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Staining and Visualisation of Mitochondria by Janus Green Stain

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Staining and Visualisation of Mitochondria by Janus Green Stain Experiment 8 Staining and Visualisation of Mitochondria by Janus Green Stain EXPERIMENT 8 STAINING AND VISUALISATION OF MITOCHONDRIA BY JANUS GREEN STAIN Structure 8.1 Introduction 8.3 Procedure Expected Learning Outcomes 8.4 Observations and Results 8.2 Materials Required 8.5 Precautions 8.1 INTRODUCTION Do you know which organelle is referred to as the “power house of the cell”? It is the mitochondrion. The major function of the mitochondria is ATP production by oxidative phosphorylation. ATP is the “energy currency of the cell”. Recall the structure and function mitochondria that you learnt in BBCCT103 Block II. The complete breakdown of carbon to carbon dioxide also takes place in the mitochondria by citric acid cycle. The oxidation of biomolecules is accompanied by the production of NADH and FADH2. The reduced pyridine and flavin nucleotides are in turn reoxidised by the mitochondrial electron transport chain (ETC). The ETC is organised in the inner mitochondrial membrane into five complexes, namely complex I (NADH: ubiquinone oxidoreductase), complex II (succinate CoQ reductase), complex III (CoQ- cytochrome c reductase), complex IV (cytochrome c oxidase) and complex V (ATP synthase). The complex IV is reoxidised by reducing oxygen to water. Janus Green 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 mitochondriain living cells without killing them. Janus Green B is chemically diethyl safranin-azo-dimethyl aniline. It is oxidized to become colored. The cytochrome c oxidase helps to maintain the dye in an oxidised state in the mitochondria. In its oxidized state it is bluish green in colour. The stain is 51 BBCCL-104 Cell Biology Laboratory Manual permeable and colorless before oxidation. Therefore, the rest of the cell is colorless due to its reduction to a leuco compound facilitating visualisation of mitochondrion as a bluish green spots in the colourless cytoplasm. Expected Learning Outcomes At the end of lab exercise, you will be able to: prepare a stained slide for visualisation of mitochondrion and; visualise and describe the structure of mitochondria. 8.2 MATERIALS REQUIRED Tooth pick Ethanol Janus green stain Phosphate buffer saline (PBS) Slides Pasteur pipettes Cover slips Watch glass Filter paper, Distilled water Microscope. 8.3 PROCEDURE 1. Take out the tooth pick soaked in 70% ethanol and wipe with the tissue paper. 2. Gently scrape of inner side of cheek with the rounded end of the tooth pick. 3. Uniformly spread the cheek cells that have come onto the tooth pick to form a thin smear. 4. Leave it for few minutes for air drying. In this way, cells do not wash away during processing. 5. Add 2-3 drops of Janus Green B to the smear and wait for 5-10 minutes. 6. The slide is washed with PBS to remove excess stain. 7. Place the cover slip on the slide with few drops of PBS. 8. The slide is ready for microscopic observation. 52 Experiment 8 Staining and Visualisation of Mitochondria by Janus Green Stain The procedure is presented below as a flow chart. 8.4 OBSERVATIONS AND RESULTS Under the microscope small round or bacteria like bodies are visible in the cytoplasm. They are mitochondria that are bluish green in color and are concentrated near the nucleus (Fig. 8.1). Fig.8.1: Schematic diagram of Human cheek cells (mitochondria) stained with Janus Green B. 53 BBCCL-104 Cell Biology Laboratory Manual 8.5 PRECAUTIONS Slides and coverslips to be used should be clean and free from dust and fingerprints. The material and fluid should be placed centrally on the slide. The amount of fluid should be appropriate. With too much of fluid the coverslip will move around and with too little of fluid there is high likelihood of trapping air bubbles. The excess mounting fluids should be removed carefully from the sides of the coverslip using the edge of a blotting paper. Place the coverslip gently to prevent trapping of air bubbles. You should keep the specimen moist with water. 54 .
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