Enumeration of soil bacteria with the green fluorescent nucleic acid dye Sytox green in the presence of soil particles.
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.
Klauth
Peter
P
Agrosphere Institute (ICG-IV), Research Centre Juelich, Leo-Brandt Str., Juelich 52425, Germany. p.klauth@fz-juelich.de
Wilhelm
Ralf
R
Klumpp
Erwin
E
Poschen
Lothar
L
Groeneweg
Joost
J
D000538:Aluminum Silicates; D001412:Bacillus subtilis / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D000077215:Clay; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D009930:Organic Chemicals; D011549:Pseudomonas / Q000302:isolation & purification* / Q000378:metabolism; D043367:Ralstonia / Q000302:isolation & purification* / Q000378:metabolism; D012988:Soil Microbiology; D013056:Spectrophotometry, Ultraviolet
D000538:Aluminum Silicates; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C402795:SYTOX Green; D000077215:Clay