Akt. Kirschner et B. Velimirov, Modification of the H-3-leucine centrifugation method for determining bacterial protein synthesis in freshwater samples, AQUAT MIC E, 17(2), 1999, pp. 201-206
The validity of the H-3-leucine centrifugation method for determining bacte
rial secondary production in oligotrophic and eutrophic fresh- and seawater
samples was examined. For freshwater samples, we found that the establishe
d protocol developed by Smith & Azam (1992) led to significantly lower valu
es (up to 57%) than a novel protocol presented here, where bacterial protei
ns are precipitated under acidic conditions (trichloroacetic acid) at 4 deg
rees C with a humic extract and solubilizing DNA and RNA at 100 degrees C f
or 30 min. For seawater samples, no difference was found when an ethanolic
washing step was included in the novel protocol. We also used different sal
t solutions instead of humic extract; these both act as co-precipitants for
the precipitation of the proteins. An unbuffered 3.5 % (final cone.) NaCl
solution was found to be highly effective and gave consistent results and l
ower blank values. Incorporation rates obtained with our protocol showed go
od agreement with the commonly used filtration method. Therefore, we argue
that for freshwater samples an NaCl or humic extract addition is necessary
for an efficient precipitation of the proteins when the centrifugation meth
od for determining bacterial secondary production via H-3-leucine incorpora
tion is applied.