[Histonet] RE: Warthin Starry Help

Wed Jan 15 08:14:14 CST 2014

Thank you so much.  These tips seem very helpful.  Could you give me your protocol?  Times in temps in the impregnation step.  And your measurements for each reagent in the developing solution.  I use acidulated water and I usually get the pH at about 3.8.  I am staining for h.Pylori.  Like I said, when I first started making my own reagents, I actually made up enough of each for a week and the stain was working beautifully. But after a couple months, it has just mysteriously stopped working.  I just don't get it. I can use a kit and the sections will stain wonderfully but just cant get my own reagents to work and I am trying to save money since I am such a small lab.  

Tasha Campbell, B.S.,HTL(ASCP)
Frederick Gastroenterology Associates
310 W. 9th St.
Frederick, MD 21701
301-695-6800 ext. 144 (w)
304-685-9307 (c)
-----Original Message-----
From: Tony Reilly [mailto:Tony.Reilly <@t> health.qld.gov.au] 
Sent: Tuesday, January 14, 2014 7:38 PM
To: Campbell, Tasha M.; histonet <@t> lists.utsouthwestern.edu
Subject: RE: Warthin Starry Help

Hi Tasha

I have been performing the Warthin-Starry successfully for nearly 40 years.  Without seeing your method these are a few tips that should be followed.

1.	Make sure all solutions are made fresh each time using an acetate buffer or acidulated water as diluent.  I prefer an acetate buffer.
2.	Because of the difficulty in controlling development with this method, more than one section and control should be stained, with shorter 	and longer development times.
3.	Sections must not be allowed to dry before flooding with the developer solution, as this will cause precipitation of silver, making results 	difficult to interpret.  
4.	Do not wash slides between pouring off the silver solution and adding the developer.
5.	Ensure that the developer is made up just before use and is up to temperature.  Add silver nitrate to dissolved gelatin, stirring continuously.  	Ensure this is done slowly to prevent the formation of precipitate.  Just before time of use, add hydroquinone.  
6.	Allow sections on the first pair of slides to develop until the background appears yellow to light golden brown.  This will take approximately 	5-7 minutes.  Allow the second pair of slides to develop until the background appears brown to dark brown.  This may take up to an 	additional 3-5 minutes.  Regular microscopic examination is required to check for reaction endpoints.  Flood slides with acetate buffer or 	acidulated water once endpoints are reached to prevent further silver development.
7.	Fixatives containing chrome and mercuric salts should not be used as they can interfere with the reaction, giving false negative results.
8.	Discard developer once it becomes too dark.  If sections are underdeveloped they can be placed into a second batch of fresh developer 	solution for another 3-5 minutes.
9.	The method will demonstrate a number of argyrophilic organisms.  If a particular infectious agent is suspected, use a control section 	containing that organism.  If the cause of infection is in doubt, use a control containing Spirochaetes.  If this is successfully impregnated it can 	virtually be assumed that all other argyrophilic organisms would be demonstrated.
10.	Melanin and other argentaffin or argyrophilic substances may also be demonstrated.
11.	Some methods wash in hot tap water following development, I prefer to rinse in hot acetate buffer or acidulated water first to avoid any 	background precipitation of silver.

I hope this is of some use.

Regards
Tony

Tony Reilly  B.App.Sc. , M.Sc.
Chief Scientist, Anatomical Pathology
Pathology Queensland-PA Laboratory
________________________________________________
Health Services Support Agency | Department of Health
Level 1, Building 15,Princess Alexandra Hospital
Ipswich Road,WOOLLOONGABBA  Qld 4102
Ph: 07 3176 2412
Mob: 0402 139411
Fax: 07 3176 2930
Email: tony.reilly <@t> health.qld.gov.au
Web:  www.health.qld.gov.au/qhcss/

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tasha M. Campbell
Sent: Wednesday, 15 January 2014 12:55 AM
To: Tony Reilly; histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Warthin Starry Help

I need help with warthin starry stain please.  I am a very small lab trying to save money so I want to make the reagents myself.  I have sigma Aldrich dry reagents and have been making the reagents and following the instructions correctly and I did get the stain to work wonderfully when I first started.  Then suddenly it quit working and I cannot get it to work again.  I have made fresh reagents day after day and I have a pH of 3.8 for the acidulated water.  The tissue will stain normally with the yellow to brown background and I the developing solution turns black when it is left on paper but the h.pylori organisms are not staining.  I am getting very frustrated and need some help please.

Thank you.

Tasha Campbell, HTL

tmcampbell <@t> fmh.org

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