Culture medium for identifying gram-negative and positive bacteria and use method

A technology for gram-negative and positive bacteria, applied in the field of culture medium for identifying gram-negative and positive bacteria, can solve problems such as unsolved diffusion problems, and achieve the effects of simple result judgment, strong specificity and unique chemical structure

Inactive Publication Date: 2013-12-11
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, the technical purpose of the present invention is to solve the problem that the method of measuring aminopeptidase to distinguish bacterial Gram-negative / positive bacteria does not solve the diffusion problem in the prior art, and obtain the method of distinguishing Gram-negative / positive bacteria by plate culture method ways to improve

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  • Culture medium for identifying gram-negative and positive bacteria and use method
  • Culture medium for identifying gram-negative and positive bacteria and use method
  • Culture medium for identifying gram-negative and positive bacteria and use method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Nutritive capacity evaluation and effect observation of embodiment 1 culture medium of the present invention

[0038] (1) Culture medium preparation: Weigh each component according to the formula in Table 1, add each component of the above medium into 1000mL deionized water, stir, heat and boil until completely dissolved, adjust the pH to 6.8±0.2, After cooling to 45-55°C, add L-Alanine-AMC-TFA, mix evenly, pour into a plate, and set aside.

[0039] (2) Inoculation and result observation: Pick fresh pure cultures of the test strains on the plates, prepare them in sterilized saline to a McFarland turbidity of 0.5, take 0.3 mL of bacterial suspensions and spread them on the above plates, and incubate at 36°C After 18 hours, observe whether the colonies produce fluorescence at a wavelength of 365 nm. The experimental strains that produce fluorescence are Gram-negative bacteria, and the experimental strains that do not produce fluorescence are Gram-positive bacteria.

[00...

Embodiment 2

[0045] Embodiment 2 The effect test of culture medium streak inoculation method of the present invention

[0046] Each 1000mL medium contains tryptone 10g, plant peptone 6g, yeast powder 7g, sodium chloride 5g, agar 16g, L-alanine-7-amino-4-methylcoumarin trifluoroacetate 0.1g ( L-Alanine-AMC-TFA), β-cyclodextrin 14g, the balance is water, pH6.8±0.2. Use a sterile loop to transfer the overnight broth culture of the test bacteria to the above-mentioned plate by streaking in four zones, culture at 35±1°C for 18 hours, observe whether the colonies produce fluorescence at a wavelength of 365nm, and produce fluorescence experiments The strains are Gram-negative bacteria, and the experimental strains that do not produce fluorescence are Gram-positive bacteria. Experimental results show that the streaking method for distinguishing Gram-negative and positive bacteria is also applicable to the culture medium of the present invention.

[0047] Table 3. Effect test of streak culture me...

Embodiment 3

[0049] Embodiment 3 The effect of distinguishing Gram-negative and positive bacteria after the culture medium of the present invention is made into a blood plate

[0050] Each 1000mL medium contains tryptone 10g, beef powder 5g, sodium chloride 5g, agar 16g, L-alanine-7-amino-4-methylcoumarin trifluoroacetate 0.1g (L-Alanine- AMC, TFA), β-cyclodextrin 14g, the balance is water, pH6.8±0.2, refer to the blood plate production method, add 60mL of sterile defibrated sheep blood to make a blood plate. After the medical experiment samples are processed according to the national clinical inspection operating procedures or other general methods, they are streaked and inoculated on the plate, cultured at 35±1°C for 18 hours, and observed at a wavelength of 365nm to see if the colonies produce fluorescence. The experimental strain that produces fluorescence is Gram Gram-negative bacteria, and the experimental strains that do not produce fluorescence are Gram-positive bacteria. Experime...

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Abstract

The invention relates to a culture medium for identifying gram-negative and positive bacteria and a use method, particularly, a culture medium for identifying the gram-negative and positive bacteria through stronger fluorescence produced by strains on a flat plate by expressing the property of L-alanine aminopeptidase by using gram-negative bacteria, taking L-Alanine-AMC-TFA (L-alanine-7-Amino-4-Methylcoumarin Trifluoroacetic Acid) as a substrate of the enzyme, blocking the diffusion of produced 4-methylcoumarin by using cyclodextrin, and a use method thereof. According to the culture medium and the method, the same result as the conventional gram staining method can be obtained during strain separation; the culture medium and the method have the advantages of high specificity, high sensitivity, easiness in operation and simple result judgment and the like, are suitable for the fields such as medical clinical microorganism examination, food safety microorganism examination and environmental protection and have broad application prospect.

Description

technical field [0001] The invention relates to a culture medium for distinguishing Gram-negative and positive bacteria and a method for using it. Specifically, the present invention relates to a culture medium for identifying Gram-negative and positive bacteria on a plate by using Gram-negative bacteria specifically expressing aminopeptidase and a use method thereof. Background technique [0002] By Gram staining, bacteria are divided into two categories, Gram-negative bacteria and Gram-positive bacteria, which are the basis for the identification, classification and identification of bacteria. Therefore, how to accurately and conveniently distinguish bacteria It is important to classify. [0003] At present, the distinction between Gram-negative and positive bacteria is mainly based on the staining method. First, gentian violet (also known as crystal violet) is used to stain the bacteria purple, and then coated with Gram's iodine solution to strengthen the combination of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04
Inventor 赵贵明陈颖赵勇胜杨海荣刘洋王娉胡玥
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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