Re: [Histonet] RE: purple blue haze

From:"John A. Kiernan"

I think Kemlo has to be right here. If the tap
water is very "hard" (too much calcium, usually
but not always as bicarbonate), it might
precipitate calcium sulphate, by metathesis with
sulphate ions from the haemalum. 

Hardness of water due to calcium bicarbonate is
called "temporary" because it's lost on heating
the water; carbon dioxide is released, solid
calcium carbonate deposits in the pipes, kettle
etc, and the water becomes "soft," meaning that
you don't need to mix much soap with it to wash
your feet, socks etc. With hard water you need
more soap because some is wasted in forming a
useless scum, composed of insoluble calcium salts
of higher fatty acids. Water from Lori's hot tap
may therefore not contain enough calcium to make a

Having made this convincing argument, I must
confess that I frequently add a splash of
limewater (saturated calcium hydroxide) to
accelerate blueing of a haemalum nuclear stain,
and have never seen a "haze" artifact. However, I
do all staining manually, and the diluted 
limewater follows 30-60 seconds in running (cold, 
rather hard) tap water - enough to quickly wash 
away all the surplus haemalum. If I went straight 
from haemalum into a less generous amount of hard 
tap water, especially with an added Ca salt, a 
hazy calcium sulphate precipitate might well 
happen. I've never done the test.

The phrase "run through the stainer" in Lori's
report indicates a mechanized H&E (hemalum and
eosin) procedure, in which slides may well be
exposed to mininimal passage through the steps of
the method, to save time and solvents. Rapid 
slide transfers carry over materials that don't 
mix well. Bad mixings often make precipitates or 
emulsions, which are clouds under the microscope.

Anyone who follows up this line of enquiry should
write it up for publication. There's probably a
3-page paper in this, for 2 days work in the lab.
Widely read technique-oriented journals are eager
to publish papers that investigate and explain
artifacts that occur with routine methods. It 
helps everyone. 

John Kiernan
Anatomy, UWO
London, Canada
kemlo wrote:
> Obviously something in the water! If you restain using hot water does the
> haze disappear? You must assume I suppose that something crystallises out of
> cold water and that the hot water is either 'cleaner' or is sufficiently
> warm to dissolve (or keep dissolved) what ever is doing it.
> In London we never blued in tap water, but then you ever did anything with
> London water but use it in the toilet.
> -----Original Message-----
> From:
> [] On Behalf Of
> Sent: 10 February 2006 22:02
> To:
> Subject: [Histonet] RE: purple blue haze
> We had a purple blue haze on our H&E's when we used cold tap water run
> through the stainer. We had to use hot water and the haze went away. I've
> tried to return to using cold water and we get the haze every time.
> Lori A. Harris, HT (ASCP)
> Histology Section Leader
> GSRMC - Pathology
> 3600 NW Samaritan Drive
> Corvallis, OR 97330
> 1-541-768-6078

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