Borrelia BSK storage liquid culture medium and single colony separating and purifying method and application thereof

A technology of culture medium and storage solution, applied in the field of medical microbiology, can solve the problems of difficulty in applying Borrelia diagnosing, unfavorable growth of Borrelia, and other problems, and achieves convenient cell growth, simple and rapid separation and purification method, and high degree of cross-linking. Moderate effect

Active Publication Date: 2013-11-27
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

More importantly, Borrelia is an aerobic bacterium. When the conventional coating plate method and plate streaking method are used to isolate single colonies, the surface of the plate is completely exposed, which is not conducive to the growth of Borrelia.
These all make the conventional microbiological single colony isolation method difficult to apply in the research and diagnosis of Borrelia.

Method used

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  • Borrelia BSK storage liquid culture medium and single colony separating and purifying method and application thereof
  • Borrelia BSK storage liquid culture medium and single colony separating and purifying method and application thereof
  • Borrelia BSK storage liquid culture medium and single colony separating and purifying method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] A medium for Borrelia BSK stock solution, which is made of the following raw materials by weight:

[0055]

[0056]

[0057] A preparation method of Borrelia BSK storage medium, its steps are:

[0058] Add the above components into distilled water one by one in order, stir well at room temperature (20-30℃) until completely dissolved, and then add the next component after the previous component is completely dissolved. Finally adjust the pH and constant volume. Filter and sterilize with a 0.22 micron pore filter, and store it in a refrigerator at -20°C.

Embodiment 2

[0060] Borrelia burgdorferi B31 solid plate pouring and single colony separation and purification:

[0061] (1) Preparation of agarose gel: prepare 5% (weight to volume ratio, the same below) agarose gel with low melting point agarose, sterilize at 121°C, 103.4 kPa for 20 minutes, and place at 25°C for use.

[0062] (2) Preparation of the bottom solid medium: adjust the temperature of the water bath to 55°C, and gradually melt the 5% agarose gel in it. Then mix with the BSK stock solution medium prepared in Example 1 at a volume ratio of 1:1, and add kanamycin (sigma) to a final concentration of 200 μg / ml. Pour into a petri dish. For a petri dish with a diameter of 10 cm, each requires 10 ml of bottom solid medium. Leave it at room temperature for 12 hours, and store it in a refrigerator at 4°C until the culture medium has completely solidified and there is no condensation on the surface.

[0063] (3) The Borrelia burgdorferi B31 strain was cultured in BSK-H regular liquid medium (...

Embodiment 3

[0067] The application of a method for separating and purifying Borrelia single colony in the preparation of pure culture of Borrelia burgdorferi, the application process is:

[0068] (1) Preparation of pure culture of Borrelia burgdorferi:

[0069] Use a toothpick to carefully pick 5 Borrelia burgdorferi B31 strains, and inoculate them into 12 ml of BSK-H conventional liquid medium (product of sigma), and cultivate in a 37°C carbon dioxide incubator (5%) for 3-4 days. The growth of Borrelia, the strain grows to the logarithmic growth phase, and the colony concentration reaches 10 7 Cells / ml or more, a pure culture of a single colony of Borrelia burgdorferi can be obtained.

[0070] (2) Identification of the endogenous plasmid of Borrelia burgdorferi:

[0071] Borrelia burgdorferi contains 20 linear or circular plasmids ranging in size from 5-56 kb. Some plasmids are unstable in the in vitro subculture process, but they encode important phenotypic characteristics. The identification ...

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Abstract

The invention discloses a borrelia BSK storage liquid culture medium and a single colony separating and purifying method and application thereof. The borrelia BSK storage liquid culture medium comprises CMRL-1066, bovine serum albumin V, new peptone, hydroxyethyl piperazine ethanesulfonic acid, sodium citrate, glucose, yeast powder, sodium bicarbonate, sodium pyruvate, acetyl glucosamine, rabbit serum, sodium hydroxide, and the like. The single colony separating and purifying method comprises the following steps of: preparing the BSK storage liquid culture medium, preparing sepharose gel, sterilizing at high pressure, gradually melting, uniformly mixing with the BSK storage liquid culture medium in proportion, and pouring to prepare a lower solid culture medium; mixing a borrelia burgdorferi bacterium suspension diluted in a gradient way and the BSK storage liquid culture medium, pouring into an upper culture medium so that a macroscopic single colony appears; raising the single colony by using a toothpick, inspecting by using a dark-field microscope to observe a representative burgdorferi bacterium strain. The method disclosed by the invention has the advantages of easiness and fastness for operation and clarity in result. According to the invention, the representative burgdorferi bacterium strain is uniform in genetic background without heterogeneity, suitable for subsequent scientific research and used as a standard strain.

Description

Technical field [0001] The present invention relates to the field of medical microbiology, in particular to a Borrelia BSK storage medium, and also relates to a method for separating and purifying a single colony of Borrelia by using the storage medium, and also relates to the method of separation and purification in the preparation of microbiology scientific research, Use of single colonies and pure cultures in clinical diagnosis. Background technique [0002] Single colony separation and microbial pure culture technology is one of the most common and important technologies in microbiology scientific research, medical treatment, and industrial production. A culture containing more than one kind of microorganisms is called a mixed culture. If all cells in a colony come from a parental cell, then the colony is called a pure culture. In the identification of strains, industrial production and scientific research, the microorganisms used are generally required to be pure cultures. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/02C12R1/01
Inventor 叶美萍楼永良
Owner WENZHOU MEDICAL UNIV
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