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Formulae for Reagents used in Microscope Slide Preparation

Reagents, Dyes and other solutions used in microscopy and microtomy.

Brilliant Green, 1% (w/v) Aqueous Indicator Solution

Aceto Carmine...
There are several versions of this stain. a stain containing iron is often used because it produces a darker/stronger bluish-red colour.

Non-iron version...
Heat a solution of 45% acetic acid (45ml glacial acetic acid/55ml of distilled water) to boiling Add 0.5g of Carmine and continue heating for 15-20 minutes while stirring, Cool the resulting solution and filter to remove any precipitate.

Iron containing version...
Make up the non iron version as above.
Separately make a solution of 45ml glacial acetic acid/55ml distilled water/5g Ferric oxide, Slowly (drop by drop) add the Ferric oxide solution to 50ml of the Carmine solution until a precipitate starts to appear then Promptly add 50ml of Carmine solution to the titrated mixture. Filter to remove any precipitate.

Pampel's Fluid... (for preserving specimens)
This is prepared as follows, all quantities by parts by volume. Take 30 parts distilled water, 15 parts 95% ethyl alcohol; 6 parts 40%(w/v) formaldehyde; and 4 parts glacial acetic acid. add the acid slowly after mixing the other ingredients. let the mixture stand for several days before use to allow the unpleasant smells of the acid and aldehyde to disperse.

Acridine orange reagent...
Dissolve 5 g of colourless phenol crystals in a solution containing 50 ml of deionised water, 25 ml of glycerol and 25 ml of 95% ethanol.
Prepare acid-alcohol de-staining solution by mixing 74 ml of 95% ethanol, 26 ml of deionised water, 0.5 ml of concentrated hydrochloric acid and 0.2 g of methylene blue.

Carbol Fuchsin...
To prepare a 10% Basic Fuchsin Solution Weigh 10g of Basic Fuchsin powder add 100 mL of 90% ethanol and mix, allow to dissolve on a magnetic stirrer over a period of 3 or 4 days. Shelf life of the solution at room temperature is 6 months.
To Prepare Carbol Fuchsin itself...
Take 100 mL of the Basic Fuchsin solution in a 1000 mL flask and add 50 mL of Liquefied Phenol, then 850 mL of deionised or distilled water. Mix thoroughly and filter the solution, which has a shelf life of up to 3 months at room temperature.

Carbol Fuchsin (Ziehl's Magenta) Solution...
Grind 1g of the Basic fuchsin dye with 5g of phenol using a pestle and mortar, add the 10 ml of 90% ethanol in small amounts while grinding to dissolve the dye. When dissolved, add 200ml of water in several small amounts to wash out the mortar, then filter.

Oudemans' fluid... (for preserving specimens)
87 parts of 80% ethanol. 5 parts of glycerin. and 8 parts of glacial acetic acid.

Nile red...
Is prepared by boiling Nile blue A under reflux for 2 hr in 0.5% H₂SO₄, and extracting the product into xylene. For staining, the purified dye is dissolved in 75% glycerol (1-5 micrograms/ml)

Written...2005 Revised... 18 August 2009,