Obtaining Three-Dimensional of Cell Structure

In this article we will discuss about the processes to obtain a clear three-dimensional of cell structure.

A. Maceration:

For obtaining a clear three-dimensional impression of the cell structure maceration of tissues is done with the help of certain chemical and mechanical separations. In this process the stem, roots, bark or other organs are treated with some chemicals which dissolve the middle lamellae and thus allow the cells to become separated from one another.

In operating this process, divide the material into small and thin pieces. Dry material should be boiled and then processed further.

Following are some of the commonly used macerating processes:

(a) Schultze’s Method:

1. Take the pieces of the material in a test tube.

2. Cover them with concentrated nitric acid.

3. Now add a few crystals of potassium chlorate and heat gently on a sand bath so far the material is bleached white.

4. Wash the material thoroughly and disintegrate it with a glass rod.

5. Observe the different types of cells (Fig.100) under a microscope.

(b) Jeffrey’s Method:

1. Take the pieces of material in a test tube.

2. Add in the test tube the macerating fluid which consists of 10% chromic acid and 10% nitric acid.

3. Now keep the test tube at 30 to 40°C for one to two days.

4. Wash it thoroughly, disintegrate gently with a glass rod and observe under the microscope.

(c) Harlow’s Method:

1. Take the boiled material and keep in chlorine water for two hours.

2. Wash with water thoroughly.

3. Now boil for about 15 minutes in 3% sodium sulphite.

4. Wash it thoroughly, disintegrate with glass rod and observe under the microscope.

B. Clearing:

(a) Requirements:

Clearing material, KOH, lactophenol or chloral hydrate, hydrogen peroxide, alcohol, xylol, basic fuschin, Canada balsam, test tube, water.

(b) Procedure:

1. Keep the material in 5% KOH solution for about 24 hours in a test tube.

2. Wash it thoroughly with the water and keep in a solution of equal quantity of chloral hydrate and hydrogen peroxide for about six hours.

3. Now keep the material first in 50% alcohol and then in 70% alcohol for about 2-3 minutes in each.

4. Stain in basic fuschin prepared in 90% alcohol.

5. Pass the material in 90% alcohol and then in absolute alcohol.

6. Now pass it through the different Xylol- grades, i.e., 25%, 50%, 75% and pure xylol, for about 2-3 minutes in each, one after the other.

7. Mount the material in Canada balsam. In place of Canada balsam, D.P.X. or Euparol may also be used.

8. Study the slide (Fig.101) thus prepared, under the microscope.

C. Microtomy:

(a) Killing and Fixation:

1. Cut the material into small pieces. Remove as many hairs, if present, as possible because they create difficulty in fixation as well as section cutting.

2. Fix the pieces of material in small vials filled with fixative.

3. Place a small label in the tube along with the material. On the label write the name of the material, date and time of the collection and other datas.

(b) Dehydration:

5. Wash the material very thoroughly with water and pass it through brief changes of alcohol of the same concentration as that present in the fixative.

6. Then pass the material in TBA (Tertiary butyl alcohol)-series consisting of mixtures of water, ethyl alcohol and TBA.

Different solutions of TBA-series can be prepared in the following ways:

50 → 10 ml. TBA, 50 ml. H₂O, 40 ml. 95% alcohol

70 → 20 ml TBA, 30 ml. H₂O, 50 ml. 95% alcohol

85 → 35 ml. TBA, 15 ml. H₂O, 50 ml. 95% alcohol

95 → 55 ml. TBA, 45 ml. 95% alcohol

100 → 75 ml. TBA, 25 ml. absolute alcohol

TBA → 100% TBA.

In each solution of the series keep the material for at least 2 to 4 hours. From the above TBA series it is clear that water and ethyl alcohol are replaced by TBA and the ultimate part of the series is 100% TBA. 100% TBA is used only once and that is in the end of series. By keeping the material in 100% TBA for overnight, it is now completely dehydrated.

(c) Infiltration and Embedding:

7. Transfer the material, with 100% TBA, in the another vial containing solid paraffin.

8. Place the vial or bottle, containing material and solid paraffin, in an oven and fix the temperature of the oven 5° higher than the melting point of the paraffin used.

9. Replace the molten paraffin with a fresh one after about 4 hours and repeat the process once or preferably twice.

10. Take a clean and dry porcelain embedding dish and coat its inner side with a thin layer of glycerine.

11. Bring out the vial containing material from oven and transfer the material into the embedding dish along with the molten paraffin.

12. Arrange the pieces of material in the embedding dish in parallel lines with the help of hot needle.

13. Place the embedding dish on the surface of a large dish containing ice water. This will solidify the ‘paraffin’ and a “block” of the shape of the inner side of the embedding dish will come out and float in the ice water.

(d) Sectioning:

Sectioning is done with a special instrument called microtome. It is used for section cutting of definite thickness. Two types of microtomes commonly used are sliding and rotary. Some other special microtomes meant for specialized work are wood microtome, freezing microtome, cyclone microtome, etc.

Following is the procedure of section cutting from a rotary microtome (Fig. 103):

14. Cut the paraffin block into different pieces, each having the material to be sectioned.

15. Fix the piece of paraffin containing the material to be sectioned on a wooden block by heating the bottom of the paraffin and. the top of wooden block with a warm needle. Press slightly the bottom of the paraffin to fix and allow them to solidify.

16. To remove the excess paraffin around the material trim the sides of the paraffin block. Surfaces of the trimmed block should ordinarily be parallel to one another.

17. Fix the wooden block (with the paraffin block containing the material) in the microtome with the point in mind that the edge of the block, that hits the knife, should be parallel to the edge of the knife.

18. Fix the microtome on the desired section thickness and release the safety catch for cutting some trial sections and continue until the ribbon begins to form.

19. Keep a dissecting needle under the ribbon, and when the latter is long enough place it on a clean black cardboard. Keep the ribbons in parallel lines on the cardboard for maintaining a series of sections.

20. Mount the ribbons serially on the clean slides coated by some good adhesive and then transfer the slide to a slide warmer, and when the material is fully extended allow to cool the slides.

(e) Staining and Mounting:

21. The slides are immersed in xylol in coplin jars or staining jars to dissolve wax and then in the mixture of xylol and absolute alcohol and then in absolute alcohol. These are then passed through lower alcoholic grades.

Any stain combination such as safranin-fast-green, safranin-haematoxylin, safranin cotton blue, etc. can be used. Dehydration in done and then immerse in alcohol-xylol mixture and finally in xylol, and mount with canada balsam.