Permanent slide preparation
Aim:
To study how to prepare a permanent slide.
Theory:
If a mounted slide is to be preserved for a longer time, a permanent slide is made by the double staining technique.
Double Staining Technique:
A permanent slide is useful for the preservation of section cuttings for study and for the preparation of standards, with which the samples can be compared. This process generally involves staining with two reagents, hence it is called the double staining technique One of the stains imparts colour to the lignified tissue and the other to the cellulose part. Two different techniques are involved in the preparation of a permanent slide.
Method I
Staining: In this method, safranin and haematoxylin are used.
Safranin solution: Prepare a 0.5 – 1% solution in water or 1% solution 1n 50% alcohol.
Lignin + Safranin = Deep red colour
Delafield’s Haematoxylin:
Cellulose + Haematoxylin = Purplish violet colour
Procedure:
1. Take a clean watch glass. Add safranin solution to it and transfer a thin uniform section to this solution and keep it for 10 minutes.
2. Take one watch glass containing 50% alcohol. Transfer the section from safranin to 50% alcohol, keep it for 5 minutes.
3. Transfer the section to a watch glass containing water and keep it for 5 minutes. This washing shall remove the stain from the cellulose part.
4. Transfer this safranin stained section to a watch glass containing dilute haematoxylin and treat for 2 minutes.
5. Transfer it to a watch glass containing water for washing.
This section is double-stained and now requires dehydration otherwise, over a period of time it may develop a foggy appearance and the observations shall not be clear.
Dehydration:
For dehydration, a double-stained and washed section is treated with increasing strengths of alcohol for 1 minute in each strength, starting with 30% alcohol, followed by 50%, 75%, 90% and 100%. This removes all moisture from the section. If we directly put the section cutting in absolute alcohol, it will shrink because of a sudden loss of water.
Mounting:
For mounting, select a 1 to 1.2 mm thick glass slide and a thin coverslip. Place the section in the centre of the slide and add a few drops of clove oil. This makes the section clear, as it removes unwanted debris. After 5 minutes, dry the section with blotting paper.
To this section, now add a few drops of Canada balsam dissolves in xylol. And place a coverslip carefully with the help of a needle.
Slightly warm the slide or keep it for drying in sun in a dust-free place. Natural drying is a time-consuming process and takes 2 to 3 days to complete. The solvent evaporates and the balsam fixes the section. Label the slide accordingly.
Method II
In this method, safranin and fast green solution are used.
Safranin solution: Prepare a 0.5 – 1% solution in water or 1% solution in 50% alcohol.
Lignin + Safranin = Deep red colour
Cellulose + Fast green = Bright green colour
First, stain the section with safranin and treat it with 50% alcohol as in Method I (steps 1 and 2). Later dehydrate the section as in Method I.
The second stain is added after dehydration. Fast green is dissolved in clove oil and treated with the section for 2 to 3 minutes.
The section is then transferred to a clean slide, treated with plain clove oil for 5 minutes for clearing the section. Remove excess clove oil and fix the section in Canada balsam as in Method I. Dry and store in a slide box until required for observation.
Lignified tissue, nuclei and cutinized walls get stained red; cytoplasm and cellulose walls get stained green. This is a good double stain and has the merit that the fast green solution keeps well, whereas Delafield’s haematoxylin deposits badly and requires frequent filtering.
Preparation of Canada balsam Solution for Fixing:
Canada balsam is a semi-solid, resinous substance, and translucent in nature.
Take a small quantity of Canada balsam in a test tube, warm on a water bath to remove volatile matter. Warm until a hard mass is left behind, cool and dissolve by constant stirring in xylol or benzene. A clear transparent viscous fluid 1s obtained which may be filtered to remove particle matter. This fluid is used for fixing the section.
Thus, sections are ready for observation under a microscope, no matter which technique is used for the preparation of a slide.
Remedial Biology Practicals
- Introduction to experiments in biology a) Study of Microscope b) Section cutting techniques c) Mounting and staining d) Permanent slide preparation 2. Study of cell and its inclusions 3. Study of Stem, Root, Leaf, seed, fruit, flower and their modifications 4. Detailed study of frog by using computer models 5. Microscopic study and identification of tissues pertinent to Stem, Root Leaf, seed, fruit and flower 6. Identification of bones 7. Determination of blood group 8. Determination of blood pressure 9. Determination of tidal volume
First Year B Pharm Notes, Syllabus, Books, PDF Subjectwise/Topicwise
First Year B Pharm Notes, Syllabus, Books, PDF Subjectwise/Topicwise
