Examination of smears for tubercle bacilli by Fluorescence Microscopy

IN underdeveloped countries, laboratory facilities for the bacteriological diagnosis of tuberculosis are at present, very limited. Cultural methods are unlikely to be used on a large scale for many years to come. It is, therefore, important to investigate the most economical method of examining smears for tubercle bacilli. Fluorescence microscopy was introduced by Hagemann (1937) and has since been described by many authors, including Tanner (1941, 1948), Lind and Shaughnessy (1941), Lempert (1944), Norman and Jelks (1945), Clegg and Foster-Carter (1946), Wilson (1952), Von Haebler and Murray (1954), and Needham (1957). The great advantage claimed for this method is that stained bacilli can be detected using a much lower magnification than with the usual Ziehl-Neelsen method. Considerable time is saved in examining smears and larger areas can be searched. The method has not been widely employed for two reasons. In the first place, the light source must be very bright and many of the optical systems described previously have only supplied sufficient light if the equipment was used in a darkened room. Secondly, some workers (Ritterhoff and Bowman, 1945; Kuster, 1939; Holm and Plum, 1943) consider that false positive results can be obtained, since some smears may contain small naturally fluorescent particles which can be confused with bacilli. Equipment for fluorescence microscopy that can be used in normal daylight has been in use at the Tuberculosis Chemotherapy Centre, Madras, for over two years. When it was first introduced, a comparison between this method and the conventional Ziehl-Neelsen method was undertaken to test their relative sensitivities, and to see whether fluorescence microscopy yielded false positive results. The results of this comparison are described

The Susceptibility to Hydrogen Peroxide of Indian and British Isoniazid-Sensitive and Isoniazid- Resistant Tubercle Bacilli

The present work describes an attempt to modify the method of Kreis and Le Joubioux (1957a) so that it would accurately estimate the relative proportions of catalase-positive and catalase-negative organisms in strains containing mixtures of the two types. A bactericidal test was chosen in preference to a bacteriostatic test, since it is difficult to obtain quantitative measurement with the latter technique. In performing a bactericidal test residual peroxide must be inactivated or removed by dilution so that it does not inhibit the growth of surviving organisms. Knox, Meadow and Worssam (1956) removed peroxide by centrifugation and washing, but this method was considered impracticable if this test were to be used on a large scale, and likely to produce inaccurate counts on the surviving organisms. In the present work the method of removal of peroxide was studied as well as the determination of the optimal peroxide concentration and period of exposure which would kill all catalase-negative organisms, but would leave catalase-positive organisms unaffected. In addition, the method of Kreis & Le Joubioux (1957a) was modified by reducing the inoculum of organisms exposed to peroxide so that catalase-positive bacilli would not be able to destroy peroxide during the test itself. The standardised bactericidal test was then employed in comparing the susceptibility to peroxide of isoniazid-sensitive strains from British and Indian patients, and in investigating the relationship between the peroxide susceptibility and the catalase activity of their isoniazid-resistant mutant strains

The detection of airborne transmission of tuberculosis from HIV-infected patients, using an in vivo air sampling model

Background. Nosocomial transmission of tuberculosis remains an important public health problem. We created an in vivo air sampling model to study airborne transmission of tuberculosis from patients coinfected with human immunodeficiency virus (HIV) and to evaluate environmental control measures. Methods. An animal facility was built above a mechanically ventilated HIV‐tuberculosis ward in Lima, Peru. A mean of 92 guinea pigs were continuously exposed to all ward exhaust air for 16 months. Animals had tuberculin skin tests performed at monthly intervals, and those with positive reactions were removed for autopsy and culture for tuberculosis. Results. Over 505 consecutive days, there were 118 ward admissions by 97 patients with pulmonary tuberculosis, with a median duration of hospitalization of 11 days. All patients were infected with HIV and constituted a heterogeneous group with both new and existing diagnoses of tuberculosis. There was a wide variation in monthly rates of guinea pigs developing positive tuberculin test results (0%–53%). Of 292 animals exposed to ward air, 159 developed positive tuberculin skin test results, of which 129 had laboratory confirmation of tuberculosis. The HIV‐positive patients with pulmonary tuberculosis produced a mean of 8.2 infectious quanta per hour, compared with 1.25 for HIV‐negative patients with tuberculosis in similar studies from the 1950s. The mean monthly patient infectiousness varied greatly, from production of 0–44 infectious quanta per hour, as did the theoretical risk for a health care worker to acquire tuberculosis by breathing ward air. Conclusions. HIV‐positive patients with tuberculosis varied greatly in their infectiousness, and some were highly infectious. Use of environmental control strategies for nosocomial tuberculosis is therefore a priority, especially in areas with a high prevalence of both tuberculosis and HIV infection

Storage capacity of correlated perceptrons

We consider an ensemble of $K$ single-layer perceptrons exposed to random inputs and investigate the conditions under which the couplings of these perceptrons can be chosen such that prescribed correlations between the outputs occur. A general formalism is introduced using a multi-perceptron costfunction that allows to determine the maximal number of random inputs as a function of the desired values of the correlations. Replica-symmetric results for $K=2$ and $K=3$ are compared with properties of two-layer networks of tree-structure and fixed Boolean function between hidden units and output. The results show which correlations in the hidden layer of multi-layer neural networks are crucial for the value of the storage capacity.Comment: 16 pages, Latex2

Quantitative predictions on auxin-induced polar distribution of PIN proteins during vein formation in leaves

The dynamic patterning of the plant hormone auxin and its efflux facilitator the PIN protein are the key regulator for the spatial and temporal organization of plant development. In particular auxin induces the polar localization of its own efflux facilitator. Due to this positive feedback auxin flow is directed and patterns of auxin and PIN arise. During the earliest stage of vein initiation in leaves auxin accumulates in a single cell in a rim of epidermal cells from which it flows into the ground meristem tissue of the leaf blade. There the localized auxin supply yields the successive polarization of PIN distribution along a strand of cells. We model the auxin and PIN dynamics within cells with a minimal canalization model. Solving the model analytically we uncover an excitable polarization front that triggers a polar distribution of PIN proteins in cells. As polarization fronts may extend to opposing directions from their initiation site we suggest a possible resolution to the puzzling occurrence of bipolar cells, such we offer an explanation for the development of closed, looped veins. Employing non-linear analysis we identify the role of the contributing microscopic processes during polarization. Furthermore, we deduce quantitative predictions on polarization fronts establishing a route to determine the up to now largely unknown kinetic rates of auxin and PIN dynamics.Comment: 9 pages, 4 figures, supplemental information included, accepted for publication in Eur. Phys. J.

Ectopic A-lattice seams destabilize microtubules

Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe

Islands of conformational stability for Filopodia

Filopodia are long, thin protrusions formed when bundles of fibers grow outwardly from a cell surface while remaining closed in a membrane tube. We study the subtle issue of the mechanical stability of such filopodia and how this depends on the deformation of the membrane that arises when the fiber bundle adopts a helical configuration. We calculate the ground state conformation of such filopodia, taking into account the steric interaction between the membrane and the enclosed semiflexible fiber bundle. For typical filopodia we find that a minimum number of fibers is required for filopodium stability. Our calculation elucidates how experimentally observed filopodia can obviate the classical Euler buckling condition and remain stable up to several tens of . We briefly discuss how experimental observation of the results obtained in this work for the helical-like deformations of enclosing membrane tubes in filopodia could possibly be observed in the acrosomal reactions of the sea cucumber Thyone, and the horseshoe crab Limulus. Any realistic future theories for filopodium stability are likely to rely on an accurate treatment of such steric effects, as analysed in this work

Modeling oscillatory Microtubule--Polymerization

Polymerization of microtubules is ubiquitous in biological cells and under certain conditions it becomes oscillatory in time. Here simple reaction models are analyzed that capture such oscillations as well as the length distribution of microtubules. We assume reaction conditions that are stationary over many oscillation periods, and it is a Hopf bifurcation that leads to a persistent oscillatory microtubule polymerization in these models. Analytical expressions are derived for the threshold of the bifurcation and the oscillation frequency in terms of reaction rates as well as typical trends of their parameter dependence are presented. Both, a catastrophe rate that depends on the density of {\it guanosine triphosphate} (GTP) liganded tubulin dimers and a delay reaction, such as the depolymerization of shrinking microtubules or the decay of oligomers, support oscillations. For a tubulin dimer concentration below the threshold oscillatory microtubule polymerization occurs transiently on the route to a stationary state, as shown by numerical solutions of the model equations. Close to threshold a so--called amplitude equation is derived and it is shown that the bifurcation to microtubule oscillations is supercritical.Comment: 21 pages and 12 figure

Deterministic mechanical model of T-killer cell polarization reproduces the wandering of aim between simultaneously engaged targets

T-killer cells of the immune system eliminate virus-infected and tumorous cells through direct cell-cell interactions. Reorientation of the killing apparatus inside the T cell to the T-cell interface with the target cell ensures specificity of the immune response. The killing apparatus can also oscillate next to the cell-cell interface. When two target cells are engaged by the T cell simultaneously, the killing apparatus can oscillate between the two interface areas. This oscillation is one of the most striking examples of cell movements that give the microscopist an unmechanistic impression of the cell's fidgety indecision. We have constructed a three-dimensional, numerical biomechanical model of the molecular-motor-driven microtubule cytoskeleton that positions the killing apparatus. The model demonstrates that the cortical pulling mechanism is indeed capable of orienting the killing apparatus into the functional position under a range of conditions. The model also predicts experimentally testable limitations of this commonly hypothesized mechanism of T-cell polarization. After the reorientation, the numerical solution exhibits complex, multidirectional, multiperiodic, and sustained oscillations in the absence of any external guidance or stochasticity. These computational results demonstrate that the strikingly animate wandering of aim in T-killer cells has a purely mechanical and deterministic explanation. © 2009 Kim, Maly

The Virulence in the Guinea-pig of Tubercle Bacilli Isolated before Treatment from South Indian Patients with Pulmonary Tuberculosis: 1. Homogeneity of the Investigation and a Critique of the Virulence Test

A series of studies on the virulence in the guinea-pig of tubercle bacilli isolated before treatment from Indian tuberculous patients admitted to a controlled comparison of different regimens of domiciliary chemotherapy has recently been undertaken by the Tuberculosis Chemotherapy Centre, Madras. The main object of these studies was to determine whether the differences in virulence of the tubercle bacilli obtained from Indian patients before the start of chemotherapy were related to the severtiy or type of the patients’ disease at that time and to the subsequent response to treatment. Before these relationships could be‘ investigated, however, it was necessary to find out whether the results of the virulence tests, which were carried out over a period of two-and-a-half years at the Centre and at the Microbiological Research Establishment, Porton, England, could be considered as a unified whole-that, is, as if they had all been done on the same day in the same laboratory. A proportion of the cultures was stored at – 20°C for 44-78 weeks, but this did not affect their virulence. Inter-experimental variation was found to be small in the Porton series of tests and undetectable in the Madras series, and the results in the latter series could be successfully adjusted to those in the former by allowing for differences in the means and standard deviations of the distributions for the two series. The measure of virulence used was found to be reasonably acceptable for the analysis of variance technique. Suggestions are made as to ways of improving the efficiency of the experimental design in future studies