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

A technology for separating and purifying Borrelia, applied in the field of medical microbiology, can solve problems such as unfavorable growth of Borrelia, poor quality, and difficult application of Borrelia diagnosis, etc., to achieve convenient cell growth, moderate cross-linking degree, and simple separation and purification method fast effect

Active Publication Date: 2015-04-08
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 kind of Borrelia BSK stock solution culture medium, it is made of the raw material of following weight portion:

[0055]

[0056]

[0057] A preparation method for Borrelia BSK stock solution culture medium, the steps are:

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

Embodiment 2

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

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

[0062] (2) Preparation of the bottom solid medium: adjust the temperature of the water bath to 55°C, and gradually melt 5% agarose gel in it. Then mix evenly with the BSK stock solution culture medium prepared in Example 1 according to the volume ratio of 1:1, and add kanamycin (sigma company) to a final concentration of 200 micrograms per milliliter. Pour into Petri dishes, each requiring 10 ml of bottom solid medium for 10 cm diameter Petri dishes. Stand at room temperature for 12 hours, and store in a refrigerator at 4°C until the medium is completely solidified and there is no condensed water on the surface.

[0063] (3) Borrelia burgdorferi B31 strain was cult...

Embodiment 3

[0067] A kind of Borrelia single colony separation and purification method is used in the application of preparation Borrelia burgdorferi pure culture, and its application process is:

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

[0069] Carefully pick 5 strains of Borrelia burgdorferi B31 with a toothpick, inoculate them into 12 ml of BSK-H conventional liquid medium (product of sigma company), culture them in a 37-degree carbon dioxide incubator (5%) for 3-4 days, and observe the Borrelia growth, strain growth to logarithmic growth phase, colony concentration reached 10 7 A single colony pure culture of Borrelia burgdorferi can be obtained with more than cells / ml.

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

[0071] Borrelia burgdorferi contains 20 linear or circular plasmids ranging in size from 5 to 56kb. Some plasmids are unstable during subculture in vitro, but they encode important phenotypic characteristics. Therefore,...

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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 stock solution culture medium, a method for separating and purifying a Borrelia single colony using the stock solution medium, and also relating to the use of the separation and purification method in the preparation of microbiological scientific research, Use in single colonies and pure cultures in clinical diagnostics. Background technique [0002] Single colony isolation and microbial pure culture technology is one of the most common and important technologies in microbiological scientific research, medical treatment and industrial production. A culture containing more than one type of microorganism is called a mixed culture, and if all cells in a colony come from one parent 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...

Claims

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Application Information

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