Collection and analysis of kinetic data from a stopped-flow spectrophotometer using a microcomputer

A method of interfacing an inexpensive microcomputer to a stopped-flow kinetics spectrophotometer is described. It allows software-selectable sampling frequencies between 0.1 ms and 8 s and large numbers of data points to be collected. Machine language routines to use the interface are described and these allow the sampling frequency to be altered during data collection to ensure adequate numbers of points in critical regions of the kinetic profile. BASIC programs for collection and analysis of multicomponent kinetic data using this system are also described. Due to the large number of data points that can be collected and the ability to selectively sample transmittance values in regions where the signal is rapidly changing with time, relatively unsophisticated methods of data analysis can be used. These methods are suitable for use by microcomputers and mean that data analysis and acquisition can be performed on the same microcomputer in real time. To illustrate this, multicomponent analysis of kinetic transients is performed on simulated data and on the dissociation kinetics of the ethidium-DNA complex.     

Blood collection for biochemical analysis in adult zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies. There are relatively few publications related to adult zebrafish physiology of organs and systems, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. Recently, Gupta et al. described how to dissect adult zebrafish organs, Kinkle et al. described how to perform intraperitoneal injections, and Pugach et al. described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish. The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.     

Fibreoptic injection of oesophageal varices.

A new technique for injecting sclerosant into oesophageal varices uses a flexible gastroscope. Once the gastroscope has been inserted a flexible tube with a window cut into its distal end is pushed down over the gastroscope until a varix protrudes through the window. The varix can then be injected with a needle passed down the biopsy channel of the gastroscope. Once the varices have all been injected in this way the tube is advanced to compress the injection sites and so help control bleeding. This method, using a flexible gastroscope, has proved easier and safer than the traditional method using a rigid gastroscope.     

A fluorometric assay for dihydrofolate reductase

A recording fluorometric assay for dihydrofolate reductase is described. The technique measures the appearance of product, tetrahydrofolate, as the reaction proceeds. The assay can be accomplished with as little as 2 pmol of enzyme and is sufficiently sensitive to allow accurate kinetic studies using very low concentrations of substrate. Kinetic studies of both purified and crude preparations of enzyme are reported.     

A molecular approach to the identification of S-alleles in Brassica oleracea

A molecular technique for the identification of S-alleles involved in self-incompatibility has been used to analyse the S-allele reference collection of Brassica oleracea. The reference collection contains nearly 50 different lines each with a different S-allele present in the homozygous state. The technique consists of amplifying by the polymerase chain reaction (PCR) sequences belonging to the S multigene sequence family using a single pair of conserved primers. PCR products are then analysed further by digestion with six restriction enzymes followed by gel electrophoresis of the digestion products. A simple method of estimating the band sizes of the digestion products is described. The S-locus-related sequences can be distinguished from S-locus glycoprotein and S-receptor kinase genes by the restriction patterns. Furthermore, with any one restriction enzyme, several alleles showed the same restriction pattern. Alleles could therefore be grouped together. With two exceptions, each member of the S-allele reference collection showed a unique set of restriction patterns. Investigation of the exceptions using pollen tube growth tests showed that these accessions represented duplications within the collection. This technique therefore provides a simple and useful method for identifying different S-alleles.     

A Method for Collection of Bile in the Conscious Sheep

A method to collect bile directly from the hepatic duct is described for use in the sheep. The technique is a combination of the intestinal re-entrant cannulae and a catheter from the duodenal lumen to the hepatic duct. The cystic duct is ligated near its junction with the common bile duct. The catheter is fixed in the proximal visible end of the hepatic duct. One plastic cannula is fixed to the duodenum opposite to the opening of the common bile duct and the other is fixed in the same way about 15 cm posterior to the first one. The two plastic cannulae fixed together with a plastic tube serve as an extra-abdominal anastomosis. During the collection periods the bile duct catheter is passed through an opening in the wall of the connection tube into a collection bag that is fixed to the plastic cannulae. Between the collection periods the catheter ends in the lumen of the anastomosis.     

Subpicogram analysis of sweat proteins using two-dimensional polyacrylamide gel electrophoresis

Sweat collected from six normal volunteers was analyzed to determine if reproducible protein patterns could be obtained using two-dimensional polyacrylamide gel electrophoresis of ¹²⁵I-labeled sweat proteins. This method has the capability of easily detecting picogram quantities of protein. Once the methods of collection of the sweat had been standardized, reproducible patterns were obtained from these volunteers. Over 100 discrete spots were revealed by a combination of fluorography and rare earth screen radioautography of dried two-dimensional gels. This method will allow analysis of sweat for qualitative and quantitative variations in protein content in pathologic conditions such as cystic fibrosis, renal failure, and diabetes.     

一项小鼠采血新技术——颌下静脉丛采血法

目的介绍小鼠颌下静脉丛采血方法。方法选用2月龄昆明小鼠,固定小鼠后,将采血注射针头刺入颌下静脉丛血管取血。结果单人操作约1 min内可完成小鼠颌下静脉丛采血,采血量达到0.3-0.5 mL。结论此方法无需麻醉、创伤小、采血量大并可重复采血,是一种简便、安全、快速、采血量多的采血方法。     

Rapid identification of sex in birds by flow cytometry

A rapid method to identify sex in birds is described. The method requires microliter volumes of blood, and, under appropriate conditions, results can be available within an hour of sample collection. Samples can be stored at 4 degrees C or -20 degrees C without sacrificing the ability to discriminate sex differences in DNA content. The assay will find utility in laboratory, field, and applied studies, in other classes of vertebrates, and in studies on the dynamics of genome size within and among populations.     

Simple method for bleeding the unanaesthetized rat by tail venipuncture

A technique is described for the intermittent collection of blood from the rat tail. By using commonly available equipment, blood samples can easily be obtained from rats without the need for anaesthesia. The development of this technique makes the rat more readily available as an animal model for repeated withdrawals of small blood samples for pharmacokinetic or bioavailability evaluations.     

Simplification of rat intubation on inclined metal plate

Small-animal intubation is often necessary during inhalation anesthesia to allow steady-state conditions for large operations and in vivo experiments in all fields of experimental surgery. In rats, placing an orotracheal tube is technically difficult primarily because of the small size of the subject and the lack of equipment specifically designed for this task. We describe a simple rat intubation technique in which the animal is suspended in dorsal recumbency on an inclined metal plate. The animal, anesthetized with ether, is fixed to a 70 degrees-inclined metal plate in a dorsal position by means of a Mersilene ribbon hooked around the upper incisors. This method of positioning the animal is the most important step in the intubation process and further facilitates the technique already described by other authors. A human otoscope was used as a laryngoscope, intubation was performed using the Seldinger technique, and a 14-gauge intravenous catheter served as an endotracheal tube. This inexpensive technique is quickly learned and can be used in any laboratory. Safe and reliable airway management can thus be achieved, permitting in vivo examinations and operations.     

Determination of the volume of sweat accumulated in a sweat-patch using sodium and potassium as internal reference

In the present work, we assessed the suitability of sodium and potassium physiologically present in sweat, as internal reference allowing to re-calculate the corresponding volume of sweat collected on a PharmChek Patch. A method using capillary electrophoresis with indirect ultra-violet detection was developed for the determination of sodium and potassium in sweat. The concentrations determined in specimens collected from 12 females and 10 males, using a home-made system composed of polypropylene copolymer bag, were 1039+/-89 mg/L and 711+/-45 mg/L for sodium, and 489+/-293 mg/L and 474+/-196 mg/L for potassium, respectively. In parallel, for seven females and eight males, the comparison of the volume of sweat collected in the same way to the re-calculated volume of sweat accumulated in a patch using sodium as internal standard, gave an average agreement of 98.4+/-15.0%. Results demonstrated the usefulness of sodium as internal standard to determine the volume of sweat accumulated in a patch, and confirm the suitability of PharmChek patch for the collection and determination of cations in sweat.     

Phosphorus-32 beta gauge for measuring the water relations of fleshy fruits

A technique for studying the water relations of fleshy fruits such as strawberry by measuring the beta ray penetration through the tissue is described. As a source of beta radiation, an active preparation of³²P melted into a capillary glass tube was used. The technique described permits a study of the changes in the water content of fruitsin vivo.     

The Education and Training of Biology Teachers

A technique for the numerical identification of bacteria using normalized likelihoods calculated from a probabilistic database is described, and the principles of the technique are explained. A simple computer program, which can be used to perform the calculations and identifications on a microcomputer in undergraduate classes, is presented. The program is annotated so that the steps in the calculation can be linked to the equivalent steps in the program to assist in teaching the principles of programming. Specimen results from the program, and examples of how they should be interpreted and explained, are given. It is expected that the program will be of use to teachers of undergraduates to teach the principles of numerical identification and an important use of computers in biology.     

A device to improve the Schleger and Turner method for sweating rate measurements

The objective of this study was to test a device developed to improve the functionality, accuracy and precision of the original technique for sweating rate measurements proposed by Schleger and Turner [Schleger AV, Turner HG (1965) Aust J Agric Res 16:92–106]. A device was built for this purpose and tested against the original Schleger and Turner technique. Testing was performed by measuring sweating rates in an experiment involving six Mertolenga heifers subjected to four different thermal levels in a climatic chamber. The device exhibited no functional problems and the results obtained with its use were more consistent than with the Schleger and Turner technique. There was no difference in the reproducibility of the two techniques (same accuracy), but measurements performed with the new device had lower repeatability, corresponding to lower variability and, consequently, to higher precision. When utilizing this device, there is no need for physical contact between the operator and the animal to maintain the filter paper discs in position. This has important advantages: the animals stay quieter, and several animals can be evaluated simultaneously. This is a major advantage because it allows more measurements to be taken in a given period of time, increasing the precision of the observations and diminishing the error associated with temporal hiatus (e.g., the solar angle during field studies). The new device has higher functional versatility when taking measurements in large-scale studies (many animals) under field conditions. The results obtained in this study suggest that the technique using the device presented here could represent an advantageous alternative to the original technique described by Schleger and Turner.     

Blood collection from the facial (maxillary)/musculo-cutaneous vein in true frogs (family Ranidae)

Collection of blood from amphibians, as in other classes of vertebrate animals, is essential to evaluate parameters of health, diagnose hemoparasitism, identify viral and bacterial pathogens, and measure antibodies. Various methods of blood collection have been described for amphibians. Most can be cumbersome (venipucture of femoral vein, ventral abdominal vein or lingual venus plexus) or result in pain or deleterious health consequences (cardiac puncture and toe-clipping). We describe an easy and practical technique to collect blood from frogs and toads that can be used in multiple species and is minimally invasive. The technique consists of puncturing either the facial or, less commonly, the musculo-cutaneous vein and collecting the blood with a capillary tube. These veins run dorsal and parallel to the maxillary bone and can be accessed by quick insertion and withdrawal of a needle through the skin between the upper jawline and the rostral or caudal side of the tympanum. The needle should be of 27 or 30 gauge for anurans weighing more or less than 25 g, respectively. Although the technique has been used by some amphibian researchers for years, it is little known by others and has never been fully described in a peer-reviewed publication.     

A novel method to calculate the G+C content of genomic DNA sequences.

The base composition of a DNA fragment or genome is usually measured by the proportion of A+T or G+C in the sequence. The G+C content along genomic sequences is usually calculated using an overlapping or non-overlapping sliding window method. The result and accuracy of such an approach depends on the size of the window and the moving distance adopted. In this paper, a novel windowless technique to calculate the G+C content of genomic sequences is proposed. By this method, the G+C content can be calculated at different "resolution". In an extreme case, the G+C content may be computed at a specific point, rather than in a window of finite size. This is particularly useful to analyze the fine variation of base composition along genomic sequences. As the first example, the variation of G+C content along each of 16 yeast chromosomes is analyzed. The G+C-rich regions with length larger than 5 kb sequences are detected and listed in details. It is found that each chromosome consists of several G+C-rich and G+C-poor regions alternatively, i.e., a mosaic structure. Another example is to analyze the G+C content for each of the two chromosomes of the Vibrio cholerae genome. Based on the variations of the G+C content in each chromosome, it is shown that some fragments in the Vibrio cholerae genome may have been transferred from other species. Especially, the position and size of the large integron island on the smaller chromosome was precisely predicted. This method would be a useful tool for analyzing genomic sequences.     

The effect of pre-analytical variability on the measurement of MRM-MS-based mid- to high-abundance plasma protein biomarkers and a panel of cytokines

Blood sample processing and handling can have a significant impact on the stability and levels of proteins measured in biomarker studies. Such pre-analytical variability needs to be well understood in the context of the different proteomics platforms available for biomarker discovery and validation. In the present study we evaluated different types of blood collection tubes including the BD P100 tube containing protease inhibitors as well as CTAD tubes, which prevent platelet activation. We studied the effect of different processing protocols as well as delays in tube processing on the levels of 55 mid and high abundance plasma proteins using novel multiple-reaction monitoring-mass spectrometry (MRM-MS) assays as well as 27 low abundance cytokines using a commercially available multiplexed bead-based immunoassay. The use of P100 tubes containing protease inhibitors only conferred proteolytic protection for 4 cytokines and only one MRM-MS-measured peptide. Mid and high abundance proteins measured by MRM are highly stable in plasma left unprocessed for up to six hours although platelet activation can also impact the levels of these proteins. The levels of cytokines were elevated when tubes were centrifuged at cold temperature, while low levels were detected when samples were collected in CTAD tubes. Delays in centrifugation also had an impact on the levels of cytokines measured depending on the type of collection tube used. Our findings can help in the development of guidelines for blood collection and processing for proteomic biomarker studies.     

Determination of inhibitory effects in fermentation processes by microcalorimetric techniques

A microcalorimetric kinetic method for determining the inhibition effects in fermentation is described. In order to determine the possibilities of this methodology, the inhibitory effect of ethanol on the microaerobic fermentation of xylose by Pichia stipitis is studied. Results are compared with those ones obtained by using the classic kinetic method. Microcalorimetry appears to be an interesting technique for accurately determining inhibitory effects, specially in very slow processes.