Determination of restriction endonucleases in Streptomyces and Nocardia colonies]

A simple technique is proposed for the detection of restriction endonucleases in Streptomyces and Nocardia cells. The analysis was performed directly in the cells collected from colonies cultivated on Petri dishes with an inoculation loop. The cells were treated with lysozyme, EDTA and Triton X-100. The lysates were tested for restriction endonucleases. The technique enables the detection of enzymes Nco I, Not I, Nru I, Sfr 3031, and Sfi I in the lysates of the respective strains-producers.     

Formation of Mononuclear Phagocyte (Macrophage) Colonies by Mouse Spleen Cells in Liquid Culture

When spleen cells of the adult mouse were tested for the formation of mononuclear phagocyte (macrophage) colonies by the liquid culture technique with an incubation period of 7–8 days, about 100 macrophage colonies were produced from 1 × 10⁶ cells. The number of macrophage colonies appearing after 2 days of incubation was small, but thereafter increased progressively up to at least 8 days. In the later stages of incubation (after day 6) large colonies consisting of more than 100 cells appeared. Macrophage colonies in the early stages consisted almost solely of macrophages. On day 6 significant numbers of small round mononuclear cells with no detectable phagocytic activity were seen in the center of large colonies, and by day 8 marked crowding of these cells had occurred. The peripheral region of the large colonies consisted mainly of macrophages and the intermediate region of middle-sized round or slightly stretched cells with weak phagocytic activity. Approximately two-thirds of the colony-forming cells still remained after glass-adherent cells were removed from the spleen cells by passing over a glass-bead column. In cultures of glass-nonadherent cells macrophage colonies were not generated in the early stage. The number of colony-forming cells did not change significantly even after actively phagocytic cells were rigorously removed from the spleen cells. In addition, no macrophage colonies were generated in cultures of spleen cells treated with mitomycin C.     

Honey bee hygienic behaviour does not incur a cost via removal of healthy brood

In the honey bee, hygienic behaviour, the removal of dead or diseased brood from capped cells by workers, is a heritable trait that confers colony‐level resistance against brood diseases. This behaviour is quite rare. Only c. 10% of unselected colonies show high levels of hygiene. Previous studies suggested that hygiene might be rare because it also results in the removal of healthy brood, thereby imposing an ongoing cost even when brood diseases are absent. We tested this hypothesis by quantifying hygienic behaviour in 10 colonies using a standard technique, the freeze‐killed brood (FKB) bioassay. At the same time, we also quantified the removal of untreated brood. The study colonies showed a wide range in hygienic behaviour, removing 19.7–100% of the FKB. The removal of untreated brood ranged from 2% to 44.4%. However, there was no correlation between the two removal rates for any of the four age groups of untreated brood studied (eggs, young larvae, older larvae from uncapped cells and larvae/pupae from capped cells). These results do not support the cost‐to‐healthy‐brood hypothesis for the rarity of hygienic behaviour.     

Disaggregation of colonies of Microcystis (Cyanobacteria): efficiency of two techniques assessed using an image analysis system

Disaggregation of colonies of Microcystis (Cyanobacteria) into smalleraggregates or single cells is a usefulprocess for improving the accuracy andprecision of cell abundance estimates innatural populations. An image analysissystem was therefore used to assess theefficiency of two techniques,heating/vortexing and grinding, fordisaggregating fixed and live colonies. Eight environmental samples, consisting ofeither colonial or non-colonial strains,were tested in time course experimentsusing each technique by analysing thenumber and total area of the objects (i.e.,cells, clumps of cells, colonies) acquired. The results show that i) image analysis isan efficient tool to compare the efficiencyof disaggregation techniques on colonies ofcyanobacteria, ii) the grinding techniqueis more efficient in disaggregating alltypes of Microcystis colony, withoutsignificant loss, and iii) the heatingtechnique does not work for all types ofcolonies, in particular, compact coloniesfixed with Lugol's iodine solution.     

Detection of multiple-strain carriers: the value of re-sampling

Bacteria may colonize a carrier with more than one strain of a species at any one time. Attempts to determine multiple colonization are labour intensive because of the large number of colonies per carrier which need to be tested. A possible solution -- in which only 3 colonies per carrier are initially tested and only multiple-strain carriers are re-sampled -- was recently described. We evaluated the accuracy of the "re-sampling method" devised by Cespedes et al. with 500,000 stochastic simulations per scenario. Re-sampling is acceptable where > or = 5 colonies are initially tested or where one strain predominates over others in colony counts. The method introduces bias towards overestimation which decreases with the number of available carriers, increases with the proportion of truly multiple carriers, with decreasing number of colonies available for testing, and with decreasing number of colonies tested per carrier. Initial testing of 5-8 colonies tested with re-sampling are adequate for a large study (>100 carriers), or a small study where it is suspected that no strain predominates over the other in colony counts. Testing 9-20 colonies with re-sampling is necessary for small studies where one strain predominates over others. Re-sampling is unnecessary where >20 colonies are tested.     

A technique for the direct identification of haemolytic-pathogenic listeria on selective plating media

A technique based on the addition of a red cells top layer to a selective plating medium after listeria growth is proposed in order to detect directly the haemolytic activity of pathogenic listeria colonies. It was applied to different selective plating media (modified McBride agar, lithium chloride-phenylethanol-moxalactam, listeria selective medium–Oxford formulation, polymyxin-acriflavine-lithium chloride-ceftazidime-aesculin-mannitol and LSAMM). The haemolytic activity of listeria colonies was more easily detected with the top layer than when red cells were incorporated in the selective plating medium. The LSAMM was the best medium for the recovery and identification of Listeria monocytogenes colonies by this technique (three Listeria monocytogenes colonies were distinguished among 2520 Listeria innocua colonies in raw milk).     

In vitro studies to prove the effect of sera of eosinophilia in colony formation

Serum samples from four patients with reactive eosinophilia and two patients with eosinophilic leukaemia were compared with normal sera with respect to formation of eosinophil colonies after addition of the sera to mononuclear cells from peripheral blood of healthy subjects. Supernatants from ConA stimulated guinea-pig spleen cells and human lymphocytes were tested in a similar way. Grown colonies were placed on glass slides and after staining with luxol fast blue the percentage of eosinophils was counted. The serum samples of the patients with reactive eosinophilia produced the greatest number of eosinophil colonies while supernatants of spleen and lymphocytes produced the greatest number of eosinophilic granulocytes. Our findings suggest the existence of a factor stimulating eosinophil colonies in the tested serum fractions. Beyond that an indication is given for a substance in the supernatants of spleen and lymphocyte suspensions which stimulates more intensively the maturing into eosinophilic granulocytes than the formation of colonies.     

Human erythroid colony formation in vitro: Evidence for clonal origin

Human marrow cells, suspended in methylcellulose medium containing erythropoietin, give rise to discrete colonies of hemoglobin synthesizing cells. The presumption that such colonies originate from single progenitor cells has been tested directly in females with X-chromosome inactivation mosaicism using glucose-6-phosphate dehydrogenase (G-6-PD) as a marker. When individual colonies were grown from marrow cells obtained from two black females heterozygous for G-6-PD, only one or the other isoenzyme type was observed, but not both. These results are most consistent with the interpretation that human erythroid colonies arise from single cells.     

The abundance of microcystin-producing genotypes correlates positively with colony size in Microcystis sp. and determines its microcystin net production in Lake Wannsee

The working hypotheses tested on a natural population of Microcystis sp. in Lake Wannsee (Berlin, Germany) were that (i) the varying abundance of microcystin-producing genotypes versus non-microcystin-producing genotypes is a key factor for microcystin net production and (ii) the occurrence of a gene for microcystin net production is related to colony morphology, particularly colony size. To test these hypotheses, samples were fractionated by colony size with a sieving procedure during the summer of 2000. Each colony size class was analyzed for cell numbers, the proportion of microcystin-producing genotypes, and microcystin concentrations. The smallest size class of Microcystis colonies (<50 microm) showed the lowest proportion of microcystin-producing genotypes, the highest proportion of non-microcystin-producing cells, and the lowest microcystin cell quotas (sum of microcystins RR, YR, LR, and WR). In contrast, the larger size classes of Microcystis colonies (>100 microm) showed the highest proportion of microcystin-producing genotypes, the lowest proportion of non-microcystin-producing cells, and the highest microcystin cell quotas. The microcystin net production rate was nearly one to one positively related to the population growth rate for the larger colony size classes (>100 microm); however, no relationship could be found for the smaller size classes. It was concluded that the variations found in microcystin net production between colony size classes are chiefly due to differences in genotype composition and that the microcystin net production in the lake is mainly influenced by the abundance of the larger (>100- microm) microcystin-producing colonies.     

Stimulation of retroviral vector infection of murine hematopoietic progenitors

Conditioned media (CM) from a cloned murine marrow-derived stromal cell line, AC6.21 (ALC), was shown to stimulate retroviral vector infection of hematopoietic progenitors in culture. Inclusion of ALC CM during cocultivation of normal murine bone marrow (BM) with vector-producing fibroblasts improved infection efficiency of day 13 spleen colony-forming cells (CFU-s) from 63% (15 provirus-positive spleen colonies/24 total), without added growth factor, to 90% (36 provirus-positive colonies/40 total). In addition, stimulation of BM cells with ALC CM during cocultivation improved retroviral infection of stem cells capable of repopulating the hematopoietic system of irradiated recipient animals. Because ALC CM was found to have 50 to 100 U/ml of IL-6 activity, purified recombinant human IL-6 was tested for an effect in this system. Stimulation with IL-6 alone increased retroviral infection efficiency of CFU-s from 15% (17 colonies provirus-positive/111 total analyzed) without added growth factor to 66% (97 provirus-positive colonies/148 total analyzed). These experiments support and extend previous studies which have demonstrated the necessity for growth factor stimulation in optimizing retroviral vector transduction of hematopoietic precursors.     

A Membrane Filtration Technique for the Enumeration of Escherichia coli in Seawater

S ummary . Membrane filtration has become an accepted method for enumerating Escherichia coli in water, but little published evidence could be found to judge the specificity of the method to assess faecal contamination in either fresh or saline waters. The method is used in our laboratory to monitor the extent and degree of sewage pollution in coastal areas, but there is need for information on what proportion of lactose-fermenting colonies from seawater, developing at 44° on a 4% enriched Teepol medium, are E. coli type I. A total of 1352 colonies from seawater was tested for production of indole and for gas from lactose at 44°. In addition, 46% of the colonies were screened by the IMVEC series of tests. The proportion of colonies tested ranged from 10–100%, depending on the number of colonies on the membrane. Many of the colonies (81.9%) to which IMVEC tests were applied were E. coli type I; a further 10.9% were Irregular type I. The practical implications of these findings are discussed.     

Colony formation of mammalian cells on agar plates and its application to Lederberg's replica plating

In this paper we describe a new technique of cloning by use of agar plates and its application to replica plating. It was found that most cell lines form colonies on the surface of solid agar, although the plating efficiency and size of colony is dependent on specimens and concentrations of agar and agarose used. When 0.5% Noble-agar was used as substrate, plating efficiencies were obtained comparable to those of conventional cloning techniques in liquid medium and of agar suspension cultures. In some cases, including the primary culture of Yoshida sarcoma, the efficiency of plating was apparently higher than that obtained by the already established procedures. In an experiment with a series of BHK-21 cells, it was found that virally transformed cells could form colonies on agar plate, whereas untransformed and reverted cells could not divide, suggesting that agar plate culture, as well as agar suspension culture, can be used for a selective assay of transformation.Two methods of replica plating were employed. Method I is that devised by Lederberg in which colonies on the master plate are imprinted on pile fabrics and then transferred to the replica plates. With FM3A cells, the fidelity of replica plating was around 95%. Method II is inoculation of clones by applying a glass rod to the replica plates on which positions of inocula were identified by a grid. Fidelity of replica plating of FM3A, L5178Y and YSC cells was 99.7, 100 and 100% respectively.     

DNA topoisomerase inhibitors block erythropoiesis and delay hemoglobinization in vitro

To examine the importance of topological constraints on DNA during erythroid development, we measured the effects of camptothecin and teniposide, two tumoricidal agents which are also specific inhibitors of type I and type II topoisomerases respectively, on the formation of hematopoietic colonies by cultured human bone marrow cells. When added to bone marrow culture, each inhibitor alone impairs the formation of early BFU-E-derived colonies, late CFU-E-derived colonies and mixed hematopoietic (CFU-GEMM-derived) colonies by up to 100%. Inhibition of colony formation is directly related to the time of inhibitor addition and the inhibitor concentration tested. Although either inhibitor alone reduces colony formation by 90%, when added together at a submaximal concentration, camptothecin and teniposide exert a synergistic suppressive effect. Furthermore, addition of topoisomerase inhibitors to culture impairs hemoglobinization of colony erythroblasts in a time-dependent fashion. In contrast to the effects of topoisomerase inhibitors, the antiproliferative agent aphidicolin reduces erythroid colony number and size without altering hemoglobinization of colony erythroblasts. Since neither topoisomerase inhibitor alters the morphology of cultured cells, the capacity of cells to exclude trypan blue or the potential to form erythroid colonies through the interval required for the first progenitor cell division, it is unlikely that camptothecin or teniposide are cytotoxic to hematopoietic cells. Human mononuclear cells enriched in bone marrow lymphocytes and nucleated erythroblasts from both human and mouse sources release DNA into the detergent soluble fraction. Release requires functional topoisomerases and is altered by acute exposure to topoisomerase inhibitors. Our results suggest that topoisomerases are critical not only to proliferation but also to differentiation of human marrow erythroid progenitor cells and stem cells in culture.     

Possible demonstration of multipotential nature of embryonic neural retina by clonal cell culture.

The possible multipotential nature of the neural retina of early chick embryos was examined by the technique of clonal cell culture. Cultures were prepared from cells dissociated from freshly excised neural retinas of 3.5-day-old chick embryos or from cells harvested from primary highdensity cultures. The following four colony types were obtained: colonies differentiating into “lentoid bodies”; colonies with pigment cells; colonies with both “lentoid bodies” and pigment cells; and colonies comprised entirely of unidentifiable cells. Neuronal differentiation occurred frequently in the early stages of culture (up to about 10 days). In some of these neuronal colonies, “lentoid bodies” and, rarely, both “lentoid bodies” and pigment cells differentiated after a further culture period of up to 30 days. Secondary colonies established from primary colonies after 9–10 days demonstrated that these original colonies fell into four different categories: those giving rise to secondary colonies containing only “lentoid bodies,” those giving rise to pigmented colonies only, those developing both lentoid and pigmented colonies, and finally those which gave rise to secondary colonies of all three types, lentoid, pigmented, and mixed colonies. When primary pigmented colonies were recloned at about 30 days after inoculation, the differentiated pigment cells transdifferentiated into lens. Whether multispecific colonies were really of clonal origin or not is discussed. The possible presence of a multipotent progenitor cell able to give rise to multispecific clones in the neural retina of 3.5-day-old chick embryos is suggested. A sequence of differentiation starting from multipotent neural retinal cells to be terminated with lens through the differentiation of neuronal and pigment cells is hypothetically proposed.     

The cellular composition of hemopoietic spleen colonies

The cellular composition of individual hemopoietic spleen colonies has been studied using techniques which tested primarily for cell function rather than cell morphology. Erythroblastic cells were recognized by their capacity to incorporate radioiron, granulocytic cells by their content of peroxidase-positive material, and hemopoietic stem cells by their ability to form spleen colonies in irradiated hosts. It was found that, 14 days after the initiation of spleen colonies, the distribution of these cell types among individual colonies was very heterogeneous, but that most colonies contained detectable numbers of erythroblasts, granulocytes and colony-forming cells. An appreciable proportion of the cells in the colonies could not be identified as any of these three cell types. No strong correlations between numbers of erythroblasts, granulocytes and colony-forming cells in individual colonies were observed, though there was a tendency for colonies containing a high proportion of erythroblasts to contain a low proportion of granulocytes, and for colonies containing a high proportion of granulocytes to contain a higher proportion of colony-forming cells. An analysis of colonies which contained cells bearing radiation-induced chromosomal markers indicated that 83–98% of the dividing cells within 14-day spleen colonies were derived from single precursors.     

Reversible and permanent effects of the carbon sources and various antibiotics on the morphology and metabolic properties of Ustilago cynodontis cells

The effects of various carbon sources and of antibiotics on the morphology of hypha cells of the fungus Ustilago cynodontis is described. Nonfermentable substrates promote readily reversible yeastlike colonies from hypha cells: all the hypha cells spread on these substrates give rise to yeastlike colonies that revert to the mycelial phenotype when transferred to glucose medium. Among the antibiotics tested, chloramphenicol (CAP) is found to promote, under certain circumstances, a long-lasting, even permanent modification on the morphology of the colonies: the colonies developed on CAP-glucose media are yeastlike, and a percentage of them give rise to colonies whose morphology remains yeastlike even on drug-free media: this effect is also obtained with cells cultivated in liquid medium. This permanent morphological modification is accompanied by a change of metabolic properties. Similar permanent effects are obtained with ethidium bromide, suggesting that mitochondrial functioning is involved in these modifications.     

The Processing of Mammalian Haemopoietic Cells in Thin Layer Agar Cultures for Electron Microscopy

Human and mouse haemopoietic cells cultured by the thin layer agar technique have been studied with the electron microscope. To process colonies of haemopoietic cells or individual cells which appeared in these colonies, a special technique had to be developed. The technique presented covers methods of selection, isolation, and sectioning that were devised for this purpose.

Haemopoietic cells are cultured in small plastic Petri dishes containing a culture system with 0.25% agar. Cell colonies and individual cells intended for light as well as for electron microscopic study are examined and selected microscopically with the aid of a numbered grid which is placed under the closed Petri dish.

Cells in the agar gel are fixed with glutaraldehyde which is pipetted directly onto the cultures. In order to facilitate their removal from the medium, the consistency of the agar solution is increased by evaporating liquid with controlled mild warming.

Pieces of agar containing colonies or single cells are cut out with a fine trephine and postfixed in osmium tetroxide. Agar pieces are embedded cell side up in a thin layer of Epon. After polymerization, the Epon-embedded pieces of agar are appropriately oriented at the head of flat embedding molds filled with fresh Epon. After another polymerization procedure, the top of the Epon blocks containing the cells are trimmed to a smooth surface with a glass knife.

The exact distance between the smooth surface of the blocks and the cells is measured by use of the vertical micrometer of a standard light microscope. The Epon layer around the specimen is trimmed away to expose selected cells for subsequent semi-thick and ultrathin sectioning. Sections are stained and examined microscopically.

With minor modifications the technique described also enables the processing of extremely small quantities of biological materials derived from other experiments for both light and electron microscopic observation.     

Larval Apis mellifera L. (Hymenoptera: Apidae) mortality after topical application of antibiotics and dusts

Beekeepers apply various dusts to honey bee, Apis mellifera L., colonies to dislodge parasitic mites and control bacterial brood diseases. Anecdotal reports by beekeepers indicate that the antibiotic oxytetracycline (OTC) can be toxic when applied in powdered sugar to cells containing immature bee brood, but it was not known whether the purported toxicity is caused by the antibiotic or the sugar carrier. Additionally, the toxicity of various dusts, proposed for parasitic mite control, is poorly known. In the current studies, we tested OTC and two other antibiotics (tylosin and lincomycin, candidate compounds for use in honey bee colonies) in a powdered sugar carrier for larval toxicity. We also tested for larval toxicity, several dusts that have been proposed for mite control. OTC caused significant brood mortality of approximately 80% at the concentrations used in the hive (200 mg in 20 g sugar). In contrast, tylosin and lincomycin at the 200 mg dose were both similar to untreated controls, and only five times that concentration (1000 mg) caused significant brood mortality of approximately 65%. The addition of dusts, wheat flour, talc, and a commercially available protein supplement, BeePro, resulted in mortality levels between 65 and 80%, similar to that seen with OTC. The common antibiotic carrier, powered confectioners sugar, was nontoxic. The use of 100 unsealed brood cells was demonstrated to be a reliable means of assessing potential adverse affects of dry compounds on larval honey bees. Two new candidate antibiotics for use in honey bee colonies were less toxic to larval bees than the currently labeled antibiotic, OTC.     

ULTRASTRUCTURAL ANALYSIS OF HEMATOPOIETIC COLONIES DERIVED FROM HUMAN PERIPHERAL BLOOD : A Newly Developed Method

The ultrastructure of granulocyte colonies derived from normal human peripheral blood leukocytes cultured in semisolid media has been studied by a new method developed for this purpose. Fixation, dehydration, and embedding of the whole content of the Petri dish resulted in a block of Epon containing colonies made up of cells with the spatial orientation of those observed in living cultures. This permitted serial sectioning through entire colonies. Cell maturation in vitro appeared to parallel that of normal marrow. However, even the most mature cells retained cytoplasmic characteristics of more immature cells. This was particularly true for eosinophils which only rarely possessed granules with electron-dense crystalline "cores," a feature typical for mature eosinophils. In addition to the normal-appearing hematopoietic cells found within colonies, very large round or spindle-shaped cells were present between colonies and firmly attached to the bottom of the culture dish. Although the histochemical and functional characterization of these cells awaits further study, it is suggested that they are related to histiocytes or macrophages. The technique described here should prove valuable in studies of the development, differentiation, and interaction of many types of cells.     

Physical separation of hemopoietic stem cells from cells forming colonies in culture

Mouse bone marrow cells in suspension were separated into a number of fractions on the basis of cell density by equilibrium density gradient centrifugation, or on the basis of cell size by velocity sedimentation. After each type of separation, the cells from the various fractions were assayed for their ability to form macroscopic spleen colonies in irradiated recipient mice, and for their ability to form colonies in a cell culture system. The results from either separation technique demonstrate that cells in some fractions formed more colonies in vivo than in the culture system, while cells in other fractions formed more colonies in culture than in the spleen. The results of control experiments indicate that this separation of the two types of colony-forming cells was not an artifact of the separation procedures. From these experiments it was concluded that the population of cells which form colonies in culture under the conditions used is not identical to the population of cells detected by the spleen colony assay.