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Enumeration of soil bacteria with the green fluorescent nucleic acid dye Sytox green in the presence of soil particles
- Source :
- Journal of Microbiological Methods. 59:189-198
- Publication Year :
- 2004
- Publisher :
- Elsevier BV, 2004.
-
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.
- Subjects :
- Microbiology (medical)
Analytical chemistry
Ralstonia
Biology
Microbiology
chemistry.chemical_compound
Pseudomonas
Spectrophotometry
medicine
DAPI
Organic Chemicals
Molecular Biology
Soil Microbiology
Fluorescent Dyes
Chromatography
medicine.diagnostic_test
Cell Membrane
biology.organism_classification
Fluorescence
Staining
Microscopy, Fluorescence
chemistry
Soil water
Nucleic acid
Clay
Aluminum Silicates
Spectrophotometry, Ultraviolet
Soil microbiology
Bacteria
Bacillus subtilis
Subjects
Details
- ISSN :
- 01677012
- Volume :
- 59
- Database :
- OpenAIRE
- Journal :
- Journal of Microbiological Methods
- Accession number :
- edsair.doi.dedup.....86b4d68b46ec686aa9e20685790fa3ef