THE ADVANTAGES OF A PLATINUM TUBE DROPPING PIPETTE FOR MAKING COLONY COUNTS

SUMMARY: For routine purposes where a large number of tests is involved the dropping pipette technique has many advantages over other methods of determining the colony count. However, glass pipettes are fragile and much time and labour have to be used in making and calibrating replacements. A robust platinum tube dropping pipette has been found very satisfactory. No difficulty was experienced in obtaining complete sterility of the pipettes in the technique described, and reproducibility of drop size was good.     

Das Autoselect-System – ein Automatensystem zur Selektion von Antibiotikaproduzenten. IV. Achtfach-Verdünnungsautomat

As a part of the Autoselect-system an eightfold diluter is described. Cassettes with 64 tubes are placed on the carriage of the machine and moved automatically in eight steps. One of the cassettes is loaded with separated samples in 64 tubes and the other one with 64 empty tubes. When the carriage stops, a defined volume of samples is exhausted by eight pipettes, mounted on a bridge spanning over the ground unit from left to right, and after beeing moved to the second cassette the pipettes deliver the samples into a row of empty tubes. At the same time by eight syringes mounted on both sides of the machine a defined volume of buffer is delivered through tubes and canules. By this way all samples can be diluted in two minutes.     

Estimation of the size of freshwater ciliate populations by a sub-sampling technique

A technique is described for the estimation of the size of ciliate populations based on replicated counts. Sample drops were taken by automatic pipettes of different volume from activated sludge and from a small oligotrophic stream. Specific aims were: 1) the estimation of the number of species; 2) the estimation of the number of individual in each species; 3) the selection of a suitable sub-sample size and number of replicates. For each volume the cumulative increase in species taken in successive sub-samples was determined. For each species the minimum permissible sub-sample volume and the number of replicates required for an allowable error of 10% and 25% were determined. Lastly, the relationship between the mean number of individuals counted for each species and the relative coefficient of variation was visualized.     

The use of quartz patch pipettes for low noise single channel recording.

Quartz has a dissipation factor of approximately 10(-4), which is an order of magnitude less than that of the best glasses previously used to fabricate patch pipettes; it's dielectric constant of 3.8 is also lower than that of other glasses. On the basis of these electrical characteristics it is expected that patch pipettes pulled from quartz tubing will produce significantly less noise than pipettes made from other glasses. Our work confirms these expectations and we describe theoretical and practical aspects of the use of quartz pipettes for single channel patch voltage clamp measurements. Methods for pulling quartz pipettes with a laser-based puller and coating them with low-loss elastomers are discussed, as are precautions that are necessary to achieve low noise recordings. We have shown that quartz pipettes can be pulled from tubing with outer diameter to inner diameter ratios as large as 3 and a method of applying heavy elastomer coatings all the way to the tip of pipettes is presented. Noise sources arising from the pipette and its holder are described theoretically, and it is shown that measured noise is in good agreement with such predictions. With low noise capacitive feedback electronics, small geometry holders, and thick-walled quartz pipettes coated with low-loss elastomers we have been routinely able to achieve noise of 100 fA rms or less in a 5-kHz bandwidth with real cell patches and a pipette immersion depth of approximately 2 mm. On occasion we have achieved noise as low as 60 fA rms in this bandwidth.     

A simple, rapid, aseptic method for filling micropipettes by centrifugation

A centrifugation method for sterilizing, storing, and filling micropipettes is described. Each micropipette is held in a centrifuge tube by a rubber stopper which clamps the butt end of the micropipette. The pipettes are sterilized and aseptically stored. A pipette is filled by injecting solution into the butt end of the micropipette. The micropipette is returned to a suspended position in the centrifuge tube and the liquid is rapidly forced into the micropipette tip by centrifugation. The technique is simpler and more rapid than presently used centrifugation methods.     

A whole-cell patch clamp technique which minimizes cell dialysis

A variant of the whole-cell patch clamp technique is described which allows measurement of whole-cell ionic currents in small cells while minimizing cell dialysis with the pipette solution. The technique involves the application of negative pressure to the inside of small (less than 1 micron) tip diameter pipettes placed on the cell surface to achieve high resistance seals and membrane rupture. The technique has been used successfully in a variety of different types of cells to study membrane currents carried by Ca and K, currents generated by exchange carriers as well as electrical coupling between cells. Overall, the technique seems well suited for the study of ionic currents in small cells, and provides an alternative to conventional patch clamping techniques which necessitate intracellular dialysis.     

Scanning isoelectric focusing in small density-gradient colums. 3. A method for detailed mapping of biological activity of column contents applied to pea lipoxygenase

A fractionation technique is described, which enables detailed mapping of possible biological activity in a selected part of a system obtained by density gradient isoelectric focusing in a 1.5-ml column. The actual part of the column contents is pumped into 24 capillary pipettes, each holding 10 μl. The rest of the column contents is divided into 60-μl fractions. After measurement of pH in the latter, pI values of focused protein components can be estimated.Application of the technique to the isoclectric characterization of pea lipoxygenase is reported.     

A Modification of the Indirect Immunofluorescence Test for Detection of Ehrlichia phagocytophila Antibodies

A modified technique for production of antigen and performance of the test is described. A suspension of infected neutrophils was directly applied to multiwell slides. Multichannel pipettes may be used for dilution and application of sera. The modification inreases the capacity both by production of the antigen and by performance of the test. This paper also gives a quantitative determination of the antibodies.     

Errors inherent in the use of piston activated pipettes

The accuracy and reproducibility of the volume of solution delivered from a number of piston-activated pipettes of different makes and sizes has been tested under a variety of experimental conditions. The results indicate that these devices deliver the nominal volume only when both the solution to be sampled, and the whole pipette are at the same temperature. When a temperature differential exists, the absolute accuracy is affected, and unless particular care is taken, the reproducibility is relatively poor. Thus it is only under well-controlled conditions that the behaviour of piston-activated pipettes falls within the specifications quoted by the manufacturers.     

Das Autoselect-System – ein Automatensystem zur Selektion von Antibiotikaproduzenten. V. Achtfach-Pipettierautomat

An automatic eightfold-pipetter has been developed with the same basic unit as the diluter of the Autoselect-system. The pipetter is fitted with eight pipettes on a bridge over the basic unit. By means of the pipettes a volume of 0.05 ml from eight tubes is exhauseted and delivered in the holes of a test plate. Both the test plate and a cassette with 64 tubes are located on a moving carriage. the test plate on the first floor and the cassette on the ground floor. If the pipettes transfer the samples from the tubes to the holes the distance between the pipettes must be changed by a special device from 20 to 30 mm, because the distance between the holes on the test plate differs from that of the tubes. For the transfer of 64 samples 2 minutes only are needed.     

Membrane-pipette interactions underlie delayed voltage activation of mechanosensitive channels in Xenopus oocytes.

To investigate the mechanism for the delayed activation by voltage of the predominant mechanosensitive (MS) channel in Xenopus oocytes, currents were recorded from on-cell and excised patches of membrane with the patch clamp technique and from intact oocytes with the two-electrode voltage clamp technique. MS channels could be activated by stretch in inside-out, on-cell, and outside-out patch configurations, using pipettes formed of either borosilicate or soft glass. In inside-out patches formed with borosilicate glass pipettes, depolarizing voltage steps activated MS channels in a cooperative manner after delays of seconds. This voltage-dependent activation was not observed for outside-out patches. Voltage-dependent activation was also not observed when the borosilicate pipettes were either replaced with soft glass pipettes or coated with soft glass. When depolarizing voltage steps were applied to the whole oocyte with a two-electrode voltage clamp, currents that could be attributed to MS channels were not observed. Yet the same depolarizing steps activated MS channels in on-cell patches formed with borosilicate pipettes on the same oocyte. These observations suggest that the delayed cooperative activation of MS channels by depolarization is not an intrinsic property of the channels, but requires interaction between the membrane and patch pipette.     

A whole-cell patch clamp technique which minimizes cell dialysis

Summary A variant of the whole-cell patch clamp technique is described which allows measurement of whole-cell ionic currents in small cells while minimizing cell dialysis with the pipette solution. The technique involves the application of negative pressure to the inside of small (< 1 µm) tip diameter pipettes placed on the cell surface to achieve high resistance seals and membrane rupture. The technique has been used successfully in a variety of different types of cells to study membrane currents carried by Ca and K, currents generated by exchange carriers as well as electrical coupling between cells. Overall, the technique seems well suited for the study of ionic currents in small cells, and provides an alternative to conventional patch clamping techniques which necessitate intracellular dialysis.     

Viscoelastic properties of the human red blood cell membrane. II. Area and volume of individual red cells entering a micropipette.

Previous work demonstrated that human red cells can be drawn into cylindrical glass micropipettes of internal diameter approximately 2.0 mum without lysing. For pipettes of less than approximately 2.9 mum inside diameter, the red cell must become less spherical, that is, reduce its volume-to-area ratio. In this work measurements were made from 16-mm film records that allowed the determination of the cellular area and volume of individual erythrocytes as they were drawn into a 2.0-mum pipette with negative pressures. The results showed that the total surface area of the membrane remains constant and that the cell endures the passage into the pipette by losing volume. The volume loss was interpreted to be due to cell water and solute loss when the membrane is under stress. The loss of cell volume, rather than the stretching of the membrane, adds confirmation that although it is very deformable, the membrane is very resistant to two-dimensional strain.     

Device for safe manipulation of microorganisms with unknown properties

A simple adaptation of common laboratory pipettes and injection syringes for safe manipulation with samples of cultivation liquid is described.     

Characterization of stretch-activated calcium permeable cation channels in freshly isolated myocytes of the cricket (Gryllus bimaculatus) lateral oviduct

Stretch-activated channels (SACs) were investigated in myocytes isolated from the lateral oviduct in cricket Gryllus bimaculatus using the cell-attached or excised inside-out patch clamp technique. Application of both negative and positive pressure (10-100 cm H(2)O) into the patch pipettes induced the unitary channel current openings. The open probability (NPo) of the channel increased when negative pressure applied into the patch pipettes increased. The single channel conductance for this channel was approximately 20 pS with 140 mM Na(+), K(+), or Cs(+) in the patch pipettes and was approximately 13 pS with 100mM Ca(2+) or Ba(2+) in the patch pipettes. External application of Gd(3+), La(3+), Cd(2+) and Zn(2+)inhibited the channel with the IC(50) values of 14, 15, 28, and 18 microM respectively. Interestingly external application of TEA, a specific blocker of K(+) channel, also inhibited this channel with IC(50) value of 8.8mM. These results show for the first time the presence of stretch activated Ca(2+)-permeable nonselective cation channel in myocytes isolated from the cricket lateral oviduct. The physiological significance of this channel in oviposition behavior is discussed.     

Pneumatic, Multiple, Simultaneous Inoculator for Plastic Trays

A device is described which consists of a bonded Plexiglas-silicone rubber plate, perforated to hold capillary pipettes in register with the wells of a plastic tray to be inoculated. Pipette contents are simultaneously discharged by positive air pressure, initiated by closing an exhaust port with the thumb.     

Spatial distribution of maxi-anion channel on cardiomyocytes detected by smart-patch technique

Spatial distribution of maxi-anion channels in rat cardiomyocytes were studied by applying the recently developed patch clamp technique under scanning ion conductance microscopy, called the “smart-patch” technique. In primary-cultured neonatal cells, the channel was found to be unevenly distributed over the cell surface with significantly lower channel activity in cellular extensions compared with the other parts. Local ATP release, detected using a PC12 cell-based biosensor technique, also exhibited a similar pattern. The maxi-anion channel activity could not be detected in freshly isolated adult cardiomyocytes by the conventional patch-clamp with 2-MΩ pipettes. However, when fine-tipped 15-20 MΩ pipettes were targeted to only Z-line areas, we observed, for the first time, the maxi-anion events. Smart-patching different regions of the cell surface, we found that the channel activity was maximal at the openings of T-tubules and along Z-lines, but was significantly decreased in the scallop crest area. Thus, it is concluded that maxi-anion channels are concentrated at the openings of T-tubules and along Z-lines in adult cardiomyocytes. This study showed that the smart-patch technique provides a powerful method to detect a unitary event of channels that are localized at some specific site in the narrow region.     

Optical evaluation of red blood cell geometry using micropipette aspiration.

Although red blood cell (RBC) geometry has been extensively studied by micropipette aspiration, the small size of RBC and pipettes vs. the optical resolution of light microscopy suggests the need to consider potential errors. The present study addressed such difficulties and investigated four specific problems: (1) use of exact equations to calculate RBC membrane area and volume; (2) calibration of the pipette internal diameter (PID); (3) correction for a noncylindrical pipette barrel; (4) diffraction distortion of the RBC image. The observed PID represents a cylinder lens enlargement that can be theoretically derived from the glass/buffer refractive index ratio (1.49/1.33 = 1.12). This enlargement was experimentally confirmed by: (1) studying pipettes bent to allow measurement through the barrel (horizontal) and at the orifice (vertical), with a resulting diameter ratio of 1.12 +/- 0.01; (2) and by replacing the surrounding buffer with immersion oil and hence abolishing the lens phenomenon (ratio = 1.12 +/- 0.02). In addition, use of aspirated oil droplets demonstrated a 3.2 +/- 0.2% error when the PID is focused at a sharp, maximum diameter. The average pipette cone angle was 1.49 +/- 0.09 degrees and varied considerably with pipette pulling procedures; calculated tongue geometry inside the pipette was affected by the noncylindrical pipette barrel. The RBC diffraction error, demonstrated by touching two aspirated cells held by opposing pipettes, was 0.091 +/- 0.002 microns. The PID, cone angle, and diffraction artifacts significantly (p < 0.001) affected calculated RBC geometry (average errors up to 5.4% for area and 9.6% for volume). Two new methods to calculate, rather than directly measure, the PID from images of a single RBC, during either osmotic or pressure manipulation, were evaluated; the osmotic method closely predicted the PID, whereas the pressure method markedly underestimated the PID. Our results thus confirm the need to consider the above-mentioned phenomena when determining RBC geometric parameters via micropipette aspiration.     

Power-law rheology analysis of cells undergoing micropipette aspiration

Accurate quantification of the mechanical properties of living cells requires the combined use of experimental techniques and theoretical models. In this paper, we investigate the viscoelastic response of suspended NIH 3T3 fibroblasts undergoing micropipette aspiration using power-law rheology model. As an important first step, we examine the pipette size effect on cell deformation and find that pipettes larger than ~7 μm are more suitable for bulk rheological measurements than smaller ones and the cell can be treated as effectively continuum. When the large pipettes are used to apply a constant pressure to a cell, the creep deformation is better fitted with the power-law rheology model than with the liquid drop or spring-dashpot models; magnetic twisting cytometry measurement on the rounded cell confirms the power-law behavior. This finding is further extended to suspended cells treated with drugs targeting their cytoskeleton. As such, our results suggest that the application of relatively large pipettes can provide more effective assessment of the bulk material properties as well as support application of power-law rheology to cells in suspension.     

Simple, fast, and efficient method of manual oocyte enucleation using a pulled Pasteur pipette

Cloning mammals by somatic cell nuclear transfer entails the replacement of oocyte chromosomes with the nucleus of a somatic cell. A major step in this technique is to efficiently produce large batches of enucleated oocytes, a process that requires considerable micromanipulation skills and expensive equipments. Here, a simple, fast, and efficient method of manual oocyte enucleation was introduced that can be adopted in every laboratory with the minimum equipments. Common laboratory glass pipettes were pulled on the flame of a burner and then used for manual bisection or enucleation of sheep and goat zona-free oocytes by passing them through the discontinuous cutting border of culture medium and mineral oil. The described techniques showed a certain efficiency to conveniently bisect or enucleate large batches of sheep, and goat oocytes being pre-treated with demecolcine. The method may be straightforward for simple manipulation of oocytes of other species and for development of automated cloning methods as well.