The following points highlight the seven main processes involved in preparation of permanent slides. The processes are: 1. Killing 2. Fixing and Hardening 3. Staining 4. Dehydration 5. Clearing 6. Mounting 7. Labelling.
Process # 1. Killing:
It is the first step in the preparation of permanent mounts and is of prime importance. By killing we mean the instantaneous stoppage of all the activities of life in their respective original state without giving the tissue enough opportunity to undergo any post-mortem changes. This can only be achieved by not allowing any change in the form of the tissue through use of reagents which are quick acting.
The best killing agents are:
1. 0.1% osmic acid and
2. Ether
Another good reagent which is most commonly used is absolute alcohol. But this reagent has one major drawback, that it shrinks and contorts the tissue.
Process of Killing:
(1) The material to be killed is placed on a slide on which a very thin film of Mayer’s albumen has been spread uniformly by rubbing a drop of albumen on surface of slide vigorously with index finger.
(2) (a) The slide is either inverted for few seconds on the mouth of the bottle in which osmic acid crystals have been put in distilled water.
Or
(b) A few drops of ether or absolute alcohol are placed on the slide over the object (material) and are allowed to evaporate.
Process # 2. Fixing and Hardening:
The fixing and hardening is the second major step in slide preparation. In case of minute living organisms it is usually attained by the killing agents alone but in case of sections of larger organisms or tissues it may be done through various fixing agents. The process of fixing has multifarious importance i.e.,
(1) Fixing stops any alteration in the form of tissues.
(2) It hardens the tissue and makes them fit for extensive subsequent treatment.
(3) It makes the tissue suitable and susceptive for the action of stains and various other reagents.
(4) Fixing agents make various constituents and components of a tissue optically differentiated by changing their refractive indices.
The various important fixing agents used are:
(1) Bouin’s fluid,
(2) Mercuric chloride,
(3) 70% Alcohol,
(4) Acetic acid,
(5) Formaline (formaldehyde),
(6) Potassium dichromate and
(7) Osmic acid (osmium tetra-oxide).
After fixing in any of the aforesaid fixatives thorough washing of material is very essential otherwise the tissue will be completely spoiled, various mediums used for washing are:
(1) 70% Alcohol (for Bouin’s)
(2) Iodine+ 70% alcohol, (for mercuric chloride)
(3) 50% alcohol (for acetic acid)
(4) 70% alcohol (tor formaldehyde)
(5) Water and 0.12% chloral hydrate (for K2Cr2O7) or
(6) Water (for osmic acid and K2C2O7)
Process # 3. Staining:
The process of colouring of various components and parts of a tissue for purpose of clear and absolute differentiation through use of different dyes (strains) is called staining. The nature of dyes may be acidic, basic or neutral.
Usually the acidic dyes stain (colour) the cytoplasmic part and basic dyes colour the nuclear part. However, with a nuclear dye cytoplasm may also be stained. For the purpose of undergraduates usually general staining (single staining) is used, which may colour both nucleus and cytoplasm at the same time.
But, where specific stains are used, usually they are used in combinations of two (double staining), three (triple staining), four or even more stains.
i. Single Staining:
For purpose of single staining the dyes used are picro-indigo-carmine or borax carmine in 70% alcoholic solution or pircrocarmine in water.
Procedure:
The material after thorough washing is passed through the gradually increasing percentage of the medium in which stain is made e.g., since the borax carmine or picro-indigo-carmines are usually made in 70% alcohol the material should first be passed through 30% alcohol, 50% alcohol and 70% alcohol for at least 3 minutes in each.
After the material has been saturated with the medium, it is put in the stain for about 3 minutes or till the material becomes dark red (Borax carmine) or dark green (Picro-indigocarmine).
After staining, the material is washed with the medium or solvent (70% alcohol In case of borax carmine and Picro-indigocarmine). If the stain is over it may easily be removed from the tissue through use of acid alcohol in which material is put for few minutes (1-2 minutes) and after every one or two minutes it is washed in 70% alcohol and examined under microscope, till desired results are achieved.
ii. Specific Staining:
For the purpose of double staining usually Delafleld’s Haematoxylin in distilled water and Eosin in 70% alcohol are used.
Procedure:
The material, after thorough washing, is put in Haematoxylin for 5 minutes till it is dark or blackish blue3. The material, after staining in haematoxylin, is passed through the medium or solvent in which second stain is prepared e.g. since Eosin is prepared in 70% alcohol the material is passed through 30% ale., 50% alc. and 70% alcohol and is kept in Eosin for one minute. After staining it is just washed through a dip in 90% alcohol (never use 70% alcohol).
Process # 4. Dehydration:
The process by which the traces of water present in the tissue are removed and replaced by alcohol, in which clearing agent and solvent of mounting medium are readily soluble, is called dehydration. It is done because water is un-miscible with mounting medium, its solvent (usually Xylene) and the clearing agent (usually Xylene or Benzene).
The dehydration is achieved by passing the tissue through gradually increasing percentage of alcohol. Otherwise, if we directly put the material in absolute alcohol it will shrink because of sudden loss of water.
The material is thus passed through 30%, 50%, 70%, 90% and 100% or absolute alcohol. To achieve proper dehydration the material after absolute alcohol, should once again be placed in fresh absolute alcohol for 3-5 minutes.
The alcohol may be better substituted by Cello solve (Ethylene glycol-mono-ethyl- ether) because it freely mixes with alcohol, water, xylol and clove oil etc. and it doesn’t shrink the tissue. As such it avoids repeated changes of alcohol in gradually increasing strength.
1. It is always advisable to use regressive staining method i.e., the tissue should be first over stained and then destained till desired depth of colour is achieved.
2. After single and double staining the excessive stair is removed by washing the tissue in acid alcohol (or alcohol for prepared stains) or acid water (for water prepared stains) for very short periods repeatedly till desired differentiation is achieved.
3. It is always advisable to use either staining tube or cavity blocks (covered) or any thing else with a lid to avoid entry of atmospheric moisture, excessive evaporation of alcohol and reagent and to safeguard against the loss of material.
Process # 5. Clearing (Dealcoholization):
The substitution of dehydrating agent (alcohol or cello solve) by the solvent of mounting medium is called clearing. The term clearing is also used because of the fact that the solvent or clearing agent imparts transparency to tissue.
The best clearing agents are Cedar wood oil and Clove oil but the most commonly used reagent is Xylene. In its place Benzene may also be used Xylene makes the tissue hard and brittle and also causes its shrinkage. As such it may be avoided if possible and should be replaced by Cedar wood oil or Clove oil.
Procedure:
The material after absolute alcohol is placed in xylene or any other clearing agent. If the clearing agent turns turbid or white, it shows that dehydration is not complete. Put the material back in absolute alcohol for 5 minutes and then in clearing agent for 5 minutes or till it becomes transparent. Still if turbidity comes give the material 2-3 changes in clearing agent.
Process # 6. Mounting:
The material after it has been made transparent is transferred to a drop of mounting medium which is placed in the centre of slide and is covered by a cover slip.
The mounting medium should be of the same refractive index as crown glass (R.I. 1.5).
The best mounting mediums are:
(1) Canada balsam dissolved in xylene (1.4 refractive index) or
(2) Euparol (1.4 refractive index)
Procedure:
With a glass rod put a small drop of anada balsam in the centre of slide. Transfer the material from Xylene to this drop with a brush. Take a cover slip and put its one edge on the slide touching the balsam. The other end of cover slip should be held obliquely through a needle as shown in fig. 41.
Now, bring down slowly the edge which is held with needle as shown in fig. 42. This will prevent the air bubble from entering in between the balsam and coverslip. Clean the oozing balsam with the help of a blotting paper.
(1) There should be no air bubble in balsam.
(2) The material should be in the centre of coverslip.
(3) The coverslip should be in the centre of slide.
(4) The Canada balsam should not ooze out of coverslip.
Process # 7. Labelling:
Now, put down your name on one edge of the slide and identification of material on the other edge of the slide and put it under microscope for examination.