Disc Plate Method of Microbiological Antibiotic Assay: I. Factors Influencing Variability and Error 1

Several factors are investigated that normally cause variation in zone diameters in conventional disc plate diffusion assay procedures. Of these factors the most serious is the unequal exposure of the individual plates at top or bottom of stacks to temperatures above and below room temperature. This unequal temperature exposure is avoided by novel handling and incubation procedures. A major variable, but one which can be controlled, is the varying time interval between pouring seeded agar and the time of applying the pads with antibiotic to the plates. This influence of time of setting and the effects of several other sequential operations are combined into a composite variable. This variable is then accounted for and normalized by interposing "external" reference plates set with a reference solution in the sequence of approximately 100 plates. No "internal" reference zones are employed. Such factors as volume of agar poured, wedge shape of agar in a dish, volumetric errors in dilutions, and timing considerations are studied and discussed. The results of this study form the basis for a test protocol which is presented in a following paper.     

Growth of Chikungunya Virus in Baby Hamster Kidney Cell (BHK-21-Clone 13) Suspension Cultures

This report describes the methods used to obtain high titers of chikungunya virus with suspension cultures of BHK-21-clone 13 cells. The cells were grown at 37 C to a cell concentration of 10(6) to 2 x 10(6) per ml. After maximum cell growth, the cells were inoculated with chikungunya virus at a multiplicity of 1 to 2 50% suckling mouse intracerebral lethal doses (SMICLD(50)) per cell in the spent Eagle's minimum essential medium for suspension cultures (MEMS), or the cell cultures were centrifuged at 200 x g and resuspended in either fresh MEMS or medium 199 prior to inoculation. The medium used had no effect on virus titer. The inoculated cultures were incubated at 34 C until the cell viability dropped to 30%, which usually occurred 28 to 30 hr postinoculation. After these procedures, chikungunya virus titers of log(10) 10.3 to 11.8 SMICLD(50) per ml were obtained.     

Active Transport of Iron in Bacillus megaterium: Role of Secondary Hydroxamic Acids

Kinetics of radioactive iron transport were examined in three strains of Bacillus megaterium. In strain ATCC 19213, which secretes the ferric-chelating secondary hydroxamic acid schizokinen, ⁵⁹Fe³⁺ uptake from ⁵⁹FeCl₃ or the ferric hydroxamate Desferal-⁵⁹Fe³⁺ was rapid and reached saturation within 3 min. In strain SK11, which does not secrete schizokinen, transport from ⁵⁹FeCl₃ was markedly reduced; the two ferric hydroxamates Desferal-⁵⁹Fe³⁺ or schizokinen-⁵⁹Fe³⁺ increased both total ⁵⁹Fe³⁺ uptake and the ⁵⁹Fe³⁺ appearing in a cellular trichloroacetic acid-insoluble fraction, although 10 min was required to reach saturation. Certain characteristics of transport from both ferric hydroxamates and FeCl₃ suggest that iron uptake was an active process. The growth-inhibitory effect of aluminum on strain SK11 was probably due to the formation of nonutilizable iron-aluminum complexes which blocked uptake from ⁵⁹FeCl₃. Desferal or schizokinen prevented this blockage. A strain (ARD-1) resistant to the ferric hydroxamate antibiotic A22765 was isolated from strain SK11. Strain ARD-1 failed to grow with Desferal-Fe³⁺ as an iron source, and it was unable to incorporate ⁵⁹Fe³⁺ from this source. Growth and iron uptake in strain ARD-1 were similar to strain SK11 with schizokinen-Fe³⁺ or the iron salt as sources. It is suggested that the ferric hydroxamates, or the iron they chelate, may be transported by a special system which might be selective for certain ferric hydroxamates. Strain ARD-1 may be unable to recognize both the antibiotic A22765 and the structurally similar chelate Desferal-Fe³⁺, while retaining its capacity to utilize schizokinen-Fe³⁺.     

Effect of LSD on mitotic and meiotic plant chromosomes

LSD was found to induce chromosomal aberrations in root tip cells of Allium cepa, Hordeum vulgare and Secale cereale. Aberrations occurred in the form of chromatid and isochromatid breaks with most of these breaks failing to rejoin. The distribution of chromosome breaks was not uniform over the length of chromosomes, and a majority of the breaks were localized at the centromeric regions. For a given dose of LSD (30 g/ml), onion appeared to be more susceptible than barley or rye. The diploid and tetraploid rye used in the study showed no appreciable difference in sensitivity to LSD treatment. — A preliminary study on meiotic chromosomes in LSD-treated diploid rye revealed the presence of univalents, chromosome breaks and fragments, suggesting that LSD can induce meiotic abnormalities in plant material.Contribution from the Department of Agronomy, University of Kentucky. The investigation reported in this paper (73-3-75) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director.     

The reduction of cystine during its transport into rat jejunal everted segments and sacs

1. Everted segments and sacs of rat jejunum were incubated in buffer containing [(35)S]cystine. 2. Concentration gradients were achieved by both segments and sacs, and the effects of duration of incubation and of cystine concentration on the isotope distribution ratios were determined. 3. Kinetic constants were determined for the uptake of cystine by both segments and sacs, and the differences between the two systems are discussed. 4. Reduction to cysteine was virtually complete intracellularly and in the sac lumen. Extensive reduction in the medium occurred only when segments were incubated. 5. Anaerobiosis prevented a concentration gradient being obtained between the medium and the tissue, but had little effect on the extent of reduction to cysteine in the tissue and sac lumen. 6. It is concluded that cystine is transported by an active process into rat jejunum, where it is present almost entirely in the reduced form, and that efflux of cysteine occurs through the serosal surface.