Author: Tuan Hsu-chuan
J Integr Plant Biol 1959, 8 (1): -.
In this paper, the author reports on a series of improved methods to fixing, hydrolyze, separating, staining anti mounting of plant cells together with their organoids. In the end, he succeeded in studying single isolated cells and recorded his observations in photomicrographs.
Ⅱ. Material and method
Material used in this experiment consists of several bulbous plants. They are: Narcissus tazetta var. chinensis Hippeaestrum vittatum Herb., Ornithogalum caudatum Ait., and Allium cepa L. Both old and young leaves of Narcissus and Hippeastrum are good material for the study of chloroplasts and mitochondria.
Ceratain chemicals and dyes employed in fixation, staining, hydrolysis, etc., found in the literature, are tested and analyzed. Then these chemicals and dyes are repeatedly tried singly or in various combinations for their usefulness in this experiment .Those combinations proved to give consistent good results, are chosen as the ones to be used throughout this study.
Ⅲ. The technique
For conveneince, the technique is presented here in seven procedures.
1. Fixation: Material is fixed for 24 hours in either one of the three fluids listed below.
These fluids harden the cytoplasm and walls of the parenchymatous cells, thus the cell as a whole is rendered more resistent to the subsequent treatment.
10% ammonium dichromate .... 4 ml 4 ml 4 ml
10% potassium dichromate .... 12 ml 8 ml 16 ml
Neutral formalin (add before use) 16 ml 16 ml 20 ml
Water 68 ml 72 ml 60 ml
2. Oxidation: After fixation, oxidation of the material is accomplished in a solution with the following ingredients for12–24 hours. This is a preliminary treatment for the differential hydrolysis immediately to follow.
10% ammonium dichromate .............. 20 ml
10% potassium dichromate ............. 80 ml
3. Differential hydrolysis: When oxidatien is completed, the material is transferred into a mixture of formalin and adds for12–24 hours. The purpose of this treatment is to remove the ground-cytoplasm of the cells. The mixture is prepared as the following.
Concentrated formalin ................. 8 ml
Sulphuric acid .......... ........ 8 ml
Nitric acid .............. ......... .. 3 ml
Water ...................... 81 ml
Usually, it is better to keep the nitric acid in a separate bottle, add it to the formalin and sulphuric acid mixture before use.
4. Separation: The term separation used here is to mean the disintegration of the middle lamella of the cells, which may or may not associated with the softening or disintegration of the tissue and cell walls. Separation of the cells is done by coaking the material in the following solution.
Chromic acid .................... 2 gms
Sulphuric acid .................... 8 ml
Nitric acid ..................... 3 ml
Water ................. . ..... 89 ml
The chromic acid used in this solution, may cause shrinkage of the cells, therefore too long a treatment of the material must be avoided. 4–6 hours at 25 ℃ is usually more than enough.
Formerly, the term "maceration" is used to mean the separation of the cells by means of various macerating agents, but now it is widely used to denote the fractional centrifugation of finely ground cells with their components such as the mitochondria, plastids, microsomes etc. blended into a suspension ready for enzyme and biochemical studies. Therefore, now, it is necessary to adopt a new term to mean the disintegration of the middle lamella of the cells to take the place left by "maceration". The author suggests the term "separation" as Gray (11) used it to mean the disintegration of the middle lamella.
5. Preparation of a slide: Following separation, a small piece of the material is teased into threads by means of two needles on a slide, with a drop of acidified floating fluid. The material is then squashed into a fine cell suspension. Blot off the excess fluid by inserting a small piece of blotting paper at the edge of the suspension. The floating fluid is prepared according to the following formula.
Pure glycerine .................... 10 ml
10% nitric acid ................. 1 ml
1% hydrochloric acid ............... 1 ml
1% acetic acid ................... 1 ml
Water ................ ..... 87 ml
6. Staining: With the foregoing pretreatment of differential hydrolysis, the staining of the material is then a simple matter, since the stain is a mixture of several dyes and differentiation is accomplished immediately when a drop of the stain mixture is given to the cell suspension. Stir the suspension with a needle and put a coverglass on the suspension after 1–5 minutes. Draw away the excess fluid from the preparation by inserting again a piece of blotting paper to the edge of the coverglass. The slide can be examined immediately under the microscope or kept in this manner for days and months. The stain can be prepared in three concentrations according to the following formulas. Generally the stain is purplish-red when mixed, however, the quality of the dyes may affect the coloration of the mixture. If the mixture is too red or blue, the effect of mix-staining may lost.
1% aq. sol. of acid fuchsia ..... 30 ml 20 ml 10 ml
1% aq. sol. of fast green ..... 12 ml 12 ml 12 ml
1% aq. sol. of anilin blue .... . 12 ml 12 ml 12 ml
Pure glycerine ..... 10 ml 10 ml 10 ml
Glacial acetic acid ........ 12 ml 12 ml 12 ml
Water ........... 24 ml 34 ml 44 ml
7. Dehydration and mounting: Not less than 6 hours after staining, the coverglass of the preparation can be dropped into a horizontal staining jar filled with enough acidified 95% alcohol. It is important to see that the preparation is submerged in the alcohol with the cover-glass facing the bottom of the jar. There is a slight advantage by suspending the slide in the jar so that the coverglass will drop off from the slide flatly down to the bottom. Plenty of time may be allotted to this stage of dehydration, for the acid alcohol will keep enough stains in the cells. From here on, the dehydration and clearing is carried on as usual in absolute alcohol and xylol-alcohols. Mounting is completed in balsam. The acid alcohol is prepared according to the following formula.
95% alcohol ..................... 97 ml
10 % nitric acid .................. 1 ml
1% hydrochloric acid ....... 1 ml
1% acetic acid .................... 1 ml
Ⅳ. Observation and conclusion
Examing one of the preparations with the microscope, it is quite interesting to see the various cell types in a single field. The relative position and sizes of the nuclei, chloroplasts, and mitochondria give the cell a solid contour which is not seen in any of the sectioned preparations. From the numbers, shapes and the sizes of the chloroplasts and mitochondria of the cells, the author is able to identify most them to the positions they occupy in the tissues. The development of the chloroplasts observed in some of the material can be followed clearly through from the thread like proplastids to the mature chloroplasts.
In conclusion, the author recommends the technique as a convenient method to study the relationship of the cells and their organoids. It is the hope of the author, that the technique may be applied more widely to certain branches of cell research for the promotion of better understanding or all types of cells and cell organoids with their environments.