Re: [Histonet] Cloudy MMA embedded block
Ooi, Somehow you are getting moisture/water mixed in with the MMA solution(s). I only remember visiting the IMCB facility, so if this is not your facility then I am not sure of your laboratory environment (air temperature/humidity). The best thing I can tell you at this point is to try and find ways to reduce the chance for moisture accumulation. I am pretty sure that at a minimum you are storing your catalyst and monomer (MMA) at 4C and your softner (DBP) at room temp. Maybe you should take extra steps to ensure your chemicals are at room temp before any use. Lastly, your polymerization is taking too long for your small 20mL vials. My specimens of similar size require only 3-5 days to completely polymerize. Now, I dont remember your concentration of catalyst, but if it is around 2.5g/1L embedding solution, try skipping the 4C step and use room temp (23C) until the specimens are polymerized. Then finish them off in the oven when they are tacky or mostly hard on top. The 4C step is mostly to slow the polymerization for larger specimens requiring a larger volume of solution and/or when the lab temp cannot be controlled routinely at or below 23C. The water bath then also helps to regulate the temperature of the polymerization in the case where the lab environment is too cool (20C) and retarding the rate of polymerzation. The main thing to keep in mind is that the lab environment pretty much controls the process and is probably the main variable that will contrast labs that perform the exact same method. Jack On Apr 21, 2009, at 12:33 AM, ooi.ting.h...@nhc.com.sg wrote: Hi, I am trying very hard to get a clear MMA embedded block. I am appreciate if there is any advice on doing it. I am using 100% MMA (liquid) and 0.5% Perkadox 16 (powder) to do the embedding. I have tried to embed the sample in different conditions. However, it seems to be extremely hard to get a clear embedded block. Below are the methods that I have tried. 1. Before embedding, bring the MMA and perkadox 16 to room temperature. After a few mix it by invertion, I aliquot the solution to the glass vials that contain samples or simply glass vials that without sample. I put the glass vials (both with and without sample) in 4 degree, room temperature and even 37degree oven. 2. Mix the MMA and perkadox when it is cold, invert it a few times. Aliquot it to glass vials that contain sample or without sample. Put in 4degree, room temperature and 37degree oven. I have also tried to speed up the polymerization process by using vacumm for a few samples... Throughout all the sample that I have, I only manage to get a clear empty glass vial which I put in the 4degree for one month and a clear embedded sample which I put in 4 degree overnight and bring it out to room temperature. The rest of the glass vials (with or without sample) are cloudy. I have repeated the methods that I used to get a clear embedded block, I did not get any clear block (for both with and without sample). I am glad if there is any expert advice or guidance. We need a clear embedded sample. Thank you very much! Regards, Ooi _ Confidential information. Unauthorized use or disclosure prohibited. Refer http://www.singhealth.com.sg/ContactUs/#Disclaimer ___ 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] Cloudy MMA embedded block
Hi, We don't store our MMA or DBP in the refirgerator as the movement between 4C and room temperature can cause condensation in the bottles. This just adds water to the material and as it is cooled and re-warmed you are adding more each time and in both directions. When it comes to room temperature and then is replaced in the refrigerator it again will develop condensation. Over time the MMA will have enough water in it to become discolored. I am surprised you are not seeing the discoloration in the MMA liquid as it poured as a warning not to use it. We have fair control of temperature in the laboratory and are generally between 68F to 75F with the average as around 70F. We have never had a problem with the MMA polymerizing or developing a water content. It is stored in a dark cabinet that is in an area I would say is a little cooler than the room. We generally don't polymerize in the cold at 4C however, you timing seems long for the process. It is best not to disturb the vials too much once you are in the embedding phase. Movement or inversion of the vials can cause a problem as the MMA will polymerize from the bottom up and remixing it can slow the process. Also make sure you tops are tight at this step. Pam Marcum UPENN Vet Sch New Botlon Center - Original Message - From: Jack Ratliff ratliffj...@hotmail.com To: Ooi Ting Huay ooi.ting.h...@nhc.com.sg, histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Tuesday, April 21, 2009 9:24:29 AM GMT -05:00 US/Canada Eastern Subject: Re: [Histonet] Cloudy MMA embedded block Ooi, Somehow you are getting moisture/water mixed in with the MMA solution(s). I only remember visiting the IMCB facility, so if this is not your facility then I am not sure of your laboratory environment (air temperature/humidity). The best thing I can tell you at this point is to try and find ways to reduce the chance for moisture accumulation. I am pretty sure that at a minimum you are storing your catalyst and monomer (MMA) at 4C and your softner (DBP) at room temp. Maybe you should take extra steps to ensure your chemicals are at room temp before any use. Lastly, your polymerization is taking too long for your small 20mL vials. My specimens of similar size require only 3-5 days to completely polymerize. Now, I dont remember your concentration of catalyst, but if it is around 2.5g/1L embedding solution, try skipping the 4C step and use room temp (23C) until the specimens are polymerized. Then finish them off in the oven when they are tacky or mostly hard on top. The 4C step is mostly to slow the polymerization for larger specimens requiring a larger volume of solution and/or when the lab temp cannot be controlled routinely at or below 23C. The water bath then also helps to regulate the temperature of the polymerization in the case where the lab environment is too cool (20C) and retarding the rate of polymerzation. The main thing to keep in mind is that the lab environment pretty much controls the process and is probably the main variable that will contrast labs that perform the exact same method. Jack On Apr 21, 2009, at 12:33 AM, ooi.ting.h...@nhc.com.sg wrote: Hi, I am trying very hard to get a clear MMA embedded block. I am appreciate if there is any advice on doing it. I am using 100% MMA (liquid) and 0.5% Perkadox 16 (powder) to do the embedding. I have tried to embed the sample in different conditions. However, it seems to be extremely hard to get a clear embedded block. Below are the methods that I have tried. 1. Before embedding, bring the MMA and perkadox 16 to room temperature. After a few mix it by invertion, I aliquot the solution to the glass vials that contain samples or simply glass vials that without sample. I put the glass vials (both with and without sample) in 4 degree, room temperature and even 37degree oven. 2. Mix the MMA and perkadox when it is cold, invert it a few times. Aliquot it to glass vials that contain sample or without sample. Put in 4degree, room temperature and 37degree oven. I have also tried to speed up the polymerization process by using vacumm for a few samples... Throughout all the sample that I have, I only manage to get a clear empty glass vial which I put in the 4degree for one month and a clear embedded sample which I put in 4 degree overnight and bring it out to room temperature. The rest of the glass vials (with or without sample) are cloudy. I have repeated the methods that I used to get a clear embedded block, I did not get any clear block (for both with and without sample). I am glad if there is any expert advice or guidance. We need a clear embedded sample. Thank you very much! Regards, Ooi
RE: [Histonet] Cloudy MMA embedded block
In our lab we do store our MMA solutions in a flammable storage refrigerator per EHS requirements; however we put Molecular Sieves in all solutions to keep moisture from contaminating them. We rarely have any problems with cloudy blocks. We order them from VWR, catalog # JT2708-1, 500 g, ~$40 each; manufacturer is J.T. Baker. They last a very long time and are useful for any open non-aqueous solution. Just add a few (6-12) to each container. Hope this helps! Lori -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Pamela Marcum Sent: Tuesday, April 21, 2009 6:45 AM To: Jack Ratliff Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Cloudy MMA embedded block Hi, We don't store our MMA or DBP in the refirgerator as the movement between 4C and room temperature can cause condensation in the bottles. This just adds water to the material and as it is cooled and re-warmed you are adding more each time and in both directions. When it comes to room temperature and then is replaced in the refrigerator it again will develop condensation. Over time the MMA will have enough water in it to become discolored. I am surprised you are not seeing the discoloration in the MMA liquid as it poured as a warning not to use it. We have fair control of temperature in the laboratory and are generally between 68F to 75F with the average as around 70F. We have never had a problem with the MMA polymerizing or developing a water content. It is stored in a dark cabinet that is in an area I would say is a little cooler than the room. We generally don't polymerize in the cold at 4C however, you timing seems long for the process. It is best not to disturb the vials too much once you are in the embedding phase. Movement or inversion of the vials can cause a problem as the MMA will polymerize from the bottom up and remixing it can slow the process. Also make sure you tops are tight at this step. Pam Marcum UPENN Vet Sch New Botlon Center - Original Message - From: Jack Ratliff ratliffj...@hotmail.com To: Ooi Ting Huay ooi.ting.h...@nhc.com.sg, histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Tuesday, April 21, 2009 9:24:29 AM GMT -05:00 US/Canada Eastern Subject: Re: [Histonet] Cloudy MMA embedded block Ooi, Somehow you are getting moisture/water mixed in with the MMA solution(s). I only remember visiting the IMCB facility, so if this is not your facility then I am not sure of your laboratory environment (air temperature/humidity). The best thing I can tell you at this point is to try and find ways to reduce the chance for moisture accumulation. I am pretty sure that at a minimum you are storing your catalyst and monomer (MMA) at 4C and your softner (DBP) at room temp. Maybe you should take extra steps to ensure your chemicals are at room temp before any use. Lastly, your polymerization is taking too long for your small 20mL vials. My specimens of similar size require only 3-5 days to completely polymerize. Now, I dont remember your concentration of catalyst, but if it is around 2.5g/1L embedding solution, try skipping the 4C step and use room temp (23C) until the specimens are polymerized. Then finish them off in the oven when they are tacky or mostly hard on top. The 4C step is mostly to slow the polymerization for larger specimens requiring a larger volume of solution and/or when the lab temp cannot be controlled routinely at or below 23C. The water bath then also helps to regulate the temperature of the polymerization in the case where the lab environment is too cool (20C) and retarding the rate of polymerzation. The main thing to keep in mind is that the lab environment pretty much controls the process and is probably the main variable that will contrast labs that perform the exact same method. Jack On Apr 21, 2009, at 12:33 AM, ooi.ting.h...@nhc.com.sg wrote: Hi, I am trying very hard to get a clear MMA embedded block. I am appreciate if there is any advice on doing it. I am using 100% MMA (liquid) and 0.5% Perkadox 16 (powder) to do the embedding. I have tried to embed the sample in different conditions. However, it seems to be extremely hard to get a clear embedded block. Below are the methods that I have tried. 1. Before embedding, bring the MMA and perkadox 16 to room temperature. After a few mix it by invertion, I aliquot the solution to the glass vials that contain samples or simply glass vials that without sample. I put the glass vials (both with and without sample) in 4 degree, room temperature and even 37degree oven. 2. Mix the MMA and perkadox when it is cold, invert it a few times. Aliquot it to glass vials that contain sample or without sample. Put in 4degree, room temperature and 37degree oven. I have also tried to speed up
