Preparation method of TEM (transmission electron microscope) ultrathin section samples of phosphorus-accumulating bacteria

A technique of transmission electron microscopy and ultra-thin sectioning, which is applied in the field of preparation of TEM ultra-thin section samples, can solve the problems of complex sample pretreatment process, difficult to clearly display the ultrastructure of PAOs cells, and strong toxicity of drugs.

Inactive Publication Date: 2015-07-01
DONGHUA UNIV
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a method for preparing ultra-thin section samples of phosphorus-accumulating bacteria through transmission electron microscopy. Difficult to display the problem clearly, using this sample preparation method to prepare PAOs samples, in TEM observation, the cell structure of the bacteria, especially the poly-Ps in the bacteria can be clearly and completely displayed

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  • Preparation method of TEM (transmission electron microscope) ultrathin section samples of phosphorus-accumulating bacteria

Examples

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Effect test

Embodiment 1

[0034] The specific steps of the preparation method of purely cultured phosphorus accumulating bacteria transmission electron microscope ultrathin section samples are as follows:

[0035] (1) Take materials

[0036] The sample was collected from the pure PAOs obtained by isolation, screening and purification, and identified as Pseudomonas putida; the pure culture PAOs sample (volume less than 1mm 3 ) into a 1.5ml centrifuge tube, and an appropriate amount of 0.1M sodium cacodylate (SCB) buffer solution was added dropwise to submerge the purely cultured PAOs sample. The above-mentioned process needs to be completed within 1-2 minutes after the sample is collected. At the same time, in order to prevent the autolysis of purely cultured PAOs cells, the above-mentioned process needs to be operated at a low temperature of 0-4°C.

[0037] (2) cleaning

[0038] Centrifuge the 1.5ml centrifuge tube in the above steps at a speed of 4000-5000r / min, discard the supernatant, and repeat t...

Embodiment 2

[0051] The specific steps of the preparation method of mixed culture phosphorus accumulating bacteria transmission electron microscope ultrathin section sample are as follows:

[0052] (1) Take materials

[0053] The sample was collected from the biofilm in the anaerobic-aerobic alternating biofilter (AABF) with stable laboratory operation, and the mixed culture PAOs sample (volume less than 1mm 3 ) into a 1.5ml centrifuge tube, and an appropriate amount of 0.1M sodium cacodylate (SCB) buffer solution was added dropwise to submerge to mix and culture PAOs samples. The above-mentioned process needs to be completed within 1-2 minutes after the sample is collected, and at the same time, in order to prevent the autolysis of the mixed culture PAOs cells, the above-mentioned process needs to be operated at a low temperature of 0-4°C.

[0054] (2) cleaning

[0055] Centrifuge the 1.5ml centrifuge tube in the above steps at a speed of 4000-5000r / min, discard the supernatant, and rep...

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Abstract

The invention relates to a preparation method of TEM (transmission electron microscope) ultrathin section samples of phosphorus-accumulating bacteria, which comprises the following steps: after a sample is taken, carrying out centrifugal cleaning on the sample, putting the phosphorus-accumulating bacteria PAOs sample into a glutaraldehyde solution with a pH value being 7.4 and a volume percentage concentration of 2.5% to fix; dyeing the phosphorus-accumulating bacteria sample by using a Pb(NO3)2 solution with a mass percentage concentration of 2-10%; carrying out dehydration by using a gradient ethanol-acetone series dehydration method; and carrying out resin impregnation and embedding, and sectioning finally. The method disclosed by the invention can be used for solving the problem that, the sample pretreatment process is complicated, the toxicity of used medicaments is strong, and the ultrastructure in a PAOs cell is difficult to clearly display in existing methods; and once the preparation method is adopted for preparing PAOs samples, in TEM observation, the cell structures of bacteria, especially poly-Ps in bacteria, can be clearly and completely revealed.

Description

technical field [0001] The invention belongs to the field of preparation of TEM ultra-thin section samples, in particular to a method for preparing phosphorus-accumulating bacteria transmission electron microscope ultra-thin section samples. Background technique [0002] Phosphorus accumulating organisms (PAOs) play a key role in the process of biological phosphorus removal. The phosphorus removal function of PAOs is closely related to the number of intracellular polymers such as polyphosphate particles (poly-Ps). In view of the importance of intracellular poly-Ps in PAOs, qualitative and quantitative analysis and determination are often carried out, usually by staining and observing the distribution of poly-Ps in PAOs with the help of optical or electron microscopes (staining conditions for intracellular polymers of PAOs Optimization and comparison of dyeing methods [J]. Environmental Science and Technology, 2014, (2): 1-6). However, the resolution of the optical microscop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28
Inventor 田晴倪兵朱艳彬杨波李方孟丹杰秦华星庄林杰王琦张成王康伟
Owner DONGHUA UNIV
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