Rugosity in Grimontia hollisae

Grimontia hollisae, formerly Vibrio hollisae, produces both smooth and rugose colonial variants. The rugose colony phenotype is characterized by wrinkled colonies producing copious amounts of exopolysaccharide. Cells from a rugose colony grown at 30 degrees C form rugose colonies, while the same cells grown at 37 degrees C form smooth colonies, which are characterized by a nonwrinkled, uncrannied appearance. Stress response studies revealed that after exposure to bleach for 30 min, rugose survivors outnumbered smooth survivors. Light scatter information obtained by flow cytometry indicated that rugose cells clumped into clusters of three or more cells (average, five cells) and formed two major clusters, while smooth cells formed only one cluster of single cells or doublets. Fluorescent lectin-binding flow cytometry studies revealed that the percentages of rugose cells that bound either wheat germ agglutinin (WGA) or Galanthus nivalis lectin (GNL) were greater than the percentages of smooth cells that bound the same lectins (WGA, 35% versus 3.5%; GNL, 67% versus 0.21%). These results indicate that the rugose exopolysaccharide consists partially of N-acetylglucosamine and mannose. Rugose colonies produced significantly more biofilm material than did smooth colonies, and rugose colonies grown at 30 degrees C produced more biofilm material than rugose colonies grown at 37 degrees C. Ultrastructurally, rugose colonies show regional cellular differentiation, with apical and lateral colonial regions containing cells embedded in a matrix stained by Alcian Blue. The cells touching the agar surface are packed tightly together in a palisade-like manner. The central region of the colony contains irregularly arranged, fluid-filled spaces and loosely packed chains or arrays of coccoid and vibrioid cells. Smooth colonies, in contrast, are flattened, composed of vibrioid cells, and lack distinct regional cellular differences. Results from suckling mouse studies showed that both orally fed rugose and smooth variants elicited significant, but similar, amounts of fluid accumulated in the stomach and intestines. These observations comprise the first report of expression and characterization of rugosity by G. hollisae and raise the possibility that expression of rugose exopolysaccharide in this organism is regulated at least in part by growth temperature.     

Growth of human acute myeloblastic leukemic (aml) cells in vitro.

Human normal and granulocytic leukemic progenitor cells produce colonies and clusters when cultured in the plasma clot system in vitro. The number of colonies formed and their cellular composition was comparable to that reported for the system (soft agar) usually used for these studies. The plasma clot system has the advantage of permitting in situ morphologic and cytochemical characterizaion of the cells within the colonies. A comparison was also made of the growth of leukemic cells in suspension cultures placed within conventional and modified Marbrook flasks. These studies demonstrated no advantage for the Marbrook system in cultures incubated for up to 7 days.     

Hypothesis: the target cell of GM-CSF is a macrophage precursor capable to produce cells with the property to secrete a G-CSF like activity.

The induction of granulocyte and macrophage colony formation by the granulocyte-macrophage colony stimulating factor (GM-CSF) on bone marrow cells (BMC) was evaluated as a function of time in agar cultures. We found that while macrophage cell clusters were very abundant on the first two days of culture, granulocytic cell clusters did not appear until the third day. We also found that macrophage colonies were present from the fourth day of culture, while granulocyte colonies did not appear until the fifth day. When two day cell clusters were transferred to cultures with GM-CSF we observed that only macrophage-colonies developed. On the other hand, when four day clusters were transferred, both granulocyte and macrophage colony formation was obtained in a similar way as the one obtained when using GM-CSF with fresh BMC. Two day clusters did not respond to granulocyte colony stimulating factor (G-CSF) while fourth day clusters generated granulocytic colonies in a similar way as when G-CSF was used with fresh BMC. In order to test the hypothesis that granulocyte colony formation in these assays could be a result of the secretion of G-CSF by the macrophages previously induced by GM-CSF, lysates from macrophage colonies were used to induce colony formation on BMC. We observed that colonies, mainly granulocytic, were induced in a similar way as when G-CSF was used. Finally, the possibility that GM-CSF is just a macrophage inducer with the property to produce cells that secrete G-CSF is discussed.     

Rugosity in Grimontia hollisae

Grimontia hollisae, formerly Vibrio hollisae, produces both smooth and rugose colonial variants. The rugose colony phenotype is characterized by wrinkled colonies producing copious amounts of exopolysaccharide. Cells from a rugose colony grown at 30°C form rugose colonies, while the same cells grown at 37°C form smooth colonies, which are characterized by a nonwrinkled, uncrannied appearance. Stress response studies revealed that after exposure to bleach for 30 min, rugose survivors outnumbered smooth survivors. Light scatter information obtained by flow cytometry indicated that rugose cells clumped into clusters of three or more cells (average, five cells) and formed two major clusters, while smooth cells formed only one cluster of single cells or doublets. Fluorescent lectin-binding flow cytometry studies revealed that the percentages of rugose cells that bound either wheat germ agglutinin (WGA) or Galanthus nivalis lectin (GNL) were greater than the percentages of smooth cells that bound the same lectins (WGA, 35% versus 3.5%; GNL, 67% versus 0.21%). These results indicate that the rugose exopolysaccharide consists partially of N-acetylglucosamine and mannose. Rugose colonies produced significantly more biofilm material than did smooth colonies, and rugose colonies grown at 30°C produced more biofilm material than rugose colonies grown at 37°C. Ultrastructurally, rugose colonies show regional cellular differentiation, with apical and lateral colonial regions containing cells embedded in a matrix stained by Alcian Blue. The cells touching the agar surface are packed tightly together in a palisade-like manner. The central region of the colony contains irregularly arranged, fluid-filled spaces and loosely packed chains or arrays of coccoid and vibrioid cells. Smooth colonies, in contrast, are flattened, composed of vibrioid cells, and lack distinct regional cellular differences. Results from suckling mouse studies showed that both orally fed rugose and smooth variants elicited significant, but similar, amounts of fluid accumulated in the stomach and intestines. These observations comprise the first report of expression and characterization of rugosity by G. hollisae and raise the possibility that expression of rugose exopolysaccharide in this organism is regulated at least in part by growth temperature.     

Colony formation of granulocyte (CFU-g) and macrophage (CFU-m) precursors in serum- and albumin-free culture: effect of transferrin on clonal growth

Clonal growth of mouse granulocyte and macrophage precursors were assayed in serum-free cultures without albumin. The number of granulocyte/macrophage colonies and clusters increased as transferrin (Trf) concentrations were increased in cultures containing serum-free L-cell-conditioned medium (LCM). On the other hand, cultures with LCM but without Trf produced relatively fewer colonies and clusters. These results indicate that Trf is one of the factors promoting the clonal growth of granulocyte and macrophage precursors in vitro. Although the presence of linoleic acid, alpha-thioglycerol, and dextran in the culture medium increased the number of granulocyte/macrophage colonies and clusters, these factors were not essential. Serum-free culture of mouse granulocyte and macrophage precursors provides a very useful system with which the activity and function of biological regulators of hematopoietic progenitors may be studied.     

Rodent cell transformation assays-a brief historical perspective

In vitro cell transformation is a process characterized by a series of progressive distinctive events that often emulate manifestations occurring in vivo and which are associated with neoplasia. Attendant cellular and sub-cellular alterations include, among others: cellular immortality, phenotypic changes, aneuploidy, genetic variability, cellular disarray, anchorage-independent growth, and tumorigenicity in vivo. Early chemically induced neoplastic transformation studies involved the use of normal diploid (Syrian) hamster embryo (SHE) cells and monitored the formation of morphologically altered colonies. Later investigations employed primarily two established mouse cell lines, i.e. the BALB/c 3T3 A31 cell line and the C3H 10T 1/2 cell line, and monitored the induction of morphologically aberrant foci. In either case, such transformed cellular clusters (colonies and foci) could induce tumors upon inoculation in vivo. Some subsequent noteworthy advancements using these systems included pH adjustments, metabolic supplementation, amplification of expression of formerly latent transformed foci, concurrent detection of mutagenesis and transformation, and use of a Bhas 42 cell line (v-Ha-ras transfected BALB/c 3T3 cells) to detect both tumor initiators and promoters. Over time, such transformation assay systems have been found useful in academic, industry and regulatory laboratories, generally for research purposes, but also occasionally as screening tools for potential chemical carcinogens. Nevertheless, to date, use of these assays for decision-making purposes in the regulatory arena remains elusive and will require comprehensive validation to gain universal acceptance.     

Influence of hydrocortisone on the survival and cluster and colony forming characteristics of normal human granulocyte-macrophage progenitors

This study was performed to determine the colony and cluster forming ability of granulocyte-macrophage (CFU-GM) progenitors of normal human blood low density cells cultured in a liquid culture system in the presence and absence of physiological doses of hydrocortisone (Hc). The CFU-GM recovered from the liquid cultures were assayed in soft agar medium. The results of the assays indicated that time-related development of clusters and colonies over 1-16 days, proliferative responsiveness to a source of colony stimulating activity, number of cells developed per colony, and the cellular composition of clusters and colonies produced from CFU-GM recovered from 14-day-old liquid cultures with 1.0 microM Hc, were all similar to those that developed from the normal human blood low density cells. However, a higher fraction of the CFU-GM in day 14 liquid cultures with 1.0 microM Hc were in DNA synthesis phase compared with the CFU-GM from the peripheral blood. This study confirmed the results of previous studies showing lower numbers of recognizable neutrophilic granulocytes and improved survival/proliferation of CFU-GM at day 14 in liquid cultures with 1.0 microM Hc compared with cultures without Hc. The present results suggest that the normal human blood CFU-GM which persists and proliferates under the influence of Hc in a liquid culture system is similar in ontogeny to the blood CFU-GM, and that the recovery of CFU-GM from liquid cultures under the influence of Hc appears to be exerted through stimulation of proliferation and controlled differentiation.     

Automated scanning of bone marrow cell colonies growing in agar-containing glass capillaries

An optical scanning system was developed to determine the growth of clusters and colonies of granulocytes and macrophages from mouse bone marrow cells in agar capillary tubes. The system consists of a commercially available photometer with a densitometer attachment, a two-mirror set to receive the light scattered by the cell colonies, a multiple capillary holder and an automatic sample changer. Parameters affecting scanning were examined and optimized: background scatter, instrument adjustments (e.g. signal damping) and threshold settings for clusters and colonies. Combined with the advantageous agar capillary technique, the complete scanning system provides an easy, accurate and sensitive method for rapid quantitation of hemopoietic cell colony formation in vitro.     

Cell kinetic study on GM colonies using autoradiography and collagen gel culture with special reference to unique proliferation of eosinophils

Using a newly developed technique of autoradiography and collagen gel culture, a kinetic study on human GM colonies was attempted. Colonies of immature cells appeared first on day 5. The number of mixed colonies (mixture of immature cells, neutrophils, and/or monocyte-macrophages) and neutrophil colonies attained a maximum on days 8 to 10 and a broad peak of monocyte-macrophage colonies was observed on days 11 to 16. Eosinophil colonies appeared first on day 12, reached a maximum on day 18, and then gradually decreased. A detailed analysis of the order of appearance of the colonies suggests that mixed, neutrophil and monocyte-macrophage colonies originate from immature cell colonies or clusters, while eosinophil colonies do not. An autoradiographic study was designed to study the proliferation characteristics of each colony. Labeling indices (LI) with 3H-TdR of the cells in immature cell colonies were always high. LI of the cells in differentiated colonies such as neutrophil, monocyte-macrophage, and mixed colonies were low throughout the observation period. In contrast, LI of the cells in eosinophil colonies were constantly high regardless of the size of cell aggregates and the duration of the culture period. Both mitotic indices and mean grain counts on the nuclei of eosinophils were similar to those of immature cells. These results suggest that eosinophil colonies develop from their own small clusters and that eosinophils retain a fairly good proliferative capacity even when differentiated to the level in which specific granules appear in the cytoplasm.     

Cinemicrographic Study of the Development of Subsurface Colonies of Staphylococcus aureus in Soft Agar

Cinemicrographic studies revealed that the development of elongated subsurface colonies of Staphylococcus aureus in soft agar (<0.2% agar) originated with a colony-forming unit of about 10 to 20 cells. It was then observed that small clusters of 3 to 12 cells broke off from the main colony unit and drifted away under the combined influence of gravity and Brownian motion. Once the downward or slightly sideward motion of the small clusters ceased, the clusters would continue to increase in size; at the same time, additional small clusters broke off, and the cycle was repeated until the entire colony was formed. Displacement and velocity measurements were made on the drifting small clusters. When compared with the dimensional growth rate and geometry of the subsurface colony, these showed that a correlation existed between the movement and velocity of the small clusters and the subsequent colony development. A relationship between the role of gravity reported in these results and the development of spherical colonies after rotation on a clinostat is suggested.     

Evidence of clonal budding in a radial cluster of Paraconularia crustula (White) (Pennsylvanian: ?Cnidaria)

Monospecific conulariid clusters have been interpreted as products of gregarious behavior or as clonal colonies, with members of a colony originally inclined at a high angle to the substrate and connected to each other at their apices. However. individuals in all previously documented clusters are preserved parallel, or nearly parallel, to bedding, and their apices are not preserved. We describe a cluster of three Paraconularia crustula (White) (Pennsylvanian of Missouri, USA) in which the individual specimens exhibit an original steeply inclined orientation. The individuals in this cluster converge adapically on an extremely thin. crumpled sheet of apatitic material that is similar to conulariid test material. The conulariids and associated sheet bear a striking resemblance to regenerated, clonal colonies of polypoid coronatid scyphozoans. Such colonies arise from a thin, periderm-covered sheet of non-differentiated soft tissue, produced following severance of a single parent polyp. We suggest that the conulariid cluster originated through a similar process. Should this interpretation be corroborated by the discovery of better preserved conulariid clusters, it would provide strong additional evidence for the hypothesis of a scyphoroan affinity for conulariids     

Enhancement of colony-stimulating-factor--dependent clonal growth of murine macrophage progenitors and their phagocytic activity by retinoic acid

The effect of retinoic acid (RA) on the colony-stimulating-factor-dependent clonal growth of myeloid progenitors was assessed in semisolid agar cultures of mouse bone marrow cells using L-cell-conditioned medium that gave rise to macrophage colonies, granulocyte colonies, and mixed macrophage-granulocyte colonies and clusters. RA was found to enhance the overall formation of myeloid colonies (about 50%) and clusters in 7-day cultures. The increase was due to an enhanced formation of macrophage colonies (70-250%) and clusters which reached a maximal value at about 3 microM RA. In 4-day cultures, the effect of RA on macrophage colony formation was biphasic with a maximal enhancement at 10 nM. RA suppressed granulocyte-colony formation in 4-day cultures. RA increased the phagocytic activity of bone-marrow-derived macrophages at all stages of differentiation and/or maturation in culture. The Fc-receptor-mediated erythrophagocytosis as well as the phagocytosis of heat-killed yeast cells (HK-yeast) and starch particles increased by RA treatment in a dose-dependent manner, reaching an increase of 100-200% of the activity expressed in the absence of RA. Peritoneal exudate macrophages likewise exhibited an increased phagocytic response to a variety of particles, at both physiological and pharmacological concentrations of RA. Expression of an RA-mediated increase in phagocytic activity required a prolonged incubation with RA (greater than 19 hr). The data suggest that RA may be of physiological relevance in the regulation of proliferation and function of hemopoietic cells. Therapeutic doses of RA may potentiate macrophage proliferation and function, elements that are crucial at all phases of the various defense mechanisms that the organism possesses.     

Influence of hydrocortisone on the survival and cluster and colony forming characteristics of normal human granulocyte-macrophage progenitors

Abstract. This study was performed to determine the colony and cluster forming ability of granulocyte-macrophage (CFU-GM) progenitors of normal human blood low density cells cultured in a liquid culture system in the presence and absence of physiological doses of hydrocortisone (He). The CFU-GM recovered from the liquid cultures were assayed in soft agar medium. The results of the assays indicated that time-related development of clusters and colonies over 1-16 days, proliferative responsiveness to a source of colony stimulating activity, number of cells developed per colony, and the cellular composition of clusters and colonies produced from CFU-GM recovered from 14-day-old liquid cultures with 1·0 µM. He, were all similar to those that developed from the normal human blood low density cells. However, a higher fraction of the CFU-GM in day 14 liquid cultures with 1·0 µM He were in DNA synthesis phase compared with the CFU-GM from the peripheral blood. This study confirmed the results of previous studies showing lower numbers of recognizable neutrophilic granulocytes and improved survival/proliferation of CFU-GM at day 14 in liquid cultures with 1·0 µM He compared with cultures without He. The present results suggest that the normal human blood CFU-GM which persists and proliferates under the influence of He in a liquid culture system is similar in ontogeny to the blood CFU-GM, and that the recovery of CFU-GM from liquid cultures under the influence of He appears to be exerted through stimulation of proliferation and controlled differentiation.     

STUDIES OF HEMATOPOIESIS IN MURINE SPLEENS: SIGNIFICANCE OF HEMATOPOIETIC COLONIES FORMED ON THE SURFACE OF SPLEENS

Conies of hematopoietic tissue are formed in spleens of lethally irradiated mice by the injection of small numbers of hematopoietic cells. Some of these colonies appear as surface colonies, others can be identified only in serial sections of the spleen. The present studies have related the number and cellular composition of total hematopoietic colonies in the murine spleen to their visual recognition on the splenic surface. These studies demonstrate that only 50% of the total colonies in a spleen are recognized as surface colonies and that of those colonies on the surface, approximately 80% contain erythroid elements. At least four factors play important roles in the recognition of hematopoietic colonies as splenic surface colonies: (1) dose of repopulating cells or hematopoietic stem cells injected into the irradiated animal; (2) location of colonies within the spleen; (3) size of colonies; and (4) cellular content of the colonies. These studies demonstrate that surface colony formation reflects primarily erythropoiesis and not total hematopoiesis.     

The suppressive influences of human tumor necrosis factors on bone marrow hematopoietic progenitor cells from normal donors and patients with leukemia: synergism of tumor necrosis factor and interferon-gamma

The influences of human tumor necrosis factor (TNF) (LuKII), recombinant human TNF-alpha, natural human interferon-gamma (HuIFN-gamma), recombinant HuIFN-gamma, and natural HuIFN-alpha were evaluated alone or in combination for their effects in vitro on colony formation by human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells incubated at 5% CO2 in lowered (5%) O2 tension. TNF (LuKII) and recombinant TNF-alpha caused a similar dose-dependent inhibition of colony formation from CFU-GM, BFU-E, and CFU-GEMM. Day 7 CFU-GM colonies were more sensitive than both day 14 CFU-GM colonies and day 7 CFU-GM clusters to inhibition by TNF. BFU-E colonies and CFU-GEMM colonies were least sensitive to inhibition with TNF. The suppressive effects of TNF (LuKII) and recombinant TNF-alpha were inactivated respectively with hetero-anti-human TNF (LuKII) and monoclonal anti-recombinant human TNF-alpha. The hetero-anti-TNF (LuKII) did not inactivate the suppressive effects of TNF-alpha and the monoclonal anti-recombinant TNF-alpha did not inactivate TNF (LuKII). The suppressive effects of TNF did not appear to be mediated via endogenous T lymphocytes and/or monocytes in the bone marrow preparation, and a pulse exposure of marrow cells with TNF for 60 min resulted in maximal or near maximal inhibition when compared with cells left with TNF for the full culture incubation period. A degree of species specificity was noted in that human TNF were more active against human marrow CFU-GM colonies than against mouse marrow CFU-GM colonies. Samples of bone marrow from patients with non-remission myeloid leukemia were set up in the CFU-GM assay and formed the characteristic abnormal growth pattern of large numbers of small sized clusters. These cluster-forming cells were more sensitive to inhibition by TNF than were the CFU-GM colonies and clusters grown from the bone marrow of normal donors. The sensitivity to TNF of colony formation by CFU-GM of patients with acute myelogenous leukemia in partial or complete remission was comparable with that of normal donors. When combinations of TNF and HuIFN were evaluated together, it was noted that TNF (LuKII) or recombinant TNF synergized with natural or recombinant HuIFN-gamma, but not with HuIFN-alpha, to suppress colony formation of CFU-GM, BFU-E, and CFU-GEMM from bone marrow of normal donors at concentrations that had no suppressive effects when molecules were used alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bone marrow cryopreservation: biological aspects

Ten bone marrow suspensions have been cryopreserved by a Programmed Freezer Planer R 201. Total cellularity, viability, differential myelograms, cytochemical pattern and CFU-GM growth "in vitro", have been evaluated on the cellular suspensions both before and after 1 and 18 months of storage in liquid nitrogen. Total cellular recovery and viable cell recovery were satisfactory, cellular loss being due, almost entirely to death of the more mature cells. NASDA reaction did not vary after freezing, on the contrary peroxidase reaction and overall PAS reaction showed respectively a slight and an almost complete disappearance. LAP reaction was not valuable, after freezing, because of the more mature myeloid cell loss. CFU-GM recovery was satisfactory and clusters and colonies growth in methylcellulose appeared quite similar before and after 1 and 18 months of storage at very low temperature. Our cryopreservation technique cannot prevent some cellular loss or some qualitative cellular damage, but colonizing ability is almost completely preserved.     

Role of B cell stimulatory factor 1/interleukin 4 in clonal proliferation of B cells

B cell stimulatory factor 1 (BSF-1)/interleukin 4 (IL-4) has striking effects on colony formation in soft agar by small resting B lymphocytes. BSF-1 alone induces colony formation in this cell population, presumably in costimulation with a mitogenic substance present in bacto-agar. In costimulation with anti-IgM antibodies, BSF-1 caused initial proliferation of 8 to 10% of B cells, resulting in a large number of cell clusters (10 to 40 cells/clone) after 3 to 4 days of incubation. However, substantial number of colonies (greater than 40 cells/clone) developed only from these clusters when IL-1 was added to the cultures. Using a modified immunoperoxidase staining technique for the determination of IgM allotype, evidence was obtained that B cell colonies stimulated with BSF-1 are derived from a single progenitor cell. Neutralization of BSF-1 with 11B11 after a culture period of 1 to 4 days inhibits further proliferation of B cell colonies, indicating that the action of BSF-1 extends for several cell generations beyond initial stimulation from the resting state. Furthermore, it is demonstrated that the synergistic action of IL-1 with BSF-1 is confined to the late culture period, indicating a growth-promoting effect by IL-1 for activated B cells.     

Studies on colony formation in vitro by mouse bone marrow cells. I. Continuous cluster formation and relation of clusters to colonies

Colony formation in vitro by mouse bone marrow cells following stimulation by human urine was analysed over a 7-day incubation period. There was a linear increase with time in the number of cell aggregates (clusters) developing in such plates. Early in the incubation period all clusters were granulocytic although later macrophage clusters developed. Although most fully developed colonies were composed of macrophages, mapping and transfer studies showed that at least half of these had initially arisen early in the incubation period as granulocytic clusters.     

Defending the nest: variation in the alarm aggression response and nest mound damage in the harvester ant Pogonomyrmex occidentalis

A number of studies have found that ant colonies vary in many colony-level phenotypes, including the level of aggression towards non-nestmates. The extent of a colony’s aggression and defense of the nest in response to attacks by predators is likely to affect its survival and reproduction, but the degree to which colonies vary in their defensive response is poorly known. We documented considerable variation in damage to the external nest mound of Pogonomyrmex occidentalis (Cresson) at our long-term study site in 2012. Heavily damaged colonies formed several spatial clusters, consistent with predation within a home range. We tested two competing hypotheses for the variation in nest damage: (1) colonies vary in their level of aggression, those with stronger defensive responses are better protected, and suffer less damage, versus (2) colonies have similar levels of aggression, but those that suffer predator-induced damage subsequently display a stronger defense response. We measured the alarm/aggression response in colonies exposed to a standardized stimulus and determined whether the level of aggression was correlated to the level of damage. Colonies with strong defensive responses were significantly less likely to exhibit damage than those with weak responses, suggesting that aggression level is a colony phenotype.     

Fractal bird nest distribution produces scale-free colony sizes

The spatial distribution of organisms often differs across scales. For instance, colonial bird populations could be described, from large to small scale, as scattered clumps of otherwise regularly distributed breeding pairs. We analysed the distribution of nests of a large colonial population of white storks (Ciconia ciconia) and found a fractal pattern in each of the 4 study years. Moreover, we found that the often-observed, long-tailed frequency distribution of colony sizes was well described by a power law, regardless of the cut-off used to define colonies (from 16 to 1024 m). Thus, although storks were locally highly clumped even with tens of nests in a single tree, the population was not structured in colonies (a simple clustered distribution) as previously thought. Rather, they were distributed in a continuous hierarchical set of clusters within clusters across scales, clusters lacking the commonly assumed characteristic mean size. These quantitative solutions to previously perceived scaling problems will potentially improve our understanding of the ecology and evolution of bird coloniality and animal spacing patterns and group living in general.