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Combination of Fluorescent Substrate and pH-Sensitive Fluorophore for Fluorometric Detection of Microbial Growth Medium

A technology of growth medium and fluorescent substrate, which is applied in the field of growth medium for the detection of microorganisms by fluorescent method in combination with fluorescent substrates and pH-sensitive fluorophores.

Active Publication Date: 2016-08-03
MILLIPORE CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

As such, they cannot distinguish between microorganisms present in the same growth medium, nor can they determine their relative proportions

Method used

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  • Combination of Fluorescent Substrate and pH-Sensitive Fluorophore for Fluorometric Detection of Microbial Growth Medium
  • Combination of Fluorescent Substrate and pH-Sensitive Fluorophore for Fluorometric Detection of Microbial Growth Medium
  • Combination of Fluorescent Substrate and pH-Sensitive Fluorophore for Fluorometric Detection of Microbial Growth Medium

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Experimental program
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Embodiment Construction

[0122] 1 Preparation of growth medium

[0123] 100 mL of liquid TSB (Biomerieux) medium was prepared and sterilized according to the manufacturer's recommendations. Then, the following solutions of pH-sensitive fluorophores and fluorogenic substrates were added under aseptic conditions to the TSB medium according to each combination described below.

[0124] pH-sensitive fluorophores:

[0125] HTPS:

[0126] - 30 μL of 10 mM stock solution of 8-hydroxypyrene 1,3,6-trisulfonic acid, or

[0127] CF:

[0128] - 20 μL of a sterile stock solution of 100 μM 5,6-carboxyfluorescein;

[0129] Fluorescent substrate:

[0130] Aldols:

[0131] - 200 μL of a sterile mixture of Aldol derivatives (Aldol470 phosphate, Aldol470 ester, aldol455β-D-glucopyranoside, aldol455β-D-galactopyranoside), where the stock concentration of each derivative is 25 mg / mL ;or

[0132] 4-MU:

[0133] - 200 μL of 4-methylumbelliferyl derivatives (4-MU phosphate, 4-MUβ-D-glucopyranoside, 4-MUα-D-glucopy...

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Abstract

The invention relates to a growth medium allying a fluorogenic substrate and a pH-sensitive fluorophore, in particular the combination of 4-methylumbelliferone (4-MU) and derivatives of fluorescein. This growth medium is used for the detection by fluorescence of microorganisms by coupling the fluorescence measurements relating to the pH-sensitive fluorophor and the fluorescence measurements relating to the activation of the fluorogenic substrate(s) by the microorganisms.

Description

technical field [0001] The present invention relates to methods and devices for testing the sterility of aqueous or gaseous fluids used in particular in the manufacture of pharmaceutical products and devices. [0002] In particular, the present invention relates to a growth medium comprising a fluorogenic substrate and a pH-sensitive fluorophore, in particular a combination of 4-methylumbelliferone (4-MU) and a fluorescein derivative, for use by fluorescent , in particular for the detection of microorganisms by combining fluorescence detection of pH-sensitive fluorophores with fluorescence detection of fluorescent substrates activated by the microorganisms. Background technique [0003] The pharmaceutical industry is one of those areas where sterility restrictions are very high, forcing continuous monitoring of product quality and all activities related to these products. [0004] For microbial contamination, tests are performed using microbiological methods in accordance w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/04C12N1/20
CPCC12Q1/045
Inventor F·马克
Owner MILLIPORE CORP