The comparison of isolation techniques for nematophagous fungi from soil

Using a single soil, a comparison was made of six well-known techniques for the isolation of nematophagous fungi. Each technique was tested for its ability to isolate the fungi present with regard to the number of species recovered, the number of plates needed to be 95% sure of isolating all the species possible and the amount of soil required. The advantages and disadvantages of each method are discussed. The results show that specific techniques are required for the isolation of endoparasites and predators respectively. The soil sprinkling technique and the Baermann funnel technique are shown to be the most efficient methods overall, with six and seven replicates required for 95% probability of isolating all the predator and endoparasite species respectively.     

Leukocyte isolation by sedimentation: the effect of rouleau-promoting agents on leukocyte differential count.

The rouleau-promoting agents dextran and polyvinylpyrrolidone (PVP) were used to accelerate erythrocyte sedimentation in order to harvest the leukocyte rich plasma (LRP). The objective of the work was to determine if agent concentration or blood: agent ratio had any effect on the leukocyte differential count and if so at what agent concentration and agent:blood ratio did the LRP leukocyte differential count most closely match the whole blood leukocyte differential count. With both sedimentation agents the data clearly indicate that both parameters effect LRP differential counts and that low concentrations of sedimentation agents are most important in obtaining LRP differential counts which most closely match the whole blood differential counts.     

Sheared DNA fragment sizing: comparison of techniques.

DNA fragmented by conventional French press shearing procedures (30,000 lbs/in2) has a number-average fragment size of 230 base pairs. This is considerably smaller than the 450 base pairs typically reported for DNA sheared by this method. Comparison of 5 sizing techniques indicates that sheared DNA fragment size is overestimated by either measurement of velocity sedimentation or Kleinschmidt Electron Microscopic visualization. Both adsorption grid electron microscopic visualization and gel electrophoresis yield the most reliable estimates of the mean size of small DNA fragment populations. In addition, the assessment of fragment size distribution (not possible from sedimentation analysis) potentially allows more critical evaluation of DNA hybridization and reassociation kinetic and measurement parameters.     

Poster Sessions BP03: Myelin,Demyelination and Diseases of Myelin. Myelin and myelination in PLP‐null mice

The myelin proteolipid protein (PLP) is the major structural protein of CNS myelin, accounting for approximately half of total myelin protein. We studied synthesis and accumulation of myelin components for two months postnatally in PLP‐null mice and age‐matched controls. Accumulation of myelin, as assayed by levels of whole brain cerebroside and myelin basic protein, was normal in the knockout mice. The rate of cerebroside synthesis in the knockout mice was also normal. Myelin was isolated at several ages during development, using a standard subcellular fractionation protocol. The yield of ‘purified myelin’ isolated from a large particle (crude mitochondrial) fraction was reduced in PLP‐null mice, but increased amounts of ‘myelin’ were obtained in the small particle (crude microsomal) fraction. This ‘myelin’ in the crude microsomal fraction was identified as such by flotation on 0.85 m sucrose and the myelin‐characteristic 2 : 1 molar ratio of cholesterol to cerebroside. This suggests myelin from PLP‐null mice is physically more fragile than normal myelin, and that during tissue dispersion, much more PLP‐null myelin is fragmented into small vesicles than is the case for normal myelin. Three hours after intracranial injection of tritiated acetate into PLP‐null mice, cerebroside in myelin isolated from the large particle fraction was at a similar specific radioactivity to that isolated from the small particle (crude microsomal) fraction, suggesting that the most recently deposited PLP‐null myelin is not preferentially unstable. The increased fragility evident during tissue dispersion is indicative of an underlying structural abnormality in PLP‐null myelin. Whether this inherent structural instability affects myelin metabolism is under investigation. Acknowledgements: Supported by USPHS & NMSS grants.     

Diagnosis of hypothyroidism: a comparison of statistical techniques.

From replies to a postal questionnaire used in a radioactive iodine follow-up scheme patients had to be classified as either "suspected hypothyroid" or "euthyroid." A comparisons has been made of the effectiveness of published statistical techniques in making this classification. Two main conclusions emerged. Firstly, all except one of the methods identified an acceptable proportion of the hypothyroid patients, and, secondly, the results given by these methods were remarkably similar. Thus the simplest, which required only a count of the number of symptoms present, was selected for use.     

The diagnostic process: a comparison of scanning techniques.

In choosing between various scanning techniques the factors to be considered include availability, cost, the type of equipment, the expertise of the medical and technical staff, and the inherent capabilities of the system. Although it is difficult to state dogmatically which scanning technique is best for each patient and condition, one or other technique is clearly preferable in some areas of medicine. Ultrasound, for example, should be used in obstetrics, while computerized tomography has revolutionised neuroradiological diagnosis. Nevertheless, there is still no substitute for good history taking and a thorough physical examination. The most important factor determining the choice of technique is the system''s ability to answer the specific question required for the management of the patient.     

Cryptosporidium merozoite isolation and purification using differential centrifugation techniques.

Simple modifications to a recently published merozoite purification procedure (Bjorneby et al., J. Immunol. 145:298, 1990) increased yields 3- to 5-fold. Calves were infected with 2.5 x 10(8) Cryptosporidium parvum oocysts and sacrificed 65 h post-infection. The ilium and caecum were removed. The tissue was sieved through a large strainer (2 mm2) to produce a homogeneous suspension. Red blood cells were removed by differential centrifugation (600 g); merozoites remained in the supernatant. The merozoites were pelleted (2,100 g) and washed in modified Hank's balanced salt solution deficient in Mg+2 and Ca+2. Percoll purification (density 1.070 g/ml and centrifugation speed of 22,000 g for 30 min) yielded 8 x 10(8) merozoites. Nineteen monoclonal antibodies (MAb) detected by either an enzyme-linked immunosorbent assay or an immunofluorescence assay, have been generated against the merozoite stage. Gels of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver-stained showed that sporozoites and merozoites have many common lower molecular weight proteins. Western blots of sporozoite and merozoite antigens reacted with anti-sporozoite MAb showed several cross-reacting antigens shared by these life-cycle stages.     

Characterization of macromolecular heterogeneity by equilibrium sedimentation techniques

New graphical procedures have been developed to investigate the heterogeneity of protein preparations using sedimentation equilibrium. The heterogeneous systems that can be studied include self-associating systems contaminated by incompetent monomer, self-associating systems contaminated by non-dissociating oligomer and simple non-interacting monomer-oligmer disperse systems. The new procedures are based on the concentration dependence of the apparent association constants estimated by a non-linear least square fitting program (NONLIN), on the assumption of conservation of mass during sedimentation and on the applications of several standard techniques for statistical inferences of NONLIN estimations. The procedures outlined here can detect various types of heterogeneity, discriminate amongst different types of heterogeneity, estimate the amount of contaminant causing heterogeneity and determine the true equilibrium constant of the self-associating components. The procedures appear to be sensitive, accurate and easily applicable when tested using both protein samples and computer simulated data.     

Processing liquid-based gynecologic specimens: comparison of the available techniques.

OBJECTIVE: To develop a cytopreparation protocol suitable for satisfactory processing by the AutoCyte PREP System with the gynecologic specimens collected in PreservCyt fluid for the ThinPrep machine. STUDY DESIGN: The residue of a number of gynecologic specimens collected in PreservCyt and processed by ThinPrep were processed by AutoCyte PREP. Some modifications were made in the cytopreparatory protocol in order to obtain satisfactory specimens. RESULTS: The ThinPrep and AutoCyte PREP specimens were examined independently. The results were comparable, with a high degree of concordance between the two techniques. CONCLUSION: Gynecologic specimens collected in PreservCyt and following the ThinPrep specimen collection protocol can be processed using the AutoCyte PREP System. Minor protocol modifications provided comparable diagnostic material. Additional studies are needed to explore the feasibility of this approach and fulfill the various U.S. regulatory agency requirements for the liquid-based Pap test.     

An efficient preparative isopycnic flotation method for the isolation of chitosomes

Summary Chitin synthetase, a key enzyme in fungal cell wall biosynthesis, is located in chitosomes (microvesicles). To produce large quantities of chitosomes for immunochemical and biochemical characterization, we developed a two-step purification procedure in which isopycnic sucrose density gradients were centrifuged at ultra-high gravitational forces (fixed-angle rotor at 361,000×g R_av). Chitosomes from yeast cells ofMucor rouxii were separated from the soluble proteins and from the larger membranes by isopycnic centrifugation of the cell-free extract. The resulting crude chitosome sample was adjusted to a higher sucrose concentration, and a sucrose gradient was layered over the sample. Upon recentrifugation, the chitosomes moved up into the gradient and equilibrated at their buoyant density (1.15–1.16). This accelerated flotation separated contaminating particles of higher buoyant density (larger vesicles, ribosomes, and other miniorganelles) and yielded a large population of microvesicles with a mean diameter of 48.9±13.8 nm. This preparation contained vesicles essentially free of other particulate contaminants; more than 99% of the vesicles were smaller than 100 nm. When required, an additional velocity centrifugation step was added to remove the larger vesicles from the chitosome samples. This streamlined method for chitosome isolation was much simpler and faster than earlier isolation procedures, gave a high yield of functional chitosomes, and made the large scale isolation of these organelles possible.     

A comparison of cell wall disruption techniques for the isolation of intracellular metabolites from Pleurotus and Lepista sp

Different techniques were compared for their effectiveness in the disruption of the rigid cell walls of Basidiomycetes. Grinding under liquid nitrogen, stirred glass bead milling and enzymatic cell lysis were applied to the mycelia of Pleurotus sapidus and Lepista irina grown submerged. Each of the disruption procedures was evaluated by testing the quantity and quality of released intracellular metabolites: DNA, RNA, enzymes, and secondary metabolites. The most suitable method for nucleic acid isolation was grinding under liquid nitrogen, while bead mill homogenization was the superior technique for isolation of active enzymes. A new effective method is proposed for isolation of secondary metabolites with the aid of bead milling of fungal mycelia.     

Preparative isolation of human galactosyltransferase. A comparison of methods

Human galactosyl transferase (EC 2.4.1.38), a potential tumour marker, was isolated from the ascite fluid of patients with ovary cancer by three methods based on the use of affinity adsorbents. The highest degree of purification was achieved during chromatography on galactosyl transferase-specific monoclonal antibodies immobilized on agarose. A comparison of physico-chemical properties of purified preparations was carried out. It was found that the enzyme purified on the immunoadsorbent did not practically differ in terms of molecular mass and isospectra from other enzyme preparations, which suggests that the antigenic epitope recognized by these monoclonal antibodies on the galactosyl transferase molecule is common for all enzyme isoforms.     

An approach to the isolation of biological particles using sedimentation analysis

A systematic, general approach for the design of an initial purification procedure for any biological particle is described in this communication. A series of centrifugations in fixed angle rotors has been used to obtain information on the sedimentation behavior of particles of interest (Anderson, N.G. (1967) Anal. Biochem. 23, 72-83). Refinements of this technique have facilitated the determination of sedimentation profiles of subcellular organelle markers in suspensions of murine spleen and brain. The degree of homogeneity of several particles with respect to size can be ascertained from the sedimentation profiles. Alterations in these profiles after mechanical disruption and treatment with detergents are readily measurable and have been found to be useful in both the characterization and isolation of subcellular particles. Because fixed angle rotors are used in these studies, the data obtained can be directly applied to the development of a preparatory scheme for purification of a desired particle. These methods for sedimentation analysis are readily applicable to subcellular organelles, macromolecular complexes, viruses, viral-like agents, and a variety of macromolecules.