A kind of Bozeman Legionella isolation culture medium and preparation method thereof

Abstract

The present invention provides a Legionella Bozemania isolation culture medium and a preparation method thereof, which are used for selectively isolating and culturing Legionella Bozemania, and belong to the field of microbial technology. Its components include: sodium pyruvate, yeast extract powder, α-ketoglutaric acid, ferric nitrate, activated carbon, carbon black, L-cysteine ​​hydrochloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, glycine, bromocresol purple, bromocresol green, agar, vancomycin, natamycin, polymyxin B, and potassium hydroxide. By simple dissolution and mixing, high-temperature sterilization, and aseptic filling, a culture medium suitable for the isolation and cultivation of Legionella Bozemania is prepared. The culture medium of the present invention can accurately and quickly screen out Legionella Bozemania through simple isolation and cultivation, has high efficiency and specificity, is easy to obtain raw materials, effectively reduces costs, is convenient and quick to use, reduces the contamination risks and cumbersome procedures brought by self-prepared culture media, and is suitable for standardization and specialized research.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Legionella Bozemansis separation culture medium and a preparation method thereof. Background Art

[0002] Legionella is a common Gram-negative bacillus found in freshwater, drinking water, and artificial waterways, particularly in cooling tower water used in central air conditioning systems. It is the primary pathogen causing Legionnaires' disease and Pontia fever. Currently, 90% of known Legionnaires' disease outbreaks are caused by Legionella pneumophila, but cases caused by non-pneumophila Legionella have also attracted significant attention.

[0003] Legionella bozemanii, a species of Legionella bacteria, can cause Legionnaires' pneumonia. In addition to lung inflammation, patients can also suffer liver, kidney, heart, and neuropsychiatric damage. From late 1995 to early 1996, four cases of Legionnaires' pneumonia occurred among recruits in a unit in Shunyi County, Beijing. In January 2000, an outbreak of Legionnaires' disease occurred at a new recruit training camp in suburban Beijing. The bacteria reproduces slowly, requiring a long culture time, making it difficult to conduct laboratory research and clinical testing. Currently, there is a need for a culture medium that can quickly and effectively isolate and culture Legionnaires' bacteria. Summary of the Invention

[0004] The present invention provides a Legionella Bozemanii isolation medium and a method for preparing the same. The isolation medium is a disposable, pre-filled, sterile product that is convenient, quick, and highly effective to use. Application of this isolation medium in clinical research in hospitals can shorten the time it takes to translate research findings into reality and significantly improve work efficiency.

[0005] In order to achieve the purpose of the present invention, the following technical solutions are adopted:

[0006] A Bozeman Legionella isolation medium is characterized by comprising basic nutritional components and additives; the basic nutritional components comprising yeast extract, sodium pyruvate, α-ketoglutaric acid, activated carbon, carbon black, ferric nitrate, agar, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, L-cysteine ​​hydrochloride, bromocresol purple, bromocresol green, and potassium hydroxide; and the additives comprising glycine, vancomycin, polymyxin B, and natamycin.

[0007] A further preferred technical solution: per liter of the Bozeman Legionella isolation medium contains 6-8g yeast extract, 2-4g sodium pyruvate, 1-2g activated carbon, 0.01-1g carbon black, 0.2-0.3g ferric nitrate, 0.8-1.2g α-ketoglutaric acid, 16-18g agar, 8.2g potassium dihydrogen phosphate, 1.9g dipotassium hydrogen phosphate, 0.5-1g L-cysteine ​​hydrochloride, 0.01-0.02g bromocresol purple, 0.01-0.02g bromocresol green, 2.8g potassium hydroxide, and also includes additives 3-5g glycine, 0.8-1mg vancomycin, 50,000-100,000 U polymyxin B and 60-80mg natamycin.

[0008] A method for preparing a Bozeman Legionella isolation medium, characterized in that 6-8g yeast extract powder, 2-4g sodium pyruvate, 1-2g activated carbon, 0.01-1g carbon black, 0.2-0.3g ferric nitrate, 0.8-1.2g α-ketoglutaric acid, 16-18g agar, 8.2g potassium dihydrogen phosphate, 1.9g dipotassium hydrogen phosphate, 0.5-1g L-cysteine ​​hydrochloride, 0.01-0.02g bromocresol purple, and 0.01-0.02g bromocresol green are dissolved in deionized water, and the pH is adjusted to 6.7-7.1 with 2.8g potassium hydroxide, and the volume is adjusted to 1000mL; the mixture is sterilized at a high temperature of 118-125°C and a high pressure of 100-108kPa for 13-17 minutes, and the mixture is cooled to 40-60°C, and 3-5g glycine, 0.8-1mg vancomycin, 50,000-100,000U polymyxin B, and 60-80mg natamycin are added and mixed, and the mixture is aseptically filled into a disposable sterile culture dish and sterilized by high-energy irradiation to prepare the Bozeman Legionella isolation medium.

[0009] The application is for specific separation and culture of Legionella Bozemansis.

[0010] The disposable sterile culture dish is a 90*15 mm disposable sterile culture dish.

[0011] The culture time is 24-168 h.

[0012] A method for isolating and culturing Legionella Bozemanii comprises the following steps: utilizing the above-mentioned preparation method to prepare a separation medium; inoculating a sample to be separated into the separation medium to obtain a Legionella Bozemanii sample medium; and culturing the sample medium.

[0013] Compared with existing technologies, the advantages of the present invention are as follows: It provides a special isolation culture medium formula for isolating Legionella bozemanii. Compared with other Legionella isolation culture media, this culture medium not only inhibits the growth of other bacteria and fungi, but also significantly shortens the culture time of the target strain, thereby obtaining a culture of Legionella bozemanii in a shorter time and improving isolation efficiency. The present invention uses a simple processing technique to produce a disposable pre-filled product, avoiding the contamination risks and cumbersome procedures associated with self-prepared culture media. It is convenient and quick to use, significantly shortening the research cycle and improving R&D efficiency. The prepared culture medium can be used to isolate target microorganisms. Through simple culture steps, the target bacterial species can be accurately isolated, achieving good separation results. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 (a) is the culture medium of Example 1 of the present invention;

[0015] Figure 1 (b) is the culture medium of the control group of Example 1;

[0016] Figure 2 (a) is the culture medium of Example 2 of the present invention;

[0017] Figure 2 (b) is the culture medium of the control group of Example 2;

[0018] Figure 3 (a) is the culture medium of Example 3 of the present invention;

[0019] FIG3( b ) is the culture medium of the control group of Example 3. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in detail below with reference to the examples. The specific examples described herein are only used to illustrate the present invention and are not intended to limit the present invention. Where specific conditions are not specified in the examples, conventional conditions were used. Where the manufacturers of the instruments or reagents used are not specified, they are all commercially available conventional products.

[0021] Yeast extract powder, α-ketoglutaric acid and sodium pyruvate provide basic carbon and nitrogen sources and energy for microbial growth; dipotassium hydrogen phosphate and potassium dihydrogen phosphate can stabilize the pH of the culture medium within a certain range; activated carbon and carbon black have the functions of detoxification, collecting carbon dioxide and changing surface tension; L-cysteine ​​hydrochloride and ferric nitrate are essential growth factors for Legionella; potassium hydroxide can effectively regulate the pH of the culture medium; agar, as a coagulant, is a basic component of solid culture medium; bromocresol purple and bromocresol green are acid-base indicators; glycine can inhibit the growth of miscellaneous bacteria but not Legionella; vancomycin and polymyxin B, as antibiotics, can effectively inhibit the growth of most Gram-negative and Gram-positive bacteria; natamycin, as a fungal inhibitor, can effectively inhibit the growth of fungi.

[0022] A formula and culture method for a Bozeman Legionella isolation culture medium comprises the following steps:

[0023] 1. Isolation medium formula

[0024] 6-8g yeast extract powder, 2-4g sodium pyruvate, 1-2g activated carbon, 0.01-1g carbon black, 0.2-0.3g ferric nitrate, 0.8-1.2g α-ketoglutaric acid, 16-18g agar, 8.2g potassium dihydrogen phosphate, 1.9g dipotassium hydrogen phosphate, 0.5-1g L-cysteine ​​hydrochloride, 0.01-0.02g bromocresol purple, 0.01-0.02g bromocresol green, 2.8g potassium hydroxide, additives include 3-5g glycine, 0.8-1mg vancomycin, 50,000-100,000 U polymyxin B and 60-80mg natamycin, and the solvent is deionized water.

[0025] 2. Cultivation methods

[0026] (1) Preparation of separation culture medium

[0027] Weigh 6-8g yeast extract powder, 2-4g sodium pyruvate, 1-2g activated carbon, 0.01-1g carbon black, 0.2-0.3g ferric nitrate, 0.8-1.2g α-ketoglutaric acid, 16-18g agar, 8.2g potassium dihydrogen phosphate, 1.9g dipotassium hydrogen phosphate, 0.5-1g L-cysteine ​​hydrochloride, 0.01-0.02g bromocresol purple, and 0.01-0.02g bromocresol green; dissolve in deionized water, adjust the pH to 6.7-7.1 with 2.8g potassium hydroxide, and dilute to 1000ml. mL; sterilize at 121°C and 104 kPa for 15 minutes, cool to 50°C, add 3-5 g of filter-sterilized glycine, 0.8-1 mg of vancomycin, 50,000-100,000 U of polymyxin B, and 60-80 mg of natamycin, mix well, aseptically fill into a disposable sterile culture dish, and sterilize by high-energy irradiation to prepare the Bozeman Legionella isolation medium.

[0028] (2) Vaccination

[0029] Under sterile conditions in a clean bench, cooling tower water samples from central air conditioners were inoculated onto the separation medium.

[0030] (3) Cultivation

[0031] Place the inoculated separation culture medium in an incubator at 34-38°C and incubate for 24-168 hours.

[0032] Example 1

[0033] This embodiment provides a formulation of a separation medium.

[0034] A culture medium and culture method for isolating Legionella Bozemanii, comprising the following steps:

[0035] 1. The formula of separation medium is as follows:

[0036] 8g yeast extract powder, 2g sodium pyruvate, 0.5g L-cysteine ​​hydrochloride, 1.5g activated carbon, 1g carbon black, 0.25g ferric nitrate, 1g α-ketoglutaric acid, 16g agar, 8.2g potassium dihydrogen phosphate, 1.9g dipotassium hydrogen phosphate, 0.01g bromocresol purple, 0.01g bromocresol green, 2.8g potassium hydroxide, additives include 3g glycine, 1mg vancomycin, 80,000U polymyxin B and 80mg natamycin, and the solvent is deionized water.

[0037] 2. Cultivation methods

[0038] (1) Preparation of separation culture medium

[0039] 8 g yeast extract powder, 2 g sodium pyruvate, 0.5 g L-cysteine ​​hydrochloride, 1.5 g activated carbon, 1 g carbon black, 0.25 g ferric nitrate, 1 g α-ketoglutaric acid, 16 g agar, 8.2 g potassium dihydrogen phosphate, 1.9 g dipotassium hydrogen phosphate, 0.01 g bromocresol purple, and 0.01 g bromocresol green were weighed and dissolved in deionized water. The pH was adjusted to 6.7-7.1 with 2.8 g potassium hydroxide, and the volume was adjusted to 1000 mL. The mixture was sterilized at 121° C. and 104 kPa for 15 minutes, cooled to 50° C., and filter-sterilized 3 g glycine, 1 mg vancomycin, 80,000 U polymyxin B, and 80 mg natamycin were added and mixed. The mixture was aseptically filled into a disposable sterile culture dish and sterilized by high-energy irradiation to prepare a Bozeman Legionella isolation medium.

[0040] (2) Vaccination

[0041] Under sterile conditions in a clean bench, cooling tower water samples from central air conditioners were inoculated onto the separation medium.

[0042] (3) Cultivation

[0043] Place the inoculated separation culture medium in an incubator at 36°C and incubate for 24-168 hours.

[0044] 3. Set up controls

[0045] Using GVPC agar as a control, water samples collected from the outlet of a central air conditioning cooling tower were inoculated onto the control medium under sterile conditions on a clean bench. The inoculated control medium was placed in an incubator at 36°C and incubated for 24-168 hours.

[0046] The above control group is the same as Example 1 of the present invention except where otherwise noted.

[0047] The results are shown in Table 1

[0048] Table 1 Comparison of the isolation and culture effects of the present invention and the control group on Legionella Bozemansis

[0049]

[0050] Note: +: small colony; ++: larger colony; +++: large colony

[0051] As shown in Table 1, Legionella bozemanii grows well in the culture medium prepared according to the present invention, while other fungi and bacteria do not grow. The culture results of the control group show poor growth of Legionella bozemanii. Figure 1(a) shows the culture medium prepared according to the present invention. The color of the colonies forms a sharp contrast with that of the culture medium. Furthermore, within the same culture time, Legionella bozemanii grows rapidly, with a large and numerous colony size, making it easy to count and observe the characteristics of the colonies. Figure 1(b) shows the culture medium prepared according to the control group. Legionella bozemanii grows slowly, with small and few colonies, and the color contrast is not clear, making it difficult to observe and hindering the rapid isolation and culture of Legionella bozemanii.

[0052] This embodiment 1 is the best embodiment of the present invention.

[0053] Example 2

[0054] This embodiment provides a formulation of a separation medium.

[0055] A culture medium and culture method for isolating Legionella Bozemanii, comprising the following steps:

[0056] 1. The formula of separation medium is as follows:

[0057] 6g yeast extract powder, 4g sodium pyruvate, 2g activated carbon, 0.5g carbon black, 0.2g ferric nitrate, 1.2g α-ketoglutaric acid, 17g agar, 8.2g potassium dihydrogen phosphate, 1.9g dipotassium hydrogen phosphate, 0.7g L-cysteine ​​hydrochloride, 0.015g bromocresol purple, 0.015g bromocresol green, 2.8g potassium hydroxide, additives include 4g glycine, 0.8mg vancomycin, 100,000 U polymyxin B and 72mg natamycin, and the solvent is deionized water.

[0058] 2. Cultivation methods

[0059] (1) Preparation of separation culture medium

[0060] 6 g yeast extract powder, 4 g sodium pyruvate, 2 g activated carbon, 0.5 g carbon black, 0.2 g ferric nitrate, 1.2 g α-ketoglutaric acid, 17 g agar, 8.2 g potassium dihydrogen phosphate, 1.9 g dipotassium hydrogen phosphate, 0.7 g L-cysteine ​​hydrochloride, 0.015 g bromocresol purple, and 0.015 g bromocresol green were weighed and dissolved in deionized water, and the pH was adjusted to 6.7-7.1 with 2.8 g potassium hydroxide, and the volume was adjusted to 1000 mL. The mixture was sterilized at 121°C and 104 kPa for 15 minutes, cooled to 50°C, and filter-sterilized 4 g glycine, 0.8 mg vancomycin, 100,000 U polymyxin B, and 72 mg natamycin were added and mixed. The mixture was aseptically filled into a disposable sterile culture dish and sterilized by high-energy irradiation to prepare the Bozeman Legionella isolation medium.

[0061] (2) Vaccination

[0062] Under sterile conditions in a clean bench, cooling tower water samples from central air conditioners were inoculated onto the separation medium.

[0063] (3) Cultivation

[0064] Place the inoculated separation culture medium in an incubator at 36°C and incubate for 24-168 hours.

[0065] 3. Set up controls

[0066] Using GVPC agar as a control, water samples collected from the outlet of a central air conditioning cooling tower were inoculated onto the control medium under sterile conditions on a clean bench. The inoculated control medium was placed in an incubator at 36°C and incubated for 24-168 hours.

[0067] The above control group is the same as Example 1 of the present invention except where otherwise noted.

[0068] The results are shown in Table 2

[0069] Table 2 Comparison of the isolation and culture effects of the present invention and the control group on Legionella Bozemansis

[0070]

[0071] Note: +: small colony; ++: larger colony; +++: large colony

[0072] As shown in Table 2, the culture medium prepared according to the present invention promotes good growth of Legionella bozemanii, while other fungi and bacteria do not grow. The culture results from the control group indicate that the growth of Legionella bozemanii is poor. Figure 2(a) shows the culture medium prepared according to the present invention. The colony color contrasts sharply with the culture medium. Furthermore, within the same culture time, Legionella bozemanii grows rapidly, with a large and abundant colony count, making it easy to count and observe the colony characteristics. Figure 2(b) shows the culture medium prepared according to the control group. Legionella bozemanii grows slowly, with small and few colonies. The color contrast is not clear, making it difficult to observe and hindering the rapid isolation and culture of Legionella bozemanii. As can be seen from these results, while the culture performance of Example 2 is slightly inferior to that of Example 1, with fewer colonies, Example 1 still significantly outperforms the control group and demonstrates a certain degree of effectiveness in isolating Legionella bozemanii.

[0073] Example 3

[0074] This embodiment provides a formulation of a separation medium.

[0075] A culture medium and culture method for isolating Legionella Bozemanii, comprising the following steps:

[0076] 1. The formula of separation medium is as follows:

[0077] 7g yeast extract powder, 3g sodium pyruvate, 1g activated carbon, 0.01g carbon black, 0.3g ferric nitrate, 0.8g α-ketoglutaric acid, 18g agar, 8.2g potassium dihydrogen phosphate, 1.9g dipotassium hydrogen phosphate, 1g L-cysteine ​​hydrochloride, 0.02g bromocresol purple, 0.02g bromocresol green, 2.8g potassium hydroxide, additives: 5g glycine, 0.9mg vancomycin, 50,000U polymyxin B and 60mg natamycin, solvent: deionized water.

[0078] 2. Cultivation methods

[0079] (1) Preparation of separation culture medium

[0080] 7 g yeast extract powder, 3 g sodium pyruvate, 1 g activated carbon, 0.01 g carbon black, 0.3 g ferric nitrate, 0.8 g α-ketoglutaric acid, 18 g agar, 8.2 g potassium dihydrogen phosphate, 1.9 g dipotassium hydrogen phosphate, 1 g L-cysteine ​​hydrochloride, 0.02 g bromocresol purple, and 0.02 g bromocresol green were weighed and dissolved in deionized water. The pH was adjusted to 6.7-7.1 with 2.8 g potassium hydroxide, and the volume was adjusted to 1000 mL. The mixture was sterilized at 121°C and 104 kPa for 15 minutes, cooled to 50°C, and filter-sterilized 5 g glycine, 0.9 mg vancomycin, 50,000 U polymyxin B, and 60 mg natamycin were added and mixed. The mixture was aseptically filled into a disposable sterile Petri dish and sterilized by high-energy irradiation to prepare Bozeman Legionella isolation medium.

[0081] (2) Vaccination

[0082] Under sterile conditions in a clean bench, cooling tower water samples from central air conditioners were inoculated onto the separation medium.

[0083] (3) Cultivation

[0084] Place the inoculated separation culture medium in an incubator at 36°C and incubate for 24-168 hours.

[0085] 3. Set up controls

[0086] Using GVPC agar as a control, water samples collected from the outlet of a central air conditioning cooling tower were inoculated onto the control medium under sterile conditions on a clean bench. The inoculated control medium was placed in an incubator at 36°C and incubated for 24-168 hours.

[0087] The above control group is the same as Example 1 of the present invention except where otherwise noted.

[0088] The results are shown in Table 3

[0089] Table 3 Comparison of the isolation and culture effects of the present invention and the control group on Legionella Bozemansis

[0090]

[0091] Note: +: small colony; ++: larger colony; +++: large colony

[0092] Table 3 shows that the culture medium prepared according to the present invention grows Legionella bozemanii well, while other fungi and bacteria do not grow. The culture results in the control group show that Legionella bozemanii grows poorly. Figure 3(a) shows the culture medium prepared according to the present invention. Within the same culture time, Legionella bozemanii grows rapidly, with a large and abundant colony count, making it easy to count and observe the characteristics of the colonies. Figure 3(b) shows the culture medium prepared according to the control group. Legionella bozemanii grows slowly, with small colonies and a low number of colonies, making it difficult to observe and count, and thus not conducive to the rapid isolation and culture of Legionella bozemanii. As can be seen from these results, Example 3 exhibits slightly inferior culture performance to Example 1. Compared to Example 1, the number of colonies is low, the size is small, and there is no clear color contrast. However, the culture medium is still significantly superior to the control group, demonstrating a certain degree of effectiveness in isolating Legionella bozemanii.

[0093] In summary, the isolation culture medium provided by the embodiments of the present invention absorbs toxic products such as peroxides and superoxides produced during Legionella growth by adding activated carbon and carbon black; provides essential factors for Legionella growth by adding ferric nitrate and L-cysteine ​​hydrochloride; inhibits fungal growth by adding natamycin; and inhibits the growth of other bacteria by adding vancomycin, glycine, and polymyxin B. This results in a culture medium capable of effectively isolating and culturing Legionella bozemanii. This culture medium has high practicality and application value.

[0094] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but rather represents only selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

Claims

1. A method for preparing a Legionella Bozemansis isolation culture medium, characterized in that: The invention comprises basic nutrient components and additives; the basic nutrient components include yeast extract, sodium pyruvate, α-ketoglutaric acid, activated carbon, carbon black, ferric nitrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, L-cysteine ​​hydrochloride, bromocresol purple, bromocresol green, agar, and potassium hydroxide; the additives include glycine, vancomycin, polymyxin B, and natamycin; each liter of the Bozeman Legionella isolation medium contains 6-8g yeast extract, 2-4g sodium pyruvate, 1-2g activated carbon, 0.01-1g carbon black, 0.2-0.3g ferric nitrate, 0.8-1.2g α-ketoglutaric acid, 16-18g agar, 8.2g potassium dihydrogen phosphate, 1.9g dipotassium hydrogen phosphate, 0.5-1g L-cysteine ​​hydrochloride, 0.01-0.02g bromocresol purple, 0.01-0.02g Bromocresol green, 2.8g potassium hydroxide, with additives of 3-5g glycine, 0.8-1mg vancomycin, 50,000-100,000 units polymyxin B, and 60-80mg natamycin; this medium is used for the specific isolation and culture of Legionella bozemanica. Preparation method of the culture medium: Weigh 6-8g yeast extract powder, 2-4g sodium pyruvate, 1-2g activated carbon, 0.01-1g carbon black, 0.2-0.3g ferric nitrate, 0.8-1.2g α-ketoglutaric acid, 16-18g agar, 8.2g potassium dihydrogen phosphate, 1.9g dipotassium hydrogen phosphate, 0.5-1g L-cysteine ​​hydrochloride, 0.01-0.02g bromocresol purple, and 0.01-0.02g bromocresol green, dissolve in deionized water, adjust the pH to 6.7-7.1 with 2.8g potassium hydroxide, and make up to 1000mL. Sterilize at 118-125°C and 100-108kPa for 13-17 minutes. Cool to 40-60°C, add 3-5g sterile-filtered glycine, 0.8-1mg Vancomycin, 50,000-100,000 U of polymyxin B and 60-80 mg of natamycin are mixed, aseptically filled into a disposable sterile culture dish, and sterilized by high-energy irradiation to prepare the Bozeman Legionella isolation medium.

2. The method for preparing the Bozeman Legionella isolation culture medium according to claim 1, wherein: The disposable sterile culture dish is a 90*15mm disposable sterile culture dish.

3. The method for preparing the Bozeman Legionella isolation culture medium according to claim 2, wherein: The culture time is 24-168 hours.

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