Improved rapid identification of mycobacteria by combining solid-phase extraction with high-performance liquid chromatography analysis of BACTEC cultures.

Identification of mycobacteria from BACTEC 12B cultures is achieved in 7 to 21 days by reverse-phase high-performance liquid chromatography (HPLC) using a UV spectrophotometer to detect nonpolar p-bromophenylacyl mycolic acid derivatives. However, cultures grown in BACTEC and other liquid media seldom contain sufficient mycolic acids to permit reliable identification under usual HPLC assay conditions, so the sample size must be increased. Unfortunately, samples prepared from cultures in liquid media such as BACTEC cultures also contain large amounts of extraneous polar and strongly nonpolar contaminants that interfere with the analysis and hasten deterioration of the HPLC column. The contaminants were removed from 10 samples simultaneously by solid-phase extraction (SPE), i.e., by passing the crude suspension containing the mycolic acid derivatives into disposable 500-mg tC18 SPE columns in place of the usual final filtration step used to prepare specimens for HPLC. Fifteen milliliters of 20% (vol/vol) dichloromethane in methanol was passed through the columns (< 3 ml/min) to wash through the undesired contaminants and bind the mycolic acid derivatives. The mycolates were quantitatively eluted in 3 ml of dichloromethane for analysis by HPLC. Treating a panel of 31 strains of frequently isolated mycobacteria by SPE reduced the content of contaminants by 89.3 to 99.9% without altering the chromatographic patterns compared with the same strains grown on conventional solid media and processed without SPE. Peak heights of mycolates prepared from BACTEC cultures were increased from < or = 6 to > or = 25 absorbance milliunits with SPE, sufficient for reliable interpretation by visual inspection of chromatograms obtained with a UV detector. Also, removal of the contaminants improved column longevity.

The dust distribution within the inner coma of comet P/Halley 1982i: encounter by Giotto's impact detectors.

Analysis of the data from Giotto's Dust Impact Detection System experiment (DIDSY) is presented. These data represent measurement of the size of dust grains incident on the Giotto dust shield along its trajectory through the coma of comet P/Halley on 1986 March 13/14. First detection occurred at some 287000 km distance from the nucleus on the inbound leg; the majority of the DIDSY subsystems remained operational after closest approach (604 km) yielding the last detection at about 202000 km from the nucleus. In order to improve the data coverage (and especially for the smallest grains, to approximately 10(-19) kg particle mass), data from the PIA instrument has been combined with DIDSY data. Flux profiles are presented for the various mass channels showing, to a first approximation, a 1/R2 flux dependence, where R is the distance of the detection point from the cometary nucleus, although significant differences are noted. Deviations from this dependence are observed, particularly close to the nucleus. From the flux profiles, mass and geometrical area distributions for the dust grains are derived for the trajectory through the coma. Groundbased CCD imaging of the dust continuum in the inner coma at the time of encounter is also used to derive the area of grains intercepted by Giotto. The results are consistent with the area functions derived by Giotto data and the low albedo of the grains deduced from infrared emission. For the close encounter period (-5 min to +5 min), the cumulative mass distribution function has been investigated, initially in 20 second periods; there is strong evidence from the data for a steepening of the index of the mass distribution for masses greater than 10(-13) kg during passage through dust jets which is not within the error limits of statistical uncertainty. The fluences for dust grains along the entire trajectory is calculated; it is found that extrapolation of the spectrum determined at intermediate masses (cumulative mass index alpha = 0.85) is not able to account for the spacecraft deceleration as observed by the Giotto Radio Science Experiment and by ESOC tracking operations. Data at large masses (>10(-8) kg) recently analysed from the DIDSY data set show clear evidence of a decrease in the mass distribution index at these masses within the coma, and it is shown that such a value of the mass index can provide sufficient mass for consistency with the observed deceleration. The total particulate mass output from the nucleus of comet P/Halley at the time of encounter would be dependent on the maximum mass emitted if this change in slope observed in the coma were also applicable to the emission from the nucleus; this matter is discussed in the text. The flux time profiles have been converted through a simple approach to modeling of the particle trajectories to yield an indication of nucleus surface activity. There is indication of an enhancement in flux at t approximately -29 s corresponding to crossing of the dawn terminator, but the flux detected prior to crossing of the dawn terminator is shown to be higher than predicted by simple modelling. Further enhancements corresponding to jet activity are detected around +190 s and +270 s.