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Culture medium for separating clostridium difficile in intestinal microorganisms and preparation method

A technology for Clostridium and intestinal microorganisms, applied in the field of microorganisms, can solve the problems of indistinguishable, insufficient sequencing depth, low growth and detection content, etc., and achieve the effect of high survival rate

Pending Publication Date: 2022-05-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, information obtained from molecular methods alone is also limited, requiring isolation of organisms to determine the role of specific bacteria in causing or maintaining states of health and disease
Traditional culture methods determine the number of living cells in a colony, while most molecular methods do not distinguish between DNA obtained from live or dead cells
Furthermore, cultivation using selective media allows for the growth and detection of lower abundance bacteria that might be missed in studies based on molecular methods due to insufficient sequencing depth

Method used

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  • Culture medium for separating clostridium difficile in intestinal microorganisms and preparation method
  • Culture medium for separating clostridium difficile in intestinal microorganisms and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Fecal Microbiota Culture

[0028] Specific steps are as follows:

[0029] (1) Preparation of fecal suspension

[0030] In an anaerobic chamber (condition: 37° C.), 0.1 g of feces sample was diluted tenfold with 900 μL of pre-reduced brain heart infusion (BHI) medium and 0.05% cysteine ​​hydrate, and diluted five gradients.

[0031] (2) 100 μL of 10 3 and 10 5 The dilution was evenly spread on a 100mm agar plate of brain-heart infusion (BHI) broth, and incubated in an anaerobic chamber at 37°C for 5 days. After incubation, add 1ml of BHI broth to the agar plate, scrape the surface with a cell scraper, collect colonies, freeze in pre-cooled 10% skimmed milk, and store at -80°C.

Embodiment 2

[0032] Example 2: Culture isolation and identification of Clostridium difficile

[0033] Specific steps are as follows:

[0034] (1) Preparation of Clostridium difficile isolation agar medium

[0035] Table 1: Components of Clostridium difficile isolation medium

[0036]

[0037] According to the formula configuration in the above-mentioned Table 1, a culturable Clostridium difficile isolation medium was obtained. When the medium was liquid, the amount of agar added was: 12g.

[0038] (2) Spread the feces samples stored at -80°C in Example 1 evenly on Clostridium difficile isolation agar medium, and incubate anaerobically at 37°C for 48 hours.

[0039]Since Clostridium difficile develops color under fluorescent illumination after growth in Clostridium difficile isolation medium, color-developed colonies grown on plates were streaked on Brain Heart Infusion Solid Medium to obtain purity.

[0040] (3) Rinse with 1 mL of PBS and scrape off all the cells on the plate after t...

Embodiment 3

[0045] Example 3: Static culture and dynamic culture of infant intestinal flora in large intestine bioreactor

[0046] Take the thalli that develop color under the fluorescence of Example 2 and insert them into the Clostridium difficile isolation liquid medium, and culture them anaerobically at 37° C. for 48 hours to prepare a culture.

[0047] Static culture: Inject the culture into a large intestine bioreactor filled with 150 mL Clostridium difficile isolation medium, and keep the large intestine bioreactor free of peristalsis.

[0048] Dynamic culture: Inject the culture into a large intestine bioreactor filled with 150 mL Clostridium difficile isolation medium, set the peristaltic frequency to 3 times / min, and control the pH to 6.5.

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Abstract

The invention discloses a culture medium for separating clostridium difficile in intestinal microorganisms and a preparation method, and belongs to the technical field of microorganisms. The culture medium contains glucose, maltose, a yeast extract, peptone, L-cysteine, isovaleric acid, butyric acid, acetic acid, KH2PO4, NaHCO3, NaCl, MgSO4-7H2O, a growth factor and a pigment, and clostridium difficile can develop color under the culture medium. The culture medium is used for separating and culturing fecal flora in combination with a bionic gastrointestinal tract reactor, a real intestinal environment is simulated, and a high survival rate of drug-resistant intestinal microorganisms can be achieved. The method can be applied to the recovery of specific flora, and also highlights the capabilities of culturing and recovering low-abundance bacteria and revealing diversity. Obtained cultured human microbiota will provide detailed functional characteristics of the bacteria, contributing to the discovery of their biological activity during host bacteria and inter-bacterial interactions.

Description

technical field [0001] The invention relates to a culture medium and a preparation method for isolating Clostridium difficile in intestinal microorganisms, belonging to the technical field of microorganisms. Background technique [0002] The gastrointestinal microbiota is a highly diverse community, but most bacteria are considered unculturable. Sequencing-based studies have revealed greater diversity of microbial populations in the gastrointestinal microbiota compared to previous culture methods. Consequently, most studies characterizing the human gut microbiome have relied on culture-independent sequencing methods. These studies provide new insights into the community composition of the gut microbiota in healthy individuals, the altered behavior of the GI microbiota in response to environmental perturbations, and its potential role in various diseases. However, information obtained from molecular methods alone is also limited, requiring isolation of organisms to determin...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/02C12R1/145
CPCC12N1/20C12N1/02Y02A50/30
Inventor 王玉迎詹晓北徐星海李志涛高敏杰蒋芸张倚菲张梓健史诚文
Owner JIANGNAN UNIV
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