[Histonet] Coverslipping mystery

Discussion:

Adam Boanas

2015-07-09 13:15:33 UTC

Hello,

We are having a problem that is developing into a big issue in our lab and I was wondering if anybody could shed any light on it. Our CV5000 coverslipper has recently started introducing microscopic air bubbles onto the slides during coverslipping. We have been told by our engineer that it is a consequence of the age and use of the motor and that sourcing another for an instrument that old (15yrs) will be v difficult. As such, we have been forced to manually coverslip using DPX and a pipette - manually applying the coverslips to the slide, thus mirroring the action of the coverslipper. This is fine at first and for the next few days the slides look great and very clean. However, after about day 4 -5 days post coverslipping, the slides develop an odd appearance down the microscope which looks like very fine `parched earth / crazy paving` all over the slide - including the section. The excess mountant around the edge of the coverslip also has a very faint, cloudy appearance when this occurs. This of course renders the slide un-useable. Does anyone have a clue what this might be down to / how we can stop it?
We are struggling for ideas with this one! - this occurs with fresh DPX also.

Many thanks
Adam

Adam Boanas
Senior Research Associate
Epistem Ltd
48 Grafton Street
Manchester, M13 9XX

Rene J Buesa

2015-07-09 13:47:14 UTC

Permalink

Adam:
Just guessing, but I think the "mystery" is caused by how fluid the DPX is. Guessing again, but it probably is more dense as it should.I would dilute it to the lowest density it can be used in your coverslipper. Give it a try.René

On Thursday, July 9, 2015 9:40 AM, Adam Boanas <***@epistem.co.uk> wrote:

Hello,

We are having a problem that is developing into a big issue in our lab and I was wondering if anybody could shed any light on it. Our CV5000 coverslipper has recently started introducing microscopic air bubbles onto the slides during coverslipping. We have been told by our engineer that it is a consequence of the age and use of the motor and that sourcing another for an instrument that old (15yrs) will be v difficult. As such, we have been forced to manually coverslip using DPX and a pipette - manually applying the coverslips to the slide, thus mirroring the action of the coverslipper. This is fine at first and for the next few days the slides look great and very clean. However, after about day 4 -5 days post coverslipping, the slides develop an odd appearance down the microscope which looks like very fine `parched earth / crazy paving` all over the slide - including the section. The excess mountant around the edge of the coverslip also has a very faint, cloudy appearance when this occurs. This of course renders the slide un-useable. Does anyone have a clue what this might be down to / how we can stop it?
We are struggling for ideas with this one! - this occurs with fresh DPX also.

Many thanks
Adam

Adam Boanas
Senior Research Associate
Epistem Ltd
48 Grafton Street
Manchester, M13 9XX

_______________________________________________
Histonet mailing list
***@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Simmons, Christopher

2015-07-09 13:49:00 UTC

Permalink

Look in your manual, near the back. There should be mountant type calibration settings for yo to adjust the machine to.
That, hopefully will solve your issue!

Chris Simmons B.S., A.S., HTL(ASCP)
Supervisor, UPP Dermatopathology
412.864.3880 office
412.612.0881 cell

-----Original Message-----
From: Rene J Buesa [mailto:***@yahoo.com]
Sent: Thursday, July 09, 2015 9:47 AM
To: Adam Boanas; ***@lists.utsouthwestern.edu
Subject: Re: [Histonet] Coverslipping mystery

Adam:
Just guessing, but I think the "mystery" is caused by how fluid the DPX is. Guessing again, but it probably is more dense as it should.I would dilute it to the lowest density it can be used in your coverslipper. Give it a try.René

On Thursday, July 9, 2015 9:40 AM, Adam Boanas <***@epistem.co.uk> wrote:

Hello,

We are having a problem that is developing into a big issue in our lab and I was wondering if anybody could shed any light on it. Our CV5000 coverslipper has recently started introducing microscopic air bubbles onto the slides during coverslipping. We have been told by our engineer that it is a consequence of the age and use of the motor and that sourcing another for an instrument that old (15yrs) will be v difficult. As such, we have been forced to manually coverslip using DPX and a pipette - manually applying the coverslips to the slide, thus mirroring the action of the coverslipper. This is fine at first and for the next few days the slides look great and very clean. However, after about day 4 -5 days post coverslipping, the slides develop an odd appearance down the microscope which looks like very fine `parched earth / crazy paving` all over the slide - including the section. The excess mountant around the edge of the coverslip also has a very faint, cloudy appearance when this occurs. This of course renders the slide un-useable. Does anyone have a clue what this might be down to / how we can stop it?
We are struggling for ideas with this one! - this occurs with fresh DPX also.

Many thanks
Adam

Adam Boanas
Senior Research Associate
Epistem Ltd
48 Grafton Street
Manchester, M13 9XX

_______________________________________________
Histonet mailing list
***@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

_______________________________________________
Histonet mailing list
***@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Goins, Tresa

2015-07-09 13:52:08 UTC

Permalink

I am not familiar with DPX, but is there a compatibility issue between it and the slide clearant you are using?

Tresa

-----Original Message-----
From: Adam Boanas [mailto:***@epistem.co.uk]
Sent: Thursday, July 09, 2015 7:16 AM
To: ***@lists.utsouthwestern.edu
Subject: [Histonet] Coverslipping mystery

Hello,

We are having a problem that is developing into a big issue in our lab and I was wondering if anybody could shed any light on it. Our CV5000 coverslipper has recently started introducing microscopic air bubbles onto the slides during coverslipping. We have been told by our engineer that it is a consequence of the age and use of the motor and that sourcing another for an instrument that old (15yrs) will be v difficult. As such, we have been forced to manually coverslip using DPX and a pipette - manually applying the coverslips to the slide, thus mirroring the action of the coverslipper. This is fine at first and for the next few days the slides look great and very clean. However, after about day 4 -5 days post coverslipping, the slides develop an odd appearance down the microscope which looks like very fine `parched earth / crazy paving` all over the slide - including the section. The excess mountant around the edge of the coverslip also has a very faint, cloudy appearance when this occurs. This of course renders the slide un-useable. Does anyone have a clue what this might be down to / how we can stop it?
We are struggling for ideas with this one! - this occurs with fresh DPX also.

Many thanks
Adam

Adam Boanas
Senior Research Associate
Epistem Ltd
48 Grafton Street
Manchester, M13 9XX

Jennifer MacDonald

2015-07-09 16:53:06 UTC

Permalink

What type of clearing agent are you using? The aliphatic hydrocarbons are
not compatible with all mounting media.

From: Adam Boanas <***@epistem.co.uk>
To: "***@lists.utsouthwestern.edu"
<***@lists.utsouthwestern.edu>
Date: 07/09/2015 06:16 AM
Subject: [Histonet] Coverslipping mystery

Hello,

We are having a problem that is developing into a big issue in our lab and
I was wondering if anybody could shed any light on it. Our CV5000
coverslipper has recently started introducing microscopic air bubbles onto
the slides during coverslipping. We have been told by our engineer that it
is a consequence of the age and use of the motor and that sourcing another
for an instrument that old (15yrs) will be v difficult. As such, we have
been forced to manually coverslip using DPX and a pipette - manually
applying the coverslips to the slide, thus mirroring the action of the
coverslipper. This is fine at first and for the next few days the slides
look great and very clean. However, after about day 4 -5 days post
coverslipping, the slides develop an odd appearance down the microscope
which looks like very fine `parched earth / crazy paving` all over the
slide - including the section. The excess mountant around the edge of the
coverslip also has a very faint, cloudy appearance whe
n this occurs. This of course renders the slide un-useable. Does anyone
have a clue what this might be down to / how we can stop it?
We are struggling for ideas with this one! - this occurs with fresh DPX
also.

Many thanks
Adam

Adam Boanas
Senior Research Associate
Epistem Ltd
48 Grafton Street
Manchester, M13 9XX

John Kiernan

2015-07-11 05:55:28 UTC

Permalink

DPX is a polystyrene mounting medium. In principle you can make your own from published recipes. In practice, everyone buys commercial resinous mounting media.

In the 1990s we had trouble similar to what you describe. The commercial DPX was cloudy, and not because of alcohol in our xylene. The Canadian supplier acknowledged the bad DPX and urged us to buy Entellan instead. Entellan is a poly(methacrylate) plastic and is an excellent but expensive mounting medium. Another poly(methacrylate) mountant called CytoSeal was less expensive and also came in a squeeze-easy plastic bottle for delivery onto the slide or coverslip. It's now my routine resious mountant.

Good DPX returned to the market in the 2000s, but in old-fashioned bottles and not easy to apply to slides or coverslips.

John Kiernan
= = =

Post by Adam Boanas
Hello,
We are having a problem that is developing into a big issue in our lab and I was wondering if anybody could shed any light on it. Our CV5000 coverslipper has recently started introducing microscopic air bubbles onto the slides during coverslipping. We have been told by our engineer that it is a consequence of the age and use of the motor and that sourcing another for an instrument that old (15yrs) will be v difficult. As such, we have been forced to manually coverslip using DPX and a pipette - manually applying the coverslips to the slide, thus mirroring the action of the coverslipper. This is fine at first and for the next few days the slides look great and very clean. However, after about day 4 -5 days post coverslipping, the slides develop an odd appearance down the microscope which looks like very fine `parched earth / crazy paving` all over the slide - including the section. The excess mountant around the edge of the coverslip also has a very faint, cloudy appearance wh!
en this occurs. This of course renders the slide un-useable. Does anyone have a clue what this might be down to / how we can stop it?
We are struggling for ideas with this one! - this occurs with fresh DPX also.
Many thanks
Adam
Adam Boanas
Senior Research Associate
Epistem Ltd
48 Grafton Street
Manchester, M13 9XX
_______________________________________________
Histonet mailing list
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Caroline Miller

2015-07-11 14:17:30 UTC

Permalink

I really like DPX, although funnily enough we used cytoseal in my lab in London but always called it the DPX! I think I remember by boss telling me about the bad DPX time.....

When I moved to the USA the lab I started in had a bottle of DPX and i loved it! I always decant some of the DPX into a 100ml glass bottle, put in a plastic squeeze pipette and then screw a lid on it to stop it drying out (with the pipette still inside) when not in use. Surprisingly the pipette doesn't melt! Which is good because I am a recycle freak and i couldn't stand using a new one every time I mounted something!

Yours,
mills

Caroline Miller (mills)
Director of Histology
3Scan, Inc
415-2187297

Post by John Kiernan
DPX is a polystyrene mounting medium. In principle you can make your own from published recipes. In practice, everyone buys commercial resinous mounting media.
In the 1990s we had trouble similar to what you describe. The commercial DPX was cloudy, and not because of alcohol in our xylene. The Canadian supplier acknowledged the bad DPX and urged us to buy Entellan instead. Entellan is a poly(methacrylate) plastic and is an excellent but expensive mounting medium. Another poly(methacrylate) mountant called CytoSeal was less expensive and also came in a squeeze-easy plastic bottle for delivery onto the slide or coverslip. It's now my routine resious mountant.
Good DPX returned to the market in the 2000s, but in old-fashioned bottles and not easy to apply to slides or coverslips.
John Kiernan
= = =

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Histonet mailing list
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Adam Boanas

2015-07-13 07:30:44 UTC

Permalink

Hello again,

Thought I might offer an update on the coverslipping issue as it might be of use in future.
I ran a test last week of manual coverslipping using blank charged and un-charged slides and using DPX and Pertex as the mountant. I also used 2 methods of application.
1) Mountant applied using plastic Pasteur pipette
2) Mountant applied using aluminium screw cap tube.
Following immersion in xylene for 5 mins the coverslips were applied. From viewing this morning, all slides were clear with the exception of those coverslipped using DPX applied with the Pasteur. In each case, these slides had the 'parched earth' artefact having been left to dry over the weekend.
I suspect that the DPX has had a reaction with the plastic of the pipette during application and the artefact is caused by residual `molten` plastic from the pipette that only reveals itself over time.
Does this sound plausible? No problem with the pertex and pipettes (which is what I've used for years with no issue)
Thanks
Adam

-----Original Message-----
From: Caroline Miller [mailto:***@3scan.com]
Sent: 11 July 2015 15:18
To: John Kiernan
Cc: Adam Boanas; ***@lists.utsouthwestern.edu
Subject: Re: [Histonet] Coverslipping mystery

I really like DPX, although funnily enough we used cytoseal in my lab in London but always called it the DPX! I think I remember by boss telling me about the bad DPX time.....

When I moved to the USA the lab I started in had a bottle of DPX and i loved it! I always decant some of the DPX into a 100ml glass bottle, put in a plastic squeeze pipette and then screw a lid on it to stop it drying out (with the pipette still inside) when not in use. Surprisingly the pipette doesn't melt! Which is good because I am a recycle freak and i couldn't stand using a new one every time I mounted something!

Yours,
mills

Caroline Miller (mills)
Director of Histology
3Scan, Inc
415-2187297

_______________________________________________
Histonet mailing list
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Caroline Miller

2015-07-13 13:32:37 UTC

Permalink

It sounds totally plausible, but just to put a spanner in the works; I have a plastic pipette in my DPX that I leave in there for at least 6 months and I don't get that artifact. We may have different pipettess though.

Yours,
mills

Caroline Miller (mills)
Director of Histology
3Scan, Inc
415-2187297

Post by Adam Boanas
Hello again,
Thought I might offer an update on the coverslipping issue as it might be of use in future.
I ran a test last week of manual coverslipping using blank charged and un-charged slides and using DPX and Pertex as the mountant. I also used 2 methods of application.
1) Mountant applied using plastic Pasteur pipette
2) Mountant applied using aluminium screw cap tube.
Following immersion in xylene for 5 mins the coverslips were applied. From viewing this morning, all slides were clear with the exception of those coverslipped using DPX applied with the Pasteur. In each case, these slides had the 'parched earth' artefact having been left to dry over the weekend.
I suspect that the DPX has had a reaction with the plastic of the pipette during application and the artefact is caused by residual `molten` plastic from the pipette that only reveals itself over time.
Does this sound plausible? No problem with the pertex and pipettes (which is what I've used for years with no issue)
Thanks
Adam
-----Original Message-----
Sent: 11 July 2015 15:18
To: John Kiernan
Subject: Re: [Histonet] Coverslipping mystery
I really like DPX, although funnily enough we used cytoseal in my lab in London but always called it the DPX! I think I remember by boss telling me about the bad DPX time.....
When I moved to the USA the lab I started in had a bottle of DPX and i loved it! I always decant some of the DPX into a 100ml glass bottle, put in a plastic squeeze pipette and then screw a lid on it to stop it drying out (with the pipette still inside) when not in use. Surprisingly the pipette doesn't melt! Which is good because I am a recycle freak and i couldn't stand using a new one every time I mounted something!
Yours,
mills
Caroline Miller (mills)
Director of Histology
3Scan, Inc
415-2187297

_______________________________________________
Histonet mailing list
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Bernice Frederick

2015-07-13 12:41:42 UTC

Permalink

We have a CV5030 and use the suggested Surgipath micromount. It works well. As has been previously mentioned, you can adjust the amount of mountant dispensed as desired.
Bernice

Bernice Frederick HTL (ASCP)
Senior Research Tech
Pathology Core Facility
Robert. H. Lurie Cancer Center
Northwestern University
710 N Fairbanks Court
Olson 8-421
Chicago,IL 60611
312-503-3723
b-***@northwestern.edu

-----Original Message-----
From: Caroline Miller [mailto:***@3scan.com]
Sent: Saturday, July 11, 2015 9:18 AM
To: John Kiernan
Cc: ***@lists.utsouthwestern.edu
Subject: Re: [Histonet] Coverslipping mystery

I really like DPX, although funnily enough we used cytoseal in my lab in London but always called it the DPX! I think I remember by boss telling me about the bad DPX time.....

When I moved to the USA the lab I started in had a bottle of DPX and i loved it! I always decant some of the DPX into a 100ml glass bottle, put in a plastic squeeze pipette and then screw a lid on it to stop it drying out (with the pipette still inside) when not in use. Surprisingly the pipette doesn't melt! Which is good because I am a recycle freak and i couldn't stand using a new one every time I mounted something!

Yours,
mills

Caroline Miller (mills)
Director of Histology
3Scan, Inc
415-2187297

_______________________________________________
Histonet mailing list
http://lists.utsouthwestern.edu/mailman/listinfo/histonet