Selective culture medium and method for screening or separating aeromonas
By designing a selective culture medium containing yeast extract, nitrogen source, carbon source, acid-base indicator and antibiotic, the problem of low isolation efficiency of Aeromonas in the prior art has been solved, realizing rapid and efficient isolation and detection of Aeromonas, which is suitable for monitoring aquaculture and food samples.
Patent Information
- Application Number
- CN202511409337.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2025-11-21
AI Technical Summary
Existing technologies struggle to quickly and efficiently isolate Aeromonas from aquaculture environments and food samples, especially when Aeromonas concentrations are low, as detection methods are cumbersome and inefficient.
A selective culture medium was designed, containing components such as yeast extract, nitrogen source, carbon source, acid-base indicator, antibiotics, and identification reagents. By using a combination of specific culture media, selective culture and isolation of Aeromonas can be achieved.
It achieves rapid and efficient isolation of Aeromonas, with a short detection cycle and simple result interpretation. It is suitable for monitoring aquaculture and food samples and has broad application prospects.
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Figure CN120988935A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial culture technology, and more specifically, to a selective culture medium and method for screening or isolating Aeromonas. Background Technology
[0002] Aeromonas is one of the major pathogens causing bacterial infectious diseases in my country's freshwater aquaculture industry. Widely distributed in aquatic animals and the ecological environment, it is a typical zoonotic pathogen. These bacteria can remain dormant in the aquaculture environment for extended periods, causing persistent infections and impacting aquaculture efficiency. They can trigger various intraintestinal and extraintestinal infectious diseases in animals, and in severe cases, can even cause explosive mortality in various farmed aquatic animals, leading to significant economic losses. Pathogenic Aeromonas can also cause acute diarrhea, bacteremia, meningitis, endocarditis, and other diseases in humans, posing a threat to public health and safety and impacting human health.
[0003] Currently, the detection methods for Aeromonas are basically as follows: streaking fish samples directly onto TSA medium, culturing and isolating suspected strains, and then identifying whether they belong to the Aeromonas genus through a series of biochemical reactions; or extracting DNA from suspected strains and then identifying them by 16S rRNA sequence analysis. Because Aeromonas is present in relatively high quantities in diseased samples, non-selective media can be used for isolation and culture. However, this method is not suitable for other samples (environmental water samples, healthy aquatic tissues, and food samples, etc.) where the concentration of Aeromonas is low.
[0004] In view of this, the present invention is proposed. Summary of the Invention
[0005] The purpose of this invention is to provide a selective culture medium and method for screening or isolating Aeromonas bacteria, which can be used for rapid and efficient isolation of Aeromonas bacteria from a sample.
[0006] This invention is implemented as follows: Currently, the most commonly used culture media for isolating Aeromonas include MacConkey agar with added ampicillin and RS medium. MacConkey agar is used to isolate Enterobacteriaceae bacteria, not a selective isolation medium for Aeromonas spp. Enterobacteriaceae bacteria ferment lactose in the medium, producing acid, which, combined with a neutral red indicator, turns the colonies red, thus isolating suspected Enterobacteriaceae strains. Aeromonas does not ferment lactose, appearing grayish-white, distinguishing it from Enterobacteriaceae bacteria. The national standard test method for pathogenic Aeromonas hydrophila (National Standard GB / T 18652-2002) stipulates that uncontaminated samples and water samples are streaked onto ordinary agar medium for Aeromonas isolation, while contaminated samples are isolated using RS medium. A series of biochemical tests are then performed on suspected Aeromonas hydrophila to confirm its identifiable status. While this method has high accuracy, it is cumbersome and inefficient. Currently, there is no dedicated selective culture medium for Aeromonas spp.
[0007] Based on this, on the one hand, the present invention provides a selective culture medium for screening or isolating Aeromonas, comprising the following components: yeast extract, nitrogen source, carbon source, sodium citrate, sodium deoxycholate, sodium thiosulfate, ferric citrate, sodium chloride, agar, acid-base indicator, ampicillin, and diaminopterin.
[0008] The selective culture medium of the present invention can rapidly and efficiently isolate Aeromonas bacteria from samples with different background concentrations of Aeromonas.
[0009] Optionally, in some embodiments of the present invention, the nitrogen source is fish peptone.
[0010] Nitrogen source: Aeromonas is a bacterium belonging to the Vibrio family, widely found in water and aquatic animals. Fish peptone can be selected as the nitrogen source. Other similar nitrogen source components can also be used in this invention.
[0011] Optionally, in some embodiments of the present invention, the carbon source is dextrin.
[0012] Carbon source: Dextrin is a polysaccharide obtained through the partial hydrolysis of starch. It has good solubility and stability and is added to the culture medium to provide a carbon source, promoting microbial growth and metabolism. Amylase-positive Aeromonas colonies can form a clear ring around their colonies. Maltose is also a carbon source in this culture medium; Aeromonas can break down maltose to produce acid.
[0013] Optionally, in some embodiments of the present invention, the acid-base indicator is bromothymol blue.
[0014] Acid-base indicator: Adding bromothymol blue, an acid-base indicator, to selective culture media causes Aeromonas to break down maltose and produce acid. Under the influence of bromothymol blue, the colonies turn yellow. Strains that cannot ferment maltose and produce acid can only utilize sodium citrate or sodium citrate as a carbon source, then generate carbonates, raising the pH of the culture medium and appearing blue-green (or dark green) on the medium. If the strain can produce H₂S, encountering sodium thiosulfate and ferric citrate in the culture medium can cause the center of yellow or green colonies to turn black; these colored colonies are non-Aeromonas.
[0015] This invention adds the antibiotic ampicillin to a selective culture medium, which can inhibit the growth of most bacteria, while Aeromonas produces β-lactamase and is resistant to carbapenem antibiotics.
[0016] Vibrio cholerae (a type of Vibrio) and Aeromonas have similar biochemical characteristics, both capable of breaking down maltose to produce acid. Diaminopterin (O / 129) was added to this culture medium as a distinguishing reagent. Because Vibrio cholerae is sensitive to O / 129, its growth is inhibited; while Aeromonas exhibits resistance and can grow normally on this medium. This characteristic can be used to differentiate between the two types of bacteria.
[0017] The addition of sodium deoxycholate can inhibit the growth of Gram-positive bacteria.
[0018] Optionally, in some embodiments of the present invention, each 1 L of the selective culture medium comprises the following components: 2 g yeast extract, 6 g fish peptone, 10 g dextrin, 3.5 g maltose, 10 g sodium citrate, 1 g sodium deoxycholate, 10 g sodium thiosulfate, 1 g ferric citrate, 3 g sodium chloride, 15 g agar, 0.08 g bromothymol blue, 10 mg ampicillin, and 50 mg diaminopterin, with the balance being water, and a pH of 7.6 ± 0.2; the aforementioned components may fluctuate within the range of ± (5% to 1%).
[0019] This invention creatively combines different components (carbon source, nitrogen source, acid-base indicator, antibiotics, etc.) and dosages to achieve selective culture of Aeromonas bacteria by utilizing the entire selective culture medium. It can culture samples with Aeromonas background, allowing for the screening or isolation of Aeromonas bacteria, and the detection cycle is short. It has the advantages of high specificity, high sensitivity, ease of operation, simple result interpretation, and / or short detection cycle, making it suitable for monitoring aquaculture products, environmental samples, and food samples, with broad application prospects.
[0020] It should be noted that the amount of components in the above selective culture medium may fluctuate based on the aforementioned usage, for example, within the range of 5%, 4%, 3%, 2% or 1%, and the effect obtained is similar. The culture effect within the fluctuation range is easily expected by those skilled in the art.
[0021] On the other hand, the present invention provides a method for screening or isolating Aeromonas bacteria, comprising: The sample to be tested is inoculated into the selective culture medium described above to screen for or isolate Aeromonas.
[0022] Optionally, in some embodiments of the present invention, the presence of yellow colonies with transparent rings indicates that the colonies are Aeromonas.
[0023] Aeromonas-selective culture media inhibit contaminating bacteria and highlight target bacteria using antibiotics, bile salts, or chromogenic substrates. The appearance of yellow colonies with a clear ring indicates that the colony is Aeromonas. Based on this indicator, the identification of Aeromonas chromogenicity can be easily achieved.
[0024] Optionally, in some embodiments of the present invention, the culture temperature is 22 ℃-36 ℃.
[0025] Aeromonas bacteria belong to the Vibrio family and are widely found in water and aquatic animals. They are divided into two main groups: one is thermophilic and motile, including five species: *Aeromonas hydrophila*, *Aeromonas guinea pig*, *Aeromonas temperate*, *Aeromonas vannamei*, and *Aeromonas schubertii*. The other group is psychrophilic and non-motile, containing one species, *Aeromonas robini*, which includes three subspecies: *Aeromonas robini*, *Aeromonas robini*, and *Aeromonas robini*. The thermophilic group of Aeromonas bacteria are mostly pathogenic to humans, with an optimal growth temperature range of 22 ℃–37 ℃, while the psychrophilic group of Aeromonas bacteria are mostly pathogenic to aquatic animals, with an optimal growth temperature of 22 ℃–25 ℃. The culture temperature can be selected according to different samples. For aquatic products and environmental samples, culture at 25 ℃ ± 1 ℃ is suitable; for food samples, culture at 36 ℃ ± 1 ℃ is recommended.
[0026] Optionally, in some embodiments of the present invention, the Aeromonas is selected from Aeromonas hydrophila, Aeromonas guinea pig, Aeromonas salmonicida, and Aeromonas intermedius.
[0027] It should be noted that the selective culture medium of the present invention is not limited to the screening or isolation of Aeromonas bacteria described above.
[0028] Optionally, in some embodiments of the present invention, the sample to be tested is taken from aquatic products, environmental samples, and food samples.
[0029] It should be noted that the samples to be tested can be from a wide range of sources, and any sample to be tested is suitable for the selective culture medium of this invention. Attached Figure Description
[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.
[0031] Figure 1 The colony morphology of Aeromonas hydrophila on selective culture media.
[0032] Figure 2 The colony morphology of Escherichia coli on selective culture media.
[0033] Figure 3 The colony morphology of Salmonella Typhimurium on selective culture medium.
[0034] Figure 4 The colony morphology of *Pseudomonas fluorescens* on selective culture media.
[0035] Figure 5 The morphology (inhibition) of Staphylococcus aureus on selective culture media. Detailed Implementation
[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.
[0037] The features and performance of the present invention will be further described in detail below with reference to embodiments.
[0038] Example 1 1) Activation of standard strains The test standard strains were activated, including *Aeromonas hydrophila* ATCC 7966, *Aeromonas guinea pig* ATCC 15468, *Aeromonas salmonicida* ATCC 7965, *Aeromonas intermedius* ATCC 33907, *Salmonella typhimurium* ATCC 14028, *Escherichia coli* ATCC 25922, *Staphylococcus aureus* ATCC 6538, *Pseudomonas fluorescens* ATCC 13525, *Vibrio parahaemolyticus* ATCC 17802, and non-O1 *Vibrio cholerae* FSCC 232004 (VBO). All strains underwent biochemical validation before testing and were used only after confirmation.
[0039] After activation, the above-mentioned bacterial strains were inoculated onto BHI medium. Vibrio parahaemolyticus was inoculated onto 3% sodium chloride tryptone soy agar medium and cultured at the respective optimal temperatures for 18-24 hours. Three to five colonies were then picked from each strain and dispersed in sterile physiological saline to prepare bacterial suspensions. The turbidity of the bacterial suspensions was adjusted to 0.5 McFarland turbidity, resulting in a concentration of 1.5 × 10⁻⁶. 8 CFU / mL, for later use.
[0040] 2) Culture medium preparation Prepare 1 L of culture medium according to the following formula: 2 g yeast extract, 6 g fish peptone, 10 g dextrin, 3.5 g maltose, 10 g sodium citrate, 1 g sodium deoxycholate, 10 g sodium thiosulfate, 1 g ferric citrate, 3 g sodium chloride, 15 g agar, 0.08 g bromothymol blue, 10 mg ampicillin, and 50 mg diaminopterin. Weigh and mix all components except the antibiotics, add 1 L of deionized water, stir to dissolve, and adjust the pH to 7.6 ± 0.2. Heat to boiling, and when cooled to about 50 °C, add the corresponding antibiotics. Stir aseptically until homogeneous, pour the medium, and allow it to solidify before use.
[0041] 3) Vaccination Prepare bacterial suspensions of the above 9 standard strains. Using a 1 μL inoculation loop, take one loopful of the selective test bacterial suspension and streak six parallel straight lines on the surface of the test medium. When streaking, the angle between the inoculation loop and the agar plane should be 20°-30°. The pressure of the inoculation loop on the agar surface and the streaking speed should be consistent throughout. The entire streak should be rapid and continuous. When transferring the liquid culture, the inoculation loop should be submerged in the lower part of the culture medium to prevent air bubbles or foam from forming on the loop. After streaking, incubate upside down at 36 ℃ ± 1 ℃ for 18 h-24 h and observe the color development of the colonies.
[0042] 4) Results Analysis After culturing, calculate the growth index G on the culture medium as follows (GB 4789.28 Test Method for Selective Separation and Counting of Solid Culture Media). Each streak with relatively dense, continuous growth of colonies is scored as 1 point, with a maximum of 6 points per dish. If only half of the streak has dense colony growth, it is scored as 0.5 points. If there is no colony growth on the streak, the growth is less than half the length of the streak, or the colony growth is weak, it is scored as 0 points. Record the sum of the scores for each culture medium to obtain G.
[0043] Aeromonas grows well on selective media, and develops color within 18 hours. The colonies are yellow with a clear ring around them, and the colony diameter is about 2 mm. Non-target bacteria are inhibited or appear as colonies of different colors.
[0044] Table 1. Test Results of Example 1 Example 2 1) Culture medium preparation Prepare 1 L of culture medium according to the formula. Weigh and mix all components except the antibiotic, add 1 L of deionized water, stir to dissolve, and adjust the pH to 7.6 ± 0.2. Heat to boiling, and when cooled to about 50 ℃, add the corresponding antibiotic, stir evenly under aseptic conditions, pour the culture medium, and let it solidify before use.
[0045] 2) Preparation of artificially contaminated samples Purchase fish samples to prepare 25 g of sample, add the prepared solution of approximately 10... 2 100 μL of CFU / mL Aeromonas hydrophila (ATCC7966), Pseudomonas fluorescens (ATCC 13525), and Salmonella (ATCC 14028) standard strain suspensions.
[0046] 3) Sample testing Add 225 ml of prepared sterile buffered peptone water to the sample, homogenize and incubate at 36 ℃±1 ℃ for 12 h-18 h. Streak the enrichment broth onto selective medium, MacConkey agar with ampicillin, and RS agar, and incubate at 25 ℃±1 ℃ for 18 h-24 h, observing the results.
[0047] 4) Results Analysis Single colonies with typical Aeromonas colony morphology were picked from various culture dishes. The genome of the strain was extracted using a DNA extraction kit and used as a template. PCR amplification was performed using universal primers for the bacterial 16S rDNA gene. The PCR products were identified by 1.0% agarose gel electrophoresis. Positive products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the NCBI database to determine the species of the isolated strain. The accuracy of Aeromonas detection was calculated by comparing the number of colonies identified as Aeromonas with the number of single colonies picked. Table 2 shows that the accuracy of Aeromonas selective medium was 85%, significantly higher than that of MacConkey agar and RS agar supplemented with ampicillin.
[0048] Table 2. Test results of Example 2 Example 3 1) Culture medium preparation Prepare 1 L of culture medium according to the formula. Weigh and mix all components except the antibiotic, add 1 L of deionized water, stir to dissolve, and adjust the pH to 7.6 ± 0.2. Heat to boiling, and when cooled to about 50 ℃, add the corresponding antibiotic, stir evenly under aseptic conditions, pour the culture medium, and let it solidify before use.
[0049] 2) Preparation of artificially contaminated samples Purchase fish samples and prepare four 25 g samples. Add the prepared solution at a concentration of approximately 10... 2 -10 3 1 mL of CFU / mL standard bacterial suspension was added to sample A, Aeromonas hydrophila suspension to sample B, Aeromonas guinea pig suspension to sample C, and Aeromonas salmonicidae suspension to sample D.
[0050] 3) Sample testing Four samples were added to 225 ml of prepared sterile buffered peptone water, homogenized, and enriched at 36 ℃ ± 1 ℃ for 12 h–18 h. The enrichment broth was then inoculated onto selective culture medium and incubated at 25 ℃ ± 1 ℃ for 18 h–24 h, and the observed phenomena were observed.
[0051] 4) Results Analysis Aeromonas grows vigorously on culture media, and the colonies are yellow with a transparent ring around them.
[0052] Table 3. Test results of Example 3 The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A selective culture medium for screening or isolating Aeromonas bacteria, characterized in that, The culture medium contains the following components: yeast extract, nitrogen source, carbon source, sodium citrate, sodium deoxycholate, sodium thiosulfate, ferric citrate, sodium chloride, agar, acid-base indicator, ampicillin, and diaminopterin.
2. The selective culture medium according to claim 1, characterized in that, The nitrogen source is fish peptone.
3. The selective culture medium according to claim 1, characterized in that, The carbon source is dextrin or maltose, or a combination of dextrin and maltose.
4. The selective culture medium according to claim 1, characterized in that, The acid-base indicator is bromothymol blue.
5. The selective culture medium according to any one of claims 1-4, characterized in that, Each 1 L of the selective culture medium contains the following components: 2 g yeast extract, 6 g fish peptone, 10 g dextrin, 3.5 g maltose, 10 g sodium citrate, 1 g sodium deoxycholate, 10 g sodium thiosulfate, 1 g ferric citrate, 3 g sodium chloride, 15 g agar, 0.08 g bromothymol blue, 10 mg ampicillin, and 50 mg diaminopterin, with the balance being water, and a pH of 7.6 ± 0.2; the aforementioned components may fluctuate within the range of ± (5% ~ 1%).
6. A method for screening or isolating Aeromonas bacteria, characterized in that, It includes: The sample to be tested is inoculated into the selective culture medium according to any one of claims 1-5 to screen or isolate Aeromonas.
7. The method according to claim 6, characterized in that, If yellow colonies appear with a clear ring, it indicates that the colony is Aeromonas.
8. The method according to claim 6, characterized in that, The cultivation temperature is 22-36℃.
9. The method according to claim 6, characterized in that, The aeromones are selected from Aeromonas hydrophila, Aeromonas guinea pig, Aeromonas salmonicida, and Aeromonas intermedius.
10. The method according to any one of claims 6-9, characterized in that, The samples to be tested were taken from aquatic products, environmental samples, and food samples.