CYTOCHEMICAL DEMONSTRATION OF MUCOPOLYSACCHARIDE IN BLEPHARISMA BY PERIODIC ACID SCHIFF (PAS) REACTION.

AIM: CYTOCHEMICAL DEMONSTRATION OF MUCOPOLYSACCHARIDE IN BLEPHARISMA BY PERIODIC ACID SCHIFF (PAS) REACTION.

MATERIAL REQUIRED

Biological – Slides pre-fixed with Blepharisma (Protozoa) in Carnoy’s fixative,
Chemicals – Schiff’s stain, 1% Periodic acid, Coplin jars, 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),
Glassware/Plasticware etc – Coverslips, blade, distilled water, forceps, needle, droppers, black paper (for covering the bottle containing the stain), and microscope.

Theory

Periodic Acid Schiff’s (PAS) reaction is used for staining the mucopolysaccharides. This reaction was devised by Mc Manus (1946). In this reaction, polysaccharides and their derivatives which contain hydroxyl groups or a hydroxyl adjacent to an amino group react with the periodic acid to produce aldehydic groups. These aldehydic groups react with Schiff’s reagent to bring out the magenta color. Therefore, PAS reaction is also used for demonstrating glycogen, mucin, starch, cellulose, hemicellulose, and pectin in plant cells and in animal cells for mucin, mucoproteins, hyaluronic acid, and chitin. As the majority of these polysaccharides are present in the cytoplasm, therefore the cytoplasm picks up the stain and appears magenta in color (Figure 1).

CYTOCHEMICAL DEMONSTRATION OF MUCOPOLYSACCHARIDE IN 
BLEPHARISMA BY PERIODIC ACID SCHIFF (PAS) REACTION.
Figure 1

PROCEDURE

  1. Rinse the slides pre-fixed with Blepharisma (in Carnoy’s fixative) in distilled water for about 15-20 minutes to remove the fixative completely. Air-dry the slide.
  2. Oxidation: Place the slides in 1% periodic acid for 15 minutes at room temperature. This step produces free aldehydic groups in the carbohydrates.
  3. Rinse gently in fresh distilled water to remove extra periodic acid.
  4. Air-dry the slides.
  5. Staining: Stain the slides in Schiff’s reagent for 45min to 1h at room temperature. Protect from light as the stain is light sensitive.
  6. Rinse gently in fresh distilled water to remove the extra stain.
  7. Dehydration: Dehydrate in increasing grades of alcohol i.e. from 30% to absolute alcohol with 2-3 minutes in each grade.
  8. Treat with two washes of xylene for 2 min (clearing agent).
  9. Mount in DPX and observe under the microscope.

OBSERVATION

When the slide is observed under the microscope at 10X, a magenta-colored cytoplasm is seen in the slide depending on the concentration of mucopolysaccharides that have undergone oxidation to produce aldehydic groups. Since the aldehydes are produced in the cytoplasm as a result of the binding of Schiff’s reagent, therefore, cytoplasm appears magenta in color while the nuclei remain unstained. Therefore, the entire Blepharisma appears magenta in color. (Figure 2)

Unstained Nucleus
Figure 2
CYTOCHEMICAL DEMONSTRATION OF MUCOPOLYSACCHARIDE IN BLEPHARISMA BY PERIODIC ACID SCHIFF (PAS) REACTION.
Figure 2, Here Cytoplasm is stained with magenta color whereas Nucleus is Unstained

DISCUSSION

Here in this experiment i.e “CYTOCHEMICAL DEMONSTRATION OF MUCOPOLYSACCHARIDE IN BLEPHARISMA BY PERIODIC ACID SCHIFF (PAS) REACTION” you will find that Cytoplasm is stained with magenta color whereas Nucleus is Unstained.

PRECAUTIONS

  1. Washing of slides in distilled water prior to hydrolysis in 1N HCl is important because the fixative may interfere in the reaction.
  2. 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.
  3. Always keep the stain in dark and so cover the bottle with black paper.

Also Read

Conclusion

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