The effect of FISH and CARD-FISH on the isotopic composition of (13)C- and (15)N-labeled Pseudomonas putida cells measured by nanoSIMS.

The use of nanoSIMS for the exploration of microbial activities in natural habitats often implies that stable isotope tracer experiments are combined with in situ hybridization techniques (i.e. fluorescence in situ hybridization (FISH) or catalyzed reporter deposition (CARD)-FISH). In this study, Pseudomonas putida grown on (13)C- and (15)N-labeled carbon and nitrogen, collected in exponential growth and stationary phases, was hybridized and analyzed by nanoSIMS. It was shown that (13)C and (15)N fractions decreased after FISH and CARD-FISH in comparison to chemically untreated cells. However, the fractions were influenced differently by various treatments. After paraformaldehyde fixation of exponentially growing cells, a reduction of the (13)C and (15)N fractions was measured from 94±1.2% and 89.5±3.8% to 90.2±0.8% and 64±4.6%, respectively, indicating that nitrogen isotopic composition was most influenced. A further decrease of the (13)C and (15)N fractions to 80.7±6.5 and 59.5±4.1%, respectively, was measured after FISH, while CARD-FISH decreased the fractions to 57.4±3.0% and 47.1±4.1%, respectively. The analysis of cells collected in different growth phases revealed that the effect of various treatments seemed to be dependent on the cell's physiological state. In addition, a mathematical model that can be used in further studies was developed in order to calculate the amount of carbon introduced into the cells by chemical treatments. These results can be valuable for environmental FISH-nanoSIMS studies where the isotopic composition of single cells will be used to quantitatively assess the importance of specific populations to certain biochemical processes and determine budget estimations.