bacterial/footrot tissue
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Calvo-Bado, Leonides |
bacterial/footrot tissue
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Dear all,
I am postdoc at Warwick University working on project on the microbial ecology of the footrot in sheep. Two major things that I want to do is:
1.- Apply FISH to detect bacteria population colonizers in the skin tissue of the foot. To do this, I need to develop a protocol for sectioning skin tissue for FISH (microtome sectioning, laser microdisection etc..). I have some ideas but it would be great if any of you have some experience on this.
2.- I want to do a metagenomic libraries of the colonizers of the footrot tissue. To do this, I need to separate bacterial cells from within the skin tissue and then use nycodenz to fractionate the cells for DNA extraction follow by the cloning into fosmid-metagenome. I was thinking in using collagenase treatment to degrade skin tissue then nycodenz but I would like to ask you guys for any suggestion.
Any help, suggestion or comments I would really appreciate!
Regards
Leo
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Boswell, Carl A - (cboswell) |
Re: bacterial/footrot tissue
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Hi Leo,
The commercial collagenase preps I'm familiar with
were actually mixes of enzymes extracted from bacterial cultures.
Commonly they included enzymes that work on specific types of collagen, i.e.
collagenase Type IV, collagenase Type I, plus elastases and proteolytic and
glycolytic enzymes. The blend/concentration of these enzymes were what
give a particular batch or lot of "collagenase" it's potentency and
specificity for particular tissues. When we needed new enzyme, we tested
several lots, then bought large amounts so we had some hope of
consitency. A few years ago, I believe it was Boehringer-Mannheim
had a method of making pure collagenases. The problems were two fold, most
likely because it was so pure. It was difficult to get a concentration
that worked dependably, and if it was too high, everything disappeared in a
couple of minutes. It was very hot stuff. My interpretation of all
this was that it probably requires a proper blend of enzymes to open up
connective tissue enough to release cells, or in my case, intact
microvessels. Because you are interested in bacteria, rather than tissue
cells, it may be easier to loosen up the tissue and get some bacteria out
without digesting the bacterial also.
I haven't worked with it in several years, so can't
speak to what is currently availble. However since you didn't seem to be
getting much feedback from your question, I thought I'd add my two cents
worth.
Good luck,
Carl
Carl A. Boswell, Ph.D.
Molecular and Cellular Biology University of Arizona 520-954-7053 FAX 520-621-3709
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Calvo-Bado, Leonides |
Re: bacterial/footrot tissue
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear Carl,
Many thanks for your comments. Yes, loosen up the tissue and release the bacterial cells followed by nycodenz separation of the cells from the cell tissue will be better. I am waiting for some free samples from a company that produce collagenase so I will see what happens!
Many thanks
leo From: Confocal Microscopy List on behalf of Carl Boswell Sent: Wed 09/01/2008 01:01 To: [hidden email] Subject: Re: bacterial/footrot tissue Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Leo,
The commercial collagenase preps I'm familiar with were actually mixes of enzymes extracted from bacterial cultures. Commonly they included enzymes that work on specific types of collagen, i.e. collagenase Type IV, collagenase Type I, plus elastases and proteolytic and glycolytic enzymes. The blend/concentration of these enzymes were what give a particular batch or lot of "collagenase" it's potentency and specificity for particular tissues. When we needed new enzyme, we tested several lots, then bought large amounts so we had some hope of consitency. A few years ago, I believe it was Boehringer-Mannheim had a method of making pure collagenases. The problems were two fold, most likely because it was so pure. It was difficult to get a concentration that worked dependably, and if it was too high, everything disappeared in a couple of minutes. It was very hot stuff. My interpretation of all this was that it probably requires a proper blend of enzymes to open up connective tissue enough to release cells, or in my case, intact microvessels. Because you are interested in bacteria, rather than tissue cells, it may be easier to loosen up the tissue and get some bacteria out without digesting the bacterial also.
I haven't worked with it in several years, so can't speak to what is currently availble. However since you didn't seem to be getting much feedback from your question, I thought I'd add my two cents worth.
Good luck,
Carl
Carl A. Boswell, Ph.D.
Molecular and Cellular Biology University of Arizona 520-954-7053 FAX 520-621-3709
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In reply to this post by Calvo-Bado, Leonides
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Regarding #1
See Moter A et
al. (1988) Fluorescence in situ hybridization shows spatial
distribution of as yet uncultured treponemes in biopsies from digital
dermatitis lesions. Microbiology 144:2459
Regarding #2 - I'd recommend contacting Dr. Moter. She has
papers on which she is the corresponding author where you can find her
e-mail. She may not be able to answer your question from her
experience, but she can point you in the right direction.
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, I am postdoc at Warwick University working on project on the microbial ecology of the footrot in sheep. Two major things that I want to do is: 1.- Apply FISH to detect bacteria population colonizers in the skin tissue of the foot. To do this, I need to develop a protocol for sectioning skin tissue for FISH (microtome sectioning, laser microdisection etc..). I have some ideas but it would be great if any of you have some experience on this. 2.- I want to do a metagenomic libraries of the colonizers of the footrot tissue. To do this, I need to separate bacterial cells from within the skin tissue and then use nycodenz to fractionate the cells for DNA extraction follow by the cloning into fosmid-metagenome. I was thinking in using collagenase treatment to degrade skin tissue then nycodenz but I would like to ask you guys for any suggestion. Any help, suggestion or comments I would really appreciate! Regards Leo -- 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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Calvo-Bado, Leonides |
Re: bacterial/footrot tissue
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear Robert,
Many thanks for your help!
Regards
Leo From: Confocal Microscopy List on behalf of Robert J. Palmer Jr. Sent: Wed 09/01/2008 13:34 To: [hidden email] Subject: Re: bacterial/footrot tissue Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Regarding #1
See Moter A et al. (1988) Fluorescence in situ hybridization shows spatial distribution of as yet uncultured treponemes in biopsies from digital dermatitis lesions. Microbiology 144:2459
Regarding #2 - I'd recommend contacting Dr. Moter. She has papers on which she is the corresponding author where you can find her e-mail. She may not be able to answer your question from her experience, but she can point you in the right direction.
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, I am postdoc at Warwick University working on project on the microbial ecology of the footrot in sheep. Two major things that I want to do is: 1.- Apply FISH to detect bacteria population colonizers in the skin tissue of the foot. To do this, I need to develop a protocol for sectioning skin tissue for FISH (microtome sectioning, laser microdisection etc..). I have some ideas but it would be great if any of you have some experience on this. 2.- I want to do a metagenomic libraries of the colonizers of the footrot tissue. To do this, I need to separate bacterial cells from within the skin tissue and then use nycodenz to fractionate the cells for DNA extraction follow by the cloning into fosmid-metagenome. I was thinking in using collagenase treatment to degrade skin tissue then nycodenz but I would like to ask you guys for any suggestion. Any help, suggestion or comments I would really appreciate! Regards Leo -- 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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