Specimen preparation and observation experiments of biofilm system of whole cultured cells
Specimen preparation and observation experiments of biofilm system of whole cultured cells
Cellular biofilm system refers to the organelles surrounded by membranes such as the cell membrane, the nuclear membrane, and the endoplasmic reticulum, Golgi apparatus, mitochondria, etc., which are structurally and functionally closely linked as a unified whole. Since the cell membrane, the nuclear membrane, and the organelles surrounded by membranes such as the endoplasmic reticulum, the Golgi apparatus, and the mitochondria are all involved in the cell membrane or the organelle membranes, it is often referred to as the biofilm system.
Operation method
basic program
Principle
Potassium permanganate fixative can get rid of the protein components in the cell, while retaining the lipid components of the membrane, as a result, the cytoskeleton and soluble proteins in the cell, etc. are removed, increasing the transparency of the specimen, while the endoplasmic reticulum, mitochondrion, Golgi apparatus, lysosomes and other bio-membrane systems are preserved intact. Potassium permanganate fixative can form manganese dioxide in the process of fixing the specimen, which can form microscopic precipitation at the hydrophilic end of lipids of various membrane structures of the cell, and as a result, the specimen does not need to be stained, i.e., it can be clearly shown the whole picture of the cellular biofilm system under the phase contrast microscope and transmission electron microscope.
Materials and Instruments
Renal epithelial cells Move I. Potassium permanganate fixation method showing the preparation and observation of light microscopic specimens of CV-1 cell biofilm system before and after drug administration (colchicine treatment). For more product details, please visit Aladdin Scientific website.
Potassium permanganate Colchicine PBS Formvar solution
Microscope Phase contrast microscope Transmission electron microscope Nickel mesh Slides Coverslips Petri dishes CO2 incubator
1. Take a sterile coverslip and put it into two 5 cm diameter petri dishes, inoculate CV-1 cells with 3 ml of culture medium. 2.
2. Cover with a lid and label well, and transfer to a CO2 incubator for 24 hours. Add 10 ug/ml colchicine 0.3 ml to one of the dishes 4 hours before termination of incubation. 3.
3. Observe under an inverted microscope the selected coverslip with well spread cells, terminate the culture and pour off the culture medium.
4. Rinse the coverslips twice with PBS solution to remove the culture medium and impurities on the cell surface.
5. Remove the coverslip and place the coverslip with well-spreading cells, cell side up, on the slide.
6. Apply a drop of freshly prepared potassium permanganate fixative to the coverslip and fix for 10 minutes.
7. Gently rinse the cell side of the specimen 5 times with distilled water to remove any residual fixative.
8. Remove the coverslip, absorb the excess water with filter paper, put 1 drop of PBS solution on the slide, and mount the coverslip with the cell side facing down.
Second, potassium permanganate fixative showed the preparation and observation of electron microscopy specimens of CV-1 cell biofilm system before and after drug administration (colchicine treatment).
1. Supporting membrane was made with 0.5% Formvar solution.
2. The nickel mesh covered with Formvar membrane was sterilized by ultraviolet light and put into a sterile petri dish of 5 cm in diameter with 3 ml of culture medium.
3. CV-1 cells were inoculated on the nickel mesh with the supporting membrane, one group without drug treatment as the control group, and the other group treated with 10 ug/ml colchicine as the experimental group.
4. Cultured cells in the experimental group were added with 0.3 ml of 10 ug/ml colchicine 4 hours before termination of culture, and put into a CO2 incubator for cell culture.
5. After the cells were spread, the nickel net was removed, and 1 drop of potassium permanganate fixative was dropped onto the nickel net with a nipple pipette to fix the cells for 10 minutes.
6. Dehydrate via ascending gradient ethanol series: 30%→50%→70%→85%→90%→95%→100%, 3 minutes for each dehydration.
7. Allow the nickel mesh to dry and then observe under a transmission electron microscope (75 KV voltage).
III. Results
1. The endoplasmic reticulum was spread as a net in the whole cytoplasm under light microscope and transmission electron microscope.
2. The endoplasmic reticulum around the nucleus formed a dense three-dimensional structure, while the endoplasmic reticulum far away from the nucleus was in the form of a loose mesh stretching to the edge of the cell. 3.
3. The endoplasmic reticulum consists of tubules and flat vesicles as seen under the electron microscope. In addition to the endoplasmic reticulum, you can also see the thick mitochondria in the form of cords, can also be observed in the Golgi apparatus and lysosomes and other membrane structures.
4. After colchicine treatment, the endoplasmic reticulum of CV-1 cells aggregated around the nucleus, and the endoplasmic reticulum was no longer observed at the edge of the cell.
5. Colchicine destroys the microtubule structure, therefore, the distribution of endoplasmic reticulum may be closely related to the presence of microtubules.