acinetobacter, bifidobacterium, flavobacterium, bacillus, and the like, or any combination thereof. Exemplary cocci may include, but are not limited to, staphylococcus aureus, staphylococcus epidermidis, staphylococcus hemolyticus, staphylococcus intermedia, staphylococcus capitis, staphylococcus saccharolyticus, micrococcus, enterococcus chromeli, streptococcus bovis, streptococcus blood, streptococcus intermedia, streptococcus pyogenes, streptococcus beta hemolyticus, streptococcus angina, streptococcus twins, streptococcus pneumoniae, streptococcus turquoise, neisseria, and the like, or any combination thereof. Preferably, the bacteria may be selected from one of escherichia coli, staphylococcus aureus, or a combination thereof. Commercially available standard strains can be used in the compositions disclosed herein. By way of example only, the bacteria may be selected from the ATCC25922 standard strain of Escherichia coli, and/or the ATCC 29213 standard strain of Staphylococcus aureus. In some embodiments, the total concentration of bacteria may be 105-109/ml. More preferably, the total concentration of bacteria may be 106-108 bacteria/ml. More preferably, the total concentration of bacteria may be 106 bacteria/ml. More preferably, the total concentration of bacteria may be 107 bacteria/ml. Particularly preferably, the total concentration of the bacteria may be 108 bacteria/ml.

The additive may be a substance for adjusting the pH so that the components of the composition maintain their intact properties during storage. In some embodiments, the additive may be selected from inorganic salts including, but not limited to, sodium phosphate, potassium phosphate, calcium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, calcium chloride, potassium chloride, copper sulfate, iron sulfate, magnesium chloride, magnesium sulfate, sodium bicarbonate, zinc sulfate, and the like, or any combination thereof. Preferably, the additive may include one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium chloride, or any combination thereof. More preferably, the additive may include potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride. In some embodiments, the amount of potassium dihydrogen phosphate included in the additive is 0.02% to 0.03% by weight of the total composition. More preferably, the amount of potassium dihydrogen phosphate included in the additive is 0.02% -0.027% of the total mass of the composition. More preferably, the amount of potassium dihydrogen phosphate included in the additive is 0.02% by weight of the total composition. More preferably, the potassium dihydrogen phosphate is included in the additive in an amount of 0.023% by mass of the total mass of the composition. More preferably, the amount of potassium dihydrogen phosphate included in the additive is 0.025% by weight of the total composition. Particularly preferably, the mass of potassium dihydrogen phosphate included in the additive is 0.027% of the total mass of the composition. The mass of the disodium hydrogen phosphate contained in the additive accounts for 0.1-0.2% of the total mass of the composition. More preferably, the disodium hydrogen phosphate is included in the additive in an amount of 0.11% to 0.18% by mass of the total mass of the composition. More preferably, the disodium hydrogen phosphate is included in the additive in an amount of 0.12% to 0.16% by mass of the total mass of the composition. More preferably, the disodium hydrogen phosphate is included in the additive in an amount of 0.13% to 0.15% by mass of the total mass of the composition. More preferably, the disodium hydrogen phosphate is included in the additive in an amount of 0.13% by mass of the total mass of the composition. More preferably, the disodium hydrogen phosphate is included in the additive in an amount of 0.14% by mass of the total mass of the composition. More preferably, the disodium hydrogen phosphate is included in the additive in an amount of 0.142% by mass of the total mass of the composition. The mass of sodium chloride contained in the additive accounts for 0.07% -0.1% of the total mass of the composition. More preferably, the sodium chloride is included in the additive in an amount of 0.08% -0.09% by mass of the total mass of the composition. More preferably, the sodium chloride is included in the additive in an amount of 0.08% by mass of the total mass of the composition. Particularly preferably, the sodium chloride is included in the additive in an amount of 0.09% by mass of the total mass of the composition.

It should be noted that the above description of the compositions, solvents, bacteria, fixatives, additives is for convenience of description only and is not intended to limit the application to the scope of the illustrated embodiments. It will be appreciated that any modifications and alterations to the parts are possible without departing from the principles of the present application, as will be apparent to those skilled in the art after having appreciated the principles. For example, the fixative may be replaced by a physical fixation method. Exemplary physical fixation methods may include paraffin infiltration methods, cryogenic freezing methods. Such variations are within the scope of the present application.

{ { preparation method }

One general method of preparing the media composition may include, for example, preparing a vessel for cleaning, weighing raw materials as required by the formulation, dissolving, filtering, autoclaving, and post-sterilization culturing. The preparation of the composition is described in further detail below.

Step one, cleaning preparation: and carrying out ultrasonic cleaning and pure water flushing on the container to reduce the content of particulate matters in the container.

In some embodiments, the cleaning means may include, but are not limited to, ultrasonic cleaning and pure water rinsing, as the exemplary cleaning means is to reduce the content of particulate matter in the container.

Secondly, weighing: weighing raw materials according to the requirements of the formula.

Third step, dissolving: and fully dissolving the weighed raw materials.

In some embodiments, the dissolution may include, but is not limited to, pure water dissolution, and the solvents used are described above and are not described herein.

Fourth, filtering: the presence of particulate material is reduced by first performing a primary filtration through a 0.4 μm filter and then performing a primary, secondary or tertiary filtration through a 0.22 μm filter.

In some embodiments, the filtering means may include, but is not limited to, direct filtration, membrane filtration, and the like, depending on the actual situation. Exemplary direct filtration means may include, but are not limited to, one or a combination of cartridge filtration, fine mesh filtration, and the like. Exemplary membrane filtration modes may include, but are not limited to, filtration modes of one or any combination of microporous filtration, ultrafiltration, reverse osmosis, and the like. Preferably, the dilution liquid can be filtered by a microporous filter membrane. In some embodiments, the microporous filter membrane may include one or any combination of cellulose acetate membrane, nitrocellulose membrane, polyamide membrane, polytetrafluoroethylene membrane, polyvinylidene fluoride membrane, surfactant-free cellulose acetate membrane, asymmetric polyethersulfone, fiberglass membrane, and the like. In some embodiments, the microporous filter membrane used may have a diameter of less than or equal to 12 μm (micrometers). More preferably, the microporous filter membrane used may have a diameter of less than or equal to 5 μm. More preferably, the microporous filter membrane used may have a diameter of less than or equal to 1 μm. More preferably, the microporous filter membrane used may have a diameter of less than or equal to 0.8 μm. More preferably, the microporous filter membrane used may have a diameter of less than or equal to 0.6 μm. More preferably, the microporous filter membrane used may have a diameter of less than or equal to 0.4 μm. More preferably, the microporous filter membrane may have a diameter of less than or equal to 0.3 μm. More preferably, the microporous filter membrane may have a diameter of less than or equal to 0.22 μm. More preferably, the microporous filter membrane may have a diameter of less than or equal to 0.12 μm.

Fifth, the filtered medium is subjected to high-pressure moist heat sterilization (inactivation).

In some embodiments, the inactivation may refer to an operation that disrupts the biological activity, reproductive ability, and pathogenicity of a microorganism (e.g., bacteria) to preserve its immunogenicity as much as possible. The means of inactivation may include physical inactivation methods and chemical inactivation methods. Exemplary physical inactivation may include one or any combination of high pressure wet heat inactivation, dry heat inactivation, ultrasonic inactivation, ultraviolet light inactivation, radiation irradiation inactivation, and the like. Exemplary chemical inactivation may include an inactivating agent of one or any combination of formaldehyde solution, alkylating agent, phenol, crystal violet, and the like. It will be appreciated that the effect of inactivation may be related to one or any combination of inactivation method, selection of the inactivating agent, inactivation temperature, inactivation time, pH, microorganism species, and the like. Preferably, the bacterial suspension may be inactivated by a method of high-pressure damp-heat bacterial inactivation. Parameters adopted in the high-pressure wet heat sterilization process can be adjusted according to actual conditions. For example, the parameters for the inactivation of high-pressure moist heat bacteria may be 121℃and 0.12MPa for 30min.

Sixth, the sterilized culture medium is cultured at 35 ℃ for 48 hours.

In some embodiments, the time of the culture may be adjusted according to the specific situation, and the above-mentioned "culturing of the sterilized culture medium at 35 ℃ for 48 hours" is only a preferred example, and the culture temperature and time may be adjusted according to the specific situation.

And seventh, bacteria after sterilization and culture are subjected to sterility inspection, and the culture capacity of a culture medium is checked by inoculating clinically common bacteria.

In some embodiments, the bacteria may be the last component to be counted in the composition. It should be noted that the bacteria added to the filtered dilution are live bacteria. The added bacteria may be selected from one or any combination of bacillus, coccus, helicobacter, etc. Preferably, the bacteria may be selected from the group consisting of escherichia coli and staphylococcus aureus. In some embodiments, methods of preparing a bacterial suspension may include, but are not limited to, turbidimeter or turbidimeter. The turbidimetric method can be a method for roughly judging the concentration or the number of bacteria in bacterial suspensions, and the concentration of the bacterial suspension to be detected is obtained by adjusting the turbidity between the bacterial suspensions to be detected to enable the turbidity of the bacterial suspension to be detected to be the same as or basically the same as the turbidity of a standard tube of the turbidimetric method. By way of example only, the turbidimetry may utilize sulfuric acid and barium chloride to formulate a mahalanobis standard tube number (e.g., 0.5 mahalanobis standard tube number, 1 mahalanobis standard tube number, 2 mahalanobis standard tube number, 3 mahalanobis standard tube number, 4 mahalanobis standard tube number), and the turbidity of the resulting barium sulfate precipitate may correspond to a bacterial concentration (e.g., 0.5 mahalanobis corresponding to a bacterial concentration of 1.5X108/ml, 1 mahalanobis corresponding to a bacterial concentration of 3X 108/ml, 2 mahalanobis corresponding to a bacterial concentration of 6X 108/ml, 3 mahalanobis corresponding to a bacterial concentration of 9X 108/ml, 4 mahalanobis corresponding to a bacterial concentration of 12X 108/ml). And judging the concentration of the bacterial suspension by comparing the turbidity of the bacterial suspension to be detected with that of a Maillard standard tube. As another example, the turbidimeter can directly display turbidity values of the units of mahogany by measuring the bacterial suspension to be measured, and can obtain the bacterial suspension concentration to be measured from a table of the relationship between international universal turbidimeter values (e.g., 0.5McF, 1McF, 2McF, 3McF, 4 McF) and the concentration of the bacterial suspension (e.g., 0.5McF corresponds to 1.5×108/ml, 1McF corresponds to 3×108/ml, 2McF corresponds to 6×108/ml, 3McF corresponds to 9×108/ml, and 4McF corresponds to 12×108/ml).

It should be noted that the above description of the process for preparing the medium composition is for illustration and description only, and does not limit the scope of applicability of the application. It will be apparent to those skilled in the art having the benefit of this disclosure that many modifications and changes to this process may be made without departing from the scope of the invention. Such modifications and variations are, however, still within the scope of the present application.

{ { use of Medium composition in bacterial enumeration method or apparatus }

In some embodiments, the culture medium composition may be used for bacterial culture for bacterial enumeration. For example, bacteria are grown rapidly to meet the background-reducing, i.e., particulate matter-reducing, requirements such as resistance counts.

In the first step, a suitable vessel is selected for bacterial culture.

In some embodiments, the bacteria are as described above and are not described in detail herein.

And secondly, adding the culture medium composition into the bacteria, and culturing the bacteria for less than or equal to 4 hours.

Third, the number of bacteria in the composition (composition containing bacteria and its medium) is measured by a bacteria counting method or apparatus, to obtain a measurement of the number of bacteria in the composition.

In some embodiments, the bacterial count method may include one or any combination of electronic counting, microscopy, living cell counting, cell weight method, and the like. The bacteria counting device may be a device for performing bacteria counting using the bacteria counting method as described above. The measured value of the bacterial concentration may be a value obtained by counting the bacteria in the composition in an existing step using the bacterial counting method or in an existing state using the bacterial counting apparatus. The measured value can be obtained by implementing all the steps of the bacterial counting or operating the bacterial counting device. Preferably, the bacterial count method is a resistance count method.

Possible beneficial effects of embodiments of the present application include, but are not limited to: the composition disclosed by the application can be used for the accelerated culture of bacteria in bacteria counting equipment or methods, and has the advantages of high accuracy, good stability and high speed. It should be noted that, the advantages that may be generated by different embodiments may be different, and in different embodiments, the advantages that may be generated may be any one or a combination of several of the above, or any other possible advantages that may be obtained.

Detailed Description

The principles and spirit of the present invention will be described below with reference to several exemplary embodiments. It should be understood that these embodiments are presented merely to enable those skilled in the art to better understand and practice the invention and are not intended to limit the scope of the invention in any way. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

The present application is further illustrated by the following specific examples.

EXAMPLE 1 preparation of the Medium composition

The preparation process is as follows:

firstly, placing the container into an ultrasonic cleaner for cleaning, and flushing with pure water for three times after the cleaning is finished.

And secondly, weighing MH broth and brain heart infusion according to a formula, and dissolving with water.

And thirdly, filtering the dissolved substances, namely, firstly, performing primary filtration through a 0.4 mu m filter membrane, and then performing primary, secondary or tertiary filtration through a 0.22 mu m filter membrane to reduce the existence of particulate matters, namely, performing filtration according to the filtration conditions of different proportions identified in the table.

Fourth, the filtered culture medium composition is subjected to high-pressure moist heat sterilization.

And fifthly, culturing the sterilized culture medium composition at 35 ℃ for 48 hours, performing sterility test on the sterilized culture medium composition, and inoculating the common bacteria to test the culture capacity of the culture medium. Culturing at 35℃for 48 hours is only an example, and the temperature may be between 30℃and 40℃for 36 hours to 72 hours.

The formulation of the medium composition is shown in Table 1.

Table 1.

Main component	MH	Brain heart infusion	Pure water
				A culture medium formula	21g/L	12g/L	1L
B culture medium formula	5g/L	40g/L	1L
				C culture medium formula	27g/L	5g/L	1L
D culture medium formula	0.5g/L	60g/L	2L
				E culture medium formula	40g/L	0.5g/L	2L
F culture medium formula	4g/L	10g/L	2L
				G culture medium formula	20g/L	30g/L	2L
H culture medium formula	10g/L	50g/L	1L
				I culture medium formula	18g/L	15g/L	1L
J Medium formulation	25g/L	20g/L	1L

Example 2-preparation of the culture medium composition the preparation process was as follows:

In the second step, MH broth 21g/L (g/L) and brain heart infusion 12g/L were weighed and dissolved in 1L water.

In a third step, the solubilizate is filtered, first through a 0.4 μm (micrometer) filter, and then through a 0.22 μm filter for three times to reduce the presence of particulate material.

And fifthly, culturing the sterilized culture medium composition for 48 hours at 35 ℃, performing sterility test on the sterilized culture medium composition, and inoculating clinical common bacteria to test the culture capacity of the culture medium for later use to obtain the culture medium composition A.

Example 3-preparation of the culture medium composition the preparation process was as follows:

and firstly, placing the container into an ultrasonic cleaner for cleaning, and flushing twice with pure water after the cleaning is finished.

In the second step, 5g/L (g/L) of MH broth and 40g/L of brain heart infusion were weighed and dissolved in 1L of water.

In a third step, the solubilizate is filtered, first through a 0.4 μm (micrometer) filter, and then through a 0.22 μm filter to reduce the presence of particulate material.

And fifthly, culturing the sterilized culture medium composition for 48 hours at 35 ℃, performing sterility test on the sterilized culture medium composition, and inoculating clinical common bacteria to test the culture capacity of the culture medium for later use to obtain the culture medium composition B.

Example 4-preparation of the culture medium composition the preparation process was as follows:

In the second step, 27g/L (g/L) of MH broth and 5g/L of brain heart infusion were weighed and dissolved in 1L of water.

And fifthly, culturing the sterilized culture medium composition at 35 ℃ for 48 hours, performing sterility test on the sterilized culture medium composition, and inoculating clinical common bacteria to test the culture capacity of the culture medium for later use to obtain the composition of the culture medium composition C.

EXAMPLE 5 preparation of the Medium composition

The preparation procedure was similar to that of examples 2 to 4, and a D medium composition, an E medium composition, an F medium composition, a G medium composition, an H medium composition, an I medium composition, and a J medium composition were prepared according to the formulation composition of the medium composition of example 1.

Example 6-optimal Medium composition formulation screening purposes: through preparing the culture medium composition, the aim of ensuring the rapid growth of clinically common bacteria and achieving the bacterial count in as short a time (1-4) as possible is selected, namely the aim of rapidly counting the bacterial count by resistance is fulfilled.

The experimental steps are as follows:

placing the container into an ultrasonic cleaner for cleaning, and flushing with pure water for three times after the cleaning is finished;

weighing and dissolving according to the formula requirement;

the dissolved reagent was subjected to primary filtration (0.4 μm filter), and after completion of filtration, the reagent was subjected to filtration with 0.22 μm filter three times, and the results were recorded by resistance counting, or the test was performed directly using the medium compositions of examples 2 to 4.

The time required for inoculating 50cfu/ml of Escherichia coli (ATCC 25922), pseudomonas aeruginosa (ATCC 27853), staphylococcus aureus (ATCC 25923), enterococcus faecalis (ATCC 29212) and Streptococcus pneumoniae (ATCC 49619) to reach a value of about 200 was recorded, and the test results are shown in Table 2.

TABLE 2

Time/h is time/hour experimental result analysis:

according to five standard strains in the test result, bacterial growth can be satisfied within the range of the formula for 4 hours to reach a resistance count value of about 200, wherein different proportions of MH and brain heart infusion can lead to inconsistent growth time when the resistance count of various bacteria reaches 200, wherein the formula of the culture medium A is the optimal proportion, and the time required for the growth of the five standard strains is the shortest, namely the optimal value.

EXAMPLE 7 screening of Process for preparation of Medium composition

The purpose is as follows: by preparing the culture medium composition, the optimal filtration mode is selected to remove the particulate matter so as to ensure that the culture medium can meet the requirements of bacterial culture resistance count.

The experimental steps are as follows:

1. placing the container into an ultrasonic cleaner for cleaning, and flushing with pure water for three times after the cleaning is finished;

2. weighing and dissolving according to the requirements of the culture medium formula A;

3. the dissolved reagent was subjected to primary filtration (0.4 μm filter), and after completion of filtration, the reagent was filtered once, twice and three times with 0.22 μm filter, and the number of particulate matters was counted by electric resistance, and the results are shown in Table 3;

TABLE 3 Table 3

Analysis of experimental results: according to the test result, the filter membrane is analyzed to remove 43% of the particulate matters once, 92.6% of the particulate matters are removed twice, 98.6% of the particulate matters are removed three times, the requirement of reducing the bacterial count background is completely met, the dissolved reagent is subjected to primary filtration by a 0.4 mu m filter membrane, the particulate matters removing effect of the culture medium composition after the filtration is finished by the 0.22 mu m filter membrane for three times is optimal, the requirement of resistance bacteria count on the minimum of the particulate matters can be met, and unexpected effects are generated.

While the basic concepts have been described above, it will be apparent to those skilled in the art that the foregoing detailed disclosure is by way of example only and is not intended to be limiting. Although not explicitly described herein, various modifications, improvements, and adaptations of the present application may occur to one skilled in the art. Such modifications, improvements, and modifications are intended to be suggested within this application, and are therefore within the spirit and scope of the exemplary embodiments of this application.

Meanwhile, the present application uses specific words to describe embodiments of the present application. Reference to "one embodiment," "an embodiment," and/or "some embodiments" means that a particular feature, structure, or characteristic is associated with at least one embodiment of the present application. Thus, it should be emphasized and should be appreciated that two or more references to "an embodiment" or "one embodiment" or "an alternative embodiment" in various positions in this specification are not necessarily referring to the same embodiment. Furthermore, certain features, structures, or characteristics of one or more embodiments of the present application may be combined as suitable.

Furthermore, those skilled in the art will appreciate that the various aspects of the invention are illustrated and described in the context of a number of patentable categories or circumstances, including any novel and useful procedures, machines, products, or materials, or any novel and useful modifications thereof.

Furthermore, the order in which the elements and sequences are presented, the use of numerical letters, or other designations are used in the application and are not intended to limit the order in which the processes and methods of the application are performed unless explicitly recited in the claims. While certain presently useful inventive embodiments have been discussed in the foregoing disclosure, by way of various examples, it is to be understood that such details are merely illustrative and that the appended claims are not limited to the disclosed embodiments, but, on the contrary, are intended to cover all modifications and equivalent arrangements included within the spirit and scope of the embodiments of the present application.

Likewise, it should be noted that in order to simplify the presentation disclosed herein and thereby aid in understanding one or more inventive embodiments, various features are sometimes grouped together in a single embodiment, figure, or description thereof. This method of disclosure, however, is not intended to imply that more features than are presented in the claims are required for the subject application. Indeed, less than all of the features of a single embodiment disclosed above.

In some embodiments, numbers describing the components, number of attributes are used, it being understood that such numbers being used in the description of embodiments are modified in some examples by the modifier "about," approximately, "or" substantially. Unless otherwise indicated, "about," "approximately," or "substantially" indicate that the number allows for a 20% variation. Accordingly, in some embodiments, numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and employ a method for preserving the general number of digits. Although the numerical ranges and parameters set forth herein are approximations that may be employed in some embodiments to confirm the breadth of the range, in particular embodiments, the setting of such numerical values is as precise as possible.

Each patent, patent application publication, and other material, such as articles, books, specifications, publications, documents, etc., cited in this application is hereby incorporated by reference in its entirety. Except for application history documents that are inconsistent or conflicting with the present application, documents that are currently or later attached to this application for which the broadest scope of the claims to the present application is limited. It is noted that the descriptions, definitions, and/or terms used in the subject matter of this application are subject to such descriptions, definitions, and/or terms if they are inconsistent or conflicting with such descriptions, definitions, and/or terms.

Finally, it should be understood that the embodiments described herein are merely illustrative of the principles of the embodiments of the present application. Other variations are also possible within the scope of this application. Thus, by way of example, and not limitation, alternative configurations of embodiments of the present application may be considered in keeping with the teachings of the present application. Accordingly, embodiments of the present application are not limited to only the embodiments explicitly described and depicted herein.

Claims

1. Use of a culture medium composition for bacterial resistance counting, characterized in that the composition of the culture medium composition is: MH broth at a concentration of 0.5 g/l to 40g/l, brain heart infusion at a concentration of 0.5 g/l to 60 g/l, and water.

2. Use of a culture medium composition according to claim 1 for bacterial resistance counting, characterized in that the composition of the culture medium composition is: MH broth at a concentration of 5g/l to 27g/l, brain heart infusion at a concentration of 5g/l to 40g/l, and water.

3. Use of a culture medium composition according to claim 2 for bacterial resistance counting, characterized in that the concentration of MH broth is 21g/l, the concentration of brain heart infusion is 12g/l, and the volume of water is 1 l.

4. Use of a culture medium composition according to claim 2 for bacterial resistance counting, characterized in that the concentration of MH broth is 5g/l, the concentration of brain heart infusion is 40g/l, and the volume of water is 1 l.

5. Use of a culture medium composition according to claim 2 for bacterial resistance counting, characterized in that the concentration of MH broth is 27g/l, the concentration of brain heart infusion is 5g/l, and the volume of water is 1 l.

6. Use of a culture medium composition according to any one of claims 1 to 5 for bacterial resistance counting, characterized in that the preparation method of the culture medium composition comprises:

cleaning an instrument used for weighing and dissolving the culture medium composition;

weighing the MH broth, the brain heart infusion and the water;

dissolving the weighed MH broth, brain heart infusion and water to obtain a dissolved substance;

filtering the dissolved matter to obtain a culture medium composition;

the culture medium composition is sterilized.

7. The use of the culture medium composition according to claim 6 for bacterial resistance counting, wherein filtering the lysate comprises: the lysate is first filtered and then filtered again, wherein the number of times of the filtering again is one, two or three.

8. The use of the culture medium composition according to claim 7, wherein the primary filtration uses a filter pore size of 0.3 to 1 micron and the secondary filtration uses a filter pore size of 0.1 to 0.3 micron.

9. The use of the culture medium composition according to claim 8, wherein the primary filtration uses a filter pore size of 0.4 microns and the secondary filtration uses a filter pore size of 0.22 microns.