Immobilize bacteria
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Does anybody have a good way to Immobilize bacteria without killing. We would like to do a Live/dead stain and our bugs like to float away!
Thanks!! Mike |
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I've used CyGel for that purpose before (no commercial interest).
http://www.biostatus.com/product/cygel/ -Esteban On Thu, Oct 22, 2009 at 1:11 PM, Mike Tighe <[hidden email]> wrote: > Does anybody have a good way to Immobilize bacteria without killing. We would like to do a Live/dead stain and our bugs like to float away! > > Thanks!! > Mike > -- G. Esteban Fernandez, Ph.D. Associate Director Molecular Cytology Core Facility University of Missouri 120 Bond Life Sciences Center Columbia, MO 65211 http://www.biotech.missouri.edu/mcc (573)882-4895 (573)884-9676 fax |
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Likewise, we have used CyGel. You may need try various strengths to get the
best lifetime out of the bacteria, especially if they are motile. You will also need to seal the edges with vacuum grease to stop it drying out, but once in place. Best Peter ----- Original Message ----- From: "G. Esteban Fernandez" <[hidden email]> To: <[hidden email]> Sent: Thursday, October 22, 2009 7:17 PM Subject: Re: Immobilize bacteria > I've used CyGel for that purpose before (no commercial interest). > http://www.biostatus.com/product/cygel/ > > -Esteban > > > On Thu, Oct 22, 2009 at 1:11 PM, Mike Tighe <[hidden email]> > wrote: >> Does anybody have a good way to Immobilize bacteria without killing. We >> would like to do a Live/dead stain and our bugs like to float away! >> >> Thanks!! >> Mike >> > > -- > G. Esteban Fernandez, Ph.D. > > Associate Director > Molecular Cytology Core Facility > University of Missouri > 120 Bond Life Sciences Center > Columbia, MO 65211 > > http://www.biotech.missouri.edu/mcc > > (573)882-4895 > (573)884-9676 fax > |
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In reply to this post by Mike Tighe
I use CellTak from BD which works very well. Immobilises them well
enough for long time cours records and they still grow fine. Colin On 22 Oct 2009, at 19:11, Mike Tighe <[hidden email]> wrote: > Does anybody have a good way to Immobilize bacteria without killing. > We would like to do a Live/dead stain and our bugs like to float away! > > Thanks!! > Mike |
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In reply to this post by Mike Tighe
Hi Mike,
A trick taught to me my a microbiologist (Mark McBride) is to make a 0.7% agarose solution in water (microwave to dissolve), and while it is still hot use a Pasteur pipet to flow a layer onto a microscope slide to cover it. I usully suspend the slides on wooden applicator sticks in case the agarose runs off, otherwise the solution wicks under the slide. Once the agarose gels, apply a drop of culture on top, then coverslip it. The pad of agarose needs to be larger than the coverslip you want to use. The cells stay immobile, and actually stay alive for a long time. Sincerely yours, Heather Owen Dr. Heather A. Owen Director, Electron Microscope Laboratory Department of Biological Sciences University of Wisconsin - Milwaukee Lapham Hall 3209 N. Maryland Avenue Milwaukee, WI 53211 (414)229-6816 ----- Original Message ----- From: "Mike Tighe" <[hidden email]> To: [hidden email] Sent: Thursday, October 22, 2009 1:11:00 PM GMT -06:00 US/Canada Central Subject: Immobilize bacteria Does anybody have a good way to Immobilize bacteria without killing. We would like to do a Live/dead stain and our bugs like to float away! Thanks!! Mike |
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In reply to this post by Mike Tighe
The simplest approach is to just make a nice thin mount and seal the
edges with wax or nail polish. Should work pretty well. However, for serious counting, you filter the bacteria onto a black filter, apply oil to the filter, a coverslip on top, and then use oil immersion optics to view. Another thing you can try is to coat slides with poly-lysine (I think you can buy pre-coated slides), add a drop of the bugs, then coverslip the prep. If you want to go really low tech but get results, you can coat a slide with 0.5% agar (tilt the slide and run the nearly cool agar down it), wait for it to dry, then add a drop of bacterial suspension. The agar swells, the bacteria are immobilized, and you drop a coverglass on the top. >Does anybody have a good way to Immobilize bacteria without killing. >We would like to do a Live/dead stain and our bugs like to float >away! > >Thanks!! >Mike -- Robert J. Palmer Jr., Ph.D. Natl Inst Dental Craniofacial Res - Natl Insts Health Oral Infection and Immunity Branch Bldg 30, Room 310 30 Convent Drive Bethesda MD 20892 ph 301-594-0025 fax 301-402-0396 |
 
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B. Prabhakar Pandian |
Cell Media Preparation Debate
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In reply to this post by O'Toole, P
Hello,
In our laboratory there has been recent debates on the cell media preparation. The point of contention is the presence of serum in media. When a media mentions supplemented with 10% serum, which of the following is correct for a 500ml media bottle. 1. Add 50ml of serum to the media bottle (total volume is 550ml) 2. Remove 50ml of media and then add 50ml of serum (total volume is 500ml). Any information on the right usage will be great. Thanks, -Bala |
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In reply to this post by O'Toole, P
Is there a U.S, vendor for CyGel?
On Oct 22, 2009, at 1:31 PM, Peter O'Toole wrote: > Likewise, we have used CyGel. You may need try various strengths to > get the best lifetime out of the bacteria, especially if they are > motile. You will also need to seal the edges with vacuum grease to > stop it drying out, but once in place. > > Best > > Peter > > ----- Original Message ----- From: "G. Esteban Fernandez" <[hidden email] > > > To: <[hidden email]> > Sent: Thursday, October 22, 2009 7:17 PM > Subject: Re: Immobilize bacteria > > >> I've used CyGel for that purpose before (no commercial interest). >> http://www.biostatus.com/product/cygel/ >> >> -Esteban >> >> >> On Thu, Oct 22, 2009 at 1:11 PM, Mike Tighe <[hidden email] >> > wrote: >>> Does anybody have a good way to Immobilize bacteria without >>> killing. We would like to do a Live/dead stain and our bugs like >>> to float away! >>> >>> Thanks!! >>> Mike >>> >> >> -- >> G. Esteban Fernandez, Ph.D. >> >> Associate Director >> Molecular Cytology Core Facility >> University of Missouri >> 120 Bond Life Sciences Center >> Columbia, MO 65211 >> >> http://www.biotech.missouri.edu/mcc >> >> (573)882-4895 >> (573)884-9676 fax Michael J. Herron, U of MN, Dept. of Entomology [hidden email] 612-624-3688 (office) 612-625-5299 (FAX) |
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In reply to this post by Mike Tighe
In the past we have used thin layers of agar or agarose on the surface of the slide to immobilise either pre-stain bacteria or to actually stain bacteria by incorporating the stain into the agar. This effectively prevents floating if you lightly dry the surface of the agar once the sample has been added prior to putting on a coverslip. This works very well for both live/dead staining and for time-lapse growth studies using DAPI or membrane stained bacteria so they remain viable for long periods (assuming that they are in a humidified container!).
Helen -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mike Tighe Sent: Friday, 23 October 2009 7:11 a.m. To: [hidden email] Subject: Immobilize bacteria Does anybody have a good way to Immobilize bacteria without killing. We would like to do a Live/dead stain and our bugs like to float away! Thanks!! Mike ======================================================================= Attention: The information contained in this message and/or attachments from AgResearch Limited is intended only for the persons or entities to which it is addressed and may contain confidential and/or privileged material. Any review, retransmission, dissemination or other use of, or taking of any action in reliance upon, this information by persons or entities other than the intended recipients is prohibited by AgResearch Limited. If you have received this message in error, please notify the sender immediately. ======================================================================= |
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In reply to this post by B. Prabhakar Pandian
If total volume is 550mls (500ml media and 50mls serum you will have 9.1% serum. Not 10% but probably enough to keep most cells happy
Mike >>> "B. Prabhakar Pandian" <[hidden email]> 10/22/09 3:10 PM >>> Hello, In our laboratory there has been recent debates on the cell media preparation. The point of contention is the presence of serum in media. When a media mentions supplemented with 10% serum, which of the following is correct for a 500ml media bottle. 1. Add 50ml of serum to the media bottle (total volume is 550ml) 2. Remove 50ml of media and then add 50ml of serum (total volume is 500ml). Any information on the right usage will be great. Thanks, - Bala |
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Henn, Alicia |
Re: Cell Media Preparation Debate
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In reply to this post by B. Prabhakar Pandian
We add 55ml of serum to a 500ml bottle of medium to yield 555ml of 10% serum. No 50ml tubes of extra medium to store and still get 10% final.
Alicia D. Henn, Ph.D. Department of Nephrology University of Rochester Medical Center Rochester, NY 14624 (585) 276-4897 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of B. Prabhakar Pandian Sent: Thursday, October 22, 2009 3:11 PM To: [hidden email] Subject: Cell Media Preparation Debate Hello, In our laboratory there has been recent debates on the cell media preparation. The point of contention is the presence of serum in media. When a media mentions supplemented with 10% serum, which of the following is correct for a 500ml media bottle. 1. Add 50ml of serum to the media bottle (total volume is 550ml) 2. Remove 50ml of media and then add 50ml of serum (total volume is 500ml). Any information on the right usage will be great. Thanks, -Bala |
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Nobby Clark |
Re: Cell Media Preparation Debate
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In reply to this post by Mike Tighe
Hi,
Does anybody no of anyone who is looking for a very nice confocial? we have lost funding and must get rid of our Zeiss LSM 510NLO ready. We must find a home for this soon Thanks On Thu, 22 Oct 2009 16:40:17 -0400, Mike Tighe <[hidden email]> wrote: > If total volume is 550mls (500ml media and 50mls serum you will have 9.1% > serum. Not 10% but probably enough to keep most cells happy > > Mike > >>>> "B. Prabhakar Pandian" <[hidden email]> 10/22/09 3:10 PM >>> > Hello, > In our laboratory there has been recent debates on the cell > media preparation. The point of contention is the presence of serum in > media. > When a media mentions supplemented with 10% serum, which of the > following is correct for a 500ml media bottle. > > 1. Add 50ml of serum to the media bottle (total volume is 550ml) > > 2. Remove 50ml of media and then add 50ml of serum (total volume is > > Any information on the right usage will be great. > > Thanks, > > - Bala |
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B. Prabhakar Pandian |
Re: Cell Media Preparation Debate
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In reply to this post by Mike Tighe
Hello Mike,
I know that from the mathematical stand point what you are saying is correct, but the contention is more from what 10% actually means. Is it to add 10% of the original volume of serum (option # 1) to the media or to make sure that the final concentration is 10% (option # 2). Thanks, Mike Tighe wrote: > If total volume is 550mls (500ml media and 50mls serum you will have 9.1% serum. Not 10% but probably enough to keep most cells happy > > Mike > > >>>> "B. Prabhakar Pandian" <[hidden email]> 10/22/09 3:10 PM >>> >>>> > Hello, > In our laboratory there has been recent debates on the cell > media preparation. The point of contention is the presence of serum in > media. > When a media mentions supplemented with 10% serum, which of the > following is correct for a 500ml media bottle. > > 1. Add 50ml of serum to the media bottle (total volume is 550ml) > > 2. Remove 50ml of media and then add 50ml of serum (total volume is 500ml). > > Any information on the right usage will be great. > > Thanks, > > - Bala > > > -- |
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I think that option #2 is the intent.
>>> "B. Prabhakar Pandian" <[hidden email]> 10/22/09 4:54 PM >>> Hello Mike, I know that from the mathematical stand point what you are saying is correct, but the contention is more from what 10% actually means. Is it to add 10% of the original volume of serum (option # 1) to the media or to make sure that the final concentration is 10% (option # 2). Thanks, Mike Tighe wrote: > If total volume is 550mls (500ml media and 50mls serum you will have 9.1% serum. Not 10% but probably enough to keep most cells happy > > Mike > > >>>> "B. Prabhakar Pandian" <[hidden email]> 10/22/09 3:10 PM >>> >>>> > Hello, > In our laboratory there has been recent debates on the cell > media preparation. The point of contention is the presence of serum in > media. > When a media mentions supplemented with 10% serum, which of the > following is correct for a 500ml media bottle. > > 1. Add 50ml of serum to the media bottle (total volume is 550ml) > > 2. Remove 50ml of media and then add 50ml of serum (total volume is 500ml). > > Any information on the right usage will be great. > > Thanks, > > - Bala > > > -- |
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Phillips, Thomas E. |
Re: Cell Media Preparation Debate
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The point is moot. 10% anything in a scientific formulation generally implies it was a somewhat arbitrarily chosen concentration that works well for many things. If you really want to know the correct concentration, grow your cells in multiple concentrations and see which is best. It is foolish to agonize over an arbitrary value. If it isn't worth optimizing, it isn't worth worrying about whether you are using 9.1% or 10%. You can publish the precise value (e.g., 50 mls of FBS in 500 mls DME) so that your work can be replicated if you want to stop the ambiguity of this type of concentration. My experience is that many cells grow as well in 5% FBS as in 10%.
Thomas E. Phillips, Ph.D Professor of Biological Sciences Director, Molecular Cytology Core 2 Tucker Hall University of Missouri Columbia, MO 65211-7400 573-882-4712 (office) 573-882-0123 (fax) [hidden email] http://www.biology.missouri.edu/faculty/phillips.html http://www.biotech.missouri.edu/mcc/ -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mike Tighe Sent: Thursday, October 22, 2009 4:17 PM To: [hidden email] Subject: Re: Cell Media Preparation Debate I think that option #2 is the intent. >>> "B. Prabhakar Pandian" <[hidden email]> 10/22/09 4:54 PM >>> Hello Mike, I know that from the mathematical stand point what you are saying is correct, but the contention is more from what 10% actually means. Is it to add 10% of the original volume of serum (option # 1) to the media or to make sure that the final concentration is 10% (option # 2). Thanks, Mike Tighe wrote: > If total volume is 550mls (500ml media and 50mls serum you will have 9.1% serum. Not 10% but probably enough to keep most cells happy > > Mike > > >>>> "B. Prabhakar Pandian" <[hidden email]> 10/22/09 3:10 PM >>> >>>> > Hello, > In our laboratory there has been recent debates on the cell > media preparation. The point of contention is the presence of serum in > media. > When a media mentions supplemented with 10% serum, which of the > following is correct for a 500ml media bottle. > > 1. Add 50ml of serum to the media bottle (total volume is 550ml) > > 2. Remove 50ml of media and then add 50ml of serum (total volume is 500ml). > > Any information on the right usage will be great. > > Thanks, > > - Bala > > > -- |
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Boswell, Carl A - (cboswell) |
Re: Cell Media Preparation Debate
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In reply to this post by B. Prabhakar Pandian
Option #2 is always the intent in this context. The goal is the FINAL
concentration of whatever % you want. C Carl A. Boswell, Ph.D. Molecular and Cellular Biology University of Arizona 520-954-7053 FAX 520-621-3709 ----- Original Message ----- From: "B. Prabhakar Pandian" <[hidden email]> To: <[hidden email]> Sent: Thursday, October 22, 2009 1:54 PM Subject: Re: Cell Media Preparation Debate > Hello Mike, > I know that from the mathematical stand point what > you are saying is correct, but the contention is more from what 10% > actually means. Is it to add 10% of the original volume of > serum (option # 1) to the media or to make sure that the final > concentration is 10% (option # 2). > > Thanks, > > Mike Tighe wrote: >> If total volume is 550mls (500ml media and 50mls serum you will have >> 9.1% serum. Not 10% but probably enough to keep most cells happy >> >> Mike >> >>>>> "B. Prabhakar Pandian" <[hidden email]> 10/22/09 3:10 PM >>> >> Hello, >> In our laboratory there has been recent debates on the cell >> media preparation. The point of contention is the presence of serum in >> media. >> When a media mentions supplemented with 10% serum, which of the following >> is correct for a 500ml media bottle. >> >> 1. Add 50ml of serum to the media bottle (total volume is 550ml) >> >> 2. Remove 50ml of media and then add 50ml of serum (total volume is >> 500ml). >> >> Any information on the right usage will be great. >> >> Thanks, >> >> - Bala >> >> >> > > > -- > |
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Martin Wessendorf-2 |
Re: Cell Media Preparation Debate
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I wonder to what extent the present confusion is due to Kodak. When
making developer, fixer, etc., a "1:4" dilution always meant one part ADDED TO four parts. Martin Carl Boswell wrote: > Option #2 is always the intent in this context. The goal is the FINAL > concentration of whatever % you want. > > C > > Carl A. Boswell, Ph.D. > Molecular and Cellular Biology > University of Arizona > 520-954-7053 > FAX 520-621-3709 > ----- Original Message ----- From: "B. Prabhakar Pandian" <[hidden email]> > To: <[hidden email]> > Sent: Thursday, October 22, 2009 1:54 PM > Subject: Re: Cell Media Preparation Debate > > >> Hello Mike, >> I know that from the mathematical stand point >> what you are saying is correct, but the contention is more from what >> 10% actually means. Is it to add 10% of the original volume of >> serum (option # 1) to the media or to make sure that the final >> concentration is 10% (option # 2). >> >> Thanks, >> >> Mike Tighe wrote: >>> If total volume is 550mls (500ml media and 50mls serum you will have >>> 9.1% serum. Not 10% but probably enough to keep most cells happy >>> >>> Mike >>> >>>>>> "B. Prabhakar Pandian" <[hidden email]> 10/22/09 3:10 PM >>> >>> Hello, >>> In our laboratory there has been recent debates on the >>> cell media preparation. The point of contention is the presence of >>> serum in media. >>> When a media mentions supplemented with 10% serum, which of the >>> following is correct for a 500ml media bottle. >>> >>> 1. Add 50ml of serum to the media bottle (total volume is 550ml) >>> >>> 2. Remove 50ml of media and then add 50ml of serum (total volume is >>> 500ml). >>> >>> Any information on the right usage will be great. >>> >>> Thanks, >>> >>> - Bala >>> >>> >>> >> >> >> -- >> -- Martin Wessendorf, Ph.D. office: (612) 626-0145 Assoc Prof, Dept Neuroscience lab: (612) 624-2991 University of Minnesota Preferred FAX: (612) 624-8118 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 Minneapolis, MN 55455 e-mail: [hidden email] |
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In reply to this post by Withers, Helen
To get an humidified environment just use a glass bottom mattek dish with some water droplets on the outer edges. Cells can grow on an agarose pad for days.
b
On Thu, Oct 22, 2009 at 16:11, Withers, Helen <[hidden email]> wrote: In the past we have used thin layers of agar or agarose on the surface of the slide to immobilise either pre-stain bacteria or to actually stain bacteria by incorporating the stain into the agar. This effectively prevents floating if you lightly dry the surface of the agar once the sample has been added prior to putting on a coverslip. This works very well for both live/dead staining and for time-lapse growth studies using DAPI or membrane stained bacteria so they remain viable for long periods (assuming that they are in a humidified container!). |
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In reply to this post by Heather A Owen
Hi Mike
I can vouch for the agarose method and have taken good confocal images this way of live immobilised bacterial cells. Ensure you prepare the agarose slides on a level bench just before use as specified by Heather below Kind regards Jen Quoting Heather A Owen <[hidden email]>: > Hi Mike, > > A trick taught to me my a microbiologist (Mark McBride) is to make a 0.7% > agarose solution in water (microwave to dissolve), and while it is still hot > use a Pasteur pipet to flow a layer onto a microscope slide to cover it. I > usully suspend the slides on wooden applicator sticks in case the agarose > runs off, otherwise the solution wicks under the slide. Once the agarose > gels, apply a drop of culture on top, then coverslip it. The pad of agarose > needs to be larger than the coverslip you want to use. The cells stay > immobile, and actually stay alive for a long time. > > Sincerely yours, > Heather Owen > > > Dr. Heather A. Owen > Director, Electron Microscope Laboratory > Department of Biological Sciences > University of Wisconsin - Milwaukee > Lapham Hall > 3209 N. Maryland Avenue > Milwaukee, WI 53211 > (414)229-6816 > > ----- Original Message ----- > From: "Mike Tighe" <[hidden email]> > To: [hidden email] > Sent: Thursday, October 22, 2009 1:11:00 PM GMT -06:00 US/Canada Central > Subject: Immobilize bacteria > > Does anybody have a good way to Immobilize bacteria without killing. We would > like to do a Live/dead stain and our bugs like to float away! > > Thanks!! > Mike > -- Jennifer Clarke BSc (Hons) PhD Research Associate, Anatomy and Histology Centre for Neuroscience, School of Medicine & Facility Manager, Optical Microscopy Suite, Flinders Microscopy (available for training and assistance on Mondays only) Flinders University GPO Box 2100, Adelaide 5001 Phone: 61 8 8204 6454 / 61 8 8204 4205 Email: [hidden email] |
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In reply to this post by Mike Tighe
Dear Michael
You have two options for ordering CyGEL (Product code: CY10500) (& DRAQ5, etc): 1) our website www.biostatus.com: go to our "purchase" page, register and then if USA is used the prices come up in USD. You can order by credit card or conventional purchase order system using the guidance notes. We deliver anywhere worldwide within 48 hours. 2) Axxora.com: CyGEL is distributed by Axxora. Check the price before ordering cf. our published web prices. You may be interested to know that CyGEL was cited in a new methods paper published online last week for live parasite imaging from the laboratory of Professor Deborah Smith, University of York, UK: Validation of a New Method for Immobilising Kinetoplastid Parasites for Live Cell Imaging. Helen P. Price, et al. Molecular and Biological Parasitology http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T29-4XCYJ8M-1&_user=4306206&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000010338&_version=1&_urlVersion=0&_userid=4306206&md5=c8b8f4f1cf55421b0f7de62d46fd3af6 Don't hesitate to contact me if you have any questions regarding CyGEL or CyGEL Sustain, particularly regarding the creation of wells on slides, or imaging of cells, C elegans, Danio, etc. Best regards Roy Edward Biostatus Limited Tel: +44 (0)1509 558 163 Fax: +44 (0)1509 651 061 www.biostatus.com Company No: 3079239. Registered in England and Wales. Registered office: Biostatus Ltd, 56 Charnwood Road, Shepshed, Leicestershire, LE12 9NP, UK. -----Original Message----- From: Michael Herron [mailto:[hidden email]] Sent: 22 October 2009 20:54 Subject: Re: Immobilize bacteria Is there a U.S, vendor for CyGel? On Oct 22, 2009, at 1:31 PM, Peter O'Toole wrote: > Likewise, we have used CyGel. You may need try various strengths to > get the best lifetime out of the bacteria, especially if they are > motile. You will also need to seal the edges with vacuum grease to > stop it drying out, but once in place. > > Best > > Peter > > ----- Original Message ----- From: "G. Esteban Fernandez" > <[hidden email] > > > To: <[hidden email]> > Sent: Thursday, October 22, 2009 7:17 PM > Subject: Re: Immobilize bacteria > > >> I've used CyGel for that purpose before (no commercial interest). >> http://www.biostatus.com/product/cygel/ >> >> -Esteban >> >> >> On Thu, Oct 22, 2009 at 1:11 PM, Mike Tighe >> <[hidden email] >> > wrote: >>> Does anybody have a good way to Immobilize bacteria without killing. >>> We would like to do a Live/dead stain and our bugs like to float >>> away! >>> >>> Thanks!! >>> Mike >>> >> >> -- >> G. Esteban Fernandez, Ph.D. >> >> Associate Director >> Molecular Cytology Core Facility >> University of Missouri >> 120 Bond Life Sciences Center >> Columbia, MO 65211 >> >> http://www.biotech.missouri.edu/mcc >> >> (573)882-4895 >> (573)884-9676 fax Michael J. Herron, U of MN, Dept. of Entomology [hidden email] 612-624-3688 (office) 612-625-5299 (FAX) |
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