Live staining and dead-viable identification of plant cells
Live staining and dead-viable identification of plant cells
In vivo staining can be used to (1) show certain natural structures within the cell using certain non-toxic or minimally toxic dyes, and (2) not affect the life activity of the cell or produce any physical or chemical changes that would cause cell death.
Operation method
Live staining and dead-viable identification of plant cells
Principle
Live staining is a technique that utilizes a certain dye solution that is harmless to plants to stain living cells. One of the commonly used live dyes is neutral red, which is a weakly alkaline pH indicator that changes color between pH 6.4 and 8.0 (from red to yellow). In a neutral or slightly alkaline environment, the living cells of the plant can take up a large amount of Neutral Red and excrete it into the vesicles, due to the fact that the vesicles are in general acidic in response. As a result, the neutral red that enters the vesicle then dissociates a large number of cations and takes on a cherry red color, in which case the protoplasm and cell walls are generally not colored; dead cells cannot maintain their cytosol within the vesicle due to denaturation and coagulation of the protoplasm. Therefore, staining with neutral red does not produce vesicle coloring. On the contrary, the cation of neutral red, but with a certain negative charge of the protoplasm and cell nucleus binding, and make the protoplasm and cell nucleus staining.
Materials and Instruments
Plant material Move 1. Select onion bulbs (or the base of onion pseudostem) and wheat leaves as materials. 2. 2. cut off a piece of young onion scales, with a single-sided blade in the inner scales cut into small pieces of about 0.5cm2, with pointed tweezers will be gently torn off the inner epidermis of the small pieces, can be put into the neutral red solution staining (note that the inner epidermis should be downward). If you use wheat leaves as materials, you can put the leaf blade on a slide with the back side facing up, and then put the slide into a petri dish with a small amount of water, press the leaf blade flat with the left hand, and then gently scrape off the lower epidermis and the leaf flesh with the right hand with a razor blade in one direction, leaving only the transparent cells of the upper epidermis. When scraping to only a small number of pulp cells left should be very careful, too heavy force is easy to damage the epidermal cells, or even only a layer of cell wall, too light and will leave too many pulp cells, affecting the observation. In addition to wheat, other grass plants can also be used in this way to prepare epidermal cells, will scrape the material cut into small pieces of about 0.5cm2. 3. 3. will be prepared onion bulb inner epidermis or wheat leaf epidermis small pieces into the 0.03% neutral red solution staining 5 ~ 10min, take out 1 ~ 2 pieces, slightly rinsed in distilled water, a drop of distilled water on the slide, carefully spread the preparation to the slide, cover slips, under the microscope to observe, you can see that the cell wall is stained red, while the protoplasm and vesicles are not stained, this is because distilled water is acidic, in the case of the cell walls, the cell walls are stained red, and the protoplasm and vesicles are not stained, this is because the distilled water is acidic, in the case of the cell walls are not stained. This is because the distilled water is acidic, and the cell wall is negatively charged and the dye cation is adsorbed under weakly acidic conditions. 4. will step 3 in vivo staining of several pieces of the preparation into the pH slightly higher than 7.0 tap water immersed in 10 ~ 15min, and then placed on the slide microscopy, will find that the cell wall decolorization, while the vesicles were stained crimson, this is because in the case of solution pH higher than 7.0, neutral red molecules of the dissociation of the role of the weak, mainly in a molecular state, not easy to be adsorbed by the cell wall, but easier through the plasma membrane and the vesicle membrane into the liquid. Plasma membrane and vesicle membrane into the vesicle, and the plant cytosol is mostly acidic reaction, into the vesicle of the neutral red then dissociate, the vesicle stained cherry red. At this time, the nucleus and protoplasm do not stain. In order to confirm the site of neutral red staining, the onion inner epidermis staining film can be immersed in 1 mol/L potassium nitrate solution for about 10min, and then removed for observation. Because potassium nitrate can make the protoplasm strong expansion, "cap-like plasmic wall separation", and thus can clearly distinguish the colorless and transparent protoplasm and stained red vesicles. 5. 5. Place the vivo preparation in step 4 on the flame of an alcohol lamp and heat it slightly to kill the cells, and then observe it under the microscope, and you will see that the protoplasm condenses into an uneven gel, which is stained red together with the nucleus. 6. Careful searching in a live-stained preparation may reveal individual dead cells whose nuclei are clearly recognizable as red from staining with neutral red. Caveat If only the vesicle line of a living cell stains red when stained with neutral red, and if the dye spreads and both the cytoplasm and nucleus stain red, this marks this cell as dead. For more product details, please visit Aladdin Scientific website.
Neutral red solution Potassium nitrate solution
Microscope Petri dish Slide Tweezers Alcohol lamp Absorbent paper