Use of a fluid dispensing apparatus for bronchoalveolar lavage in mice

The volume of the mouse lung is small, so bronchoalveolar lavage (BAL) in mice is generally performed with 1 ml syringes to infuse smaller volumes of fluid. Multiple infusions are required to obtain enough recovered fluid for multiple analyses. This paper introduces the use of one type of a simple fluid dispensing apparatus as an infusion device. It proved to be a faster and a less tedious method than the syringe infusion method. The results of studies in normal mice using both infusion techniques showed no differences between the two with respect to the recovery of cells and protein and to differential leukocyte counts. Thus, the results obtained with this device can be compared with those previously obtained with syringes.     

A self-refilling syringe for dispensing anaerobic media

Summary A standard Luer-lock self-refilling syringe was modified to dispense anaerobic medium at pre-set volumes. It is suitable for transferring anaerobic medium for serial dilutions, where a consistent volume delivered is necessary. Sulfate reducing and methanogenic bacteria have been cultivated routinely on media dispensed with this apparatus.     

Apparatus for measuring rat body volume: a methodological proposition.

We propose a communicating-vessels system to measure body volume in live rats through water level detection by hydrostatic weighing. The reproducibility, accuracy, linearity, and reliability of this apparatus were evaluated in two tests using previously weighed water or six aluminum cylinders of known volume after proper system calibration. The applicability of this apparatus to measurement of live animals (Wistar rats) was tested in a transversal experiment with five rats, anesthetized and nonanesthetized. We took 18 measurements of the volume under each condition (anesthetized and nonanesthetized), totaling 90 measurements. The addition of water volumes (50-700 ml) produced a regression equation with a slope of 1.0006 +/- 0.0017, intercept of 0.75 +/- 0.81 (R(2) = 0.99999, standard error of estimate = 0.58 ml), and bias of approximately 1 ml. The differences between cylinders of known volumes and volumes calculated by the system were <0.4 ml. Mean volume errors were 0.01-0.07%. Among the live models, the difference between the volumes obtained for anesthetized and nonanesthetized rats was 0.31 +/- 2.34 (SD) ml (n = 90). These data showed that animal movement does not interfere with the volume measured by the proposed apparatus, and neither anesthesia nor fur shaving is needed for this procedure. Nevertheless, some effort should be taken to eliminate air bubbles trapped in the apparatus or the fur. The proposed apparatus for measuring rat body volume is inexpensive and may be useful for a range of scientific purposes.     

Trypsinization of Animal Tissues for Cell Culture: Theoretical Considerations and Automatic Apparatus

The theoretical background and the experimental feasibility of an automatic control for the continuous trypsinization process are discussed. An automatic apparatus is described with experimental evidence that the optimal mean residence time of monkey kidney cells in the trypsinization flask is between 5 and 8 min, in a volume of fluid approximately ten times that of the tissue processed. A temperature of 36 C and a pH of 7.8 provide optimal conditions for cell viability.     

Microapplicator and micropipette for inoculation of the embryonated chicken egg.

Aflatoxin G2 was used to test the speed and accuracy of the microapplicator and the micropipette. The 50% lethal dose of both assay systems was approximately the same, and 4.0 mug of G2 had an 85% lethal effect in both systems. The 50% lethal dose of the microapplicator and the micropipette was lower than that of the syringe but, of these, only the micropipette can combine the accuracy of the microapplicator and the speed of the syringe.     

Microapplicator and micropipette for inoculation of the embryonated chicken egg.

Aflatoxin G2 was used to test the speed and accuracy of the microapplicator and the micropipette. The 50% lethal dose of both assay systems was approximately the same, and 4.0 mug of G2 had an 85% lethal effect in both systems. The 50% lethal dose of the microapplicator and the micropipette was lower than that of the syringe but, of these, only the micropipette can combine the accuracy of the microapplicator and the speed of the syringe.     

Validation of equilibration and chromium reduction methods for deuterium measurements of fluid volumes.

Determinations of fluid volumes are of importance for correct treatment of patients subjected to shock and trauma. Gas isotope ratio mass spectrometry (GIRMS) is an advanced method for analysis of stable isotopes. These can be used as tracers for measurement of various fluid volumes. In the current in vitro study, deuterium was used to determine different volumes of water simulating a range of body fluid volumes from neonates to adults. A high-precision scale gave control weights (i.e., volumes), and two methods, equilibration (EQ) and chromium reduction (CR), were compared by use of a GIRMS. The coefficient of variation was <1% when using both EQ (0.45%) and CR (0.79%). The variability was greater at small volumes, and, when regression equations for the relation between measured and calculated volumes were used as formulas, the deviation was 0.4% using EQ and 2.8% using CR at the volume of 1,000 ml. At larger volumes, the deviation when using CR approached 1%. These variations are better than previously published data using other methods. It was concluded that GIRMS is a suitable technique for fluid volume determinations in neonates as well as in adult patients, using deuterium as a tracer. EQ and CR methods were both regarded to give acceptable variabilities in this in vitro study. GIRMS may in the future increasingly be used clinically for accurate measurements of body fluid volumes.     

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission¹ by delivering neurotransmitters and neuropeptides onto neurons reproducibly². Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation³ has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo^4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain⁶. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current^2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be ˜ 1 μm or less to minimize diffusional ''leak'', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.Fabrication of micropipettes is facilitated with sophisticated electronic pullers⁸. Micropipettes are pulled from glass capillary tubes containing a filament that ''wicks'' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). The present protocol demonstrates microiontophoresis of acetylcholine (ACh⁺ Cl^-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40^BAC-GCaMP2 transgenic mice⁹ enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.Download video file.^{(37M, mov)}     

Estimates of mouse oviductal fluid tonicity based on osmotic responses of embryos

Zygotes and early cleavage-stage embryos are very sensitive to increased osmolality in vitro, although the tonicity of their in vivo environment, oviductal fluid, is unknown. A preference for low osmolality in vitro might imply similar conditions in vivo or be specific to culture. Previous electron probe x-ray microanalysis measurements of total ion content predicted oviductal fluid osmolalities of 310-360 mOs/kg, higher than osmolalities tolerated by mouse zygotes in vitro. However, such indirect estimates may not reflect the tonicity experienced by embryos. We have now used embryos themselves as osmosensors to determine the tonicity of mouse oviductal fluid. In one method, we measured the mean volume of zygotes in undiluted oviductal fluid and compared this to the mean volumes measured for zygotes in media spanning a range of osmolalities. The osmolality corresponding to the measured mean volume in oviductal fluid was taken to be isotonic. In another, independent method, the sizes of zygotes and two-cell embryos were measured as a function of time beginning immediately after removal from oviducts. The osmolality in which the embryos neither swelled nor shrank was taken to be isotonic. Both methods yielded approximately the same range for the tonicity of oviductal fluid: around 290-300 mOs/kg.     

Lung volume and compliance in neonatal rats

Lung volumes and static lung compliance were measured in decapitated three day-old neonatal Long Evans' rat pups. Compliance was measured in situ (open chest method) using a water manometer and syringe system. Mean total lung capacity at 20 cm H2O pressure (TLC20) was 0.678 ml. Minimum lung volume after experimental inflation was 0.197 +/- 0.048 ml, and vital capacity was 0.56 ml (Vmax20). The mean lung compliance value for the approximate tidal loop (between 3 and 12 cm H2O) equalled 26.2 microliters air/cm H2O for the inflation limb and 23.1 microliters/cm H2O for the deflation limb.     

A computer-controlled multichannel micropipetter

A multichannel micropipetter was developed capable of pipetting as little as 1 μl with a reproducibility of better than ±2% and an accuracy of ±0.5%. The micropipetter consists of a precision syringe to which 13 individually valved fluid channels are connected as a bundle of segments spreading out radially from the tip of the syringe to pinch valves and further to fluid interfaces consisting of steel tubing sections for uptake or dispensing of fluid. A stepping motor drives the piston of the measuring syringe by means of a precision screw. Motor and valves are under computer control. Low dead volume (internal volume, ca. 1 μl/channel; external volume, 0.3 μl/cm of tubing), and the absence of internal valving parts ensure low cross-conamination (ca. 0.1%). These features together with the versatility provided by the large number of independent channels and the automatic operation make the instrument suitable for pipetting multicomponent mixtures in the general biochemistry laboratory (for enzyme kinetics and complex reactions) as well as in specialized routine applications (clinical diagnostics and radioimmunoassay).     

A semi-automated in vitro gas production technique for ruminant feedstuff evaluation

This technical note describes the Reading Pressure Technique (RPT) – an in vitro feed evaluation system based on a semi-automated gas production technique. Substrates are incubated together with buffered rumen fluid in sealed fermentation flasks. A pressure transducer, interfaced with a PC allows accumulated head-space gas pressure values to be directly entered into a spreadsheet. These pressure measurements are then used to generate gas volume estimates using a quadratic function derived from simultaneous pressure and volume measurements. Earlier attempts to derive volume from pressure resulted in predicted volume being under estimated. This discrepancy was most likely due to diffusion of head-space gases into the liquid phase, a factor not considered in Boyle's Law, the initial relationship used. In comparison with the syringe technique (Theodorou et al., 1994), the modifications improved both the accuracy (reduced operator error) and rate at which measurements could be taken (5–6 s per flask) greatly increasing the analytical capacity (to 336 flasks or 75 substrates per incubation series). The ability of this system to provide information not just on both rate and extent of degradation, but also fermentation efficiency and degradation kinetics offers a wide variety of applications, for example the systematic evaluation of tropical feedstuffs. In conclusion it is suggested that the simplicity, low cost and high capacity of the RPT system make it ideal for those situations where either budget constraints or the level of technical expertise required render the more complex systems inappropriate.     

Exponential gradient maker using a disposable syringe

With a simple modification, any disposable syringe can become a reliable and easy to use exponential gradient maker. The modification consists of two notches, made with a razor blade, in the borders of the rubber sealing tip of the plunger. A clamp in the tube connected to the syringe allows control over solution flow. With the clamp prohibiting drainage, the body of the syringe is filled with the desired volume of starting solution I. A magnetic stir bar, small enough to spin inside the syringe is included. The notched plunger is introduced until no air space remains. This forms the fixed volume, closed mixing chamber, while the rest of the volume of the syringe forms the open chamber. The two chambers are connected through the notches in the plunger. The ending solution II is poured after the introduction of the plunger. Opening the clamp allows solution I in the closed chamber to flow out, and the solution II in the open chamber flows through the notches and mixes with solution I. This exponential gradient maker can be reused many times, but the low cost of the components makes it potentially disposable. This feature is especially useful when using toxic chemicals, or when pouring polyacrylamide gradient gels, since the apparatus may be disposed of after contamination or eventual polymerization.     

Mechanics of edematous lungs.

Using the parenchymal marker technique, we measured pressure (P)-volume (P-V) curves of regions with volumes of approximately 1 cm3 in the dependent caudal lobes of oleic acid-injured dog lungs, during a very slow inflation from P = 0 to P = 30 cmH2O. The regional P-V curves are strongly sigmoidal. Regional volume, as a fraction of volume at total lung capacity, remains constant at 0.4-0.5 for airway P values from 0 to approximately 20 cmH2O and then increases rapidly, but continuously, to 1 at P = approximately 25 cmH2O. A model of parenchymal mechanics was modified to include the effects of elevated surface tension and fluid in the alveolar spaces. P-V curves calculated from the model are similar to the measured P-V curves. At lower lung volumes, P increases rapidly with lung volume as the air-fluid interface penetrates the mouth of the alveolus. At a value of P = approximately 20 cmH2O, the air-fluid interface is inside the alveolus and the lung is compliant, like an air-filled lung with constant surface tension. We conclude that the properties of the P-V curve of edematous lungs, particularly the knee in the P-V curve, are the result of the mechanics of parenchyma with constant surface tension and partially fluid-filled alveoli, not the result of abrupt opening of airways or atelectatic parenchyma.     

An automatic, closed-circuit oxygen consumption apparatus for small animals.

An apparatus suitable for the continuous measurement of oxygen consumption of rats and mice is described. The system uses a motorized syringe dispenser to deliver fixed volumes of oxygen to a closed animal chamber. The dispenser is controlled by a micro-differential pressure switch to maintain chamber pressure slightly above ambient. The rate of oxygen consumption is determined by timing the interval between successive operations of the dispenser. The system has proved suitable for a range of experimental conditions and treatments.     

Effects of positive end-expiratory pressure on the right ventricle

Transmural cardiac pressures, stroke volume, right ventricular volume, and lung water content were measured in normal dogs and in dogs with oleic acid-induced pulmonary edema (PE) maintained on positive-pressure ventilation. Measurements were performed prior to and following application of 20 cmH2O positive end-expiratory pressure (PEEP). Colloid fluid was given during PEEP for ventricular volume expansion before and after the oleic acid administration. PEEP significantly increased pleural pressure and pulmonary vascular resistance but decreased right ventricular volume, stroke volume, and mean arterial pressure in both normal and PE dogs. Although the fluid infusion during PEEP raised right ventricular diastolic volumes to the pre-PEEP level, the stroke volumes did not significantly increase in either normal dogs or the PE dogs. The fluid infusion, however, significantly increased the lung water content in the PE dogs. Following discontinuation of PEEP, mean arterial pressure, cardiac output, and stroke volume significantly increased, and heart rate did not change. The failure of the stroke volume to increase despite significant right ventricular volume augmentation during PEEP indicates that positive-pressure ventilation with 20 cmH2O PEEP decreases right ventricular function.     

Concentration of dilute solutions of macromolecules by electrophoresis

An electrophoresis apparatus which is used for concentrating micrograms of macromolecules from solutions as large as 250 ml is described. The recoveries were greater than 91% with three different macromolecules tested (28 to 360 kDa). Solutions with volumes in the range of 35 ml were concentrated 70-fold in less than 90 min to a final volume of 0.5 ml. Larger volumes in the range of 250 ml were concentrated 227-fold in 16 h to a final volume of 1.1 ml. Sterile concentrates can be obtained if the apparatus is constructed under sterile conditions.     

6. The Sedimenting-Droplet Procedure for Measuring Fluid Densities

ABSTRACT

An apparatus is described which is used for the determination of velocities of falling droplets of small volumes (5/xl) through hydrophobic mixtures. From calibration curves with sodium chloride solutions of different molarities and specific gravities, the densities of the fluid in the droplets were obtained by interpolation. The density of the solutions of proteins may be determined and used in the calculation of partial specific volumes. The hydrophobic fluid used was a mixture of Dow Corning 200, kerosine to reduce the viscosity, and 1.2-dichlorobenzine to increase the specific gravity to approximately that of water.     

The vacuolar potential of characean cells subjected to electromagnetic radiation in the range 200–8,200 MHz

Single giant cells of Chara braunii and Nitella flexilis were placed in a microstrip exposure apparatus and subjected to bursts of electromagnetic radiation (carrier frequencies from 200 to 8,200 MHz) at a nominal power level of 100 W/m². The vacuolar potential was monitored with a micropipette, and offsets as low as 1 μ V could be resolved in real time by suitable filtering and signal averaging; under these conditions, no offsets of the vacuolar potential were detected. At much higher power levels (corresponding to > 2 V rms between microstrip and ground plane), the slow hyperpolarizing ramp reported at lower frequencies could be seen but, because of insufficient power, could not be accurately measured. It appeared to decay beyond 500 MHz and to be absent at and above 950 MHz. To investigate reports that snail neurons irradiated for 1 h at 2,450 MHz and approximately 15.5 W/kg developed lowered membrane resistivities, Characean cells were exposed in the microstrip apparatus for 1 h at 2,450 MHz and 230 W/m²; their membrane resistivities were found to be lowered about 18.5%.     

Ultrastructural and cytochemical studies on the ventral pouch of Gastrothylax crumenifer (Digenea:Paramphistomidae)

The ventral pouch of G. crumenifer consists of an extensive atrium, opening just behind the mouth and ending blindly at the level of the testes. It is lined by a thin tegument underlain by numerous flask shaped pyriform cells. The non-glandular nature of the pyriform cell cytoplasm and the presence of haemoglobin and numerous mitochondria suggest that the pouch fulfils primarily a respiratory role. Contractions and relaxations of musculature associated with the pouch may ventilate the pouch with ruminal fluid, or with gaseous oxygen which may be present in small quantities in the rumen. Oxygen taken up by haemoglobin in pyriform cells may be transferred to haemoglobin in the lymph system, and thence distributed internally.