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Improved counting method of living bacteria

A technology for counting viable bacteria and bacteria, applied in the field of biotechnology, can solve the problems of small sampling volume, inability to truly reflect the overall situation of the number of viable bacteria in the sample, and long time, so as to reduce randomness, save materials, and improve counting efficiency.

Pending Publication Date: 2020-05-01
湖南景翌湘台环保高新技术开发有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 1. The sampling volume of the diluted sample liquid is 0.1-0.2ml, and the sampling volume is small. If the bacterial liquid is not completely dispersed and mixed, randomness is likely to occur, and it cannot truly reflect the overall situation of the actual number of viable bacteria in the sample;
[0005] 2. One plate can only detect one concentration gradient of one sample once. Generally, one concentration gradient of one sample needs to be tested in 3 parallel samples. If multiple concentrations are to be detected, a large number of plates must be prepared;
[0006] 3. After the flat plate is coated, it needs to stand for 5min-180min. After the diluted sample liquid is completely absorbed, it can be inverted and placed in the incubator for cultivation. In addition to the pre-sterilization preparation and operation time, the time required for the entire experiment longer

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 A kind of improved bacteria counting method

[0026] Include the following specific steps:

[0027] (1) Sterilize the sand core filter and microporous membrane (pore size: 0.22 μm, diameter: 50 mm) used for filtration (sterilize at 121°C for 30 minutes), and then put them into an oven to dry at 60°C until Completely dry, assemble after cooling to room temperature, and put it in the ultra-clean workbench for use;

[0028] (2) Use a vortex shaker to vibrate the diluted sample solution for 30s;

[0029] (3) The mixed diluted sample liquid is suction-filtered with a sand core filter and a microporous filter membrane. After the suction filtration is completed, the filter membrane is slowly pasted on the culture medium, which contains 0.001wt% of naphthyridone and 0.02wt% yeast extract, with the bacteria side of the filter membrane up, incubate at 25°C for 4 hours in the dark, and then fix with formaldehyde at a final concentration of 1wt%;

[0030] (4) Stain ...

Embodiment 2

[0031] Embodiment 2 A kind of improved bacteria counting method

[0032] Include the following specific steps:

[0033] (1) Sterilize the sand core filter and microporous membrane (pore size: 0.22 μm, diameter: 50 mm) used for filtration (sterilize at 121°C for 30 minutes), and then put them into an oven to dry at 120°C until Completely dry, assemble after cooling to room temperature, and put it in the ultra-clean workbench for use;

[0034] (2) Use a vortex shaker to vibrate the diluted sample solution for 90s;

[0035] (3) The mixed diluted sample liquid is suction-filtered with a sand core filter and a microporous filter membrane. After the suction filtration is completed, the filter membrane is slowly pasted on the culture medium, which contains 0.005wt% of naphthyridone and 0.025wt% yeast extract, with the bacteria side of the filter membrane up, incubate at 30°C for 8 hours in the dark, and then fix with formaldehyde at a final concentration of 3wt%;

[0036] (4) Stai...

Embodiment 3

[0037] Embodiment 3 A kind of improved bacteria counting method

[0038] Include the following specific steps:

[0039] (1) Sterilize the sand core filter and microporous membrane (pore size: 0.22 μm, diameter: 50 mm) used for filtration (sterilize at 121°C for 30 minutes), and then put them into an oven to dry at 90°C until Completely dry, assemble after cooling to room temperature, and put it in the ultra-clean workbench for use;

[0040] (2) Use a vortex shaker to vibrate the diluted sample solution for 60s;

[0041] (3) The mixed diluted sample liquid is suction-filtered with a sand core filter and a microporous filter membrane. After the suction filtration is completed, the filter membrane is slowly pasted on the culture medium, which contains 0.003wt% of naphthyridone and 0.022wt% yeast extract, with the bacteria side of the filter membrane up, incubate at 27°C for 6 hours in the dark, and then fix with formaldehyde at a final concentration of 2wt%;

[0042] (4) The f...

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Abstract

The invention discloses an improved counting method of living bacteria. The improved counting method of the living bacteria comprises the following steps: (1) sterilizing a sand-core filter and a microporous filter membrane used for performing filtration, assembling the sterilized sand-core filter and microporous filter membrane, and putting the assembled sand-core filter and microporous filter membrane into a super clean bench for standby application; (2) uniformly oscillating a diluted sample solution by using a vortex mixer so as to obtain a uniformly mixed diluted sample solution; (3) performing suction filtration on the uniformly mixed diluted sample solution by using the assembled sand-core filter and microporous filter membrane, slowly attaching the microporous filter membrane to amedium, carrying out light-shielded cultivation, and carrying out immobilization by using formaldehyde so as to obtain an immobilized filter membrane; (4) dyeing the immobilized filter membrane by using acridine orange, placing the dyed filter membrane on a slide, counting fluorescent bacteria by using a microscope, and identifying growing or thickening bacteria in the field of vision as live bacteria. The improved counting method of the living bacteria disclosed by the invention is capable of reducing randomness caused by small sample size, and avoids relatively long standing time; and moreover, the method is also capable of avoiding waste of a large amount of plates in a plate assay. In addition, direct counting by the microscope is adopted, so that living bacterium counting efficiency is greatly improved; and thus, the improved counting method of the living bacteria has the advantages of being economical of materials, plenty in parallel repeats, high in efficiency and the like.

Description

technical field [0001] The invention relates to the technical field of biotechnology, in particular to an improved method for counting live bacteria. Background technique [0002] The commonly used plate colony counting method is designed according to the principle that each living bacterium can grow a colony. After the bacteria-containing sample to be tested is diluted in a series of appropriate ratios and fully shaken evenly, the microorganisms in it are fully dispersed into single cells, and then a certain amount of diluted sample solution is applied to a plate, and after cultivation, each single cell The colonies visible to the naked eye formed by growth and reproduction, the number of cultured colonies can be counted, and the number of viable bacteria contained in the sample can be converted according to the dilution factor and the sampling inoculum volume. This method has high sensitivity and is a method for detecting the number of viable bacteria in contamination, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/06
CPCC12Q1/06
Inventor 周霜艳解付兵欧阳书径曾宪军刘宜德
Owner 湖南景翌湘台环保高新技术开发有限公司
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