Improvement of human melanoma colony formation in soft agar using boiled instead of autoclaved agar

The effect of agar sterilized by either boiling or autoclaving on human melanoma colony formation in soft agar was compared using cells from 17 biopsies of metastatic malignant melanoma. The frequency of colony formation was significantly increased for cells grown in boiled agar in 8 samples (47%), unchanged in 8 samples (47%), and decreased in only one sample (6%). There were increases in both cluster and colony formation for the melanomas which had augmented colony formation when grown in boiled agar. There was also qualitative morphological improvement, including rounder, smoother cells and less extracellular debris surrounding the colonies. These data suggest that melanoma colony formation is enhanced when cells are grown in agar which has been sterilized by boiling rather than autoclaving.     

Das Autoselect-System – ein Automatensystem zur Selektion von Antibiotikaproduzenten. I. Methodische Grundlagen und Umfang des Systems

The methodical principle of an automated selection system for antibiotic producers, the Autoselectsystem, consisting of six machines and a computer is explained. In order to work with this machines the following material is needed: Cassettes with 64 microculture cups for cultivation of colonies on agar, cassettes with glass-tubes for dilution of samples, and test-plates with 64 holes for performing the agar diffusion test. The cups, the tubes and holes are arranged in a pattern of 8×8. In a serie of papers the machines will be described.     

Task Partitioning in Insect Societies. I. Effect of Colony Size on Queueing Delay and Colony Ergonomic Efficiency

The collection and handling of colony resources such as food, water, and nest construction material is often divided into subtasks in which the material is passed from one worker to another. This is known as task partitioning. When material is transferred directly from one individual to another, queueing delays frequently occur because individuals must sometimes wait for a transfer partner. A stochastic simulation model was written to study the effect of colony size on these delays. Queueing delay decreases roughly exponentially with colony size because stochastic fluctuations in the arrival of individuals are lower in larger colonies. These results support empirical studies of Polybia occidentalis and other theoretical studies of honeybees. The effect of the relative number of individuals in the two subtask groups was also studied. There is a unique optimal ratio of the number of workers associated with each of the subtasks that simultaneously minimizes mean queueing delay and maximizes colony nectar-processing rate. Deviations from this optimal ratio, for example, as a result of forager mortality or changes in nectar productivity that affect foraging trip duration, increase mean queueing delays greatly, especially in smaller colonies.     

Quantitative trait loci influencing honeybee alarm pheromone levels.

Quantitative trait loci (QTL) mapping procedures were used to identify loci that influence the levels of alarm pheromones found in the stinging apparatus of worker honeybees. An F1 queen was produced from a cross between a queen of European origin and a drone descended from an African subspecies. Haploid drones from the hybrid queen were individually backcrossed to European queens to produce 172 colonies. Samples of stings were taken from backcross workers of these colonies. Alarm pheromone levels were determined by gas chromatography. RAPD markers were scored from the haploid drone fathers of these colonies. The multiple-QTL model (MQM) of MapQTL was used to identify QTLs that influence the levels of four alarm pheromone components. Seven independent, potential QTLs were identified with LOD scores greater than two, and one at LOD 1.88. We identified one QTL for n-decyl acetate, three for n-octanol, four for isopentyl acetate, and one for hexyl acetate. One region of linkage group XI shows a strong influence on body size and the levels of three alarm pheromone components. This locus explained 40% of the variance for the amount of n-decyl acetate (LOD 6.57). In general, the QTLs influencing alarm pheromone levels were independent of previously identified loci that influenced the stinging behavior of these colonies. The only exception was a potential locus influencing levels of n-octanol, which was inversely correlated with stinging behavior.     

Genetics of Ustilago violacea. XXXII. Genetic evidence for transposable elements

Crosses between Ustilago violacea mutant strains with different color phenotypes that were derived from the 1.A1 and 2.A2 laboratory strains yielded, as expected, bisectored teliospore colonies with the parental colors as well as the a-1 and the a-2 mating-types. Generally, wild teliospore collections usually produced sporidia of both mating-types, providing two-mating-type (TMT) strains. Occasionally, however, sporidia with only one mating-type allele, a-1 or a-2, were obtained from teliospores, providing one-mating-type (OMT) strains. Crosses between OMT and laboratory strains with different color phenotypes gave (1) bisectored teliospore colonies with the parental colors or colonies with a parental color and a nonparental color and (2) nonsectored colonies with the nonparental color or with the parental color. The frequencies for the occurrence of non-parental color ranged from 41% to 93%, depending on the strain. The yield of teliospore colonies was usually reduced for these crosses. In many of these teliospore colonies, morphologically-altered sporidia (MAS phenotype) were observed. The morphology and the size of the sporidia with the MAS phenotype differed from those of teliospore colonies of the crosses between the laboratory strains. In addition, these sporidia did not form conjugants. A cross involving the TMT strains C449 yielded the MAS phenotype as well as a high incidence of tetrad colonies with a nonparental color. The high degree of instability of the parental color phenotypes, and the high frequency of the appearance of nonparental color phenotypes as well as the appearance of the MAS phenotype, are in accord with the presence of active and inactive transposable elements in the OMT strains, TMT strains, and laboratory strains.     

A Quantal Method for Determining Numbers of Erwinia carotovora var. carotovora and E. carotovora var. atroseptica in Soils and Plant Material

SUMMARY: A quantal method is described, based on the maximum likelihood method, which was found especially suitable for quantitatively estimating small populations of Erwinia carotovora in soils and plant material. Drops from serial dilutions of the test material are placed on replicated defined areas of a solid, semiselective medium with a pectate overlayer. The presence or absence of one or more of the characteristic, deep cavities formed by E. carotovora colonies in the pectate layer is the basis of the quantal count from which estimates of the population size can be derived.     

Intercellular matrix in colonies of Candida.

The histochemistry and fine structure of typical colonies of six species of Candida were studied, using a total of 31 clinical isolates. The colonies consisted of viable and degenerate cells which lay in an intercellular matrix. This matrix was made up of amorphous, granular, and fibrillar components, the relative proportions and total amount of which varied from species to species. The cells of all species were surrounded by a zone of homogeneous amorphus material, which may be a highly cross-linked carbohydrate. This separated intact cells from irregularly distributed granular debris derived from the cytoplasm of degenerate cells. Focal cellular degeneration and associated granular debris were present within the colonies of all species and were most common in the surface layers of cells of colonies of C. albicans and C. tropicalis. The large amounts of intercellular matrix in this region formed a surface coat on colonies of these two species. Intercellular strands of cell wall material, and to a lesser extent other membranous elements from degenerate cells, formed a prominent fibrillar meshwork in the colonies of C. albicans and C. tropicalis, but were less common in those of C. pseudotropicalis and C. guilliermondii and seldom seen in those of C. parapsilosis and C. krusei.     

Degradation of biochemical activity in soil sterilized by dry heat and gamma radiation.

The activities of three enzymes present in soil, phosphatases, urease, and decarboxylase, were monitered as indicators of the loss of biochemical information occurring when soil was sterilized by dry heat (0.08% relative humidity), gamma radiation, or a combination of both. More enzymatic activity was retained in soil sterilized by a long exposure to dry heat at relatively low temperature (8 weeks at 100.5 degrees C) than by a shorter exposure to a higher temperature (2 weeks at 124.5 degrees C). No enzymatic activity was detectable in soil sterilized by an even higher temperature (4 days at 148.5 degrees C). Soil sterilized with 7.5 Mrads of radiation retained much higher enzymatic activity than with heat sterilization. Combining sublethal doses of heat radiation effectively sterilized the soil and yielded enzymatic activities higher than those of soil sterilized by dry heat alone but lower than those of soil sterilized by radiation.     

Experimental hybridization between chromosomal races in Kalotermes approximatus,a termite with extensive sex-linked translocation heterozygosity

In order to elucidate the evolutionary pathway of sex-chromosome translocations in the termite Kalotermes approximatus, reciprocal matings were made between the winged reproductives from two different colonies, one in which there was a multivalent chain of 13 in male meiosis, and one in which there was a chain of 17 (or 19, in some cells). From the cross in which the male parent came from the chain-of-13 colony, the male offspring had a chain of 13 (or 15, in some cells) in meiosis; from the reciprocal cross, the male offspring had a chain of 15. Careful analysis of the multivalent chains in the hybrid males, combined with previous observations on chromosome variation in this species (Syren and Luykx, 1981), permit the following conclusions: (i) The two parent colonies differ by five distinct translocations, involving both X and Y chromosomes. (ii) In the evolution of the sex-multivalent, all of these kinds of translocations have occurred: X-X, Y-Y, X-autosome, and Y-autosome. (iii) Out of a total of 8 sex-chromosome translocations that can now be unambiguously characterized, 6 have involved X-chromosomes, and 2 have involved Y-chromosomes. (iv) In this species, different chromosomal races in adjacent geographic locales may differ in both their males and their females (where translocations have involved X-chromosomes), or they may differ only in their males (where translocations have involved Y-chromosomes).     

银柴胡离体培养与毛状根诱导技术初步研究

以银柴胡茎段为外植体,经消毒获得无菌再生材料后,筛选发根农杆菌介导毛状根诱导产生的最适条件。结果显示：最适的无菌消毒方法为：70%酒精浸5 s,0.1%升汞消毒3 min,获得了银柴胡离体培养材料;以发根农杆菌A4菌株介导的银柴胡毛状根诱导过程中,与叶片和不带腋芽茎段相比,带腋芽茎段为最适转化外植体,用OD600=0.8的菌液侵染茎段15 min,共培养3 d,800 mg/L头孢噻肟钠除菌,其诱导率及诱导密度最高,分别为100%和4.7,为最适诱导条件。研究结果说明在适合条件下,银柴胡带腋芽茎段适于诱导毛状根。     

16S rDNA Sequence Analysis of Bacterial Isolates from Die-back Affected Sissoo Trees (Dalbergia sissoo Roxb.) in Bangladesh

A new form of disease called ‘die‐back’ has been established in Dalbergia sissoo trees. This disease has reached epidemic proportions in Bangladesh as well as in other countries of South Asia and is characterized by browning of the leaves, signs of wilting, and trunk lesions with gum flow. The trees die within a few months. In order to investigate the causes of this die‐back disease, samples were taken for a first trial in the Rajshahi division at two sites around Sherpur. For the isolation of bacteria, surface‐sterilized plant material (leaves, twigs and trunk bark) from diseased trees was transferred to LB medium and incubated. After isolation of single colonies, various bacteria species could be identified by polymerase chain reaction analysis with two primers specific for highly conserved sequence regions in the bacterial 16S rDNA and by sequencing. First indications for the presence of bacteria with phytopathogenic potential were found.     

Genetics of Ustilago violacea. XXXV. Transposition in Haploid and Diploid Sporidia and Germinating Teliospores

Ustilago violacea sporidia of the white (w) MAD strain (a-2 w lys-3 ino-1 thi) incubated on minimal medium containing 100 mM KClO3 (potassium chlorate) produced only colonies with the pink phenotype. Sporidia from these colonies retained their pink color on complex medium. Sporidia of the diploid D1 strain (a-1 y nic-1 thi/a-2 w met-1 arg-f Chl70 thi) and of the diploid D2 strain (a-1 y his-1 glu-1 thi/a-2 w lys-3 ino-1 thi) produced pink colonies on complex medium. Streaks of diploid D1 sporidia from the pink colonies were stable on complex medium. In contrast, streaks of diploid D2 sporidia, which are heterozygous for the MAD strain, were unstable, initially producing pink colonies on complex medium but then, with continued incubation, producing white termini. Sporidia from the white termini with diploid morphology continued to yield white colonies. Teliospore colonies from three crosses with the MAD strain as a common parent were uniformly pink or had a pink sector instead of the expected uniformly white colonies or colonies having a white sector. Four of 20 and 13 of 20 teliospore colonies, respectively, from two of the three crosses had both a-1 and a-2 sporidia, and the remaining colonies had only a-1 or only a-2 sporidia. All 40 teliospore colonies from the third cross had only a-1 or only a-2 sporidia. All of these observations indicated that the MAD strain may have two autonomous, transactive transposable elements in different chromosomes and that insertional mutations in at least two haplolethal loci were responsible for the teliospore colonies with only a-1 or only a-2 mating type. Crossing over between a haplolethal locus and the centromere would account for teliospore colonies with both a-1 and a-2 sporidia.     

A new species of <Emphasis Type="Italic">Discostroma</Emphasis> and its anamorph <Emphasis Type="Italic">Seimatosporium</Emphasis> with two morphological types of conidia,isolated from the stems of <Emphasis Type="Italic">Paeonia suffruticosa</Emphasis>

Conidia of two morphologically different types, one with a basal appendage only and the other with appendage at both ends, were isolated from the stems of Paeonia suffruticosa. Single conidial isolates of both types of conidia yield identical colonies, which then produced both types of conidia on agar media depending on temperature, thus showing that both types of conidia belong to the same fungus. Seimatosporium botan is described based on its morphological characteristics. The teleomorph of the fungus was first found on sterilized P. suffruticosa stems placed on water agar, when grown at 5°C for 2 months in 12-h photoperiod. Discostroma botan is described for this fungus. The teleomorph is also found on the same host in the field.     

Plasmid profiles of Lactobacillus plantarum spp. found as contaminants in Hemerocallis plant tissue cultures

Plasmid profiles of 30 strains of Lactobacillus plantarum isolated from 3-year-old tissue cultures and surface sterilized stem sections of glasshouse grown plants of three different varieties of Hemerocallis were compared to pinpoint the source of Lact. plantarum contamination in plant tissue cultures. Since the profiles of strains isolated from in vitro and in vivo plant material were generally identical, it is thought that the source of Lact. plantarum contamination is the plant material used to initiate plant tissue cultures.     

Characterization of spleen colonies derived from mice with mutations at the W locus.

Mice with mutations at the W locus have a hemopoietic stem cell defect characterized by an apparent deficiency of spleen colony forming cells (CFU-S). In the present report, we provide evidence that mutant cells form colonies and we compare the characteristics of the colonies derived from mutant and normal cells. To perform the colony-derivation studies, marrow cells were transferred into lethally irradiated congenic hosts that differed from the donors in the ubiquitous genetic marker, glucose phosphate isomerase (GPI-1). Donor GPI-1 comprised over 50% of the marker in the host spleen and marrow by 12 days post injection, regardless of whether the donor was mutant or normal. To characterize the colonies, serially sectioned host spleens were examined microscopically. Colonies are present by 8 days post-transplantation regardless of donor genotype, but mutant colonies are distinctly different from normal colonies. The proportion of blast and granulocyte colonies is always greater in W/Wv than in +/+ recipients. Unlike the W/Wv donors, the +/+ donors generate primarily erythrocyte colonies at 8, 10, and 14 days and mixed colonies at 12 days post-injection. Colonies from the mutant mice are generally smaller but visible colonies do appear by 12 days. The results are consistent with the notion that the anemia in W/Wv mice is caused by the early restriction of differentiating cells to a non-erythrocyte lineage accompanied by the delayed amplification of mutant hemopoietic cells. Whether this means erythrocyte-committed cells are absent or are present but unable to respond to the appropriate cytokines is not possible to determine from the current experiments.     

Colony formation by subpopulations of human T lymphocytes. VI. Further studies on colony phenotype, function, and cloning efficiency

Phytohemagglutinin (PHA)-induced colony formation in semisolid agar medium by human peripheral blood T lymphocytes showed an increasing cloning efficiency with decreasing numbers of cultured cells. Ninety percent of CD4+ cells (inducer/helper phenotype) and 20% of CD8+ cells (cytotoxic/suppressor phenotype) formed colonies when cultured at 10-200 cells/ml culture in the presence of sheep red blood cells (SRBC) and a source of interleukin-2 (IL-2). Probably all T-colony-forming cells, but none of the subsequent colony cells, expressed the Leu-8 antigen. The cloning efficiencies of FACS-sorted cells expressing the natural killer antigenic phenotypes Leu-7+ and CD16+ were found to be less than 1%. The costimulatory effect of red blood cells for colony formation was specific for SRBC and not observed in the presence of red cells obtained from seven other species including man. All T-lymphocyte colonies obtained from unseparated peripheral blood mononuclear cells expressed the CD25 antigen (IL-2 receptor) and colonies were always composed of either CD4+ or CD8+ cells. None of the colony cells expressed the Leu-8 or the CD16 antigens. By their specific morphology in agar culture the majority of colonies composed of CD4+ cells were easily recognized, but but approximately one-third of the CD4+ colonies could not be distinguished from colonies composed of CD8+ cells. On expansion of individual colonies in liquid subculture in the presence of interleukin-2, approximately 15% of the colonies developed natural killer (NK)-like cytotoxic activity, being capable of direct killing of K562 tumor cells. It is concluded that the present method for growing human T colonies exhibits the same cloning efficiency as the most efficient liquid culture systems. Individual T colonies are composed exclusively of T inducer/helper or T cytotoxic/suppressor cells, they are never of mixed phenotype, and they do not contain cells of natural killer phenotype. Regulatory mechanisms influencing colony formation are operating between and within the various subsets of T lymphocytes.     

Effect of high-fiber and high-oil diets on the fecal flora of swine.

Six pairs of pigs were fed a basal diet, a high-fiber diet, and a diet high in corn oil in different sequences to minimize the carry-over effect of diet. After 2 months on each diet, a fecal specimen from each pig was cultured on nonselective medium in roll tubes. Fifty colonies were randomly selected from each fecal sample, and isolates were characterized to identify a representative cross section of the fecal flora. The bacterial composition of the fecal flora differed between basal and high-fiber diets (P = 0.002) and between high-fiber and high-oil diets (P = 0.015). However, the floras were not significantly different between the basal and the high-oil diets (P = 0.135), nor were the floras of the 12 individual pigs (each on all three diets) statistically different (P = 0.103). Only 14 of the 160 observed taxa have been detected in the human fecal flora, and only 159 of 1,871 total isolates (8.5%) were members of described species. The most common isolate was a Streptococcus species similar to that reported by Robinson et al. (I. M. Robinson, S. C. Whipp, J. A. Bucklin, and M. J. Allison, Appl. Environ. Microbiol. 48:964-969, 1984), which was found in 34 of 36 samples and which represented 27.5% of all isolates. Lactobacillus, Fusobacterium, Eubacterium, Bacteroides, and Peptostreptococcus species were the next most common bacteria. Escherichia coli represented 1.7% of all fecal isolates, which is somewhat higher than the 0.1 to 0.6% observed in human feces cultured similarly with prereduced anaerobically sterilized media.(ABSTRACT TRUNCATED AT 250 WORDS)     

The morphology of colony variants of three species of Candida.

The colonies of 12 isolates of 3 Candida spp. with variant colony forms were studied by scanning and transmission electron microscopy. Small colonies were formed by 4 isolates each of C. albicans and C. parapsilosis and by 1 of C. tropicalis. These had an abnormally high proportion of degenerate yeast cells with an associated increase in granular cytoplasmic material intercellularly. The increased matrix in these small colonies formed a thick superficial coat over the organisms. Rough colonies were formed by 1 isolate each of C. albicans, C. tropicalis and C. parapsilosis. The convoluted regions of these colonies contained many pseudohyphal cells but few degenerate cells and little granular or fibrillar material in their intercellular matrices. The shape of colonies of Candida spp. may be altered by variations in the viability or the morphology of the organisms.     

Global diversity of two widespread,colony‐forming diatoms of the marine plankton,Chaetoceros socialis (syn. C. radians) and Chaetoceros gelidus sp. nov.

Marine phytoplankton samples containing diatoms of the Chaetoceros socialis group were collected from Thailand, China, Denmark, and Greenland, and cells were isolated into culture for light and electron microscopy and DNA sequencing of D1–D3 of the LSU rDNA. Species of this lineage are characterized by three short and one long setae extending from each cell, the long setae from several cells joining into a common center to form large colonies, which are sometimes visible with the naked eye. Phylogenetic analyses including sequences from other parts of the world revealed segregation into three groups. Most sequences fell into two large clades, one comprising material from cold waters, whereas the other contained material from warmer waters. Strain CCMP 172 from the Strait of Georgia, Washington State, USA, formed a separate group. The warm‐water species included Chinese and Thai material and therefore probably also material from the type locality of C. socialis, Hong Kong. It is characterized by all setae being covered by spines and the setae extending from the valve at some distance from the margin. In the resting spores, both valves are ornamented with spines. The cold‐water material is characterized by three spiny and one mostly smooth long setae, and the setae extend from the valve near the margin. Both valves of the resting spore are smooth. This material is described as C. gelidus sp. nov. C. radians, described from the Baltic in 1894, is considered a synonym of C. socialis. CCMP172 is in many ways intermediate and probably constitutes a separate species. The published evidence on this globally distributed and sometimes bloom‐forming group of species indicates higher species diversity than presently thought.     

Quantitative Determination of the Spatial Distribution of Nitrosomonas europaea and Nitrobacter agilis Cells Immobilized in kappa-Carrageenan Gel Beads by a Specific Fluorescent-Antibody Labelling Technique

A novel technique, combining labelling and stereological methods, for the determination of spatial distribution of two microorganisms in a biofilm is presented. Cells of Nitrosomonas europaea (ATCC 19718) and Nitrobacter agilis (ATCC 14123) were homogeneously distributed in a kappa-carrageenan gel during immobilization and allowed to grow out to colonies. The gel beads were sliced in thin cross sections after fixation and embedding. A two-step labelling method resulted in green fluorescent colonies of either N. europaea or N. agilis in the respective cross sections. The positions and surface areas of the colonies of each species were determined, and from that a biomass volume distribution for N. europaea and N. agilis in kappa-carrageenan gel beads was estimated. This technique will be useful for the validation of biofilm models, which predict such biomass distributions.