Today in this article we will discuss BSC 1st Year Second Semester Cell Biology Practical i.e. CYTOCHEMICAL DEMONSTRATION OF DNA IN BLEPHARISMA BY FEULGEN REACTION OR SCHIFF’S REAGENT.
Biological – Slides pre-fixed with Blepharisma (Protozoa) in Carnoy’s fixative,
Chemical – Schiff’s stain (in dark bottle), 1N HCl, Coplin jars (horizontal and vertical), Dehydrating agent i.e. alcohol (30%, 50%, 70%, 90% and absolute), Xylene (i.e. clearing agent), DPX (i.e. Distrene Plasticiser Xylene as mounting media),
Others – Coverslips, distilled water, forceps, droppers, tissue paper, needle, microscope, black paper for covering the bottle containing the stain, water bath (at 60⁰C)
Schiff’s reagent is basic fuchsin which contains parafuchsin [triamino-triphenyl-methane chloride] with sulfurous acid. Parafuchsin is converted into a colorless compound, N aminosulphuric acid. In the histochemical tests involving Schiff’s reagent, three types of aldehydes may be involved:
∙ Free aldehydes
∙ Aldehydes produced by selective oxidation
∙ Aldehydes produced by selective hydrolysis
Free aldehydes are naturally found in the tissue such as those giving the plasma reactions. Aldehydes produced by selective oxidation give the PAS reaction while aldehydes produced by selective hydrolysis give the FEULGEN reaction.
Feulgen reaction: In 1928, Rossenback and Robert Feulgen introduced a staining technique which was used in histology to identify chromosomal material (or DNA) in the cells (Figure 1). )
This reaction takes place in the following steps:
∙ The acid hydrolysis using 1N HCl at 60⁰C removes the purine (depurination) at the level of the purine-deoxyriboglycosidic bond in DNA. This unmasks the aldehydic groups of the deoxyribose.
∙ The free aldehyde group reacts with Schiff’s reagent and produces a magenta color. The specificity of the reaction can be confirmed by treating the section with deoxyribonuclease which removes DNA.
∙ As a result, the reaction is positive in the nucleus and negative in the cytoplasm.
CHEMISTRY INVOLVED IN FEULGEN REACTION IS AS UNDER (Figure 1):
PROCEDURE: 1. Rinse the slides pre-fixed with Blepharisma (in Carnoy’s fixative) in distilled water for about 10 minutes to remove the fixative completely. Air dry the slide.
2. Hydrolysis: Place the slides in 1N HCl for 8-10 minutes at 60⁰C. This step produces free aldehyde groups in the DNA by depurination.
3. Dry the slides by keeping them vertical for some time.
4. Staining: Place the slide in Schiff’s reagent for 1 hour and 15 min at room temperature. Protect from light as the stain is light sensitive.
5. Rinse gently in fresh distilled water to remove the extra stain. Dry and observe under the microscope for stained nucleus.
6. Dehydration: Dehydrate in increasing grades of alcohol i.e. from 30% to absolute alcohol with 2-3 minutes in each grade.
7. Treat with two washes of xylene for 2 min (clearing agent).
8. Mount in DPX and observe under the microscope.
When the slide is observed under the microscope at 10X, a magenta-colored nucleus in the Blepharisma is seen in the slide with clear cytoplasm. Since the aldehydes are produced in the nucleus as a result of the binding of Schiff’s reagent, therefore it appears magenta in color while the cytoplasm remains unstained.
Photograph of stained cells
Figure 2: Blepharisma (protozoan) showing cytochemical staining of the nucleus (magenta color) by Schiff’s reagent.
Write yourself. Discuss the result and your observation here. i.e Blepharisma with the magenta-colored nucleus.
1. Washing of slides in distilled water prior to hydrolysis in 1N HCl is important because the fixative may interfere in the reaction.
2. Check the temperature of 1N HCl before keeping the slides in it and keep it exactly for 8- 10 minutes.
3. The stain should be straw-colored. If it appears darker in color, it may reflect that it has undergone oxidation. In such a case, it should not be used.
4. Always keep the stain in dark.