RE: [Histonet] Bluing
Blueing will differentiate and can help crisp the stain. The pH does affect the rate and intensity of color change. You want a slightly alkaline solution to “Blue” your slides. The blueing process will differentiate the normal purplish color of the acid dye, Hematoxylin (pH 2.6-2.9) by changing the color to a blue from the reddish purple. This process produces a better contrast with the usual red counter stains, Eosin and Phloxine, used in the HE. Common blueing/differentiators I have used are; 0.1% Sodium Bicarbonate (1 g / 1000 ml distilled water), 0.2% Ammonia Water (2 ml / 1000 ml distilled water) and Saturated Lithium Carbonate (1.54 g / 100 ml distilled water). You can also use “tap water”, but be careful about how the water is treated. Rinsing in tap or distilled water also provides a crisper stain because is rinses away the alum. If alum remains, the color will fade with time. William DeSalvo, B.S., HTL(ASCP) Production Manager-Anatomic Pathology Chair, NSH Quality Management Committee Owner/Consultant, Collaborative Advantage Consulting Date: Tue, 18 Dec 2012 11:20:26 -0500 From: turke...@gmail.com To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Bluing Dear Histonetters, I wonder what is the purpose of bluing the hematoxylin. Can bluing effect the clarity of the nuclear staining? Can one achieve different bluing results varying the composition, pH or time of the bluing reagent? Mes ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] bluing
Just add a little bit more ammonia hydroxide to the water =) Sarah Goebel-Dysart, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Webb, Dorothy L Sent: Friday, June 24, 2011 2:21 PM To: 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] bluing Looking to change my bluing step in the HE process to obtain a bluer (less purple) hue to the nuclear detail. What is everyone using in their bluing step?? Thanks for all of your ideas!! This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this e-mail in error, please immediately notify the HealthPartners Support Center by telephone at (952) 967-6600. You will be reimbursed for reasonable costs incurred in notifying us. HealthPartners R001.0 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] bluing
It might just need longer time in the bluing agent. Hematoxylin solutions stain the nuclei reddish (look at your slide right after coming out of hematoxylin - tissue will look reddish). The alkaline solutions that are used for bluing remove a H+ group on the aluminum hematein (that's the staining chemical of the hematoxylin solution), and change it to a -OH group. This changes the aluminum hematein from a reddish color to a blue color. The more alkaline (higher pH) the bluing agent, the faster this reaction and color change, and the less time is needed in the bluing agent. The lower the alkalinity (not as high pH but still in the alkaline range) the bluing agent, the slower the reaction and color change, and more time is needed in the bluing agent. Dilute ammonia water usually only takes a few seconds, as it's pH is usually high (pH 10). Tap water can have a pH of around 7, and may take 5-10 minutes to blue. If the tap water is more acidic (pH 5 or below), the slides may not blue. Now, we have to get all the nuclei from the reddish color to the bluish color. If the slide is not in the bluing agent for enough time (for the type/pH of bluing agent), then some of the nuclei change to blue, while some still remain reddish (or within a single nucleus, some of the DNA has changed to blue, some remains reddish), hence a more purple color. So, the easiest thing to try right now is to stain 2 slides from the same block (serial sections) in your hematoxylin for the same time, put one in the bluing agent for the usual time, put the other in the alkaline solution for extended time, and see if the nuclei on the second slide are now more blue than the first slide. Let us know. Peggy A. Wenk, HTL(ASCP)SLS Beaumont Hospital Royal Oak, MI 48073 -Original Message- From: Webb, Dorothy L Sent: Friday, June 24, 2011 3:20 PM To: 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] bluing Looking to change my bluing step in the HE process to obtain a bluer (less purple) hue to the nuclear detail. What is everyone using in their bluing step?? Thanks for all of your ideas!! This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this e-mail in error, please immediately notify the HealthPartners Support Center by telephone at (952) 967-6600. You will be reimbursed for reasonable costs incurred in notifying us. HealthPartners R001.0 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] bluing
There are several bluing solutions in the market, or you could use lithium carbonate at different concentrations until you find one of your liking. René J. --- On Fri, 6/24/11, Webb, Dorothy L dorothy.l.w...@healthpartners.com wrote: From: Webb, Dorothy L dorothy.l.w...@healthpartners.com Subject: [Histonet] bluing To: 'histonet@lists.utsouthwestern.edu' histonet@lists.utsouthwestern.edu Date: Friday, June 24, 2011, 3:20 PM Looking to change my bluing step in the HE process to obtain a bluer (less purple) hue to the nuclear detail. What is everyone using in their bluing step?? Thanks for all of your ideas!! This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this e-mail in error, please immediately notify the HealthPartners Support Center by telephone at (952) 967-6600. You will be reimbursed for reasonable costs incurred in notifying us. HealthPartners R001.0 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] bluing
Hi Dorothy, Try Richard Allan Bluing Reagent. Here's what they say about their product: It is a buffered product that ensures the proper alkalinity (pH=8.0). Unlike ammonia and lithium carbonate, RA's Bluing Reagent does not allow for the pH shift which can affect the crispness of nuclear detail. When I first began supervising this lab 5 years ago, they made from scratch their own hematoxylin and eosin (not to mention buffered formalin.) Unfortunately, the quality of the stain was very spotty and caused the Pathologists a lot of problems. I switched us to the Richard Allan 7211 Hematoxylin, which has beautiful, crisp nuclear detail. We also went with the recommended Richard Allan Clarifier, Bluing and Eosin, as well, so that we could produce a consistently high quality HE every time. In 5 years, we've had very few complaints about the stain from the 8-10 Pathologists we've worked with, except for one occasion when there was some isolated nuclear hazing. We did some detective work and determined that the cause was due to a rack or two that had been placed in the oven without properly removing the excess water or draining of the slides before placing them in the oven. Have a great week-end everybody. It's practically the 1st sunny day we've had, here in Minneapolis, for the past 2 weeks and Saturday and Sunday's forecast looks good, too! Sandy Harrison VA-Minneapolis Supervisor, Anatomical and Surgical Pathology 612-467-2449 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Webb, Dorothy L Sent: Friday, June 24, 2011 2:21 PM To: 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] bluing Looking to change my bluing step in the HE process to obtain a bluer (less purple) hue to the nuclear detail. What is everyone using in their bluing step?? Thanks for all of your ideas!! This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this e-mail in error, please immediately notify the HealthPartners Support Center by telephone at (952) 967-6600. You will be reimbursed for reasonable costs incurred in notifying us. HealthPartners R001.0 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] bluing in tap water?
Is there such a thing as bluing too much? -Original Message- From: Smith Wanda wanda.sm...@hcahealthcare.com Date: Mon, 12 Jan 2009 15:12:12 To: Anne C Lewinanne.le...@bms.com; Eva Permaule...@georgetown.edu Cc: histonet@lists.utsouthwestern.eduhistonet@lists.utsouthwestern.edu Subject: RE: [Histonet] bluing in tap water? What should the pH of tap water be to blue just right and not too much??? WANDA G. SMITH, HTL(ASCP)HT Pathology Supervisor TRIDENT MEDICAL CENTER 9330 Medical Plaza Drive Charleston, SC 29406 843-847-4586 843-847-4296 fax -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Anne C Lewin Sent: Monday, January 12, 2009 12:04 PM To: Eva Permaul Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] bluing in tap water? When I have used tap water, I use cold running water for 5 minutes. Works fairly well, depending on the pH of your tap water. Eva Permaul wrote: Good morning, I was wondering if someone uses tap water to blue their slides after Hematoxyline. If yes, do you use warm or cold water and for how long? Thanks, Eva Georgetown University ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] bluing in tap water?
Hello, I rinse in tap water, but blue in 1-2% ammonium hydroxide for ~ minute, then rinse again in tap water. Paula From: Eva Permaul e...@georgetown.edu To: histonet@lists.utsouthwestern.edu Sent: Monday, January 12, 2009 8:35:15 AM Subject: [Histonet] bluing in tap water? Good morning, I was wondering if someone uses tap water to blue their slides after Hematoxyline. If yes, do you use warm or cold water and for how long? Thanks, Eva Georgetown University ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] bluing in tap water?
Bluing in tap water is quite often done but has the disadvantage that your protocol cannot be repeated in other Lab because not all tap waters have the same composition of bluing quality. As any other chemical reaction it is better to blue in warm tap water (it will take less time) but you can also blue in cold water (at the temperature the water comes out of the tap). As to the time that will be determined by the blue intensity you are looking for and it will be less time in summer than in winter. All these intrinsic inconsistencies is what has determined the wide acceptance of the practice of bluing in a chemically prepared solution (like lithium carbonate or weak ammonia water). René J. --- On Mon, 1/12/09, Eva Permaul e...@georgetown.edu wrote: From: Eva Permaul e...@georgetown.edu Subject: [Histonet] bluing in tap water? To: histonet@lists.utsouthwestern.edu Date: Monday, January 12, 2009, 9:35 AM Good morning, I was wondering if someone uses tap water to blue their slides after Hematoxyline. If yes, do you use warm or cold water and for how long? Thanks, Eva Georgetown University ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] bluing in tap water?
When I have used tap water, I use cold running water for 5 minutes. Works fairly well, depending on the pH of your tap water. Eva Permaul wrote: Good morning, I was wondering if someone uses tap water to blue their slides after Hematoxyline. If yes, do you use warm or cold water and for how long? Thanks, Eva Georgetown University ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] bluing in tap water?
I don't think you can blue too much. If the pH is too high then it can bleach the haematoxylin. Any mildly alkaline solution will do (in fact neutral tap water (pH 7) will slowly get there. The special blueing solutions available are many and varied: Warm tap water, phosphate buffer (pH7-8), a weak sodium hydroxide solution ( 0.5%), a lithium carbonate solution (saturated or a diluted form), a few drops of ammonia in water, Scott's blueing solution, etc. If you are worried about the alkalinity of your blueing solution check it with some pH strips. If the solution appears a slight pink (indicating that carry-over haematoxylin is in its acidic state) then the pH will be acidic and need replacing. How can you tell if the Haematoxylin is blued? Check microscopically. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Smith Wanda Sent: Tuesday, 13 January 2009 8:12 AM To: Anne C Lewin; Eva Permaul Cc: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] bluing in tap water? What should the pH of tap water be to blue just right and not too much??? WANDA G. SMITH, HTL(ASCP)HT Pathology Supervisor TRIDENT MEDICAL CENTER 9330 Medical Plaza Drive Charleston, SC 29406 843-847-4586 843-847-4296 fax -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Anne C Lewin Sent: Monday, January 12, 2009 12:04 PM To: Eva Permaul Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] bluing in tap water? When I have used tap water, I use cold running water for 5 minutes. Works fairly well, depending on the pH of your tap water. Eva Permaul wrote: Good morning, I was wondering if someone uses tap water to blue their slides after Hematoxyline. If yes, do you use warm or cold water and for how long? Thanks, Eva Georgetown University ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet * This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] bluing hematoxylin and alkaline water????
Eva: Hematoxylin is a pH indicator, it turns reddish when you differentiate it in the acid alcohol, and returns to violet-blue when you either blue it with the ammonia water or place it in tap water after the alcohol-acid solution. The differentiation you know is to make the nuclear details more visible and the bluing is essentially done to stop the acid effect of the clearing, that could end decolorizing the section. It is a good practice to blue with the alkaline (ammonia) solution or just plain tap water if it is hard enough (containing salts), but it is not good practice to let the slides in running water for long periods of time because, being the universal solvent, running water will end making the stain fainter, as you have noticed in the difference between the two types of batches you mention. It would preferable to leave the last batch in your clearing (pre-coverslipping) liquid (usually xylene), this will not affect the staining intensity at all. René J. --- On Fri, 11/21/08, Eva Permaul [EMAIL PROTECTED] wrote: From: Eva Permaul [EMAIL PROTECTED] Subject: [Histonet] bluing hematoxylin and alkaline water To: histonet@lists.utsouthwestern.edu Date: Friday, November 21, 2008, 11:11 AM Good morning out in histoland, Thank god it is Friday. Have just a small question that I am sure you all have an answer for. It is about bluing slides after hematoxylin. I read somewhere that alkaline water can blue hematoxylin and I am wondering if this is part of our problem. We use an automated stainer that we run both during the day and during the night. Normally we blue the slides in 1% ammonium after the runs for 1min. The run that was done during the day ended up less blue (more purple). The ones that run during the night continue being washed in water until we come in in the morning. They ended up more blue. I tested the pH of the water we are using. It is 7.96. Would this be enough to blue the slides if they are washed in it every hour from 9pm to 8am? Any other reason this might be happening? Should we not blue the slides that are run during the night? Why do we blue hematoxylin anyways? Thank you for all your answers Eva Permaul (still learning) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
