Observation experiments on intact cellular biofilm systems
Observation experiments on intact cellular biofilm systems
Biofilm is an important structure of the cell, in eukaryotic cells, it includes the plasma membrane wrapped around the cell and the inner membrane wrapped around the cell in the endoplasmic reticulum, mitochondria, Golgi complex, lysosomes and other organelles, the basic structure of the lipid bilayer composed of lipids, various proteins inlaid with a series of functions to accomplish such as material transport, enzyme reactions, signaling, structural connections, etc., which reflects the structure and distribution of the biofilm in different cells, is important for understanding and understanding the overall structure and function of cells. A complete reflection of the structure and distribution of biological membranes in different cells is of great significance in recognizing and understanding the overall structure and function of cells.
Principle
The basic principle of the observation of intact cellular biofilm system is based on the method developed by Jindan Song in 1984, which can show the whole structure of the cellular biofilm system. The method is based on the fixation of intact cells with potassium permanganate fixative, which removes the protein components of the cells and retains the lipid components of the membranes. As a result, the cytoskeleton and soluble proteins in the cells are removed, the transparency of the specimen is increased, and the biofilm system including the endoplasmic reticulum and mitochondrion is preserved intact. In the process of fixing the specimen with potassium permanganate, the high-valent manganese is reduced to manganese dioxide and deposited in the hydrophilic end of the lipid molecules constituting the membrane structure, which increases the contrast of the specimen, and enables the specimen to be stained without staining to show the whole picture of the cellular bio-membrane system clearly under the phase contrast microscope.
Operation method
Observation experiments on intact cellular biofilm systems
Principle
The basic principle of the observation of intact cellular biofilm system is based on the method developed by Jindan Song in 1984, which can show the whole structure of the cellular biofilm system. The method is based on the fixation of intact cells with potassium permanganate fixative, which removes the protein components of the cells and retains the lipid components of the membranes. As a result, the cytoskeleton and soluble proteins in the cells are removed, the transparency of the specimen is increased, and the biofilm system including the endoplasmic reticulum and mitochondrion is preserved intact. In the process of fixing the specimen with potassium permanganate, the high-valent manganese is reduced to manganese dioxide and deposited in the hydrophilic end of the lipid molecules constituting the membrane structure, which increases the contrast of the specimen, and enables the specimen to be stained without staining to show the whole picture of the cellular bio-membrane system clearly under the phase contrast microscope.
Materials and Instruments
Equipment: Move The basic procedure of observing the intact biofilm system of cells can be divided into the following steps, taking the observation of African green monkey kidney cells CV-1 as an example: Caveat 1 Potassium permanganate is a strong oxidizing agent, which has a strong effect on the cells and is easy to cause the cells to become brittle and affect the observation. In practice, attention should be paid to finding and controlling the optimal fixation time in order to obtain better results.2 Potassium permanganate fixative should be used now, or can be stored in brown glass bottles, 4 ℃ can be stored for 8 weeks, room temperature storage for 2 weeks. For more product details, please visit Aladdin Scientific website.
Cover slips, staining vat, carrier slips, absorbent filter paper, pipettes, ordinary light microscope, phase contrast microscope, CO
2
incubator, African green monkey kidney cells CV-1.
Reagents:
① PBS (pH 7.2).
② KMnO
4
Fixation solution
(trisodium citrate, 60 mmol/L; potassium chloride, 25 mmol/L; magnesium chloride, 35 mmol/L; potassium permanganate, 125 mmol/L).
A Cultured CV-1 cells were inoculated on sterile coverslips, and the cells were grown into a monolayer after 24-48 h.
B Take a sheet of the coverslip and rinse it with PBS for 3 times, remove the culture fluid and impurities on the surface of the cells, and blot out the liquid on the filter paper.
C Put a drop of freshly prepared KMnO4 fixing solution on the coverslip and fix it for 5-7 min.
D Carefully rinse the coverslip with PBS for 5 times, remove the residual fixative, and absorb the excess liquid with filter paper.
E Place the coverslip cell side down on a slide and observe under the microscope.