The following points highlight the six main steps in chromosome methodology. The steps are: 1. Pretreatment 2. Fixation 3. Hydrolysis 4. Processing 5. Staining 6. Mounting.
Step # 1. Pretreatment:
Pretreatment is an important step in the course of preparations of plant tissue for studying the chromosome number and their morphology in order to establish the karyotype of a species.
It is carried out for:
(i) Causing mitotic block and metaphase arrest through destruction of spindle fibre
(ii) Bringing about scattering of chromosomes by removing the binding force
(iii) Clarification of constriction regions through differential hydration in chromosome segments
(iv) Influencing chromosome condensation. In soma special cases this is done also for
(v) Clear ring the cytoplasm by removing the heavy contents to bring about the transparency of cytoplasmic background
(vi) To achieve rapid penetration of the fixative by removing undesirable deposits from the surface of the tissue
(vii) Separation of middle lamella causing softening of the tissue
The most important aspect of pretreatment is to fulfill the first two requirements which exert a direct effect on the chromosome, and the underlying principle is the viscosity change of the cytoplasm. The spindle formation depends on the viscosity balance between cytoplasmic and spindle constituents.
Therefore, a change in cytoplasmic viscosity brings about destruction of the spindle mechanism making the chromosomes free from any binding force within the cell.
Viscosity change of the cytoplasm also affects the chromosomes, which undergo differential hydration in its segments making the primary and the secondary constriction regions conspicuous and the satellite gap greatly exaggerated. Spindle inhibition also results in high frequency of metaphase stage.
Pretreating Agents:
A number of chemicals are used for the purpose of pretreatment, the most common being colchicine. These chemicals are not universally applicable to all plant materials. Generally, a particular group of plants gives better results in a particular chemical. Suitable pretreating agent must be worked out through several trials (Table 21.1).
In some special cases, pretreatment Is done for clearing the cytoplasm from its heavy contents and softening of tissue, brought about by acid treatment (NHCI), alkali treatment (NaOH), and enzyme treatment (pectinase, cytase, clarase, cellulose).
Removal of secretory and excretory deposits from the surface of the tissue or cell wall is achieved by the application of hydrofluoric acid, chloroform and also some other chemicals.
Step # 2. Fixation:
Fixation is the most critical step in chromosome study. It is the process by which tissues and their components are fixed selectively at a particular stage thereby respective divisional stages are arrested. The purpose of fixation is to kill the tissue instantaneously (lethality) without pycnosis, i.e., not causing any distortion of the components to be studied.
Fixation in chromosome study brings about blocking of cell divisions and enables the preservation of the structural integrity of nucleic acid and protein of the chromosomes.
A truly effective fixative should fulfill the following conditions:
(i) Rapid penetration to cause immediate killing of the tissue
(ii) Coagulation of the protein component and consequent precipitation causing a marked change in the refractive index of the chromosomes
(iii) Checking denaturation of protein, consequent to the death of cells. Due to lethality the medium becomes acidic which causes enzymes to act in the reverse direction, with the breakdown of complex protein molecule into simpler amino acids
(iv)Checking bacterial action with the onset of lethality, thus preventing tissue decomposition
(v) Precipitating the chromatin matter to render the chromosome visible
(vi) Increase the basophilic of the chromosomes, helping in the adherence of the acidic stain
Fixatives:
Since all the above-mentioned requirements are rarely exhibited by a single chemical, a suitable fixative is, therefore, a combination of several chemicals. Some common fixatives are presented in the Table 21.2. Even with the best fixative, the chemical changes undergone by the nuclear bodies in the entire process cannot be ignored.
This can be eliminated by freeze-drying method of fixation which involves rapid cooling of the tissue at low temperature followed by extraction of water in vacuum.
Based on ingredients, fixatives may be metallic (chromic acid, osmic acid) or non-metallic (ethanol, chloroform, acetic acid). All metallic fixatives require thorough washing with water after fixation. The most commonly used fixative is Carnoy’s fluid I and the tissue is kept in cold for overnight. For rapid fixation tissue can be treated with acetic-ethanol (1:2) for 1-2 hours at cold.
Ethanol precipitates nucleic acid, causes irreversible denaturation of protein and due to its dehydrating property causes undesirable hardening effect. Acetic acid also precipitates nucleic acid and when kept for prolonged period it dissolves histones.
However, in scheduled time, it does not cause any distortion to nucleoprotein but causes excessive swelling of chromosome segments. Therefore, acetic acid is used in combination with alcohol to resist hardening of tissues.
Carnoy’s fluid II yields good result with materials containing fatty and waxy substances as chloroform is a good solvent for them. Carnoy’s fluid II, as well as, propionic-ethanol are used for material with oil contents like Lens, as they clear the cytoplasmic background.
Step # 3. Hydrolysis:
The step hydrolysis is required in chromosome preparation to bring about the softening of tissue by dissolving the pectic substances of middle lamella causing separation of cells, also called maceration. In squash preparation, this maceration will help to get an uniform, monolayer of cells with application of slight pressure.
In acetic-orcein staining schedule, hydrolysis and staining are simultaneously achieved by warming with a mixture of 2% acetic-orcein and IN HCl (9:1).
In chromosome preparation from callus tissue, maceration is done with 5N HCI at room temperature for 1 hour. Enzymes like pectinase, cellulose, clarase, etc. are also used for maceration in chromosome preparation.
In Feulgen staining, hydrolysis has another important objective, to liberate the aldehyde group involved in the Schiff’s reaction. In this case hydrolysis is done prior to staining with 1N HCI at58-60°C for 10-12 min.
Step # 4. Processing:
Processing of tissue is required for chromosome study after suitable fixation or after staining.
Different schedules employed are:
(a) Block preparation and microtomy;
(b) Squash preparation and
(c) Smear preparation.
(a) Block Preparation and Microtomy:
For getting serial sections, instead of squashes and smears, tissues are dehydrated and embedded in a suitable medium (paraffin) which supports the material on all sides and allows sections of the material to be cut without distortion. It is useful to get the sequence of stages of growth of the tissue and divisional stages of meiosis in an anther or mitotic divisions in the somatic cells.
The procedure involves the following operations:
Washing:
After fixation the tissue is thoroughly washed in running water for 24 hours (if fixed in metallic fixative) to remove all traces of the fixing chemical.
Dehydration:
The tissue is passed through successive grades of ethanol 30%, 50%, 70%, 80%, 90%, 95% and absolute ethanol keeping for 1 hr. in each, except overnight treatment in 70% and absolute ethanol.
Clearing:
As paraffin does not mix with many dehydrating agents, an intermediate medium (ante-medium) is used. After dehydration with ethanol the tissue is passed through a series of ethanol-chloroform grades 3:1, 1:1 and 1:3, being kept for 1 hr. in each. Finally it is kept in pure chloroform for 10-30 min.
Infiltration:
Small chips of paraffin of low melting point are added to the chloroform containing the tissue and kept at 37°C on a hot plate for 48 hr. with tight corking. The mouth of the container is then opened for 24 hr. Then it is transferred to a hot bath of 45°C and kept overnight.
The tissue is finally transferred to a hot bath of 55-60°C. As the paraffin melts, it is changed with molten embedding paraffin and two more successive changes are given with the same intervals of 30 min and the final change in molten paraffin being given only when no trace of the smell of chloroform is left.
Embedding:
After infiltration of the tissue with paraffin, embedding is done by pouring the molten paraffin with the tissue into a suitable receptacle (paper tray), arranging the tissue in a proper manner with heated needles. After partial solidification in air, transfer of the tray to ice-cold water is done.
Section cutting:
Rotary microtomes are commonly used for section cutting. A block is trimmed to a suitable size with the material at the centre and it is attached to a block holder with the help of a heated scalpel. The holder is fitted to the microtome with the edge of the razor barely touches the face of the block.
Sections are cut generally of 14µ thick with uniform strokes. The ribbon is transferred to a sheet of paper and cut into short equal segments.
A minute quantity of Mayer’s adhesive (albumin 50 ml + glycerin 50 ml + sodium salicylate 1 ml) is smeared on a clean grease-free slide and flooded with water. Generally 3 or 4 strips of the ribbon are transferred to the slide and warmed on a hot plate to stretch out the strips. The slide is then tilted to drain off the excess water and is kept on hot plate for 3 hr. to overnight for drying.
Removal of embedding material:
The sections are passed through xylol for removing the paraffin and gradually bring down to the medium in which the stain is dissolved. Atypical grade of chemicals for this purpose is : Xylol, I, II and III – 1 hr. each; Ethanol (1:1) – 1 hr; Absolute ethanol – 30 min; 90%, 80%, 70%, 50% and 30% ethanol – 30 min each; and Distilled water – 10 min.
Staining:
Then the sections are stained with crystal violet (vide staining).
(b) Squash Preparation:
This method has great advantage over sectioning method because one can carry out observation on separated single cells and the entire process is rapid. Squashing is recommended for studying chromosomes in somatic tissue (root tip, leaf tip).
After fixation, NHCI treatment is carried out for dissolution of the pectic salts of the middle lamella so that monolayer of separated individual cells can be obtained from a compact mass of cells. Softening is carried out prior to staining or along with staining. Following staining, the materials are teased with the needle in the mounting medium (45% acetic acid) and covered with a square coverslip.
Then the tissue is finally squashed by applying uniform pressure over the coverslip through a piece of blotting paper and properly sealed. The pressure should be hard enough to loosen the cell mass-to form a single-layered squash. Moreover, the cells have to be flattened, so that the chromosomes are well scattered.
The best way to do it is by placing the slide on a blotting paper, the blotting paper is then folded so that the slide remains within it, then it is tapped with the blunt end of a needle and excess fluid is blotted off. The slide is moved slightly to the dry side of the blotting paper and further pressure is applied. The preparation can be kept in temporary condition for 1-2 weeks if it is perfectly sealed.
(c) Smear Preparation:
Smearing is recommended for studying chromosomes in meiotic cells (pollen mother cells). In smears, the cells are directly spread over a slide prior to fixation and no treatment is necessary to secure cell separation. The procedure involves squeezing out of fluid from anther on to a clean, grease-free, dry slide, quickly spreading it with the aid of a scalpel and immediately inverted it in a tray containing fixative.
The procedure may be modified by directly smearing the anther with a scalpel in a drop of acetic-carmine solution (vide staining) which serves the double purpose of fixing and staining. Finally, it is covered with a cover-slip and gentle pressure (as the PMCs are fragile) is applied for the spreading of cells.
Excess stain is soaked with a blotting paper and is sealed with paraffin. This temporary preparation can be kept in cool place for 1-2 weeks.
Step # 5. Staining:
Staining is done to make the cell constituents visible under the microscope helping in the study of their structure and behaviour. The colour of the chromosomes as well as of different cell constituents is usually secured through the use of different stains or dyes. Staining may be vital or non-vital.
In case of vital staining, water soluble non-toxic dyes (methylene blue) are applied to the living tissue so that the latter can be studied without being killed. In non-vital staining, the colouration of the chromosomes and other cell constituents in the killed tissue is caused by certain chemicals which are insoluble in chromosome substance.
The stains are usually termed acidic, basic or amphoteric on the basis of their chemical nature and behaviour. Most of the acidic dyes are salts of potassium or sodium, whereas basic dyes are mostly available as chlorides or sulphates. In acidic dye, the balance of the charge on the dye ion is negative and they react with basic (acidophilous) substances.
Aniline blue, lacmoid are examples of acidic dye and colour the cytoplasm which is predominantly basis. In basic dye, the ion charge is positive and it stains acidic substance (basophilic).Carmine orcein are basic dyes and stains chromatin and chromosome which is strongly acidic.
The process of staining is principally due to physical adsorption and/ chemical reaction. The colour of the dye is due to certain chemical configurations in itself, known as chromophores; and the adherence of the dye to the cellular constituents is due to auxochromes, other chemical configurations in itself.
The best example of a chromophoric group is the quinonoid ring and the auxochromes are mostly amino or hydroxyl groups. The adherence of a dye may be accelerated through the process of mordanting by the use of metallic salts (ferric chloride, ferric acetate) which form compounds with the dye and attach the dye to the cellular constituents.
Of all the different stains employed for the study of chromosomes, a few important ones are given in Table 21.3.
Preparation of Common Stains and Staining Procedure:
A. Feulgen Stain (fuchsin sulphurous acid):
Principle:
The principle underlying the preparation of Feulgen solution (Schiff’s reagent) is the conversion of β-rosaniline chloride to leucosulphinic acid by sulphurous acid which is obtained through the action of HCI on potassium metabisulphitfe. The excess of SO2 liberated by this action undergoes reaction with leucosulphinic acid to produce bi-N-aminosulphinic acid, popularly known as Schiff’s reagent.
Requirements:
Basic fuchsin = 0.5 g
NHCI = 10 ml
Potassium metabisulphite = 0.5 g
Activated charcoal = 0.5 g
Distilled water = 100 ml
Preparation:
In 100 ml boiling distilled water 0.5 g basic fuchsin is dissolved. The solution is allowed to cool at 58°C. It is then filtered and the filtrate is further cooled down to 26°C. 10 ml of NHCI and 0.5 g potassium metabisulphite are added to it. The solution is kept in a dark container, properly sealed and stored in a cool dark chamber for 24 hours.
The magenta coloured solution turns straw coloured and is ready for use. If the solution remains otherwise coloured, charcoal powder is added to it, thoroughly shaked and kept overnight in cold temperature. The solution is then filtered and ready for use.
Precaution:
The stain should always be kept in a dark container, with proper sealing under cool temperature and away from light.
Staining procedure:
1. The fixed tissue is hydrolyzed in normal HCI at 56-60°C for 10-12 min.
2. The tissue is washed with distilled water giving 3 changes to wash out excess HCI.
3. The tissue is then stained in Feulgen stain (Schiff’s reagent) for 30-45 min at 10-12°C under dark condition. The colour develops within a short time and the chromosomes take up magenta colour against a clear cytoplasmic background.
Chemical basis of Feulgen reaction:
It is a DNA specific stain. By hydrolysis with NHCI, the purine containing fraction of DNA is separated from the sugar and the breakdown of glycofuranosidic linkage occurs, unmasking the -CHO group of the deoxyribose sugar. The reactive -CHO then enters into combination with fuchsin sulphurous acid to yield the typical magenta colour.
B. Acetic-orcein and Acetic-carmine stain:
Requirements:
Orcein or = 2 g (for 2% soln.)
Carmine = 1 g (for 1 % soln.)
Glacial acetic acid = 45 ml
Distilled water = 55 ml
Preparation:
Distilled water is added to glacial acetic acid to make 45% acetic acid solution. The solution is heated in a conical flask to boiling. The dye is gradually added to the boiling solution, stirring with a glass rod. The solution is boiled at simmering point for 10 min, cooled down to room temperature, filtered and stored in a bottle with a glass stopper.
Precaution:
The mouth of the flask should be kept covered while the solution is being heated.
Staining procedure:
(a) Squash preparation (somatic tissue)
1. The fixed tissue is first kept in 45% acetic acid for 10 min.
2. Then it is transferred to a mixture of 2% aceticorcein stain and NHCI (9:1).
3. The mixture is gently heated over the flame for 5-10 seconds, taking care that the liquid does not boil.
4. It is kept at room temperature for 45 min – 1 hr. to attain perfect staining of chromosomes.
(b) Smear preparation (meiotic tissue):
The tissue is stained directly in 1% acetic- carmine stain on a slide and the slide is heated slightly over a flame. The use of a scalpel during smearing aids in the addition of iron which acts as a mordant through the formation of iron acetate.
C. Crystal violet stain:
Requirements: Crystal violet = 1 g
Distilled water = 100 ml
Preparation:
1g dye is dissolved in 100 ml boiling water with constant stirring. The solution is cooled, filtered and allowed to mature for a week before use.
Staining procedure:
1. The fixed tissue (smears, sections) is stained in 1% crystal violet solution for 30 min.
2. The excess dye is washed off by rinsing in distilled water.
3. Then the slides are processed through 1% iodine and 1% potassium iodide mixture in 80% ethanoi for 45 sec to obtain proper colour of chromosome.
Step # 6. Mounting (Permanent Slide Preparation):
Mounting of tissue (section, squash, smear) in a suitable medium (Table 21.4), after staining, is necessary to avoid drying up of tissue and not to render it opaque. The mounting media should have a refractive index closer to glass to avoid refraction. It should harden quickly in contact with air and should check de-staining.
The chief aims of mounting are:
(i) To render the tissue transparent;
(ii) To increase the visibility of tissue under microscope;
(iii) To hold it with the protecting cover-slip firmly in place; and
(iv) To preserve it for a long period.
A. Mounting of Sections and Smears after Crystal Violet Staining:
1. For dehydration, the slide is passed through absolute ethanol, II, ill keeping in each for 2 sec.
2. Differentiation is done by passing the slide through clove oil I for 2-5 min (observation under microscope is required for satisfactory staining) and then transferred to clove oil II and kept for 10-15 min.
3. For clearing, the slide is kept in xylol I, II and III for 1 hr. in each.
4. Finally, It is mounted in Canada balsam under a cover-slip and the slide is allowed to dry overnight on a hot plate (35-45°C).
B. Mounting of Squashes/Smears after Aceticorcein, Acetic-carmine and Feulgen staining:
(a) Acetic-alcohol schedule:
1. The paraffin seal of temporary preparations is carefully removed with blade after 1-2 days and inverted in a covered petridish containing glacial acetic acid – ethanol (1:1) mixture till the cover-glass is detached.
2. Both the slide and cover-glass with materials are transferred to ethanol and kept for 10 min.
3. These are then passed through ethanol-xylol (1:1) mixture, xylol I and xylol II, keeping in each for 10 min.
4. The slide and the cover-slip are mounted separately in Canada balsam (two slides will be prepared) and allowed to dry overnight on a hot plate.
(b) Butanol schedule:
1. The paraffin seal of temporary preparations is carefully removed with blade and inverted in a covered petridish containing glacial acetic acid – ethanol (1:1) mixture till the cover-glass is detached.
2. Both the slide and cover-glass with material are transferred to ethanol: n- butyl alcohol (1: 1) for 5 min.
3. Both the slide and cover-glass with material are passed through n-butyl alcohol I and n-butyl alcohol II, keeping 20-30 min in each.
4. The slide and the cover-glass are mounted separately in euparal (two slides will be prepared) and dried on a hot plate for overnight.