You counterstain with hematoxylin, rinse your slides and put them under running tap water.
Some protocols say 5 minutes. Some say 10. Others happily prescribe 15 minutes.
It works. But why?
And do we really need to leave a tap running for 15 minutes to turn hematoxylin blue?
Usually, no.
Why do we blue hematoxylin?
After staining with alum hematoxylin, nuclei don't immediately have the crisp blue colour we associate with a finished hematoxylin stain. Under acidic conditions, the hematoxylin-mordant complex appears more reddish-purple.
Bluing exposes it to a more alkaline environment and shifts the colour towards the familiar blue/blue-black.
So bluing is chemistry, not a ritual.
Why does tap water work?
Tap water is often sufficiently alkaline to blue hematoxylin, which is why running tap water has been used in histology protocols for generations.
The problem is that tap water is not a laboratory reagent.
Its pH and mineral composition depend on the local water supply. Water hardness and alkalinity are related to its mineral chemistry, but hardness itself isn't a reliable measure of bluing performance. What matters directly for bluing is having sufficiently alkaline conditions.
If your tap water provides them, bluing works nicely. If it doesn't, you may wait considerably longer or get inconsistent results.
Lukewarm tap water can therefore work perfectly well. But running it continuously for 10–15 minutes is a rather inefficient way of controlling pH.
A defined alkaline bluing solution gives you much more control - and can work in seconds rather than minutes.
Bluing solutions you can make yourself
You don't necessarily need to buy a commercial bluing reagent. Several perfectly good bluing solutions can be prepared from basic laboratory chemicals.
Saturated lithium carbonate solution - my favourite
- Lithium carbonate - 15.4 g
- Distilled water - 1,000 mL
A saturated lithium carbonate solution is a traditional alkaline bluing agent for hematoxylin. It works quickly and the recipe could hardly be simpler.
It's saturated, so don't worry if it looks precipitated. That's by design.
0.1% sodium bicarbonate - also my favourite
- Sodium bicarbonate - 1 g
- Distilled water - 1,000 mL
Mix until dissolved.
This gives a mildly alkaline solution, around pH 8. Published histology protocols use it for approximately 30 seconds to 1 minute of bluing.
If your lab has sodium bicarbonate, you already have almost everything you need.
Scott's tap water substitute
One formulation:
- Magnesium sulfate (MgSO₄) - 10.0 g
- Sodium bicarbonate - 1.4 g
- Water - 1,000 mL
Scott's solution was designed to provide a controlled alternative to variable tap water. It combines magnesium sulfate with sodium bicarbonate to provide conditions suitable for reliable hematoxylin bluing.
There are several published formulations of Scott's tap water substitute, so don't be surprised if another protocol gives you different quantities. Another commonly published formulation contains 20 g magnesium sulfate heptahydrate and 2 g sodium bicarbonate per litre of water.
Which one should you use?
Lithium carbonate and sodium bicarbonate solutions are my favourites because they are simple to prepare and relatively low-hazard laboratory solutions.
But in the end, your best bluing agent may simply be the one you already have the reagents for.
There is little point ordering another specialized bottle if you already have everything needed to make a perfectly suitable bluing solution.
Whichever one you choose, keep the process consistent: blue until the nuclei reach the desired colour, rinse in water and continue with the protocol.
These solutions can also be used as staining baths rather than sending a fresh litre down the drain for every slide. How often a bath can be reused depends on slide load, carry-over and your staining requirements, so replace it when its appearance or performance changes rather than assuming unlimited reuse.
And if your current protocol says 15 minutes under running tap water, ask what those 15 minutes are actually achieving.
Sometimes making a protocol greener doesn't require redesigning the assay.
Sometimes you just need to turn off the tap.
References
- Bancroft JD, Gamble M. Theory and Practice of Histological Techniques. Alum hematoxylins and alkaline bluing solutions, including lithium carbonate, ammonia water and Scott's tap water substitute.
- Sigma-Aldrich / Merck. Scott's Tap Water Substitute Concentrate. Scott's solution for bluing hematoxylin staining.
- Leica Biosystems. H&E Staining Overview: A Guide to Best Practices. Discussion of tap-water pH, hematoxylin bluing and Scott's tap water substitute.
- OpenWetWare. Immunohistochemistry Paraffin - Bluing Reagents. 0.1% sodium bicarbonate solution (1 g/L), approximately pH 8, with 30 s–1 min bluing.
- Alwahaibi N, et al. Effect of water softening on the colour intensity of routine haematoxylin and eosin stain. Journal of Histotechnology. Comparison of untreated and treated water in H&E staining.