In situ microscopy for on-line determination of biomass

A sensor is presented, which allows on-line microscopic observation of microorganisms during fermentations in bioreactors. This sensor, an In Situ Microscope (ISM) consists of a direct-light microscope with a measuring chamber, integrated in a 25 mm stainless steel tube, two CCD-cameras, and two frame-grabbers. The data obtained are processed by an automatic image analysis system. The ISM is connected with the bioreactor via a standard port, and it is immersed directly in the culture liquid-in our case Saccharomyces cerevisiae in a synthetic medium. The microscopic examination of the liquid is performed in the measuring chamber, which is situated near the front end of the sensor head. The measuring chamber is opened and closed periodically. In the open state, the liquid in the bioreactor flows unrestricted through the chamber. In closing, a defined volume of 2,2. 10(-8) mL of the liquid becomes enclosed. After a few seconds, when the movement of the cells in the enclosed culture has stopped, they are examined with the microscope. The microscopic images of the cells are registered with the CCD-cameras and are visualized on a monitor, allowing a direct view of the cell population. After detection, the measuring chamber reopens, and the enclosed liquid is released. The images obtained are evaluated as to cell concentration, cell size, cell volume, biomass, and other relevant parameters simultaneously by automatic image analysis. With a PC (486/33 MHz), image processing takes about 15 s per image. The detection range tested when measuring cells of S. cerevisiae is about 10(6) to 10(9) cells/mL (equivalent to a biomass of 0.01 g/L to 12 g/L). The calculated biomass values correlate very well with those obtained using dry weight analysis. Furthermore, histograms can be calculated, which are comparable to those obtained by flow cytometry.     

A root observation chamber for replicated use in a natural plant community

Summary A relatively small, low cost root observation chamber of steel construction has been designed for replicated use in a natural plant community. An undisturbed soil profile, suitable for detailed study can be obtained by avoiding conventional backfilling methods and despite such factors as the recognized need for teh chamber roof to be below ground level. Installation procedure emphasizes preparation of the observation face in established woody plant communities. The technique incorporates microscopic study of roots and simultaneous recording of major soil climatic factors.     

A simple trickle chamber for rearing aquatic invertebrates

A chamber for laboratory rearing of aquatic invertebrates is described. The animals are reared in plastic dishes, through which a recirculating water volume is passed. The design ensures identical water quality in a high number of parallels. The chamber is constructed for rearing the freshwater amphipod Gammarus pulex, but can be used in studies of growth, reproduction etc. of other aquatic invertebrates as well.     

High pressure microscopy--a powerful tool for monitoring cells and macromolecules under high hydrostatic pressure.

A high pressure chamber, which withstands a pressure up to 300 MPa has been developed. The so-called HPDS (Hartmann, Pfeifer, Dornheim, Sommer) High Pressure Cell in combination with an inverted microscope and an analysis system allows brilliant microscopic colour pictures with an optical resolution better than 0.56 microm. The pressure chamber allows the in situ observation of dynamic changes of microscopic structures in bright field, phase contrast and fluorescence microscopy. This publication should demonstrate the capabilities of the system using results of experiments with two types of Spirogyra algae. The pictures have shown significant variations of the chloroplasma and the cell wall membrane at pressures of up to 120 MPa. The new system provides a simple way to perform microscopic analyses at pressures of up to 300 MPa.     

Construction of up-and-down temperature-jump apparatus

A fast up-and-down temperature-jump apparatus whose dead time is about 60 ms was constructed. The principle of the method is to let the sample solution flow to the observation cell through a capillary in a heat-exchange chamber. Bubbling and cavitation effects in the observation cell at large up or down temperature jumps were eliminated by application of a nitrogen gas pressure of 2-5 bar. The down-temperature-jump method is especially effective for measuring temperature-induced conformational transitions of biopolymers and their assemblies.     

Structural organization of the copulation site in the medfly Ceratitis capitata (Diptera: Tephritidae) and observations on sperm transfer and storage

The copulation site of the medfly Ceratitis capitata was investigated at anatomical and ultrastructural levels. It consists of the anterior vagina, with a ventral fertilization chamber and a dorsal insemination pocket into which the two spermathecal ducts open. The fertilization chamber is an organ comprised of a number of alveoli that in virgin females are filled with a filamentous secretion, whereas in mated females contain sperm bundles. Through study of the internal morphology of the aedeagus, its position in the anterior vagina, and the direct observation of sperm transfer and storage, we confirmed that sperm are ejaculated through two gonopores at the top of the distiphallus and another at the base of the genital rod. The sperm flow dorsally into the insemination pocket and ventrally into the fertilization chamber. During copulation, the two spermathecae and the fertilization chamber are progressively filled with spermatozoa.     

A Perfusion Chamber with Temperature Regulation

When investigating microscopic preparations perfusion chambers allow exchange and regulation of different solutions and ensure their constant flow in the sample chamber. Temperature deviations, however, may be problematic. We describe a new chamber that contains an additional circulation system which regulates the inside temperature using an external thermostat. An integrated thermometer probe records the sample temperature, which appears on a monitor. The glass chamber. measuring 75 × 35 × 3 mm, provides good optical quality and is compatible with every type of microscope.     

Observations Relating to the Efficiency of Mixing in Rapid Reaction Flow Devices

The principle of dimensional analysis of liquid flow has been applied to the problem of rapid mixing in flow apparatus. A model of the Hartridge-Roughton mixing chamber and observation tube has been scaled approximately 1/1.000 so that the times after mixing are approximately 6 s, the flow velocities are the order of 2 cm/s, and the distance from mixing to observation is 120 mm. Visual observation is employed to observe the end point of the mixing of acid-base with a color indicator, thymol blue. Quantitative estimates of the effect of the number of jets, the effect of screens placed in the flow stream, and the angle of jets, one with respect to another, lead to quantitative evaluations of these parameters for the scale model. The extent to which these parameters apply to the full-scale apparatus at faster flow velocities suggest that the general principles employed in the scale model are valid, although more emphasis is placed upon turbulence generation at the low flow velocities employed in this experiment than at the faster flow velocities employed in the full-scale apparatus.     

Two-dimensional trajectory analysis of the diatom Navicula sp. using a micro chamber

We describe a trajectory analysis of diatom cell locomotion by combining a micro chamber and two-dimensional position coordinate analysis. By shutting cells in a micro chamber, continuous microscopic observation of Navicula sp. cells was possible. The trajectory of each cell was visualized once every second by using position coordinate analysis although time resolution of previous papers were range of minutes. Our data revealed frequent change of movement direction. Furthermore, the correlation between the distances moved, the velocity, and the acceleration of the cells was discussed in detail.     

Two-dimensional trajectory analysis of the diatom Navicula sp. using a micro chamber

We describe a trajectory analysis of diatom cell locomotion by combining a micro chamber and two-dimensional position coordinate analysis. By shutting cells in a micro chamber, continuous microscopic observation of Navicula sp. cells was possible. The trajectory of each cell was visualized once every second by using position coordinate analysis although time resolution of previous papers were range of minutes. Our data revealed frequent change of movement direction. Furthermore, the correlation between the distances moved, the velocity, and the acceleration of the cells was discussed in detail.     

A simple method for examining organotypic CNS cultures with nomarski optics

Summary This paper describes a method for examination of living organotypic cultures of CNS with Nomarski differential interference-contrast optics. Cultures grown in Maximow assemblies. which promote the best differentiation of the tissue but are optically faulty, are transferred for Nomarski observation to a simple sandwich chamber which combines the optical perfection of the usual sandwich chamber with the flexibility and safeguarding of sterility characteristic of the Maximow assembly. Thus cultures can be transferred repeatedly between their maintenance and observation chambers. In the resulting microscopic images, it is posible to visualize delicate unmyelinated fibers, myelinated cell bodies and other features which are normally impossible to demonstrate in living cultures as well as to improve the images of other structures such as large neuronal perikarya and myelinated axons. This work was supported in part by NIH Grant NS 11425     

Two types of assays for detecting frog sperm chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm. Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg. Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 μm diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer. The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.     

Simple flow chamber for embryos]

The chamber is made of two subject glasses arranged in such a way that nutritional medium can flow through it. In order to prevent washing out of the embryos by the stream of nutritional medium, a porous packing is inserted through which the nutritional medium is flowing. The upper subject glass has a hole through which the embryos are put into the chamber. The hole is covered with a covering glass. The chamber can be preserved for a long time "avaiting" for the cultivated material at any regime of the stream or at a static regime.     

The effect of a single burn event on the aquatic invertebrates in artesian springs

Fire can often occur in aquatic ecosystems, which may affect aquatic invertebrates. Despite the importance of aquatic invertebrates to ecosystem function, the effect of fire on these environments has been little studied. We studied the effects of fire on aquatic invertebrates in artesian springs in the arid zone of South Australia. Artesian springs are a unique and threatened ecosystem, containing several rare and endemic species. Evidence suggests these wetlands were routinely burnt by indigenous Aboriginal people before European settlement over 100 years ago. Recently, burning has been suggested as a reinstated management tool to control the dominant reed Phragmites australis. A reduction in the cover of the reed may benefit the threatened flora and fauna through enhancement of water flow. Three artesian springs were burnt and aquatic invertebrates sampled from the burnt and three unburnt springs. A single fire in late winter completely burnt the dominant vegetation, followed by recovery of Phragmites over the following 2 years. A single fire event did not deplete populations of endemic aquatic invertebrates in artesian springs, but probably did not substantially benefit these populations either. Isopods, amphipods, ostracods and three species of hydrobiid snail survived the fire event, and most had increased in number 1 month post fire but then returned to pre‐burnt numbers by 1 year post fire. Morphospecies richness of all identified invertebrates increased over time in all springs, but did not differ appreciably between burnt and unburnt springs. If burning artesian springs is to be adopted as a management tool to suppress the growth of Phragmites australis, we conclude that the endemic aquatic invertebrates will survive a single burn event, without negative effect to their populations.     

A long-term stable and adjustable osmotic pump for small volume flow based on principles of phloem loading

An adjustable pump for microfluidics employing principles of osmoregulation analogous to those of phloem loading in plant leaves has been constructed and tested. Volume flow arises in a hollow fibre with vapour-permeable hydrophobic membrane. The fibre is connected to a source chamber filled with salt crystals and saturated salt solution. The source chamber takes up water through a relatively small membrane area and delivers saturated salt solution to one end of the capillary flow path within the hollow fibre. A stationary osmotic gradient is sustained in the hollow fibre lumen by constant input of saturated salt solution and radial osmotic water absorption. The strong temperature dependence of isothermal membrane distillation enables adjustment of the flow rate up to 20 nL/s. The pump provides pulse-free flow of any liquid with constant rate for at least 26 days without recharging the source chamber. Backpressures up to 1 bar decrease the flow rate by less than 4%. The volume delivered at a constant rate is more than 40 times larger than the volume of the source chamber. Osmoregulatory pumps of the described type may be useful for microinfusion, microdialysis and analytical microsystems.     

Fluid flow in a spiral device used for irradiation of biological fluids

The manufacture of plasma‐derived therapeutics includes dedicated viral inactivation steps to minimize the risk of infection. Traditional viral inactivation methods are effective for the removal and inactivation of enveloped viruses, but less effective against small nonenveloped viruses. UV‐C irradiation has been demonstrated to be an effective means of inactivating such viruses. The UVivatec lab system consists of a spiral tube around an UV‐C irradiation source. Flow of a solution through the chamber generates and ensures controlled mixing and uniform exposure to irradiation. A detailed assessment of the effect of flow rate, alternate cross sectional design and scale up of the irradiation chamber on Dean vortices was performed using the smoothed particle hydrodynamics method. The aim was to provide a basis for setting flow rate limits and using a laboratory scale apparatus to model viral inactivation in larger manufacturing scale equipment. The effect of flow rate related changes on the fluence rate was also investigated through chemical actinometry studies. The data were consistent with the simulations indicating that Dean vortices were present at low flow rates, but dissipated at higher flow rates through the spiral chamber. Importantly, this work also allowed a correlation between the small system and large scale system to be established. This will greatly facilitate process development and viral validation studies. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 359–367, 2013     

Effect of the Environmental Factors on the Caloric Content of Benthic and Phytophilous Invertebrates in Neotropical Reservoirs in the Paraná State,Brazil

The complexity of the relations between the organisms and the environment are reflected through the energy content. So we tested the hypothesis on body energy content variation in invertebrates and its relation with some environmental factors (season, habitat type, trophic status and trophic guild). We expected higher energy values for phytophilous fauna, rainy season, in reservoirs with higher trophic levels and for herbivorous invertebrates. The results showed the influence of seasonality and trophic status of the reservoir on the energy content of invertebrates, which was higher in the rainy season and in the mesotrophic reservoirs, due to the input of allochthonous material during this season, confirming our initial prediction. A higher caloric content was recorded for carnivorous and detritivorous invertebrates. The differences between the trophic guilds were primarily related to the opportunistic feeding behavior, which may be considered as one of the main factors controlling the energy flow of benthic and phytophilous invertebrates. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Optical chamber system designed for microscopic observation of living cells under extremely high hydrostatic pressure

A novel pressure chamber system has been developed for the study of living cells under conditions of extremely high hydrostatic pressure up to 100 MPa (1 atm = 0.101325 MPa). The temperature in the chamber is thermostatically controlled in the range from 2 degrees to 80 degrees C. Two high-pressure pumps are employed for continuous perfusion of the chamber with culture medium and a chemical solution under high hydrostatic pressure conditions. The chamber has a 2-mm-thick glass window 2 mm in diameter, with a minimum working distance of 3.8 mm. The chamber system is designed to be adaptable to a variety of microscopic and imaging techniques. Using this chamber system, we successfully carried out real-time observations of elongated Escherichia coli and rounded HeLa cells under pressure.     

Water fleas (Daphnia magna) provide a separate ventilatory mechanism for their brood

The planktonic filter feeder Daphnia magna depends on a steady oxygen supply by convection. In the ventral carapace chamber, this convection is established by the feeding current which is generated by the movement of the thoracic limbs. The present study revealed that this movement can cause an additional flow of medium which passes through the brood chamber of the animal. To visualise this current, ink or fluorescent microspheres were released by a microcapillary near the posterior opening of the brood chamber. The tracks of these probes were monitored by video microscopy. Digital motion analysis was used for the determination of flow velocity and flow rate. Ambient medium entered the brood chamber at the abdomen of the animal and moved then to the anterior end of the brood chamber before entering the ventral carapace chamber. Two horizontal lamellae, which are attached at both sides of the trunk and project laterally to contact the carapace walls, almost completely separate the dorsal brood chamber from the ventral carapace chamber. Water can only pass these barriers through small depressions in these lamellae at the level of the 3rd and 4th appendages. Female daphnids with embryos at late developmental stages showed more rapid water currents in the brood chamber than those with younger embryos. Moreover, animals showed higher flow rates when exposed to hypoxic conditions. As the oxygen uptake rate of older embryos is approximately three times higher than that of younger embryos, the enhanced brood chamber current could improve the oxygen availability for both the mother and its brood under conditions of reduced oxygen availability.     

Combined imaging of bacteria and oxygen in biofilms

Transparent sensors for microscopic O(2) imaging were developed by spin coating an ultrathin (<1- to 2-microm) layer of a luminescent O(2) indicator onto coverslips. The sensors showed (i) an ideal Stern-Volmer quenching behavior of the luminescence lifetime towards O(2) levels, (ii) homogeneous measuring characteristics over the sensor surface, and (iii) a linear decline of luminescence lifetime with increasing temperature. When a batch of such coverslip sensors has been characterized, their use is thus essentially calibration free at a known temperature. The sensors are easy to use in flow chambers and other growth devices used in microbiology. We present the first application for combined imaging of O(2) and bacteria in a biofilm flow chamber mounted on a microscope equipped with a spinning-disk confocal unit and a luminescence lifetime camera system.