Assessment of pathogenic bacteria using periodic actuation

Sorin David, Cristina Polonschii, Mihaela Gheorghiu, Dumitru Bratu, Alin Dobre, Eugen Gheorghiu
2013 Lab on a Chip  
Assessment of cell concentration and capture using fluorescence assays Material and Methods To assess the concentration of cells in suspension, cells were stained with the Acridine Orange fluorophore (AO) and measured with the fluorescence module of the Multimodal GloMax Reader (Promega). The fluorescence module has the excitation range: 465-485 nm and emission detector spanning: 515-575 nm. AO is a fluorescent dye that can penetrate the cytoplasm and interact with nucleic acids (for DNA the
more » ... itation is at 502 nm and the emission at 525 nm, for RNA the excitation is at 460 nm and the emission at 650 nm) 1 . Cell labelling was performed by adding 1 µl of AO to a 50 µl volume of stock suspension and vortexing. The suspension was centrifuged at 6000 rpm for 1h and the AO containing supernatant was removed and replaced with PBS. From a stock suspension of labelled cells (7.9x10 9 cells/ml), successive dilutions were made to obtain concentrations ranging from 10 2 to 10 7 cells/ml. Each 10 fold dilution was made by adding 50 µl of concentrated cell suspension to a volume of 450 µl of buffer solution and analysed in the multimode reader. The LOD for this method is evaluated at 10 4 cells/ml (see Table 1 ). For microscopy experiments the cells and MB-clusters containing cells were observed using a Zeiss Axio Observer Z1 inverted microscope in transmission and epifluorescence mode. The fluorescence of the AO stained cells was observed using the halogen epifuorescence lamp and the 09 filter set with excitation at 450-490 nm and long pass emission filter above 515 nm. Pictures were taken using the 40x objective and an Andor EMCCD DU-885K camera. The formation of the clusters was validated using fluorescence microscopy ( figure S1 , B, C). Using AO stained cells we highlight the presence of the cells in specific MB-clusters (figure S1B inset) -cells are visible within the MB clusters. It is been shown that in the absence of target cells (figure S1A) and in the presence of nonspecific cells (figure S1C) the formation of MB-clusters is not induced.
doi:10.1039/c3lc50411e pmid:23807196 fatcat:7gbp77f5vfbc3mijnhwgeyqqz4