Does the physical disector method provide an accurate estimation of sensory neuron number in rat dorsal root ganglia?

The physical disector is a method of choice for estimating unbiased neuron numbers; nevertheless, calibration is needed to evaluate each counting method. The validity of this method can be assessed by comparing the estimated cell number with the true number determined by a direct counting method in serial sections. We reconstructed a 1/5 of rat lumbar dorsal root ganglia taken from two experimental conditions. From each ganglion, images of 200 adjacent semi-thin sections were used to reconstruct a volumetric dataset (stack of voxels). On these stacks the number of sensory neurons was estimated and counted respectively by physical disector and direct counting methods. Also, using the coordinates of nuclei from the direct counting, we simulate, by a Matlab program, disector pairs separated by increasing distances in a ganglion model. The comparison between the results of these approaches clearly demonstrates that the physical disector method provides a valid and reliable estimate of the number of sensory neurons only when the distance between the consecutive disector pairs is 60 microm or smaller. In these conditions the size of error between the results of physical disector and direct counting does not exceed 6%. In contrast when the distance between two pairs is larger than 60 microm (70-200 microm) the size of error increases rapidly to 27%. We conclude that the physical dissector method provides a reliable estimate of the number of rat sensory neurons only when the separating distance between the consecutive dissector pairs is no larger than 60 microm.

Fast and simple epidemiological typing of Pseudomonas aeruginosa using the double-locus sequence typing (DLST) method.

Although the molecular typing of Pseudomonas aeruginosa is important to understand the local epidemiology of this opportunistic pathogen, it remains challenging. Our aim was to develop a simple typing method based on the sequencing of two highly variable loci. Single-strand sequencing of three highly variable loci (ms172, ms217, and oprD) was performed on a collection of 282 isolates recovered between 1994 and 2007 (from patients and the environment). As expected, the resolution of each locus alone [number of types (NT) = 35-64; index of discrimination (ID) = 0.816-0.964] was lower than the combination of two loci (NT = 78-97; ID = 0.966-0.971). As each pairwise combination of loci gave similar results, we selected the most robust combination with ms172 [reverse; R] and ms217 [R] to constitute the double-locus sequence typing (DLST) scheme for P. aeruginosa. This combination gave: (i) a complete genotype for 276/282 isolates (typability of 98%), (ii) 86 different types, and (iii) an ID of 0.968. Analysis of multiple isolates from the same patients or taps showed that DLST genotypes are generally stable over a period of several months. The high typability, discriminatory power, and ease of use of the proposed DLST scheme makes it a method of choice for local epidemiological analyses of P. aeruginosa. Moreover, the possibility to give unambiguous definition of types allowed to develop an Internet database ( http://www.dlst.org ) accessible by all.