The Aternate Bielschowsky Stain Protocol

From:

"Featherstone, Annette"

Bielchowsky stain

Here is the Alternate Bielschowsky Stain:

1. Deparaffinize to DH2O

2. Place sections in 0.1% silver nitrate solution, containing citric acid (working silver nitrate) in a 65 degree C water bath for seven minutes. (I preheat this solution for about 5min, in a plastic coplin jar)

3. Rinse in 2 changes of DH2O, remove slides one at a time.

4. Rinse in 95% ethanol, then 2 changes of AbsoluteEthanol.

5. Immerse slides in filtered gum mastic for about 5 min. (We keep this in a coplin jar and top it off as needed)

6. Drain well and put into developing solution in a 65 degree C water bath for 12- 13 minutes. (I start to check it at around 10 minutes, I also preheat this solution for about 5 min, usually when I put the slides in the gum mastic.

7.Rinse in DH2O

8. Dehydrate clear and mount.

SOLUTIONS:

1% Silver Nitrate

Dissolve 1gm silver nitrate in 100 ml DH20. this solution last for months in a brown bottle in the frig provided it is not left

at room temp.

0.1% Silver Nitate

Take 5ml of the 1%silver nitrate, dilute with 45 ml DH2O. Prepare fresh each time

1% Citric Acid

1gm citric acid in 100 ml DH2O

Working Silver Nitrate Solution

Add 0.1 ml of 1% citric acid to 40 ml of the 0.1% silver nitrate.

Gum Mastic

Dissolve 2.5 gm of gum mastic in 100ml absolute ethanol. Filter before use. May be topped off and reused.

Hydroquinone

Dissolve 0.5gm Hydroquinone in 30 ml DH2O

Developing Solution

Combine 10 ml filtered gum mastic, 30 ml Hydroquinone, add 0.9ml of 1% silver nitrate

We got this stain from the Journal of Histotechnology/Vol. 22, No. 1/ March 1999 and we have used it ever since with absolutely beautiful results

We always use sterile water but I guess DH2O might be fine too. This saves hundreds of dollars per year, maybe more.

-----Original Message-----
From: christine lee [mailto:c.lee@uq.net.au]
Sent: Monday, February 17, 2003 05:18
To: histonet@pathology.swmed.edu
Subject: Fw: Bielchowsky stain

Annette,

Could you put the method on the net for all of us. thanks.

Christine Lee

----- Original Message -----
From: Andrew Shand

To: 'Featherstone, Annette'

Cc: Histonet (E-mail)

Sent: Monday, February 17, 2003 7:47 PM

Subject: RE: Bielchowsky stain

Annette,

We do Modified Bielschowsky which as you suggest is an expensive method, so I'd be very interested to have details of your cheap protocol.

Bob Quilty

Dept of Neuropathology

Frenchay Hospital, Bristol, UK.

-----Original Message-----
From: Featherstone, Annette [mailto:AFeatherstone@KaleidaHealth.Org]
Sent: 14 February 2003 17:04
To: 'Nymeyer, Heather'; 'histonet@pathology.swmed.edu'
Subject: RE: Bielchowsky stain

We use a modified Bielschowsky stain that uses much less silver nitrate. The stain is consistent and much, much cheaper. The whole stain only uses 5.9ml of "1%" silver nitrate. The stain is watched for development and taken out before it becomes over stained. Let me know if you would like this protocol

Annette Featherstone HT/ Kalieda Health, Buffalo NY

-----Original Message-----
From: Nymeyer, Heather [mailto:Heather.Nymeyer@interiorhealth.ca]
Sent: Thursday, February 13, 2003 19:49
To: 'histonet@pathology.swmed.edu'
Subject: Bielchowsky stain

I need some help in reducing or eliminating the amount of background silver precipitate on the slide when performing a Bielchowsky stain. The method being used is a 20 % silver nitrate solution for 20 mins followed by a 15 min incubation (in the dark) of ammoniacal silver. The reducer consisting of citric acid, formaldehyde, water. A few drops of the reducer is added to the ammoniacal silver and checked microscopically until fibers are seen. At the same time the fibers are being developed the excess background precipitate is forming. Any suggestions or solutions would be greatly appreciated.

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