Enumeration of soil bacteria with the green fluorescent nucleic acid dye Sytox green in the presence of soil particles

Journal of Microbiological Methods
Peter KlauthJoost Groeneweg

Abstract

Total counts in soils are usually determined using fluorescent dyes, such as DAPI or Sybr green, due to fluorescence enhancement if they are bound to nucleic acids. Unfortunately, these commonly used dyes stain soil particles as well. Therefore, besides fluorescence enhancement, sufficient spectral differentiation is also required. We present a new procedure that overcomes the problems of visualising bacteria on surfaces in soil and avoids the separation of soil particles to a large extent. Spectral differentiation between bacteria and soil matrix is achieved by using Sytox green and a suboptimal excitation wavelength. Bacteria exhibit a bright green fluorescence, while soil particles fluoresce blue or red. Slight homogenisation and sedimentation of the sand and coarse silt that were too big for microscopic investigations were the only separation steps required. We compared the proposed Sytox green staining with Sybr green staining. The recovery of Sybr green-stained cells amounted to 38%, whereas in samples stained by Sytox green 81% of the spiked cells were counted. Sytox green can also be combined with fluorescence in situ hybridisation (FISH) using deep red dyes such as Cy5.

References

Feb 2, 2002·Applied and Environmental Microbiology·Thomas R NeuJohn R Lawrence
Apr 12, 2002·Journal of Microbiological Methods·Lothar PoschenRalf Wilhelm
Oct 29, 2002·Journal of Microbiological Methods·Heidi L Gough, David A Stahl
Jan 25, 2003·Antimicrobial Agents and Chemotherapy·T TheisU Stahl

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Citations

Sep 16, 2006·Applied Microbiology and Biotechnology·Peter KlauthSiegfried M Schoberth
Aug 30, 2008·Astrobiology·J L NadeauR Leon
Sep 16, 2009·Journal of Microbiological Methods·Stefano Amalfitano, Stefano Fazi Alberto Puddu
Sep 9, 2006·Environmental Microbiology·Debarati PaulRakesh K Jain
Sep 29, 2012·Environmental Microbiology·T D L Irvine-FynnA J Hodson
Dec 6, 2014·Frontiers in Microbiology·Alicia M Purcell The Wissard Science Team
Jan 26, 2005·FEMS Microbiology Letters·Srinivas Reddy PallerlaSiegfried M Schoberth
Jul 7, 2016·Journal of Microbiological Methods·Gerrit G TammingaGert-Jan W Euverink
Aug 7, 2007·Nature Reviews. Microbiology·Anthony G O'DonnellJohn W Crawford
Oct 11, 2019·MSphere·Banafshe KhaliliJennifer B H Martiny

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