lacZ detection

/G=Gerhard/S=Nebe-von-Caron/OU=1890CHPE/O=TMGB.URC/@LANGATE.gb.sprint.com /G=Gerhard/S=Nebe-von-Caron/OU=1890CHPE/O=TMGB.URC/ at LANGATE.gb.sprint.com
Mon Mar 4 08:33:00 EST 1996


          I don't know about the lacz substrate, but a number of 
          microorganisms can pump out dyes like rhodamine or the 
          esterase substrates much faster than they could leak. E.coli 
          is an excellent example for that. When staining 
          Lactobacillus plantarum with carboxyfluorescein-diacetate we 
          found that dye retention improves dramatically when the 
          cells are kept on ice. See also Breeuwer et al 
          Appl-Environ-microbiol May 1994, Energy -dependent, 
          carrier-mediated extrusion of carboxyfluorescein...
          
          Gerhard.nebe-von-caron at urcgb.sprint.com


______________________________ Reply Separator _________________________________
Subject: Re: lacZ detection
Author:  dbk at aber.ac.uk at INTERNET
Date:    04/03/96 06:20


> From:          DR M AL-RUBEAI <ALRUBEAM at novell1.academic-computing-service.bi 
rmingham.ac.uk>
> Organization:  The University of Birmingham 
> To:            Cytometry Mailing List <[7f]> 
> Date:          Thu, 29 Feb 1996 09:03:58 BST 
> Subject:       Re: lacZ detection
> Reply-to:      M.Al-Rubeai at birmingham.ac.uk 
> Priority:      normal
          
> John
>
> The best flow cytometry substrate for the detection of lacZ activity 
> is a lipophillic FDG derivatves with 2, 4, 8 and 16 carbon acyls
> known as ImaGene (Molecular Probes). However, this substrate has a 
> problem for the quantification of lacZ activity. We have used it to
> assay b-gal activity in insect cells infected with baculovirus. A concentrati 
on
> equilibrium between cell and supernatant of the hydrolysed substrate, 
> irrespective of enzyme activity, meant that lacZ+/lacZ- resolution
> was impossible. Quantitative measurements proved problematic because the 
> enzyme activity showed no sign of saturation by the substrate.
> Good luck
> Mohamed Al-Rubeai, Ph.D.
> School of Chemical Engineering
> University of Birmingham
>
> es, let me agree. These probes are just not what they are cracked 
> up to be. We wasted some important time and scarce money on this 
> type of stuff in bacteria - only bugs that are in absolutely
> tip-top condition keep the product in. Most bugs are more than
> slightly leaky (and actually dormant ones of a species which is an 
> obligate respirer can be completely leaky, and can reseal as part 
> of resuscitation, which if you ever believed in the chemiosmotic
> coupling hypothesis might cause you to cease so doing,but that is 
> another story (Votyakova, T. V., Kaprelyants, A. S. & Kell, D. B. 
> (1994). Influence of viable cells on the resuscitation of dormant 
> cells in Micrococcus luteus cultures held in extended stationary 
> phase. The population effect. Appl. Env. Microbiol. 60, 
3284-3291.))
          
Bottom line: most of these things don't work if you want to be 
quantitative.
          
Even more bottom line to manufacturers: but we'd like them to.
          
          
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