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Method for membrane breaking production of protozoa single cell scanning electron microscopy sample

A technology of protozoa and scanning electron microscopy, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of internal structure damage of cells, loss of organelles, and inability to observe the structure of cytoplasm, etc., and achieve good in-situ characteristics and simple dehydration steps , the effect of reducing the experimental risk

Inactive Publication Date: 2016-11-09
HARBIN NORMAL UNIVERSITY
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Problems solved by technology

[0004] The present invention aims to solve the problem that the protozoan single-cell scanning electron microscope sample prepared by the existing method can only observe the cell surface structure, and the result of the early membrane rupture sample is unstable, and the loss of organelles and the damage of the internal structure of the cell often occur. It is limited to the structure immediately under the surface membrane, but cannot observe the structure in the cytoplasm, and provides a method for preparing protozoan single-cell scanning electron microscope samples by breaking the membrane

Method used

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  • Method for membrane breaking production of protozoa single cell scanning electron microscopy sample
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  • Method for membrane breaking production of protozoa single cell scanning electron microscopy sample

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specific Embodiment approach 1

[0057] Embodiment 1: The method for preparing a protozoan single-cell scanning electron microscope sample by breaking the membrane in this embodiment is carried out according to the following steps:

[0058] 1. Material collection and cleaning

[0059] The day before the experiment, the protozoa were treated with low temperature or starvation, and on the second day, 140-160 protozoa with normal morphology and uniform protoplasm were picked into a clean concave dish, and the protozoa bodies were rinsed with pure water to remove the protozoa. Clean the dirt attached to the surface of the body, and then suck out the water in the concave dish, leaving as little water as possible;

[0060] 2. Fixation and membrane rupture

[0061] Inject the fixed membrane-breaking agent into the fixed cylinder, use a micropipette to absorb the protozoa in the concave dish and inject it into the fixed cylinder, fix it in the refrigerator at 4°C in the dark and rupture the membrane for 10-15 minute...

specific Embodiment approach 2

[0074] Embodiment 2: The difference between this embodiment and Embodiment 1 is that the low temperature treatment in Step 1 is at 16-18° C. for 24 hours. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0075] Embodiment 3: This embodiment is different from Embodiment 1 in that: the starvation treatment in Step 1 is to stop feeding for 24-48 hours. Others are the same as in the first embodiment.

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Abstract

A method for membrane breaking production of a protozoa single cell scanning electron microscopy sample is provided to solve the problems of unstable result, frequently appearing organelle missing, cell internal structure damages, only observation of the structure cling to the lower surface of a surface membrane and unable observation of the structure in the mitochondria of protozoa single cell scanning electron microscopy samples produced through present methods. The method comprises the following steps: 1, material drawing and cleaning; 2, fixing and membrane breaking; 3, water washing; 4, dehydration; 5, CO2 critical point drying; 6, table bonding; 7, ; and 8, electron microscope observation. The method has the advantages of step simplification, simplicity in convenience, observation of the complete and meticulous cell internal structure, and realization of the 90% or above success rate, and can be used for membrane breaking production of the protozoa single cell scanning electron microscopy sample.

Description

technical field [0001] The invention relates to a method for preparing a single-cell scanning electron microscope sample by membrane rupture. Background technique [0002] The resolution of scanning electron microscope is between optical microscope and transmission electron microscope, 30-60 Angstroms. The conventional SEM sample preparation process includes five main steps: cleaning, fixing, dehydration, drying, and gold spraying. Due to the complex living environment of protozoa, various structures and high water content in cells, the preparation methods of protozoa are different from other biological samples in the steps of cleaning, fixing and dehydration. Conditions for washing, fixing and dehydrating must be determined according to specific protozoan cell characteristics. [0003] Over the years, researchers have explored a series of stable steps and conditions for the preparation of different protozoan SEM samples, which are widely used in the study of the three-dim...

Claims

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Application Information

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IPC IPC(8): G01N23/22
CPCG01N23/2202
Inventor 陈瑛邱子健潘旭明王丽高晶丁文乔胡鹏月吴禹岐
Owner HARBIN NORMAL UNIVERSITY
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