A light microscope may be used to see and examine the vacuole’s structure. While the vacuole doesn’t stain as the other organelles of the cell (because it does not contain many constituents that stain) tests have demonstrated that staining is possible for this organelle because the vacuole’s sap absorbs and stores dyes that are colored.
The majority of dyes that are employed are natural in nature. The pH of the dye will also depend on the kind of vacuole to be stained.
The dyes used to stain vacuoles are:
- Neutral red
- Brilliant cresyl blue
- Methylene blue
- Pyronin
This time, we will concentrate on staining using a Neutral red solution (pH lower that 6.0).
Requirements of Vacuole Staining
- A part of a plant (roots, leaves etc)
- Neutral red stain (7.2 in 0.08M phosphate buffer)
- Water (distilled)
- A compound light microscope
- Glass slides and cover slips
- Droppers
- Tweezers
Procedure for Vacuole Staining
- Take a thin layer of the plant by using forceps or a pair of forceps. stain the specimen with 0.01 percent neutral red
- Rinse the sample using the buffer for phosphate.
- Mount to observe
Observation of Vacuole Staining
By this method, it is possible to stain the vacuole in live plants cells without staining other organelles. If examined under the microscope, students will see the vacuoles to appear red in the color.
Yeast Vacuole Staining with FM4-64
The probe that is lipophilic, FM 4-64, does not sparkle significantly in water, but it does fluoresce in a strong manner when it is bound to the plasma membrane’s outer surface that provides clear and distinct staining of plasma membranes. The binding is quick and is reversible. Vacuoles inside yeast cells will be stained by FM4-64 dye. This permits the use of live cell imaging when needed.
Materials and Reagents
- FM4-64 (Life Technologies, Molecular Probes, catalog number: F34653 )
- DMSO (Sigma-Aldrich)
- YES medium
- FM4-64 stock solution
- EMM
- PBS
Equipment
- Water bath
- Bench-top centrifuge
Procedure
- Make yeast cells grow to exponential phase in YES media at 30°C.
- Then, spin the cells down and resuspend the cell pellets in 500 ml of YES plus 0.5 milliliters FM4-64 stock solutions (8 millimols) So the total concentration of FM4-64 is eight milligrams. Place the cells under darkness (i.e. covered with aluminum foil). Incubate the cells in a 30°C water bath for 30 minutes.
- *FM4-64 stock solution equals 8 milligrams (5 mg/ml) in DMSO (stored at -20°C).
- *FM4-64 is not able to efficiently label cells even in minimal medium. Therefore, even if you have grown cells in EMM to keep the plasmid, you need to label the cells using FM in Yes.
- Rinse cells and wash pellets by resuspending them in YES to eliminate free FM4-64. Resuspend the cells and spin them down again in 1 ml of YES. Transfer cells to a tube and add 4ml of YES. Shake at 30°C for 90 minutes.
- Transfer cells (5 mg) into the centrifuge tube and spin for 5 minutes at RT.
- Resuspend the cell pellets using EMM as well as PBS.
- EMM doesn’t show nearly the same amount of autofluorescence as the YES system, so even if you did grow cells in YES, put them back in EMM in this stage.
- Spot cells are placed on glass slides, and then cover with a coverslip.
- Examine fluorescence with a microscope using RFP filter.
Notes
This is a pulse-chase method specifically designed to mark only the vacuole membranes in yeast cells using FM4-64. It is possible to label the membranes of the compartments that are part of the entire endocytic pathways (plasma membrane and early and late endosomal membranes, as well as vacuole membranes) when you continue to pulse cells with FM4-64 over 60 to 120 minutes (i.e. do not chase in medium without label). You can, however, mark only the plasma membrane when you apply FM4-64 to cells placed on ice (of course, you’ll have to keep the cell at 0°C during the microscopy procedure, which may prove difficult).
