Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids

The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47949/1/10295_2005_Article_238.pd

Rearrangement of bacterial community structure during peat diagenesis

The relationship between microbial diagenesis of Sphagnum peat (SP) and reed-sedge peat (RSP) and the spatial organization of peat bacterial communities was studied. Peats were aerobically incubated at 18-22 °C for 4 months. Changes in molecular composition of peat organic matter were monitored with solid-state 13C NMR, and the respective amount of functional groups was determined by integration of corresponding peaks. No abiotic peat transformation was detected. SP diagenesis caused about a 4% loss of parent materials with a similar yield of ketones, phenols, aromatic, and carbonyl compounds; whereas about 20% of RSP carbohydrates, along with ketones and methoxyl compounds were gradually transformed into carbonyl and aliphatic compounds. SP and RSP substantially varied in bacterial composition. To address spatial community structure, bacterial populations were dissected by a differential elution technique into three fractions based on the degree of their attachment to peat. Community composition was surveyed with T-RFLP (HhaI, MspI, and RsaI). The fragments were further attributed to freely-dispersed (FD), particle-associated (PA), or omnipresent (OMN) bacterial fractions. In both peats, bacterial communities have gradually shifted with the progress of diagenesis. In SP, numbers of exclusively FD or PA bacteria slightly decreased while in RSP their numbers more than doubled after 4-month incubation, and the number of OMN bacteria respectively decreased. The substantially greater changes in the spatial structure of RSP bacterial community compared to SP were consistent with the chemical transformations detected in these peats suggesting the diagenesis-driven divergence of RSP bacterial community into FD and PA sub-communities