Fixatives and Stains

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  • Calberla's staining solution

Materials

  • 5ml glycerine
  • 10ml 95% ethanol
  • 15ml distilled water
  • Saturated aqueous basic fuchsin
  • Melted glycerine jelly or glycerol

Method
Mix together a 50:50 solution of fuschin and glycerol. Upon cooling the solution will form a dark pink gel. Store in an airtight jar for future use. Mix the glycerine, ethanol, distilled water and 6 drops of the melted fuschin/glycerol solution. Store the Calberla’s solution in a clear bottle with a dropper lid.

Place a drop of Calberla’s on the slide and cover with a coverslip. Allow ~ 10 minutes before observing pollen structure under the microscope. Calberla’s stains pollen grains pink, but not fungus spores nor most of the other debris usually present on samples exposed outdoors. The dye is deposited on the outer wall of pollen grains, enhancing the surface characteristics necessary for identification.
See also;


  • Carnoy's Solution

Carnoy's solution I (Farmer's solution). 1 part glacial acetic acid 3 parts 95 % or absolute ethanol (Carnoy's I is the most commonly used fixative and gives good results for a large number of different species and tissues.)


  • Fuschin Stain

This stain contains fuchsin or magenta I, a decolorized dye which has a strong affinity for DNA, producing a red colour in its presence. Also called "rosaniline," is an aniline dye which consists of three aniline rings, one meta-methylated, para-attached to a central carbon. It's positively charged nitrogen is attracted to and stains the negatively charged PO4 groups in the DNA backbone.

It is nearly colourless until it reacts with the PO4 which turns it red.

For 30 mL of the stain:

  1. Weigh out 150 mg of Basic Fuchsin into 250 mL beaker.
  2. Pour 30 mL boiling dH2O over the Basic Fuchsin, swirl to dissolve.
  3. Cool to 50C, filter through Whatman No. 1 paper.
  4. Add 4.5 mL 1N HCl, mix. (Caution: corrosive acid)
  5. Dissolve 0.45 g K2S2O5 (potassium metabisulfite) to decolorize the solution. Set in dark for 24 hours to bleach.
  6. Decant or filter through activated charcoal (e.g., Norit) to further clarify (if desired).
  7. Store in light-proof bottle in refrigerator. (I.e. wrap in aluminum foil to exclude light.)

NOTE:

It looks innocent because of its light straw color before it reacts with PO4 , but it will stain you seriously if you get in on your hands or clothes!

[Fuchsin(e) Acid is related, but meta-sulfonated on all three aniline rings.]

  • Methylene blue

Dissolve 0.5g methylene blue powder in 30cm3 ethanol and dilute to 1003 with distilled water.