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Fluorescent microscopic counting method for detecting number of bacteria in water body

A technology of bacteria counting and fluorescence microscopy, which is applied in the field of environmental microbiology, can solve the problems of complex time-consuming and uncomfortable rapid detection, and achieve the effects of reducing detection cost, good observation effect, and reducing background fluorescence

Inactive Publication Date: 2015-05-06
武汉市环境保护科学研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the reported technologies and methods for using organic membranes, the organic membranes have to undergo complex and time-consuming pretreatments to reduce the background fluorescence signal, which is not suitable for rapid detection. The filter membrane used in "Cultivation State Fluorescence Microscopy Observation and Counting Method" must be hydrophobically treated, dyed and dried before it can be used for fluorescence microscopic observation

Method used

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  • Fluorescent microscopic counting method for detecting number of bacteria in water body
  • Fluorescent microscopic counting method for detecting number of bacteria in water body
  • Fluorescent microscopic counting method for detecting number of bacteria in water body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 Preparation of anti-fading agent stock solution and anti-fading agent working solution

[0025] (1) Preparation of anti-fading agent stock solution

[0026] Dissolve 2g p-phenylenediamine, 0.8g sodium chloride, 3.2g disodium hydrogen phosphate, 0.25g Tris, 4g glycine in 80mL deionized water, adjust the pH to 7.2 with 0.1M hydrochloric acid or sodium hydroxide, add 100mL glycerol , and finally dilute to 200mL with deionized water to make anti-fading agent stock solution. Store the anti-fading agent stock solution in the dark at 4°C, and the validity period is 3 months.

[0027] (2) Preparation of anti-fading agent working solution

[0028] Take 1mL of the anti-fading agent stock solution, filter and sterilize it with a sterile needle filter, and then dilute to 10mL with sterile water to make the anti-fading agent working solution. The anti-fading agent working solution is prepared and used immediately.

Embodiment 2

[0029] Example 2 E. coli culture count

[0030] Cultivate Escherichia coli DH5α with beef extract-peptone liquid medium until the absorbance at 600nm is 0.2, take 2mL of bacterial liquid, centrifuge at 12000g for 10min, discard the supernatant, and resuspend the precipitate in 2mL of sterile saline for later use.

[0031] Take 100 μL of the above-mentioned spare bacterial suspension in a 5 mL centrifuge tube, add 10 μL of 10× SYBR Green I fluorescent dye, and stain in the dark for 5 minutes, then add 890 μL of sterile normal saline and 3 mL of anti-fading agent working solution, and keep it at room temperature in the dark 5 minutes, and then filtered through a 0.22 μm pore size cellulose acetate filter. Place the filter membrane on a clean glass slide so that the side with Escherichia coli is facing up, then drop a drop of anti-fading agent working solution on the filter membrane, cover it with a cover glass, and place it on the stage of the fluorescence microscope. Excit...

Embodiment 3

[0033] Example 3 Detection of Total Bacteria in Water Samples of Wuhan East Lake

[0034] In October 2014, 500mL mineral water bottles were used to collect water samples from four sub-lakes of East Lake in Wuhan (Guozheng Lake, Niuchao Lake, Tangling Lake, and Guanqiao Lake), and the incubators were refrigerated and transported back to the laboratory. Take 1 mL of each water sample into a 5 mL centrifuge tube, add 100 μL of 10× SYBR Green I fluorescent dye, and stain for 5 min at room temperature in the dark. Add the anti-fading agent working solution to each dyed water sample according to the volume ratio of the anti-fading agent working solution and the dyed water sample at a ratio of 3:1, mix well and keep it in the dark for 5 minutes. Afterwards, each water sample was filtered using a vacuum filtration device, and the filter membrane used was an ordinary cellulose acetate filter membrane with a pore size of 0.22 μm. After the filtration is completed, take out the filt...

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Abstract

The invention discloses a fade-resistant agent used in bacteria counting by virtue of a fluorescent microscopy and a fluorescent microscopic counting method for detecting the number of bacteria in a water body and belongs to the field of environmental microbiology. The fade-resistant agent is obtained by dissolving p-phenylenediamine, sodium chloride, disodium hydrogen phosphate, Tris, glycine and glycerol in deionized water and adjusting the pH. The fluorescent microscopic counting method for detecting the number of bacteria in the water body comprises the following steps: adding a fluorescent dye into a water sample, staining, adding a fade-resistant agent working liquid for carrying out fade-resistant treatment, carrying out suction-filtration on the sample subjected to the fade-resistant treatment with a cellulose acetate filtration membrane and observing under the fluorescent microscope. The method for detecting the number of bacteria in the water body is time-saving and effort-saving, low in cost and strong in fade-resistant capability and is free of background fluorescence interference and the number of bacteria in the water sample can be quickly and accurately detected.

Description

technical field [0001] The invention belongs to the field of environmental microbiology, and in particular relates to an anti-fading agent for counting bacteria with a fluorescent microscope and a fluorescent microscopic counting method for detecting the number of bacteria in a water body. Background technique [0002] The total number of bacteria in the water body is one of the indicators reflecting the water quality of lakes and rivers. The commonly used detection methods for total bacterial count in water are culture methods, including plate method, MPN method, etc. But not all bacteria can be cultured, therefore, the culture method will lead to large errors in the detection results. In order to overcome the shortcomings of the culture method, various direct counting methods are widely used, among which the results are relatively reliable and widely recognized methods include fluorescence microscopic counting method and flow cytometry counting method. Due to the high co...

Claims

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Application Information

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IPC IPC(8): C12Q1/06
Inventor 沙茜朱志超柯文彪何君周帆琦
Owner 武汉市环境保护科学研究院
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