RE: [Histonet] Re: Ammonia water pH and other bluing solutions[Sc anned]

From:Kemlo Rogerson

Oh I see now, so why are nuclei blue?

Kemlo Rogerson
Cellular Pathology Manager
East Lancashire Hospitals NHS Trust
DD. 01254-294162
Mobile 0774-9754194

-----Original Message-----
From: [] 
Sent: 03 December 2004 10:38
To: Kemlo Rogerson; 'Jackie M O'Connor'; Johnson, Teri
Cc: Histonet
Subject: Re: [Histonet] Re: Ammonia water pH and other bluing

Let's see if I can explain pH, hematoxylin, red vs. blue, and bluing, and
not get myself into a murky mess of chemistry.

Hematoxylin solution is actually an aluminum salt and oxidized hematoxylin,
collectively known as aluminum-hematein (Al-hematein).

In order to stain just the DNA and RNA, the pH of the Al-hematein solution
needs to be between 2.2 to 2.9 (most usually around 2.4-2.5).

At this lower (acidic) pH, there are a lot of hydrogen ions (H+) in the
Al-Hematein solution. These excess H+ bind to the Al-hematein, causing a
shift to the red.

When the stained tissue is placed in a solution more alkaline than the
2.2-2.9 stain, there are fewer H+ in the staining solution. This removes the
H+ from the Al-Hematein. This causes a color shift in the dye to the blue
range (i.e.,  "bluing the slides").

Therefore, bluing agents must have a pH higher than the mid-2.5. The higher
the pH (more alkaline), the faster the H+ are removed from the Al-Hematein,
and the faster the "bluing" will take place. Bluing at about pH 5.0 can take
up to about 10 minutes to "blue" totally (all red nuclei to some red/some
blue (= purple looking tissue) to all blue nuclei). At pH 10, it will take
30 seconds.

Tap water is more alkaline than the Al-hematein, so it can be used to blue
slides. If the tap water's pH is around 7, this should take about 5 minutes.
Some lab's water is more alkaline than other labs, so will take less time.
The pH of tap water can change depending upon what chemicals the water
treatment facility is using that day to purify the water, so using just tap
water to blue may have some variability of time.

Most labs rely on an alkaline solution (dilute ammonia, dilute lithium
carbonate, Scott's tap water) to speed up the bluing processing and to not
rely upon the variability of water pH.

Hope this helps.

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073

----- Original Message -----
From: "Kemlo Rogerson" 
To: "'Jackie M O'Connor'" ; "Johnson, Teri"

Cc: "Histonet" ;

Sent: Wednesday, December 01, 2004 3:18 AM
Subject: RE: [Histonet] Re: Ammonia water pH and other bluing

> I think you are wrong, so I'll correct you, as you requested.
> I thought the 'blue' state of haematin was, like litmus, the effect an
> alkaline condition has on the siderphilic dye. Red, like litmus, denoted
> acidic conditions. Damned if I know what colour (with the 'u') haematin
> when neutral (bluey/ red?)
> Running tap water takes longer, I concede, but good things are worth
> for and it's easier to make up; we rush too much I find. TIP: Never use
> London water, been through too many kidneys!
> -----Original Message-----
> From: Jackie M O'Connor [mailto:Jackie.O']
> Sent: 30 November 2004 19:09
> To: Johnson, Teri
> Cc: Histonet;
> Subject: Re: [Histonet] Re: Ammonia water pH and other bluing
> solutions[Scanned]
> Anyone remember using saturated lithium carbonate?  I could probably use a
> little lithium right now . . . . .
> Correct me if I'm wrong (Pandora's box) but isn't the "bluing" step just
> bringing the slides BACK to a neutral pH after treating them with acid
> which makes them purple-ish?  I like the ammonia because it 'seems' to
> make the nuclei sharper and instantaneously - Li2Co3 and PBS seemed to
> make them "blah".(Perception is in the eye of the beholder)   Running tap
> water will make the same miracle happen, but it takes longer.
> "Johnson, Teri" 
> Sent by:
> 11/30/2004 12:51 PM
>         To:     "Histonet" 
>         cc:
>         Subject:        [Histonet] Re: Ammonia water pH and other bluing
> solutions
> Way back in the day, Brigati was using PBS on his immunostainer to blue
> after the hematoxylin counterstain.  I have since used PBS to blue my
> slides instead of ammonium hydroxide/water.  It's cheap, and very easy
> on the sections, and I have plenty of it on hand.
> Teri Johnson
> Managing Director Histology Facility
> Stowers Institute for Medical Research
> 1000 E. 50th St.
> Kansas City, Missouri  64110
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