Plant Anatomy: Practical Course - Panyuta O.O. 2019

Appendices
Staining of Slides

Acid fuchsin. Acid fuchsin belongs to the group of aniline Dyes. It stains the Cytoplasm, protein crystals, and Plastids, particularly leucoplasts. Before staining, the studied specimens must first be fixed for 24 hours in a saturated solution of mercuric chloride in absolute alcohol. Staining takes 24 hours, after which the specimen is thoroughly rinsed with plain Water and mounted in glycerin. To prepare acid fuchsin, use 0.2 g of fuchsin per 100 ml of distilled water. Upon Treatment with this reagent, the cytoplasm stains red.

Eosin. Eosin is used for differential staining of specimens. Under METABOLISM/18.html">The Influence of this dye, pectic substances remain unstained, while starch grains, fats, and resins take up little stain. Structural and Reserve Proteins stain very well with eosin. When double staining with eosin and methylene blue is performed, the cytoplasm stains red, while The Nucleus, nucleolus, and Chromatin turn dark blue. In this Procedure, a 1% aqueous solution of eosin is prepared first, and the specimens are immersed in it for 5-7 minutes; after rinsing for 25 minutes, they are kept in a 4% aqueous solution of methylene blue.

Caustic potash. Potassium hydroxide solutions affect various Cell components differently. For example, starch grains gelatinize, fats undergo saponification, protein substances dissolve, and As a result, the specimen becomes clarified. Treatment with potassium hydroxide solution dissolves intercellular lamellae. This process occurs particularly fast upon heating, leading to maceration.

Carmine staining is performed using solutions of this dye. Several types of carmine stains are prepared, namely: alum carmine, acetic carmine, and borax carmine.

Alum carmine. Used for nuclear staining. To ensure a clean stain, the specimens are rinsed with a 5% alum solution and then with distilled water.

Borax carmine. Specimens stained with this dye are rinsed in acidified alcohol. For this purpose, 3 drops of concentrated Hydrochloric acid are added to 100 ml of alcohol. Such a stain is extremely stable.

Other alum carmines can be prepared, for instance, According to the following recipe: 1 g of carminic acid and 10 g of alum per 200 ml of distilled water. The ingredients are dissolved by heating, then filtered, and 0.20 g of salicylic acid is added to the solution.

Hematoxylin staining. Hematoxylin staining is used in various combinations. There are several Methods FOR PREPARING this dye, of which the most widespread are the following:

1. Take a 3% solution of iron alum and a 10% solution of hematoxylin in absolute alcohol, store it in the dark, and dilute it with 9 parts of distilled water immediately before use.

The Microscope slide with sections of the studied objects is placed in the alum solution for 2 hours and then transferred to a vessel of tap water, which is changed several times. Afterwards, the alum is rinsed off the slide, because an excess of alum spoils hematoxylin. This procedure must be performed very quickly so as not to leach the alum out of the section. Next, the specimens are placed in the hematoxylin solution for 2-2.5 hours. If they have remained in hematoxylin long enough, but further Processing has to be postponed, the specimens can be stored in 96% alcohol. Once processing is complete, any remaining hematoxylin is washed off with regular water. The bottom side of the slide must also be rinsed. Under the microscope, the section will appear black.

2. A saturated alcoholic solution is prepared from crystalline hematoxylin at room Temperature. Concurrently, a saturated aqueous solution of ammonium alum is prepared at room temperature. Then, 4 ml of the first solution and 150 ml of the second are combined in a beaker or wide-necked flask. To allow air penetration for oxidation, the vessel is covered with gauze or filter paper (to keep out dust) and left in the light for 6-8 days. Afterwards, the liquid is filtered, and 22 ml of glycerin and 22 ml of methyl alcohol are added to the filtrate. This solution is left in the light and can only be used after a month and a half to two months, once all sediment has precipitated.

Mitochondria can be stained using the iron-hematoxylin method. For this purpose, sections of the studied objects are left in an alum solution for 2-3 days, then removed, quickly rinsed, and placed in hematoxylin for 2 days. During this time, the sections become heavily overstained. Differential staining is carried out in the usual manner. The progress of differential staining is checked under the microscope by covering the sections with a coverslip. With some experience, one can determine the exact moment to stop differentiation even under low magnification. When successfully stained, the mitochondria will show, and the nucleus will be stained more intensely.

Specimens can also be stained with hematein alum.

To prepare this, 1 g of hematein is dissolved by heating in 50 ml of 90% alcohol and mixed with a solution of 50 g of alum in 1 l of distilled water. Twenty milliliters of glacial acetic acid are added to this mixture. Hematein alum can be stored for a long time, but keep in mind that it forms a precipitate on the Glass; therefore, the stain should be drawn with a pipette from the middle of the vessel. Sections are stained for a few minutes. After that, they are rinsed first with a 1% alum solution, then with distilled water, and finally with tap water. Immediately after rinsing, the sections turn blue.

Methylene blue. This dye stains specimens light blue. Different cellular elements take up methylene blue at varying rates and intensities.

Fuchsin produces a very stable red color; it is used In aqueous solutions at a concentration of 0.2 parts by weight of fuchsin per 100 parts of water.

Safranin. Dissolve 1 g of safranin in 100 ml of water. After a few hours, the solution is ready for use. Safranin stains cell walls from pink to red, depending on the degree of lignification.

Safranin and light green. To prepare the light green solution, dissolve enough of the dye in 70-90% alcohol so that the liquid appears bright green. For every 100 ml of the solution, add 2 drops of concentrated hydrochloric acid. Specimens are immersed in safranin for 8-10 hours and then rinsed with water. The acidified light green solution simultaneously stains and differentiates the previous coloration. The light green solution is handled in the same way as acidified alcohol: it is poured onto the slide with the specimen and gently rocked. Staining and differentiation are monitored under the microscope. Once the nuclei stain red and the cytoplasm stains green, the process is stopped. The acid is washed out with 96% alcohol, and the specimen is placed in oil of cloves to remove the alcohol.

For double staining, a mixture of fuchsin and methylene green is used. In this case, both dyes are dissolved separately in 50% alcohol. Then, the fuchsin solution is gradually added to the methylene green solution until it turns purple. This mixture produces a double stain: the Cell Cytoplasm colors red, while The Cell walls stain greenish or bluish.