A simplified method of tissue processing for immunostaining with good preservation of antigens and morphology

A simple and rapid method of tissue processing has been developed for immunostaining. Human and murine tissues were fixed in a PVA solution, diluted in a special buffer and embedded in paraffin or stored in a stock solution before preparing frozen sections. By indirect immunofluorescence, several antigens (collagen isotypes, laminin and fibronectin) were better demonstrated in the samples processed by the present method than with frozen or deparaffinized sections. In addition, this method allows a histological preservation quite identical to that seen in classical histology.     

A reliable method with good cell preservation for the demonstration of peroxidase activity in human platelets and megakaryocytes

Summary We have tried to improve existing methods for demonstration of platelet peroxidase (PPO) in human platelets and megakaryocytes by introducing a fixation in 0.1% glutaraldehyde prior to incubation in the DAB medium. This prefixation with low concentration of glutaraldehyde preserves excellent morphological detail and does not inhibit PPO activity. All 23 platelet-rich plasma samples show PPO reaction product in the dense tubular system after incubation in DAB medium with 0.003% H₂O₂. When 0.01% H₂O₂ is used in excessive DAB medium, PPO activity can also be demonstrated in platelets and megakaryocytes of bone-marrow cell suspensions. This method can be used for the identification of megakaryoblasts in acute non-lymphocytic leukemia, myelodysplastic syndromes and in blastic crisis of chronic myeloid leukemia. PPO cytochemistry can be combined with postfixation in a OsO₄-ruthenium red mixture. This method reveals -granules, dense bodies, microtubul,, glycogen, mitochondria, dense tubular system and invaginated membrane system in the same platelet and is useful for investigation of platelet ultrastructure.     

In vitro utilization of L-cystine by keratinophilic fungi inhabiting a gelatin factory

Twenty-six different species of keratinophilic fungi were examined to determine their ability to utilize free cystine. Of the fungi tested, the majority metabolized free L-cystine in a glucose-peptone culture medium. Cystine was used as source of sulfur, and carbon and nitrogen as well. Excess sulfur was excreted into the culture fluid, as thiosulfate and sulfate, following oxidation. The rate of cystine oxidation varied with the different fungal strains, but was maximal for Graphium penicilloideus (88.5%). Low quantities of thiols were found in the medium. Cystine oxidation and inorganic thiosulfate excretion were found to correlate significantly (r = 0.94).     

An inexpensive high-throughput method to extract high yields of good quality DNA from fungi

We developed an efficient method for high-throughput extraction of high-quality DNA from various fungi. In this method, fungal mycelia were cultured and harvested on the surfaces of membranes on media plates. We degraded cell walls using a lytic enzyme (Yatalase). Purification was performed on 96-well glass fibre filter plates. DNA was successfully extracted from various fungi provided (102 genus 132 species) at high yields and quality, and proved suitable for storage, polymerase chain reaction amplification and restriction enzyme digestion. The method described is rapid, inexpensive and automation friendly. This enables the simultaneous extraction of large numbers of samples, significantly improving the potential throughput in genomics, particularly in diagnostic and population studies.     

Filamentous fungi in good shape: microparticles for tailor-made fungal morphology and enhanced enzyme production

Filamentous fungi such as Aspergillus?niger are important biocatalysts for industrial production of various enzymes as well as organic acids or antibiotics. In suspended culture these microorganisms exhibit a complex morphology which typically has a strong influence on their production properties. In this regard, we have recently shown that the addition of inorganic micro particles to the culture medium is a straightforward and elegant approach to precisely tame fungal morphology. For A.?niger a full range of morphological forms from pellets with different diameters to free mycelium could be adjusted by supplementation with talc powder. Aluminium oxide particles similarly affected morphology, showing that this effect is largely independent of the chemical particle composition.?Exemplified for different recombinant A.?niger strains enzyme production could be strongly enhanced by the addition of microparticles. This was demonstrated for the production of fructofuranosidase, an important high-value biocatalyst for pre-biotic fructo-oligosaccharides, by recombinant A.?niger. In a microparticle enhanced fed-batch process, a highly productive mycelium could be achieved. The enzyme titre of 2800?U/mL finally reached was more then tenfold higher then that of any other process reported so far. Here we provide additional insights into the novel production process. This includes the confirmation of the highly selective production of the target enzyme fructofuranosidase using MALDI-TOF?MS analysis. Moreover, we show that the obtained enzyme suspension can be efficiently used with minimal pre-treatment for the biosynthesis of short chain fructooligosaccharides of the inulin type, such as 1-kestose and 1-nystose, prebiotics with substantial commercial interest. In particular, these compounds are highly attractive for human consumption, since they have been shown to reduce the risk of colon cancer. In summary, the use of microparticles opens a new avenue of engineering fungal morphology into the desired form for specific production processes.     

Ecology of nematophagous fungi: Methods of collection,isolation and maintenance of predatory and endoparasitic fungi

The methods employed in a recent investigation into the ecology of nematophagous fungi are examined in detail. The best sources from which fungi can be isolated are examined, and the main field collection and recovery techniques are compared and discussed. Other practical problems considered are the examination of plates, microscopy, identification, isolation and culture methods, maintenance of fungi, the production of nematodes as bait. A summary of the experimental techniques used is given.     

Polarity in filamentous fungi: establishment,maintenance and new axes

Germ tube emergence in filamentous fungi appears to be similar to bud emergence in yeast. Several key proteins (e.g. Cdc42, septins, Bni1 formin, Rho1 and Rho3) play common roles in polarity establishment and early polarity maintenance in both processes. Although germ tube extension, which can be thought of as extreme polarity maintenance, uses some of the same genes, they are likely to be regulated differently. Mutations in polarity maintenance genes often lead to a split tip in filamentous fungi, a phenotype without an analogue in yeast. Cell cycle regulation differs between tip splitting and subapical branching, but in both processes filamentous fungi maintain several axes of polar growth simultaneously.     

供体胰腺保存方法的改进

目的 探索在胰腺保存液中添加胰蛋白酶抑制剂保护受损供体胰腺的方法方法按照获取的供体胰腺分为对照组、完整组和破损组.在完整组和破损组的UW 液中添加乌司他丁,于胰腺灌注前取保存胰腺的UW 液送测淀粉酶浓度,并比较各组的胰岛得率、纯度、活率和胰岛素释放指数.结果 对照组的UW 液中的淀粉酶含量为(31 ± 21)U,完整组...     

Characterization of a lipophilized gelatin prepared by a new method

Summary A new method for the preparation of lipophilized gelatin (LG) was developed. Gelatin dissolved using a microwave oven was easily lipophilized via reaction with fatty acid anhydrides for 3 hr at 50 °C and the subsequent emulsion was prepared by mixing LG and cotton seed oil. Palmitic anhydride LG was the most useful material for preparing stable emulsions. The emulsion was a multiple water-in-oil-in-water type.     

Sclerotization as a long-term preservation method for Rosellinia necatrix strains

This work describes a simple protocol for longterm preservation of strains of Rosellinia necatrix based on sclerotia production combined with storage at 4°C in liquid substrate, without affecting the growth and pathogenic characteristics of the fungal isolates recovered. The sclerotization process was set up in both liquid and solid media, and the sclerotia-like structures (pseudosclerotia) obtained were preserved in liquid media or water at 4°C. R. necatrix pseudosclerotia viability after 6 years of preservation at 4°C was confirmed by growth and microscopic characteristics, with no differences when compared with the fungal strains routinely preserved by periodic transfers. Additionally, pathogenicity on avocado plants by the preserved R. necatrix strains showed no difference from those preserved by periodic transfers. The albino strain used in this study should continue to be preserved by periodic subculturing.     

Low-cost and low maintenance preservation of Agaricus brasiliensis cultures

Agaricus brasiliensis cultures quickly lose viability when stored at cool temperatures, even for a short period of time. We evaluated several low-cost preservation methods using varied substrates, preservation solutions, and storage temperatures. Agaricus brasiliensis was intolerant to freezing temperatures, making liquid nitrogen use and deep-freezing methods impossible for its preservation. The best preservation conditions for the A. brasiliensis CS1 strain tested in this study were obtained by using rice as substrate and water as preservation solution, with storage at room temperature or when using soil, mushroom cultivation compost, or rice and stored at 10 °C without preservation solution. Those cultures that were reactivated showed the same productivity attributes as the control. In addition, no effect on productivity or biological efficiency was observed through successive subculturing of the strain (CS1). Parboiled rice was successfully used for other A. brasiliensis strains (CS2, CS5, CS7, CS9, and CS10), and also for Pleurotus ostreatus, P. sajor-caju, and Lentinula edodes.     

Biochemical mutagens affect the preservation of fungi and biodiversity estimations

Many fungi have significant industrial applications or biosafety concerns and maintaining the original characteristics is essential. The preserved fungi have to represent the situation in nature for posterity, biodiversity estimations, and taxonomic research. However, spontaneous fungal mutations and secondary metabolites affecting producing fungi are well known. There is increasing interest in the preservation of microbes in Biological Resource Centers (BRC) to ensure that the organisms remain viable and stable genetically. It would be anathema if they contacted mutagens routinely. However, for the purpose of this discussion, there are three potential sources of biochemical mutagens when obtaining individual fungi from the environment: (a) mixtures of microorganisms are plated routinely onto growth media containing mutagenic antibiotics to control overgrowth by contaminants, (b) the microbial mixtures may contain microorganisms capable of producing mutagenic secondary metabolites, and (c) target fungi for isolation may produce “self” mutagens in pure culture. The probability that these compounds could interact with fungi undermines confidence in the preservation process and the potential effects of these biochemical mutagens are considered for the first time on strains held in BRC in this review.     

Isochoric preservation: A novel characterization method

Isochoric (constant volume) preservation is an alternative to traditional cryopreservation methods because it requires less cryoprotectant and is simple to operate. In order to validate that this method automatically minimizes the pressure for a given temperature, pressure and temperature data were collected from a specially designed pressure vessel. This vessel was then used to examine the effect of an isochoric environment on freezing point nucleation in an aqueous antifreeze protein solution, and to generate pressure-temperature phase diagrams for various cryoprotectant solutions. Our results show that the isochoric pressure vessel follows the pressure-temperature phase diagram of water, thereby minimizing the pressure for the given temperature. We also show that the nucleation temperature of the antifreeze protein in an isochoric vessel is lower than that of the isobaric method. Furthermore, the nucleation temperature decreased with increasing concentration in the isochoric vessel while the isobaric nucleation temperature showed no change. These results indicate that the isochoric environment imposes additional constraints on ice formation and warrants further study as these results may change when a different type of cryoprotectant is used. Finally, all of the cryoprotectant phase diagrams exhibited a similar pressure-temperature slope indicating that, regardless of the cryoprotectant used or the mechanism by which it suppresses freezing, isochoric freezing affects the molecules in the same manner. Together, all of these results indicate that the isochoric method of preservation is a valuable tool for characterizing the thermodynamic properties of cryoprotectants and has great potential as a cryopreservation method in the field of cryobiology.     

Three-step cooling: A preservation method for adult rat heart cells

Adult rat heart cells were exposed to two-step cooling to ?196 °C with different holding periods at different subzero temperatures between both steps. The highest survival based on the percentage of trypan blue-excluding cells was 25% with 10% DMSO and a holding period of 6 min, and 21% with 15% DMSO and a holding period of 30 min. The highest survival based on morphological intactness was about 10%; there was no difference in results after cooling with 10 and 15% DMSO, and after holding between 2 and 30 min. The optimal survival based on the percentage of contracting cells was 52%, with 15% DMSO and a holding period of 2 min.When the holding period was replaced by a programmed cooling stage, the results could be improved. With this threestep cooling method, the optimal values, based on the number of trypan blue-excluding, intact, and contracting cells, were 40, 32, and 60%, respectively. It appeared that in the presence of 10% DMSO, which provided better survival than 5 and 15%, no significantly different results were obtained when the starting temperatures of the second cooling step varied between ?10 and ?20 °C, when the end temperatures varied between ?30 and ?60 °C, or when the cooling rates of the second cooling step varied between 0.1 and 1 °C/min. Three-step cooling provided similar results as linear cooling from 0 to ?100 °C, followed by rapid cooling to ?196 °C.     

Baggasse preservation: a need for a biotechnological approach

Paper is one of the basic needs of modern life. With the consumption of paper likely to grow to 320 million MT by the year 2001, the the worldwide pulp and paper industry is gradually realizing that there is a shortage of the traditional raw material of cellulosic fiber. Bagasse--a byproduct of the sugar industry, presents a potential source of fiber for the paper industry without further compromising the environmental concern. It is cheap, perennially replenishible, presently does not have an alternative economically attractive value added usage, and has adequate chemical and mechanical properties for paper making. However, for it to be available to the paper industry throughout the year, it needs to be stored and preserved for a period of 6 to 8 months. With inherent problems associated with bagasse morphology, intricacies reflected as a result of its physicochemical characteristics, army of microbial infestants, cost-effective quality expectations of the paper industry, and the ecofriendly approach demanded by the society/pollution control boards/environmentalists, there does not seem to be any viable alternative except to use biotechnology approaches for bagasse preservation. It envisages the preservation of maximally depithed bagasse in the piles/heaps, using one-time fine misting of a preservative formulation comprised of biodegradable and nondegradable chemicals inhibiting the microbial population at selected enzyme levels. Its efficacy is improved by open, dry, windy, and moderate sunlit sites for storage. The method is simple, sustainable, and superior to the prevalent methods that are cost, capital, and energy intensive, non-eco-friendly and have adverse cost:benefit ratios. The biotechnology approach has an inherent scope for further optimization, automation, and economization.