Vibrio vulnificus selective culture medium and preparation method thereof

Abstract

The present invention discloses a Vibrio vulnificus selective culture medium and a preparation method thereof. The culture medium comprises a basal culture medium and additives; the basal culture medium comprises peptone, beef extract powder, sodium chloride, potassium chloride, a long-chain alkyl sulfate anionic surfactant or nisin, D-cellobiose, an acid-base indicator, and agar; the additive is tellurite; and the solvent is water. When the basal culture medium contains nisin, the additives further comprise vancomycin, amphotericin B, and polymyxin E. The Vibrio vulnificus selective culture medium of the present invention has high growth selectivity, effectively inhibits the growth of Gram-positive bacteria, effectively inhibits fungal growth, and is sensitive and specific for detecting Vibrio vulnificus. Furthermore, the preparation method does not require autoclaving and avoids the inconvenience of preparing the high-content component, cellobiose, into a sterile additive prior to use, making it more convenient.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and particularly relates to a Vibrio vulnificus selective culture medium and a preparation method thereof. Background Art

[0002] Vibrio vulnificus is a significant pathogen of humans and aquacultured animals. A Gram-negative, mildly halophilic bacterium, it can infect humans through exposure to seawater in seafood and wounds, causing gastroenteritis, primary sepsis, and severe wound infections. Primary sepsis has a high mortality rate in immunocompromised individuals, making it the foodborne pathogen with the highest mortality rate in humans. In recent years, with global warming and an increase in seafood consumption, Vibrio vulnificus infection rates have increased in my country and other coastal countries. In 2020, Vibrio vulnificus was included in the National Food Safety Standard for Food Microbiology (GB 4789.44-2020). In aquaculture, Vibrio vulnificus can cause disease in a variety of commercial species, including tilapia, whiteleg shrimp, golden pomfret, Anguilla eel, sturgeon, and grouper, causing significant economic losses to the aquaculture industry. Therefore, the detection of Vibrio vulnificus is of great significance for preventing foodborne diseases and aquaculture animal diseases.

[0003] At present, mCPC and CC culture media are commonly used culture media for detecting Vibrio vulnificus, and they have been recommended or prescribed for use by the U.S. FDA and my country's national food safety standard GB 4789.44-2020. However, the growth selectivity of these two culture media is still not high enough, especially the inhibitory effect on Gram-positive bacteria is poor, which can easily lead to false positive interference of Gram-positive bacteria, resulting in reduced detection specificity. In addition, due to the low selectivity, the basal culture medium of mCPC and CC culture media needs to be sterilized under high pressure during preparation, which in turn leads to the trouble of high-content cellobiose, a relatively easy-to-degrade component, needing to be made into a sterile additive before use during the high-pressure sterilization process (such as the inconvenience of sterile filtration treatment, pipetting and addition, and storage of relatively large volumes of cellobiose aqueous solution).

[0004] Therefore, it is very necessary to develop a Vibrio vulnificus culture medium that is more selective, more sensitive and specific, and easier to prepare. Summary of the Invention

[0005] The purpose of the present invention is to provide a selective culture medium with high selectivity, good inhibition of Gram-positive bacteria and fungal growth, higher sensitivity and specificity for detecting Vibrio vulnificus, no need for high-pressure sterilization, and easy preparation based on the above-mentioned shortcomings of existing culture medium technology.

[0006] The present invention is achieved through the following technical solutions:

[0007] A Vibrio vulnificus selective culture medium comprises a basal culture medium and additives; the basal culture medium comprises 5-20 g / L of peptone, 3-15 g / L of beef extract powder, 10-25 g / L of sodium chloride, 0.5-5 g / L of potassium chloride, 0.1-1 g / L of a long-chain alkyl sulfate anionic surfactant or 0.05-2 g / L of nisin, 6-25 g / L of D-cellobiose, 0.01-0.10 g / L of an acid-base indicator, and 10-20 g / L of agar; the additive comprises 0.1-3.5 mg / L of tellurite, and the solvent is water; wherein, when the basal culture medium contains nisin, the additive further comprises 1-30 mg / L of vancomycin, 0.2-5 mg / L of amphotericin B, and 1.0×10 4 ~1.0×10 6 IU / L. The above concentrations are the final concentrations of each component in the Vibrio vulnificus selective culture medium.

[0008] Preferably, the basal culture medium is 10 g / L peptone, 5 g / L beef extract powder, 15 g / L sodium chloride, 1.0 g / L KCl, 15 g / L D-cellobiose, 0.5 g / L sodium dodecyl sulfate (SDS), 0.04 g / L cresol red, 0.04 g / L bromothymol blue, and 15 g / L agar; the additive is 1.5 mg / L tellurite, and the solvent is water.

[0009] Preferably, the basal culture medium is peptone 10 g / L, beef extract powder 5 g / L, sodium chloride 15 g / L, KCl 1.0 g / L, D-cellobiose 10 g / L, Nisin Z type 0.8 g / L, cresol red 0.04 g / L, bromothymol blue 0.04 g / L, and agar 15 g / L; the additives are potassium tellurite 0.5 mg / L, vancomycin 6 mg / L, amphotericin B 2 mg / L, and polymyxin E 1.0×10 5 IU / L.

[0010] Preferably, the acid-base indicator is one or a mixture of two of cresol red, phenol red, neutral red, bromothymol blue, and thymol blue.

[0011] Preferably, the long-chain alkyl sulfate anionic surfactant is sodium dodecyl sulfate (SDS).

[0012] Preferably, the tellurite is potassium tellurite.

[0013] A second object of the present invention is to provide a method for preparing the aforementioned Vibrio vulnificus selective culture medium, comprising the following steps: mixing the components of the basal culture medium, continuously heating in a hot bath or repeatedly boiling briefly intermittently, and stirring or shaking until completely dissolved; then cooling the mixture to 45-50°C, and adding a sterile additive to prepare the culture medium.

[0014] The continuous heating of the basic culture medium in a hot bath or repeated intermittent short boiling means that the basic culture medium is continuously heated in a 100° C. hot bath or repeated intermittent short boiling, and the total boiling time does not exceed 1 minute.

[0015] Compared with existing culture medium technology, the advantages of the present invention are:

[0016] The Vibrio vulnificus selective culture medium of the present invention has high growth selectivity, good effect of inhibiting the growth of Gram-positive bacteria, and can also effectively inhibit the growth of fungi. It is sensitive and more specific in detecting Vibrio vulnificus. In addition, it does not require high-pressure sterilization during preparation and is easy to prepare, thus avoiding the trouble caused by the need to prepare the high-content component cellobiose into a sterile additive before use. DETAILED DESCRIPTION

[0017] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0018] Example 1:

[0019] The effects of different potassium tellurite concentrations on the detection sensitivity of Vibrio vulnificus were investigated using 10 g / L peptone, 5 g / L beef extract powder, 15 g / L sodium chloride, 1 g / L potassium chloride, 6 g / L D-cellobiose, and 15 g / L agar as the basal medium, with water as the solvent. The medium was prepared by adding 10 g peptone, 5 g beef extract powder, 15 g sodium chloride, 1 g potassium chloride, 6 g D-cellobiose, and 15 g agar to water, then adjusting the volume to 1 L to obtain the basal medium. The medium was then heated continuously in a 100°C hot bath or repeatedly boiled briefly (for a total boiling time of no more than 1 min) with stirring or shaking until completely dissolved. After cooling to 45-50°C, different amounts of sterile potassium tellurite were added, shaken thoroughly, and poured into sterile plates. The medium was then allowed to solidify.

[0020] The results are shown in Table 1.

[0021] Table 1. Effects of different concentrations of potassium tellurite on the detection sensitivity of Vibrio vulnificus

[0022]

[0023] Note: Take Vibrio vulnificus cultured in alkaline peptone water at 37°C under aerobic conditions for 15-18 hours, dilute it with sterile saline to the required concentration, then take 0.25 mL and spread it on the above culture medium, and culture it at 37°C under aerobic conditions for 24 hours; "+" indicates detection, and "-" indicates non-detection.

[0024] As shown in Table 1, excessively high potassium tellurite concentrations can increase the detection limit for Vibrio vulnificus, thereby reducing its sensitivity. Therefore, to achieve sensitive detection of low-concentration Vibrio vulnificus, the optimal potassium tellurite concentration is 0.1 to 3.5 mg / L.

[0025] Example 2:

[0026] The effects of different added concentrations of sodium dodecyl sulfate (SDS) on the detection sensitivity of Vibrio vulnificus and the inhibition of Gram-positive bacteria were investigated using 10 g / L peptone, 5 g / L beef extract powder, 20 g / L sodium chloride, 0.5 g / L potassium chloride, 6 g / L D-cellobiose, 0.1 mg / L potassium tellurite and 15 g / L agar as the basal culture medium.

[0027] The culture medium is prepared by adding 10 g of peptone, 5 g of beef extract powder, 20 g of sodium chloride, 0.5 g of potassium chloride, 6 g of D-cellobiose, 15 g of agar, and different amounts of sodium dodecyl sulfate to water, and then adjusting the volume to 1 L to obtain a basic culture medium. It is continuously heated in a 100°C hot bath or repeatedly boiled intermittently for a short time (the boiling time does not exceed 1 min in total), and stirred or shaken until it is completely dissolved. After cooling it to 45-50°C, 0.1 mg of potassium tellurite is added, shaken well, and poured into a sterile plate. After the culture medium is fully solidified, it is used.

[0028] The results are shown in Table 2.

[0029] Table 2. Effects of different concentrations of SDS on the detection sensitivity of Vibrio vulnificus and inhibition of Gram-positive bacteria

[0030]

[0031] Note: Vibrio vulnificus cultured in alkaline peptone water at 37°C under aerobic conditions for 15-18 hours was diluted with sterile saline to the required concentration, and 0.25 mL of each culture medium was spread on the above culture medium. Enterococcus faecalis and Staphylococcus aureus cultured in TSB broth at 37°C under aerobic conditions for 15-18 hours were streaked onto the above culture medium. After inoculation, the above culture medium was cultured at 37°C under aerobic conditions for 24 hours. "+" indicates detection or growth, and "-" indicates no detection or growth.

[0032] From the results in Table 2, it can be seen that when the concentration of SDS added is too low, the inhibitory effect on Gram-positive bacteria will be poor. Therefore, in order to inhibit the growth of Gram-positive bacteria as much as possible and not affect the sensitivity of detecting Vibrio vulnificus as much as possible, the concentration of SDS added can be 0.1-1 g / L.

[0033] Example 3:

[0034] A Vibrio vulnificus selective culture medium comprises a basal culture medium containing 10 g / L peptone, 5 g / L beef extract powder, 15 g / L sodium chloride, 1.0 g / L KCl, 15 g / L D-cellobiose, 0.5 g / L sodium dodecyl sulfate (SDS), 0.04 g / L cresol red, 0.04 g / L bromothymol blue, and 15 g / L agar; an additive is 1.5 mg / L potassium tellurite, and the solvent is water.

[0035] The culture medium is prepared by adding 10 g of peptone, 5 g of beef extract powder, 15 g of sodium chloride, 1 g of potassium chloride, 15 g of D-cellobiose, 0.5 g of sodium dodecyl sulfate (SDS), 0.04 g of cresol red, 0.04 g of bromothymol blue, and 15 g of agar to water, and then adjusting the volume to 1 L to obtain a basic culture medium. The culture medium is continuously heated in a 100°C hot bath or repeatedly boiled intermittently (the boiling time does not exceed 1 min in total) and stirred or shaken until completely dissolved. After cooling it to 45-50°C, 1.5 mg of potassium tellurite is added, shaken well, and poured into a sterile plate. The culture medium is allowed to solidify.

[0036] Example 4:

[0037] A Vibrio vulnificus selective culture medium, wherein the basal culture medium comprises 10 g / L peptone, 5 g / L beef extract powder, 15 g / L sodium chloride, 1.0 g / L KCl, 10 g / L D-cellobiose, 0.8 g / L nisin Z type, 0.04 g / L cresol red, 0.04 g / L bromothymol blue, and 15 g / L agar; and the additives include 0.5 mg / L potassium tellurite, 6 mg / L vancomycin, 2 mg / L amphotericin B, and 1.0×10 5 IU / L. When preparing culture medium plates, heat the basal culture medium in a 100°C hot bath or microwave briefly (boiling time should not exceed 1 minute in total) and stir or shake to dissolve. After cooling to 45-50°C, add sterile additives, shake well, and pour into sterile plates. Wait until the culture medium is fully solidified.

[0038] The culture medium is prepared by adding 10 g of peptone, 5 g of beef extract powder, 15 g of sodium chloride, 1 g of potassium chloride, 10 g of D-cellobiose, 0.8 g of Nisin Z type, 0.04 g of cresol red, 0.04 g of bromothymol blue, and 15 g of agar to water, and then adjusting the volume to 1 L to obtain a basal culture medium. The culture medium is heated continuously in a 100°C hot bath or repeatedly boiled intermittently (the boiling time does not exceed 1 min in total) and stirred or shaken until completely dissolved. After cooling to 45-50°C, 0.5 mg of potassium tellurite, 6 mg of vancomycin, 2 mg of amphotericin B, and 1.0×10 5 IU, shake well and pour into a sterile plate, and wait until the culture medium is fully solidified.

[0039] Example 5:

[0040] Comparison of detection effects of different culture media on bacteria and fungi (see Table 3 and Table 4)

[0041] Table 3. Comparison of detection effects of different culture media on bacteria

[0042]

[0043]

[0044] Note: Each Vibrio strain cultured in alkaline peptone water at 37°C under aerobic conditions for 15-18 h and each non-Vibrio bacteria cultured in TSB broth at 37°C under aerobic conditions for 15-18 h were streaked onto each culture medium and then cultured aerobically at 37°C for 24 h. The mCPC and CC culture media used were domestically purchased and prepared according to their instructions. "++++++" indicates good growth, 1 to 6 "+"s indicate varying degrees of growth inhibition, "+ / -" indicates sparse growth or almost no growth, and "-" indicates no growth.

[0045] Table 4. Comparison of the detection effects of different culture media on fungi

[0046]

[0047] Note: After streaking, each fungal lawn grown on MEA medium was inoculated into each culture medium and cultured aerobically at 28°C for 5 days; "+" indicates significant growth or growth into sheets, "+ / -" indicates sparse growth or almost no growth, and "-" indicates no growth.

[0048] From the results in Table 3, it can be seen that compared with the existing mCPC and CC culture media commonly used for detecting Vibrio vulnificus, the Vibrio vulnificus selective culture medium of the present invention also shows high or relatively higher growth selectivity, especially good inhibitory effect on the growth of Gram-positive bacteria, and has higher detection specificity for Vibrio vulnificus.

[0049] From the results in Table 4, it can be seen that compared with the existing mCPC and CC culture media commonly used for detecting Vibrio vulnificus, the Vibrio vulnificus selective culture medium of the present invention also shows a good growth inhibitory effect on fungi such as yeast and mold.

[0050] Example 6:

[0051] Comparison of the detection sensitivity of different culture media for Vibrio vulnificus and Vibrio cholerae (see Table 5)

[0052] Table 5. Comparison of the detection sensitivity of different culture media for Vibrio vulnificus and Vibrio cholerae

[0053]

[0054] Note: Take Vibrio vulnificus cultured in alkaline peptone water at 37°C under aerobic conditions for 15-18 hours, dilute it with sterile saline to the required concentration, and then take 0.25 mL and spread it on the above culture medium. Then, culture it at 37°C under aerobic conditions for 24 hours. "+" indicates the growth of target bacteria, and "-" indicates the absence of target bacteria growth.

[0055] From the results in Table 5, we can see that for 10 1 ~10 2 CFU / mL Vibrio vulnificus ATCC 27562. From the perspective of whether the bacteria grow or not, the Vibrio vulnificus selective culture medium of the present invention is better than mCPC in detection sensitivity and is equivalent to CC culture medium.

[0056] Example 7:

[0057] Comparison of the detection of Vibrio vulnificus in natural samples using the chromogenic culture medium of the present invention and the national standard method (see Table 6)

[0058] Table 6. Comparison of the detection of Vibrio vulnificus in natural samples using the chromogenic culture medium of the present invention and the national standard method

[0059]

[0060] Note: Five samples of marine fish (saury, salmon, large yellow croaker, grouper, turbot), marine shrimp (basa shrimp, sea shrimp, bamboo shrimp, slipper shrimp, prawn) and seafood aquaculture water were purchased from the market, and Vibrio vulnificus was detected using the Vibrio vulnificus selective culture medium of the present invention (the specific method is the same as GB 4789.44-2020, except that the culture medium is replaced with the Vibrio vulnificus selective culture medium of the present invention). At the same time, Vibrio vulnificus was detected separately according to the national standard method of GB4789.44-2020 as a control, and MALDI-TOF-MS was used for identification.

[0061] From the results in Table 6, it can be seen that for the natural actual samples collected, the detection results of Vibrio vulnificus using the Vibrio vulnificus selective culture medium of the present invention and the national standard method are consistent.

[0062] The above description is only the best embodiment of the present invention and is not intended to limit the present invention. All modifications and equivalent replacements made within the spirit of the invention are included in the protection scope of the present invention.

Claims

1. A Vibrio vulnificus selective culture medium, characterized in that The invention comprises a basal culture medium and additives; the basal culture medium comprises 5-20 g / L of peptone, 3-15 g / L of beef extract powder, 10-25 g / L of sodium chloride, 0.5-5 g / L of potassium chloride, 0.1-1 g / L of a long-chain alkyl sulfate anionic surfactant or 0.05-2 g / L of nisin, 6-25 g / L of D-cellobiose, 0.01-0.10 g / L of an acid-base indicator, and 10-20 g / L of agar; the additive comprises 0.1-3.5 mg / L of tellurite; and the solvent is water; wherein, when the basal culture medium contains nisin, the additive further comprises 1-30 mg / L of vancomycin, 0.2-5 mg / L of amphotericin B, and 1.0×10 4 ~1.0×10 6 IU / L; The acid-base indicators are cresol red and bromothymol blue; The long-chain alkyl sulfate anionic surfactant is sodium lauryl sulfate; The tellurite is potassium tellurite.

2. The Vibrio vulnificus selective culture medium according to claim 1, characterized in that The basal culture medium comprises 10 g / L peptone, 5 g / L beef extract powder, 15 g / L sodium chloride, 1.0 g / L KCl, 15 g / L D-cellobiose, 0.5 g / L sodium lauryl sulfate, 0.04 g / L cresol red, 0.04 g / L bromothymol blue, and 15 g / L agar; the additive is 1.5 mg / L tellurite, and the solvent is water.

3. The Vibrio vulnificus selective culture medium according to claim 1, characterized in that The basic culture medium comprises 10 g / L peptone, 5 g / L beef extract powder, 15 g / L sodium chloride, 1.0 g / L KCl, 10 g / L D-cellobiose, 0.8 g / L nisin type Z, 0.04 g / L cresol red, 0.04 g / L bromothymol blue, and 15 g / L agar; the additives comprise 0.5 mg / L potassium tellurite, 6 mg / L vancomycin, 2 mg / L amphotericin B, and 1.0×10 5 IU / L.

4. A method for preparing the Vibrio vulnificus selective culture medium according to claim 1, characterized in that: The steps are: after mixing the basic culture medium in claim 1, continuously heating it in a hot bath or repeatedly boiling it intermittently for a short time, and stirring or shaking it until it is completely dissolved, and then after cooling it to 45-50° C., adding the sterile additives described in claim 1 to prepare the culture medium.

5. The preparation method according to claim 4, characterized in that The continuous heating of the basic culture medium using a heat bath is continuous heating of the basic culture medium using a heat bath at 100°C.

6. The preparation method according to claim 4, characterized in that The said repeatedly boiling the basal culture medium intermittently and briefly means repeatedly boiling the basal culture medium intermittently and briefly, with the total boiling time not exceeding 1 minute.

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