CN110819689A - Culture medium and application thereof in detection of escherichia coli - Google Patents
Culture medium and application thereof in detection of escherichia coli Download PDFInfo
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Abstract
The invention relates to a culture medium and application thereof for detecting escherichia coli, belonging to the technical field of escherichia coli detection, wherein the culture medium comprises an enzyme substrate 1 of 4-methylumbelliferone- β -D-glucuronide, a nutrient 2 of tryptone or peptone and a stock solution 3, wherein the stock solution 3 comprises phosphate buffer solution with the content of 10g of potassium dihydrogen phosphate, 25g of dipotassium hydrogen phosphate, 20g of disodium hydrogen phosphate and 2g of ammonium chloride per liter, 11g/L of magnesium sulfate solution, 28g/L of calcium chloride solution and 0.15g/L of ferric chloride solution, the stock solution 3 of the culture medium is favorable for accelerating the escherichia coli to generate enzyme decomposition substrates, and the detection time can be shortened aiming at low-concentration escherichia coli, the stock solution 3 is an independent salt solution and can be stably stored at 0-4 ℃ for more than 6 months, so that online monitoring can be conveniently realized.
Description
Technical Field
The invention relates to the technical field of escherichia coli detection, and particularly relates to a culture medium and application thereof in detection of escherichia coli.
Background
The traditional detection method of escherichia coli (E.coli) mainly comprises multitubular fermentation, a filter membrane method and a plate counting method (GB4789.3-2016 replaces GB/T4789.32-2002), and the methods have the defects of complex operation, long time consumption and low sensitivity. In recent years, scientists have developed many rapid detection methods, which are mainly classified into three categories: molecular biological methods, immunoassay techniques, and metabolic techniques. Wherein, the molecular biology method is based on the detection of genetic materials, and the accuracy is guaranteed. However, the method needs professional instruments and technologies and cannot realize online monitoring; and the detection signal is based on genetic material amplification (PCR, etc.), and the detection limit is generally 102The immunoassay technology can realize rapid detection, but the technology only aims at the detection of surface antigen and antibody known bacteria, such as an escherichia coli O157: H7 kit, is aimed at the detection of certain serotype escherichia coli, and cannot complete the broad-spectrum detection of all escherichia coli in a water body, the technology based on the metabolic detection can detect most types of escherichia coli in water, at present, the most effective method is to utilize the characteristic that E.coli generates β -glucuronidase, 4-methylumbelliferone- β -D-glucuronide (MUG) can be decomposed to generate fluorescent 4-methylumbelliferone (4-MU) for detection, and 94-97% of escherichia coli have the metabolic pathway.
Prior art 1 has been listed in the national standard method (HJ1001-2018) using a minimum Medium ONPG-MUG (MMO-MUG) Medium with 97-well plates, counting the cells that fluoresce at the end of the reaction (cells where E.coli converts MUG to 4-MU) and looking up the table for the number of cells that fluoresce to give the maximum possible number of E.coli (MPN) in the sample. The method has the following defects: 1. the reaction time is longer than 24h, so that most of MUG in the culture medium can be converted into 4-MU and the MUG is accumulated to a macroscopic quantity; 2. the quantity difference of the escherichia coli contained in each grid cannot be reflected, and the differences are marked as a 'grid with fluorescence', and then a table is looked up, so that the specific concentration value of the escherichia coli in the sample cannot be obtained; i.e. the method gives a result of the maximum possible number MPN, not a specific concentration value; 3. the detection limit was 10MPN/L and no linearity was obtained since the number of E.coli in a single cell was not distinguished. 4. The solution is prepared and then needs to be used immediately, and the operation and maintenance of an online monitoring instrument are difficult to realize. The Chinese patent application No. 200410051350.7 of prior art 2 discloses a detection method for rapidly detecting escherichia coli, which is to divide prepared MUG culture solution into test tubes, inoculate a sample into the culture solution, culture the culture solution for 6-12h at 35-44 ℃, then use a fluorescence spectrophotometer to detect the fluorescence intensity of the culture solution at 366nm, and detect the number of escherichia coli in the sample according to the corresponding relation between the fluorescence intensity and the bacteria concentration under the condition. The method has the following defects: 1. the detection time of less than 10 escherichia coli samples is about 20-24 hours; 2. the preparation needs to be used immediately after the solution is prepared, and the operation can be realized in a laboratory, but the operation and maintenance of an online monitoring instrument are difficult to realize. The composition of the medium for detecting escherichia coli based on the metabolism can be summarized into three components: enzyme substrates, such as MUG and the like; nutrients such as peptone, beef extract, yeast extract, and the like; salt solution, MMO-MUG salt solution is widely used in the field. Of these three components, the enzyme substrate acts as an indicator; the nutrients provide substrates for the fission and proliferation of escherichia coli; salt solutions are effective factors affecting the activity and metabolic activity of E.coli. Therefore, the research on the salt solution can accelerate the reaction and improve the detection speed.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provides a culture medium and application thereof in detecting escherichia coli.
In order to solve the technical problems, the technical scheme of the invention is as follows:
the present invention provides a culture medium comprising: enzyme substrate 1, nutrient 2, and stock solution 3;
the enzyme substrate 1 is 4-Methylumbelliferyl- β -D-glucuronide (4-Methylumbelliferyl- β -D-glucuronide, abbreviated as MUG);
the nutrient 2 is tryptone or peptone;
the storage solution 3 is a salt solution and comprises the following components:
phosphate buffer solution: the content of each liter of the compound comprises 10g of monopotassium phosphate, 25g of dipotassium phosphate, 20g of disodium phosphate and 2g of ammonium chloride;
magnesium sulfate solution with the content of 11g per liter;
calcium chloride solution with a content of 28g per liter;
ferric chloride solution, 0.15g per liter.
In the above technical solution, it is preferable that: the culture medium comprises:
the enzyme substrate 1 is 4-methylumbelliferone- β -D-glucuronide, and the mass concentration of the enzyme substrate is 0.25-150 mg/L;
the nutrient 2 is tryptone, and the mass concentration of the tryptone is 0-10 g/L;
the dosage of each component of the stock solution 3 is 0.2-5 mL/L.
In the above technical solution, it is further preferable that: the culture medium comprises:
the enzyme substrate 1 is 4-methylumbelliferone- β -D-glucuronide (MUG), and the mass concentration of the enzyme substrate is 75 mg/L;
the nutrient 2 is tryptone, and the mass concentration of the tryptone is 10 g/L;
the stock solutions 3 each contained 1 mL/L.
In the above technical solution, it is preferable that: when fungi or gram-positive bacteria are present in the actual sample to be tested, the culture medium may also be supplemented with amphotericin b (amphotericin b) or solanum extract (solanum extract) or bile salts.
In the above technical solution, it is further preferable that: to 1L of the medium, amphotericin B (amphotericin B)1mg, or Solanum plant extract (solanum extract) 500mg, or bile salt 1.5g was added.
The invention provides an application of the culture medium in detecting escherichia coli.
The culture medium of the invention is used for detecting escherichia coli and comprises the following steps:
step 1, preparing a culture medium according to a formula.
And 2, inoculating escherichia coli into a pure culture medium by aseptic operation, culturing to a stationary phase, and performing serial dilution to prepare a standard curve.
And 3, quantifying the bacterial liquid obtained in the step 2 by using a standard plate counting method (CFU) to obtain the true concentration of the pure cultured escherichia coli.
Step 4, inoculating each bacterial liquid diluted to a series of concentrations in the step 2 into the culture medium prepared in the step 1, and calculating the number of escherichia coli contained in each bacterial liquid according to the CFU quantitative result in the step 3;
6, incubating the culture medium obtained in the step 4 and the step 5 at 35-44 ℃ for 6-19h, measuring fluorescence intensity, exciting 366nm, and reading the peak value of an emission peak at 450 nm;
step 7, drawing a standard curve according to the number of the escherichia coli measured in the step 4 and the emission peak value measured in the step 6; and (5) substituting the peak values measured in the step (5) and the step (6) into a standard curve, and calculating to obtain the concentration of the escherichia coli in the actual sample to be measured.
In the above technical solution, it is preferable that: in step 5, 1L of the culture medium is supplemented with amphotericin B (amphotericin B)1mg, or Solanum plant extract (solanum extract) 500mg, or bile salt 1.5 g.
The invention has the beneficial effects that:
1. the method for detecting escherichia coli by using the culture medium provided by the invention has the following advantages:
1) the detection time is shortened, and the detection is particularly aimed at the detection of low-concentration escherichia coli, the 3-salt solution of the storage solution of the culture medium is favorable for accelerating the escherichia coli to generate an enzymatic decomposition substrate, and the slow-up of the low-concentration escherichia coli is detected for 5 hours by comparing with the prior art, so that the result is obtained on the same day; in the prior art 1 and the prior art 2, the detection result needs to be obtained within more than 24 hours;
2) the detection result is a specific concentration value of the escherichia coli, but not a maximum possible number MPN value;
3) the sensitivity is high, and the detection of as low as 1 escherichia coli in a reaction system can be realized.
2. The storage solution 3 of the culture medium provided by the invention is an independent saline solution, and the solution can be stably stored for more than 6 months at 0-4 ℃, so that online monitoring is conveniently realized.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a graph showing the time course of fluorescence signal generation using the medium of example 1 of the present invention and the medium of prior art 2.
FIG. 2 is a plot of CFU versus fluorescence intensity for E.coli in the culture medium of example 2 of the present invention.
FIG. 3 is a plot of CFU versus fluorescence intensity for E.coli in the culture medium of example 3 of the present invention.
FIG. 4 is a plot of CFU versus fluorescence intensity for E.coli in the culture medium of example 4 of the present invention.
FIG. 5 is a plot of CFU versus fluorescence intensity for E.coli in the medium of example 5.
Detailed Description
The present invention will be described in detail with reference to specific examples, which should not be construed as limiting the scope of the present invention. For those skilled in the art to which the invention pertains, numerous deductions or substitutions may be made without departing from the spirit of the invention, which should be construed as falling within the scope of the invention.
Example 1
1. Solution 1 (labeled this) was prepared according to the medium formulation of the present invention
The formula of the culture medium comprises 7.5mg/L of enzyme substrate 14-methylumbelliferone- β -D-glucuronide (MUG) and 10.0g/L of nutrient 2 tryptone, wherein 1mL/L of each storage solution is added;
the stock solution 3 is a salt solution and comprises the following components:
phosphate buffer solution: the content of each liter of the compound comprises 10g of monopotassium phosphate, 25g of dipotassium phosphate, 20g of disodium phosphate and 2g of ammonium chloride;
magnesium sulfate solution with the content of 11g per liter;
calcium chloride solution with a content of 28g per liter;
ferric chloride solution, 0.15g per liter.
2. Solution 2 (labeled other) was prepared according to the formulation disclosed in prior art 2;
prior Art 2 Medium A commercially available product was purchased, and 1L of the medium contained the following components:
4-Methylumbelliferone- β -D-glucuronide (MUG)75.0 mg;
tryptone 10.0 g;
ammonium sulfate [ (NH)4)2SO4]5.0g；
Manganese sulfate (MnSO)4)0.5mg；
Zinc sulfate (ZnSO)4)0.5mg；
Magnesium sulfate (MgSO)4)100.0mg；
10.0g of sodium chloride (NaCl);
calcium chloride (CaCl)2)50.0mg；
Sodium sulfite (Na)2SO3)40.0mg；
KH2PO40.9g；
Na2HPO46.2g。
3. Inoculating E.coli DH5 α in LB medium by aseptic technique, culturing to stationary phase, centrifuging and cleaning the cultured E.coli 3 times with fixed amount of OD600 ═ 0.2 as bacterial liquid A, diluting the bacterial liquid A102Diluting 10 to obtain bacterial liquid B4Diluting 10 for bacterial liquid C5Diluting 10 for bacterial liquid D6Bacterial liquid E is obtained.
4. The bacterial suspension was quantified by standard plate Counting (CFU), 50. mu.L of bacterial suspension C was inoculated onto a plate, and the average bacterial suspension contained 174CFU of E.coli, and the concentration of other bacterial suspensions was estimated from this.
5. mu.L of inoculum E115 was added to 5mL of solution 1 and solution 2, respectively. From the CFU quantification result, the number of Escherichia coli contained in E115. mu.L of the bacterial suspension was 4 CFU.
6. Incubation was carried out at 37 ℃ for 0-24h, and the fluorescence intensity was measured every hour. 366nm excitation, reading the peak emission at 450 nm.
7. A standard curve was drawn based on the number of E.coli cells measured and the fluorescence intensity, and the results are shown in FIG. 1: the fluorescence generated signal using the medium formulation of this example was 5 hours earlier than using the medium formulation of prior art 2, greatly shortening the time for detection of low concentration E.coli.
Example 2
1. The solution is prepared according to the formula of the culture medium
The formula of the culture medium comprises 0.25mg/L of enzyme substrate 14-methylumbelliferone- β -D-glucuronide (MUG) and 10.0g/L of nutrient 2 tryptone, and 3 portions of stock solutions with the volume of 5mL/L are added;
stock solution 3 was a salt solution having the same composition as stock solution 3 in step 1 of example 1.
2. Coli ATCC25922 was inoculated in LB medium in a sterile operation, and the culture was carried out to a stationary phase at 37 ℃ for about 12 hours at 220 rpm. After the culture, e.coli was centrifuged and washed 3 times, and a bacterial suspension a was prepared by quantifying OD600 ═ 0.2 and diluting 10 the bacterial suspension a2 Diluting 10 to obtain bacterial liquid B4Diluting 10 for bacterial liquid C5Diluting 10 for bacterial liquid D6Bacterial liquid E is obtained.
3. The bacterial suspension was quantified by a standard plate counting method (CFU), 50. mu.L of bacterial suspension D was inoculated on a plate, and the average of the bacterial suspension D contained Escherichia coli (E.coli) of 32.5CFU, and the concentration of other bacterial suspensions was estimated from the results.
4. The cells were inoculated with E15. mu.L, 30. mu.L, 60. mu.L, 90. mu.L, 120. mu.L and 150. mu.L, respectively, in 5mL of the medium. The CFU quantification results calculate the contents of E.coli 1, 2, 4, 6, 8, 10CFU in sequence.
5. Incubation was carried out at 35 ℃ for 19h and the fluorescence intensity was measured. 366nm excitation, reading the peak emission at 450 nm.
6. A standard curve was drawn based on the number of E.coli cells measured and the fluorescence intensity, and the results are shown in FIG. 2.
Example 3
1. The solution is prepared according to the formula of the culture medium
The formula of the culture medium comprises 7.5mg/L of enzyme substrate 14-methylumbelliferone- β -D-glucuronide (MUG) and 1.0g/L of nutrient 2 tryptone, and 1mL/L of each storage solution is added.
Stock solution 3 was a salt solution having the same composition as stock solution 3 in step 1 of example 1.
2. Coli ATCC25922 was inoculated in LB medium in a sterile operation, and the culture was carried out to a stationary phase at 37 ℃ for about 12 hours at 220 rpm. After the culture, e.coli was centrifuged and washed 3 times, and a bacterial solution a was prepared by quantifying OD600 ═ 0.2 and diluted 10 with bacterial solution a2 Diluting 10 to obtain bacterial liquid B4Diluting 10 for bacterial liquid C5Diluting 10 for bacterial liquid D6Bacterial liquid E is obtained.
3. The bacterial suspension was quantified by a standard plate count method (CFU), 50. mu.L of a bacterial suspension D plate was inoculated, and the average content of Escherichia coli was 32.5CFU, and the concentration of other bacterial suspensions was estimated from the results.
4. Coli to the medium. The experiment was carried out by inoculating E15. mu.L, 150. mu.L, 300. mu.L, 450. mu.L and 600. mu.L into 5mL of culture medium, and calculating the contents of E.coli 1, 10, 20, 30 and 40CFU in sequence from the CFU quantitative results.
5. The incubation was carried out at 37 ℃ for 16h, and the fluorescence intensity was measured. 366nm excitation, reading the peak emission at 450 nm.
6. A standard curve was drawn based on the number of E.coli cells measured and the fluorescence intensity, and the results are shown in FIG. 3.
Example 4
1. The solution is prepared according to the formula of the culture medium
The culture medium formula of the invention comprises 150.0mg/L indissolvable enzyme substrate 14-methylumbelliferone- β -D-glucuronide (MUG), 1.0g/L nutrient 2 peptone and 0.2mL/L storage solution 3;
stock solution 3 was a salt solution having the same composition as stock solution 3 in step 1 of example 1.
2. Coli ATCC25922 was inoculated in LB medium in a sterile operation, and the culture was carried out to a stationary phase at 37 ℃ for about 12 hours at 220 rpm. After the culture, e.coli was centrifuged and washed 3 times, and a bacterial solution a was prepared by quantifying OD600 ═ 0.2 and diluted 10 with bacterial solution a2 Diluting 10 to obtain bacterial liquid B4Diluting 10 for bacterial liquid C5Diluting 10 for bacterial liquid D6Bacterial liquid E is obtained.
3. The bacterial suspension was quantified by a standard plate count method (CFU), 50. mu.L of a bacterial suspension D plate was inoculated, and the average content of Escherichia coli was 32.5CFU, and the concentration of other bacterial suspensions was estimated from the results.
4. Coli to the medium. The experiment was carried out by inoculating the culture medium C15. mu.L, 30. mu.L, 60. mu.L, 90. mu.L, 120. mu.L and 150. mu.L in 5mL culture medium, and then sequentially containing Escherichia coli 100, 200, 400, 600, 800 and 1000CFU according to the CFU quantitative result.
5. The incubation was carried out at 44 ℃ for 8h, and the fluorescence intensity was measured. 366nm excitation, reading the peak emission at 450 nm.
6. A standard curve was drawn based on the number of E.coli cells measured and the fluorescence intensity, and the results are shown in FIG. 4.
Example 5
1. The solution is prepared according to the formula of the culture medium
The culture medium formula of the invention comprises 150.0mg/L indissolvable enzyme substrate 14-methylumbelliferone- β -D-glucuronide (MUG), 10.0g/L nutrient 2 peptone and 0.2mL/L storage solution 3;
stock solution 3 was a salt solution having the same composition as stock solution 3 in step 1 of example 1.
2. Inoculating E.coli DH5 α in LB medium by aseptic technique, culturing to stationary phase, centrifuging and cleaning the cultured E.coli 3 times at 37 deg.C and 220 rpm, taking OD600 ═ 0.2 as bacterial liquid A, diluting the bacterial liquid A102Diluting 10 to obtain bacterial liquid B4Diluting 10 for bacterial liquid C5Diluting 10 for bacterial liquid D6Bacterial liquid E is obtained.
3. The bacterial suspension was quantified by a standard plate count method (CFU), 50. mu.L of a bacterial suspension D plate containing 17.4CFU of Escherichia coli on average was inoculated, and the concentration of other bacterial suspensions was estimated from the results.
4. Coli to the medium. In this experiment, 14.3. mu.L, 28.6. mu.L, 57.5. mu.L, 86. mu.L, 115. mu.L and 143. mu.L of the bacterial suspension C were inoculated into 5mL of the culture medium, and the bacterial suspension C contained Escherichia coli 50, 100, 200, 300, 400 and 500CFU in this order as calculated from the CFU quantification results.
5. The incubation was carried out at 37 ℃ for 10 hours, and the fluorescence intensity was measured. 366nm excitation, reading the peak emission at 450 nm.
6. A standard curve was drawn based on the number of E.coli cells measured and the fluorescence intensity, and the results are shown in FIG. 5.
Example 6
1. Culture medium
1) In solution 1 prepared in example 1, amphotericin B1mg/L or solanum extract 500mg/L was added.
2) In the prior art 1, a commercial commodity is purchased, operated according to a specification and checked to obtain a result;
the culture medium of the prior art 1 has the following formula:
a minimum Medium ONPG-MUG (MMO-MUG) culture Medium was used, and 1L of the Medium contained the following components:
ammonium sulfate [ (NH)4)2SO4]5.0g；
Manganese sulfate (MnSO)4)0.5mg；
Zinc sulfate (ZnSO)4)0.5mg；
Magnesium sulfate (MgSO)4)100.0mg；
10.0g of sodium chloride (NaCl);
calcium chloride (CaCl)2)50.0mg；
Sodium sulfite (Na)2SO3)40.0mg；
Amphotericin B (amphotericin B)1.0 mg;
o-nitrophenyl- β -D-galactopyranoside (ONPG)500.0 mg;
4-Methylumbelliferone- β -D-glucuronide (MUG)75.0 mg;
500.0mg of Solanum plant extract (solanum extract);
5.3g of N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid sodium salt (HEPES sodium salt);
6.9g of N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES);
3) prior Art 2, a commercially available product, solution 2 prepared in example 1, was purchased.
2. The standard curves for solution 1 and solution 2 were plotted, respectively, according to the methods described in examples 2 to 5.
3. Actual sample
Sample 1: laboratory sewer water sample.
Sample 2: melting ice and snow.
Sample 3: lake water.
Sample 4: the cotton balls of the table and the chair of a certain community hospital are wiped and soaked by 100mL of sterile physiological saline.
The pH of the sample is adjusted to be neutral after sampling, and no other treatment is carried out, but immediate measurement after sampling is ensured. Dividing 4 parts of the culture medium into 4 parts, and respectively detecting the number of the escherichia coli by using the culture medium of the invention, the culture medium of the prior art 1, the culture medium of the prior art 2 and a standard plate counting method. 100mL of each of the above samples was prepared according to the formula and dosage of the culture medium of the present invention, the culture medium of prior art 1, and the culture medium of prior art 2. The other was concentrated with a sterile filter paper sheet and then plate counted.
4. Incubation at 37 ℃ for 16h, excitation at 366nm, reading the peak of the emission peak at 450nm, fitting the peak into a standard curve, and calculating the concentration of Escherichia coli.
5. The results are shown in the following table:
prior art 1 and its similar methods use the "presence" or "absence" of E.coli in the culture medium of one grid at the end of the reaction, without differentiation of the amount. The result given by the prior art 1 is the maximum possible number MPN, and the accuracy is poor. Prior art 2 and the present invention establish a relationship between a specific product and the concentration of Escherichia coli in a reaction process. Because the different formulations of the salt solution lead to different reaction speeds, the invention is 2 hours faster than the prior art for low concentration samples. That is, at 16h, the medium formulation of the present invention successfully detected the amount of E.coli in samples 1 and 2, but it could not be achieved by prior art 2. The standard plate counting method has accurate result, but large error, long plate culture time and is not beneficial to preparing an online monitoring instrument.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (8)
1. A culture medium comprising: enzyme substrate 1, nutrient 2, and stock solution 3;
the enzyme substrate 1 is 4-methylumbelliferone- β -D-glucuronide (MUG);
the nutrient 2 is tryptone or peptone;
it is characterized in that the preparation method is characterized in that,
the storage solution 3 is a salt solution and comprises the following components:
phosphate buffer solution: the content of each liter of the compound comprises 10g of monopotassium phosphate, 25g of dipotassium phosphate, 20g of disodium phosphate and 2g of ammonium chloride;
magnesium sulfate solution with the content of 11g per liter;
calcium chloride solution with a content of 28g per liter;
ferric chloride solution, 0.15g per liter.
2. The culture medium according to claim 1, comprising:
the enzyme substrate 1 is 4-methylumbelliferone- β -D-glucuronide, and the mass concentration of the enzyme substrate is 0.25-150 mg/L;
the nutrient 2 is tryptone, and the mass concentration of the tryptone is 0-10 g/L;
the dosage of each component of the stock solution 3 is 0.2-5 mL/L.
3. The culture medium according to claim 1, comprising:
the enzyme substrate 1 is 4-methylumbelliferone- β -D-glucuronide (MUG), and the mass concentration of the enzyme substrate is 75 mg/L;
the nutrient 2 is tryptone, and the mass concentration of the tryptone is 10 g/L;
the stock solutions 3 each contained 1 mL/L.
4. The culture medium according to any one of claims 1 to 3, wherein amphotericin B or an extract of Solanum or bile salts is added to the culture medium when fungi or gram-positive bacteria are detected in the actual sample.
5. The medium according to claim 4, wherein the amphotericin B is added to 1L of the medium at 1mg, or the extract of the plant belonging to genus Solanum at 500mg, or the bile salt at 1.5 g.
6. Use of a culture medium according to any one of claims 1 to 3 for the detection of E.
7. Use of the culture medium according to claim 6 for the detection of E.coli, comprising the following steps:
step 1, preparing a culture medium according to a formula;
inoculating escherichia coli into a pure culture medium by aseptic operation, culturing to a stationary phase, diluting to obtain a series of concentration gradients, and preparing a standard curve;
step 3, quantifying the bacterial liquid obtained in the step 2 by using a standard plate counting method (CFU) to obtain the true concentration of the pure cultured escherichia coli;
step 4, inoculating the bacterial liquid obtained in the step 2 into the culture medium prepared in the step 1, and calculating the number of escherichia coli contained in each bacterial liquid according to the CFU quantitative result obtained in the step 3;
step 5, inoculating an actual sample to be tested into the culture medium prepared in the step 1, and adding amphotericin B or solanum plant extracts or bile salts; 6, incubating the culture medium obtained in the step 4 and the step 5 at 35-44 ℃ for 6-19h, measuring fluorescence intensity, exciting 366nm, and reading the peak value of an emission peak at 450 nm;
step 7, drawing a standard curve according to the number of the escherichia coli measured in the step 4 and the emission peak value measured in the step 6; and (5) substituting the peak values measured in the step (5) and the step (6) into a standard curve, and calculating to obtain the concentration of the escherichia coli in the sample to be measured.
8. The use of the culture medium according to claim 7 for detecting Escherichia coli, wherein amphotericin B1mg, or Solanum plant extract 500mg, or bile salt 1.5g is added to 1L of the culture medium in step 5.
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