Title:

- A modified method of preparing Feulgen

stain or Schiffs reagent.
Authors name:
Department:
Date:

- Steve Reader
- Crop Genetics
- 6th February 2003
(* fields must be filled in)

Species of plant*
Anything that has easily accessible DNA, i.e. not too woody

Tissue Type*
Any tissue

Structures Probed*
DNA in nuclei, chromosomes

Probes used*
Feulgen stain, or Schiffs reagent

Type of microscopy*
Bright field

Specialised equipment required

Fume hood, magnetic stirrer.

Origins of probes and unusual chemicals used in the protocol

Pararosaniline chloride is available from Sigma - Cat. No. P3750.
Alternatives, and almost as good, used to be available from Kodak Eastman Co, USA,
as Basic Fuchsin C8907 or Basic Fuchsin C1762.

Working safely comments (an indicator not a hazard assessment)

1 Basic fuchsin is a carcinogen.
2 Pararosaniline chloride, also a carcinogen, has a very slightly different colour
index, but is usually purer. However, it maybe worth purchasing a small amount
first to establish purity.
3 Use an anti-static gun to prevent the fine crystals of pararosaniline chloride from
flying about.

Relevant background
Adapted from Feulgen, R. & Rossenbeck, H., 1924. Hoppe Seyl Z. 25; 203-248.

Additional comments
1 The manufacture of Basic fuchsin was historically dangerous, with many tales of
woe. Commercial preparations today can still be very impure, and are often
acquired by the larger suppliers from small companies, rarely with quality testing
imposed.
2 Pararosaniline chloride has proven to be a far superior alternative.
3 Commercial preparations of Schiffs reagent do work for some tissues, but the
staining time is often excessive due to the inferior quality. This protocol has
proven far superior for the best intensity and short staining time.
4 When decolourising the stain, it is common for a small amount of charcoal to
initially pass through the filter paper. This is not a problem, but it can be removed

by returning this solution to the filter funnel again.

Representative Image (optional)

a representative image for people to compare too and dont forget to tell people what
they are looking at.

Protocol (in a list format)*

Reagents:lM Hydrochloric acid (HCL)
Potassium metabisulphite crystals (K2S2O5)
Activated Charcoal powder (NORIT)
Method:Steps 1-3 and 5-6 in a fume hood!
1 Boil 1 litre of distilled water in Pyrex beaker, using a magnetic
stirrer/heater. Remove from heat, allow boiling to stop, add the stirrer
magnet, and then add 5 g of pararosaniline hydrochloride whilst constantly
stirring. Stir until all crystals/powder have dissolved.
2 Cool solution to 50-60C and transfer to a 2.5 litre brown glass stoppered
bottle (UV light attacks the large stain molecule and can weaken the
intensity of staining). A safety glass Winchester, suitably labelled, serves
this purpose well.
3 Add 15 g of potassium metabisulphite and 150 cm3 of 1M hydrochloric
acid to bottle and replace stopper firmly.
4 Shake bottle vigorously by hand for 15 seconds and then return to the
stirrer, ensuring that the stirring magnet is spinning correctly. Leave on
magnetic stirrer for 2 hours. Alternatively, transfer to an orbiting shaker for
2 hours or place stoppered bottle in a dark cupboard (with warning label!!)
overnight. The resultant solution should have changed from semi-opaque,
purple to clear orange/brown colour.
5 To decolourise the solution, add 2.5 g of activated charcoal powder,
stopper securely and shake bottle by hand for 15 seconds.
6 Filter solution to remove charcoal (keep exposure to light to a minimum).
The resultant solution should be clear and almost colourless. Aliquot into
250 cm3 (or smaller) brown glass bottles and store at +4oC until required.
7 This solution should stain hydrolysed plant material in 3-5 minutes.
Smaller volumes can be prepared by reducing the reagent volumes
accordingly.