Separation and fluorescence observation of chloroplasts

Summary

Tissue homogenization followed by suspension in isotonic media for differential centrifugation is a common method for separating organelles. The settling rate of a particle in a centrifugal field depends on the size, shape, density, centrifugal force and medium viscosity of the particle. In the same centrifugal field, particles with different densities and sizes settle at different rates during the same time. Sequentially increase the centrifugal force and centrifugation time, can make the non-homogeneous suspension of particles according to its size, density successive batch settlement at the bottom of the centrifuge tube, so that a variety of sub-cellular components can be collected in batches.

Operation method

isotonic method

Principle

Tissue homogenization followed by suspension in isotonic media for differential centrifugation is a common method for separating organelles. The settling rate of a particle in a centrifugal field depends on the size, shape, density, centrifugal force, and medium viscosity of the particle. In the same centrifugal field, particles of different densities and sizes settle at different rates at the same time. Sequentially increase the centrifugal force and centrifugation time, can make the non-homogeneous suspension of particles according to its size, density successive batch settlement in the bottom of the centrifuge tube, so that can be collected in batches of various sub-cellular components.

Materials and Instruments

Fresh spinach
Sodium chloride
Centrifuge Tissue masher Microscope Balance Centrifuge tube Gauze Beaker Measuring cylinder Dropper Slide, coverslip

Move

I. Experimental steps

1. Select fresh young spinach leaves, wash and dry them, remove the leaf stalks and thick veins, weigh 30 g in 150 ml of 0.35 mol/L NaCI solution, and load them into a tissue masher.2. Homogenize for 3-5 min at low speed (5,000 r/min) using a tissue masher.3. Strain the homogenate through 6 layers of gauze into a 500 ml beaker.4. 4 ml of the filtrate was centrifuged at 1,000 r/min for 2 min and the precipitate was discarded.5. Centrifuge the supernatant at 3,000 r/min for 5 min, discard the supernatant, and the precipitate is chloroplasts (mixed with part of the new cell).6. The precipitate was suspended in 0.35 mol/L NaCI solution.7. Take a drop of chloroplast suspension on a slide, cover the slide and observe it under a normal light microscope and a fluorescence microscope.(1) Observed under an ordinary light microscope.
(2) Observed under a fluorescence microscope.
(3) Take a drop of chloroplast suspension on a non-fluorescent carrier, then add a drop of 0.01% acridine orange fluorescent dye, add a non-fluorescent cover slip that can be observed under a fluorescence microscope.Spinach leaves were sliced by hand for observation with a whisker cutter, fresh young spinach was cut on a slant and placed on a carrier plate, 1~2 drops of 0.35 mol/L NaCI solution was added, the plate was covered with a cover sheet and then pressed gently, and was placed under a microscope for observation.(1) Observed under an ordinary light microscope.
(2) Observed under a fluorescence microscope.
(3) Prepare the film in the same way, but add 1~2 drops of 0.01% acridine orange staining solution for 1 min, wash away the residual solution, and then cover the mounting with a cap for observation under a fluorescence microscope.II. Experimental results1. Isolation and observation of chloroplasts(1) Under an ordinary light microscope, the chloroplasts are seen to be green and olive shaped, and under high magnification the chloroplasts can be seen to contain small darker green particles, known as stromata, inside the chloroplasts.(2) In an Olympus fluorescence microscope, for example, chloroplasts emit fire-red fluorescence under the conditions of a B (blue) excitation filter, a B dichroic mirror, and an O530 (orange) blocking filter.(3) When acridine orange staining is added, chloroplasts fluoresce in orange, while the nuclei mixed with them fluoresce in green.2. Spinach leaf hand sliced for observation(1) Three types of cells can be seen under an ordinary light microscope①Surface cells: squamous cells with serrated edges.
(ii) Defense cells: pairs of kidney-shaped cells that constitute stomata.
(iii) Phloem cells: oblong and ellipsoidal cells arranged in rows of fenestrations.The chloroplasts are green and olive shaped, and the green basidia can also be seen under high magnification.(2) Under a fluorescence microscope, chloroplasts fluoresce a fiery red color, but their fluorescence intensity is weaker than that of free chloroplasts. The stomata fluoresce green, while the fire-red chloroplasts in the two guard cells are arranged in a circle around the stomata. There are fewer chloroplasts in the epidermal cells than in the chloroplasts.(3) After staining with acridine orange, the chloroplasts then fluoresce in a dead red color, the nucleus town fluoresces in green, and the stomata remain green.

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