Janus green B staining technique for mitochondria.
Aim: Janus green B staining technique for mitochondria from onion peel.
Principle:
The major function of the mitochondria is ATP production by oxidative phosphorylation. ATP is the “energy currency of the cell”.
JanusGreen B belongs to the phenazine group of dyes. It is a dark green/dark brown/dark black in color. It is a basic cationic vital dye that is used to stain mitochondria in living cells without killing them.
The cytochrome c oxidase helps to maintain the dye in an oxidized state in the mitochondria. In its oxidized state, it is bluish-green in color.
permeable and colorless before oxidation. Therefore, the rest of the cell is colorless due to its reduction to a leuco compound facilitating visualization of mitochondrion as a bluish-green spots in the colorless cytoplasm.
Requirement:
- Onion (Peel)
- Janus green stain,
- Slides,
- Droper
- Coverslips,
- Watch glass
- Filter paper
- Distilled water
- Microscope.
- Forcep
Procedure:
- Pour some distilled water into a watch glass.
- pull out a piece of transparent onion peel (epidermis) from the leaf.
- Put the peel in the watch glass containing distilled water.
- Take a few drops of Janus green- B stain in a dropper and transfer this into another watch glass.
- Using a brush, transfer the peel into the watch glass containing the safranin solution.
- Let this remain in the Janus green- B stain for 30-60 seconds so that the peel is stained.
- Take the peel from the Janus green- B stain using the brush and place it in the watch glass containing the distilled water.
- Take a few drops of glycerine in a dropper and pour 2-3 drops at the center of a dry glass slide.
- Using the brush, place the peel onto the slide containing glycerine.
- Take a coverslip and place it gently on the peel with the aid of a needle.
- Remove the extra glycerine using a piece of blotting paper.
- Place this glass side on the stage of the compound microscope and view it.
Observations
- There are a large number of rectangular-shaped cells lying side by side and each cell has a distinct cell wall.
- A distinct nucleus is present on the periphery of each cell.
- Lightly stained cytoplasm is observed in each cell.
- A large vacuole is present at the center of each cell and is surrounded by the cytoplasm.
Conclusion
As cell walls and large vacuoles are clearly observed in all the cells, the cells placed for observation are plant cell
mitochondria) stained with Janus Green B.
