Electrical detection of germination of viable model Bacillus anthracis spores in microfluidic biochips.

In this paper, we present a new impedance-based method to detect viable spores by electrically detecting their germination in real time within microfluidic biochips. We used Bacillus anthracis Sterne spores as the model organism. During germination, the spores release polar and ionic chemicals, such as dipicolinic acid (DPA), calcium ions, phosphate ions, and amino acids, which correspondingly increase the electrical conductivity of the medium in which the spores are suspended. We first present macro-scale measurements demonstrating that the germination of spores can be electrically detected at a concentration of 10(9) spores ml(-1) in sample volumes of 5 ml, by monitoring changes in the solution conductivity. Germination was induced by introducing an optimized germinant solution consisting of 10 mM L-alanine and 2 mM inosine. We then translated these results to a micro-fluidic biochip, which was a three-layer device: one layer of polydimethylsiloxane (PDMS) with valves, a second layer of PDMS with micro-fluidic channels and chambers, and the third layer with metal electrodes deposited on a pyrex substrate. Dielectrophoresis (DEP) was used to trap and concentrate the spores at the electrodes with greater than 90% efficiency, at a solution flow rate of 0.2 microl min(-1) with concentration factors between 107-109 spores ml(-1), from sample volumes of 1-5 microl. The spores were captured by DEP in deionized water within 1 min (total volume used ranged from 0.02 microl to 0.2 microl), and then germinant solution was introduced to the flow stream. The detection sensitivity was demonstrated to be as low as about a hundred spores in 0.1 nl, which is equivalent to a macroscale detection limit of approximately 10(9) spores ml(-1). We believe that this is the first demonstration of this application in microfluidic and BioMEMS devices.

Trapping and viability of swimming bacteria in an optoelectric trap.

Non-contact manipulation methods capable of trapping and transporting swimming bacteria can significantly aid in chemotaxis studies. However, high swimming speed makes the trapping of these organisms an inherently challenging task. We demonstrate that an optoelectric technique, rapid electrokinetic patterning (REP), can effectively trap and manipulate Enterobacter aerogenes bacteria swimming at velocities greater than 20 µm s(-1). REP uses electro-orientation, laser-induced AC electrothermal flow, and particle-electrode interactions for capturing these cells. In contrast to trapping non-swimming bacteria and inert microspheres, we observe that electro-orientation is critical to the trapping of the swimming cells, since unaligned bacteria can swim faster than the radially inward electrothermal flow and escape the trap. By assessing the cell membrane integrity, we study the effect of REP trapping conditions, including optical radiation, laser-induced heating, and the electric field on cell viability. When applied individually, the optical radiation and laser-induced heating have negligible effect on cells. At the standard REP trapping conditions fewer than 2% of cells have a compromised membrane after four minutes. To our knowledge this is the first study detailing the effect of REP trapping on cell viability. The presented results provide a clear guideline on selecting suitable REP parameters for trapping living bacteria.

Growth response of cottonwood roots to varied NH4:NO3 ratios in enriched patches.

Maximization of short-rotation forest plantation yield requires frequent applications of nutrients, especially nitrogen (N). Whole-plant growth is known to be sensitive to the proportion of ammonium to nitrate (NH4:NO3). However, the extent to which N form affects root growth, branching and morphology is poorly understood, and these variables may have substantial impacts on plant nutrient and water acquisition. We used rooted cuttings of cottonwood (Populus deltoides Bartr. ex Marsh.) to investigate the effect of various NH4:NO3 ratios on root growth in N-enriched patches. A sand culture study with split-root systems was carried out in which 1-3% of the total root system of each cutting was supplied with 2 mM N at NH4:NO3 ratios of 0:100, 20:80, 40:60, 60:40, 80:20 and 100:0 (molar basis), with the rest of the plant supplied with 0 mM N, resulting in the whole plant becoming N deficient. During the experiment, whole-plant growth was unaffected by the treatments. Of the NH4:NO3 ratios tested, greatest total root length, specific root length, and root N concentration of roots in enriched patches occurred in the 20:80 NH4:NO3 treatment. The largest response of roots in enriched patches was third- and fourth-order root production. We conclude that N form has a profound effect on root development and morphology in enriched patches.

Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage.

A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus.