Transfllter Studies on Neural Induction in the Newt

In order to study the transmission mechanism of neuralising signals during primary embryonic induction, the interacting components (competent newt gastrula ectoderm and dorsal lip tissues) were separated by filter membranes of varying pore size. Nuclepore filters with nominal pore size from 0.1 to 8 μm were employed and the neuralising effect was shown to traverse all of these membranes. Electron microscopic examination did not reveal any cytoplasmic processes in the pores and the authors conclude that the morphogenetic signals are carried by transmissable compounds rather than through direct cytoplasmic contacts.     

Transmission Problem in Primary Induction

It has been suggested that during the neuralization step of primary induction in the amphibian embryo the inductive signals are mediated from mesodermal cells to the responding competent neuroepithelium by means other than cell contacts. This idea corroborated by experiments in which the interacting tissues were separated by a Nuclepore filter with pores of 0.05 μm (series 1) or by a dialyzing membrane with pores of only 12,000 daltons (series 2). After 18–22 h exposure to mesoderm followed by 8–10 days' culture in isolation the ectodermal explants were neuralized in both series with about 80% differentiating into archencephalic structures. These results exclude the possibility of cell contact as a mediating mechanism in this step.
In a third series similar experiments were made using a special Nuclepore filter with dense pores of 0.6 μm and the exposure time was prolonged to 24 h. During subsequent culture in isolation the ectoderm was neuralized in every case, except forebrain, the ectoderm also differentiated in 25% to hindbrain and less frequently to spinal cord, myotomes, and in some cases even to notochord. The result is interpreted to mean that during the prolonged exposure the tissues have had time to age to an early neurula stage, and the ectodermal cells, after being neuralized, have had time to form cell contacts by cytoplasmic bridges through the pores resulting in the segregation of the preneuralized ectoderm into more caudal structures than the forebrain.     

Picoplankton size distributions in marine and fresh waters: problems with filter fractionation studies

Abstract The retention of algal picoplankton by Nuclepore polycarbonate filters of 0.2, 1.0, 2.0 and 3.0 μm pore size was tested in 2 marine and 3 freshwater sites. When 1 μm Nuclepore filters were used, the percentage of the total cyanobacterial cells passing the filter varied between sites and with increasing depth within sites. As much as 99% of the Synechococcus -like cells was retained by a 1 μm filter. This could lead to an underestimation of the picoplanktonic contribution or, more seriously, an apparent distribution pattern that is an artifact of the choice of filter pore size. Filter retention was also dependent on vaccum pressure during filtration. This study emphasizes the need for direct observation of picoplankton numbers in filter fractionation studies.     

Transfilter Lens Induction in Avian Embryo

The directive influence of the optic vesicle during lens induction of 2-day chick embryos was studied in vitro. Trunk ectoderm was chosen for the responding tissue. This uncommitted ectoderm formed distinct lentoid bodies when grown together with the optic vesicle. Crystallin synthesis was demonstrated in the lentoids with fluorescein labelled antiserum.
By interposing filters of different thicknesses and pore sizes between the interacting tissues, three questions were put forward: (1) Whether the intimate contact between the interactants was essential for induction, (2) how far the inductive influence of the optic vesicle extended, and (3) what was the smallest pore through which the inductive influence could penetrate.
The inductive influence reached across a Millipore filter with a thickness of 100 μm. It also penetrated a dialyzer membrane, allowing passage of molecules with molecular weights (MW) of less than 12,000 daltons. It was concluded that the directive, inductive signal(s) passing from the optic vesicle, diffused in the extracellular space as far as 100 μm and had a molecular weight of less than 12,000 daltons.     

VEGETALIZATION OF PRESUMPTIVE ECTODERM OF NEWT GASTRULAE IN A TRANSFILTER EXPERIMENT WITH FISH SWIMBLADDER AS THE INDUCTOR

The diffusibility of the vegetalizing factor was examined by a transfilter culture using an ethanol-fixed swimbladder of the crucian carp ( Carassius auratus ) as the inductor and presumptive ectoderm from gastrulae of Cynops pyrrhogaster as the responding tissue. Nucleopore filters, about 12–14 μm thick, with nominal pore sizes of 0.05, 0.1, 0.6, 0.8, 3.0 and 8.0 μm were interposed between the interacting tissues. The responding pieces of ectoderm were removed from the assemblies after contact for 0.5, 1, 3, or 24 hr and cultured in Holtfreter's solution for 10 days at 20°C.
The inductions observed were almost entirely mesodermal, although masses of endoderm-like yolky cells were seen in explants and neural tissues in a few cases. Filter membranes with pores of 0.05 to 8.0 μm did not interfere with the vegetalizing effect.
Under an electron microscope, small cytoplasmic cones of the responding cells of the presumptive ectoderm were observed in the pores of the interposed filter after 3 hr's contact. The cones grew longer as the cultivation time increased, but even after 24 hr there was no contact between the interacting tissues. Since 3 hr's contact between the interacting tissues was sufficient to cause full vegetalization on the transfilter culture with the swimbladder, the formation of the cytoplasmic outgrowths had no significance in the induction.     

Transfilter studies on neural induction in the newt.

In order to study the transmission mechanism of neuralising signals during primary embryonic induction, the interacting components (competent newt gastrula ectoderm and dorsal lip tissues) were separated by filter membranes of varying pore size. Nuclepore filters with nominal pore size from 0.1 to 8 mum were employed and the neuralising effect was shown to traverse all of these membranes. Electron microscopic examination did not reveal any cytoplasmic processes in the pores and the authors conclude that the morphogenetic signals are carried by transmissable compounds rather than through direct cytoplasmic contacts.     

Interference of tooth differentiation with interposed filters

The effect of interposed Nuclepore filters on the epithelio-mesenchymal interaction in embryonic mouse tooth was studied. Filters with pore sizes of 0.6 and 0.2 μm allowed differentiation of odontoblasts and ameloblasts in the bell-stage tooth germ. This differentiation progressed more rapidly when the 0.6-μm pore size filter was used. Nuclepore filters with 0.1-μm pores prevented differentiation. Electron microscopic examination revealed penetration of cell processes into the filter pores. Cytoplasmic material could be seen in the 0.6-μm pore-size filter within 3 days of cultivation, whereas, in the 0.2-μm filter pores, penetration was slight. After 6 days of cultivation, cytoplasmic material was found at all levels of the 0.2-μm pore-size filter, but not in the channels of the 0.1-μm pore-size filters, preventing differentiation. It is concluded that the 0.1-μm pore-size filter blocks tooth development at the level of mesenchymal cell differentiation into odontoblasts. It is suggested that this differentiation requires a close association between the interacting mesenchymal and epithelial cells.     

The influence of epithelia on cartilage and loose connective tissue formation by limb mesenchyme cultures

In order to explain the observation that normally nonchondrogenic limb mesenchyme forms extensive cartilage in culture, the possibility that limb ectoderm inhibits chondrogenesis is examined. Small pieces of quail or chick ectoderm are grafted onto micromass cultures of wing mesenchyme from stage 23–34 chick embryos. The presence of nonridge wing or several other epithelia results in increased collagen accumulation in the underlying mesenchyme, a delay in cell differentiation, and the eventual formation of loose connective tissue, as determined by transmission electron microscopy. The influence can occur across Nuclepore filters having 0.1-μm-diameter pores and ultrathin Millipore filters, but not across Millipore filters of standard thickness. The influence is not contact dependent since cell processes do not cross these filters. The apical ectodermal ridge has the additional effect of stimulating mesenchymal outgrowth. These results support the idea that the ectoderm plays a direct but negative role in the formation of a chondrogenic core within the developing limb.     

The importance of bacterial utilization of released phytoplankton photosynthate in two humic forest lakes in southern Finland

Bacterial utilization of photosynthetically fixed dissolved organic carbon (PDOC) released from natural phytoplankton assemblages was studied in two small, extremely humic, forest lakes in southern Finland. Bacterial activity (measured as uptake of ¹⁴C-glucose) and phytoplankton photosynthesis (measured as light uptake of ¹⁴CO₂) could be most effectively separated using Nuclepore filters of pore size 1–2 μm. Released PDOC was 10–67% of total phytoplankton carbon fixation during in situ experiments, and represented about 0.1% of total DOC. Net uptake of PDOC by bacteria was found to be about 20% during 24 hour laboratory incubations, although about 40% of PDOC present at the start of an experiment could be utilized by bacteria during a 24 hour period. PDOC does not provide a quantitatively important substrate supply for bacterial respiration in humic forest lakes.     

Transfilter analysis of the inductive influence of proventricular mesenchyme on stomach epithelial differentiation of chick embryos

Summary To clarify the precise conditions under which chick embryonic proventricular mesenchyme can induce proventricular epithelial differentiation, transfilter experiments were carried out. Six-day proventricular epithelium formed glands and expressed pepsinogen when a Nucleopore filter with a pore size of more than 0.6 m, but not 0.2 m, was inserted between the epithelium and the proventricular mesenchyme. The larger the pore size of the filter, the more elongated the glands and the more pepsinogen was induced in the explants. The quail nuclear marker and scanning electron microscopy were used to examine penetration of mesenchymal cells through the Nuclepore filter. The filter of more than 0.2 m pore size allowed cell processes of mesenchymal cells to pass through. However, only the filter with a pore size of more than 0.6 m allowed actual migration of mesenchymal cells through the filter, and the larger the pore size of the filter, the more mesenchymal cells passed through. Under the same conditions 6-day and 4.5-day gizzard epithelium formed glands and expressed pepsinogen. These results indicate that a flow of diffusible substances through a Nuclepore filter and even direct contact of a few short cell processes of mesenchymal cells with epithelial cells are not sufficient for induction, and that direct contact of mesenchymal cell processes and/or mesenchymal cells with epithelial cells over a considerably wide area may be prerequisite for the induction.     

Effect of temperature on the deformability of a lamprey, Entosphenus japonicus, and Pacific salmon, Oncorhynchus keta, red blood cells, studied using a modified filtration method

The rates of filtration through Nuclepore filters (5 or 8 μm) of blood from lampreys and Pacific salmon have been studied using a method which visualizes the flow pattern. From these measurements, passage times for single red blood cells have been calculated and serve as an index of their deformability. The deformability increases as temperature is raised in vitro , but even at 5°C the passage time of lamprey blood is relatively rapid. The increase in deformability with a rise in temperature is small relative to that found in other fish such as yellowtail and carp.
The distribution of red cell volumes has shown the presence of a secondary peak for salmon blood taken during surgery which is reduced following recovery, the main peak being at a lower volume.     

On the microparticles which pass through glass fiber filter type GF/F in coastal and open waters

The chlorophyll a content of nicroparticles which passed throughglass fiber filters Whatman type GF/F but were retained on 0.2µm Nuclepore membranes was analyzed on a weekly basisover the course of 1 year in Kaneohe Bay, Hawaii. Depth profileswere also obtained at four oceanic stations off the islandsof Maui and Molokai, Hawaii. Experimental evidence indicatedthat these microparticles were photosynthetically active. Theproportion of microparticulate chlorophyll a could be up to35% of picoplankton chlorophyll a (2.0–0.2 µm sizerange) retained on a single pass through a 0.2 p.m Nucleporefilter. The filtrate from both GFIF and 0.2µm Nucleporefilters was found to contain chlorophyll a which could be retainedon a subsequent pass through either 0.2 µm Nuclepore orGF/F filters. Only serial filtration can ensure that essentiallyall picoplankton have been filtered from the water when eitherof these types of filters is used.     

The detection and characterization of bacteria-sized protists in “Protist-free” filtrates and their potential impact on experimental marine ecology

Nuclepore filters of 0.6–1.0m pore size have been used to prepare protist-free water for a number of studies in microbial ecology. This procedure has been called into question by a recent study claiming that a significant portion of bacterial loss in filtrates could be due to uncharacterized predators passing through 0.6m filters. We were unable to directly observe protists in 0.6m filtrates using phase contrast, epifluorescence, or transmission electron microscopy. Using the culture techniques of rice grain enrichment and most probable number, however, we were able to observe and quantify several species of bacterivorous nanoflagellates that developed not only in 0.6m, but also in 0.4m seawater filtrates. The ability of predacious nanoflagellates to squeeze through bacteria-sized pores questions studies of bacterial production and chemical cycling that have assumed protist-free filtrates.     

Simultaneous measurement of phosphorus and carbon uptake in Lake Kinneret by multiple isotopic labeling and differential filtration

Differential filtration and multiple isotopic labeling were combined to study the uptake of [¹⁴C]bicarbonate, [¹⁴C]glucose, and [³²P]orthophosphate by microplakton in Lake Kinneret, Israel. Short-term (4 hr) uptake experiments showed seasonal changes in the size distributions of organisms taking up inorganic carbon, glucose carbon, and orthophosphate in the lake water. In a time-course experiment of 48 hr (Jan 1976) most, but not all, of the photosynthetic activity (average 72%) and a similar fraction of chlorophyll (72%) were associated with organisms retained on 3-m Nuclepore filters (retention on 0.4-m filters was 100%). About 90% of the organisms that assimilated glucose passed through 3-m filters. Photosynthetic carbon fixation, dark carbon uptake, and heterotrophic uptake of glucose carbon accounted for 99%, 1%, and 1%, respectively, of the total net carbon assimilated during the first 6 hours. Radioactive phosphorus showed an initial rapid uptake into particles, which was not affected by light or dark. We suggest that this methodology has a wide potential for elucidating the flux of nutrients into various components of the microplankton and in characterizing different aquatic environments.     

Comparison of two direct-count techniques for enumerating aquatic bacteria.

Planktonic bacteria from an estuary were concentrated on membrane filters and counted with both a scanning electron microscope and an epi-illuminated fluorescent microscope. Counts on 0.2 micron Nuclepore filters (polycarbonate) were significantly higher (P less than 0.001) than counts on 0.2-micron Sartorius filters (cellulose). In contrast, there was not a statistically significant difference between the two techniques when Nuclepore filters were used (0.5 less than P less than 0.9). The average cell volume from this study area was 0.047 micron3. The estimated number of bacteria ranged from 10(6) to 10(7) bacteria per ml, representing from 4 to 40 mg of C per m3.     

Comparison of two direct-count techniques for enumerating aquatic bacteria.

Planktonic bacteria from an estuary were concentrated on membrane filters and counted with both a scanning electron microscope and an epi-illuminated fluorescent microscope. Counts on 0.2 micron Nuclepore filters (polycarbonate) were significantly higher (P less than 0.001) than counts on 0.2-micron Sartorius filters (cellulose). In contrast, there was not a statistically significant difference between the two techniques when Nuclepore filters were used (0.5 less than P less than 0.9). The average cell volume from this study area was 0.047 micron3. The estimated number of bacteria ranged from 10(6) to 10(7) bacteria per ml, representing from 4 to 40 mg of C per m3.     

Electropositively charged filters for the recovery of yeasts and bacteria from beverages

The ability of electropositively charged filters to recover yeasts and lactic acid bacteria from a variety of beverages was evaluated. Filtration through 'Zeta plus', grade OSS, filters recovered nearly all of the yeast contaminants from table wines, sherry and port. Recovery of yeasts from cream liqueurs and egg-based beverages was also good but it was not possible to filter drinks containing orange juice, even through filters with nominal pore sizes of 2 to 10 μm. Lactic acid bacteria proved more difficult to recover than yeasts, even though smaller pore-sized filters (1 to 4 μm) were employed. However, a sufficiently high percentage of bacteria were recovered to justify use of these filters for quality assurance. The advantage of concentrating contaminants by using charged filters, and the influence of product composition on the efficiency of microbial adsorption are discussed. The growth of wine-spoiling yeasts and lactic acid bacteria were not inhibited by water- or ethanol-soluble extracts of the filter material.     

Collection of airborne micro-organisms on Nuclepore filters, estimation and analysis—CAMNEA method

The total number of airborne micro-organisms collected on Nuclepore filters was determined by acridine orange staining and epifluorescence microscopy. The viable fraction of the total numbers varied significantly when actinomycete and fungal spores from different environments were stored on the filter surface for 1 week, although the microflora composition was not altered. A high correlation between viable and total counts was noted in environments where the airborne flora was dominated by fungal spores, while a low correlation was found for airborne bacteria. Peak values of the total counts registered in some work environments varied between 10⁷ and 10¹¹ micro-organisms/m³. Size analysis showed a dominating fraction of respirable micro-organisms (aerodynamical diameter < 5 μm). The investigation shows that it is of the utmost importance to combine viable counts with total count enumeration in the study of exposure to micro-organisms in work-related situations.     

Binding of anti-fibronectin to early amphibian ectoderm does not result in inhibition of neural induction under in vitro conditions

Summary Antibodies directed to fibronectin (anti-FN) were injected into the blastocoel of late blastulae of Xenopus laevis. Two animal caps (ectoderm) were isolated, when control embryos reached the early gastrula stage, and were combined with untreated upper blastopore lip in the sandwich method. In two control series fibronectin or Holtfreter solution was injected into the blastocoel. The results of the experiments suggest that neural induction cannot be prevented by binding anti-FN to fibronectin, which covers the blastocoelic side of the ectoderm. The data support the view that extracellular matrix proteins are not themselves responsible for neural induction. However, in comparison with the control series a slight shift of the differentiation pattern in the spinocaudal direction could be observed in the anti-FN series. The possible role of extracellular proteins in the formation of a close juxtaposition of mesodermal and ectodermal target cells as a prerequisite for shortdistance transmission of neural inducers is discussed.     

Detection of viable, but non-culturable Pseudomonas fluorescens DF57 in soil using a microcolony epifluorescence technique

Abstract Kanamycin-resistant Pseudomonas fluorescens DF57-3 cells (Tn5 modified) inoculated in soil microcosms rapidly lost their culturability, as defined by visible colony formation on Kings B agar supplemented with kanamycin. Thus, after 40 days only 0.02–0.35% of the initial inoculum was culturable. A microcolony epifluorescence technique was developed to determine the viable, but non-culturable subpopulation. A suspension of bacteria from the soil was prepared in salt solution after a sonication procedure and a sample was filtered onto a 0.2 μm Nuclepore filter. The filter was then placed for 3–4 days on the surface of Kings B agar before staining with acridine orange for epifluorescence microscopy. By staining and washing the filters carefully, disruption of microcolonies could be avoided. A majority of the microcolonies resulted from 2–3 cell divisions during the first 2 days of the incubation period, after which the cell divisions stopped. These microcolonies were taken to represent a population of viable, but non-culturable cells and comprised about 20% of the initial inoculum. A similar recovery was obtained when the filters were incubated on the surface of citrate minimal medium or soil extract medium. A few microcolonies showed continued growth on the filters, however, and their number corresponded well with that of visible macrocolonies. Observation by microscopy of a few (2–3) cell divisions (microcolony epifluorescence technique) is proposed for determination of subpopulations of viable, but non-culturable bacteria in soil.