Presence Of Liquid Nitrogen Research Articles

Gain Characteristics of Short-Length Erbium-Doped Fiber Amplifiers at Extreme Temperatures

Erbium-doped fiber amplifiers are used for amplifying optical signals. In this type of optical amplifier, the core of a silica fiber is doped with Er3+ ions. Here, the effects of temperature on the gain parameters for short-length erbiumdoped fiber amplifiers are simulated. Temperatures were selected over a wide range (−200 to +80°C) for different ion densities to simulate extreme conditions, such as the presence of liquid nitrogen. The maximum gain occurs at temperatures close to 0°C, and the Er3+ concentration also plays an important role.

Dielectric Characteristics of PPLP with various Pressures and Intervals between Butt Gap

Superconducting cables are one of the most promising applications among several high voltage superconducting power apparatuses owing to its low energy loss and high power capacities. Superconducting cables system consists of four parts: the termination, the joint box, the core, and the cooling system. The core is responsible for power transport, and is insulated with polypropylene laminated paper (PPLP) to ensure a high dielectric strength in the presence of liquid nitrogen (LN 2 ). The insulation layer of the core is fabricated by winding PPLP in multiple layers. It is inevitable that butt gap filled with LN 2 is generated in this process. Currently, many studies on the dielectric characteristics of PPLP according to various intervals between butt gap under 0.1 MPa and those with fixed butt gap according to various pressures have been conducted. A study on determining a proper interval between butt gap according to pressure in order to develop reliable superconducting cables under high pressure should be performed because superconducting cables system in power grid is operated under high pressure. This study focuses on the evaluation of the dielectric characteristics of PPLP with interval between butt gap at various pressures. In addition, dielectric experiments on V-t characteristics for a mini-model cable are conducted and experimental results are analyzed by the inverse power model.

Changes in the Solid State of Nicergoline, a Poorly Soluble Drug, Under Different Grinding and Environmental Conditions: Effect on Polymorphism and Dissolution

Nicergoline native crystals (Form I) were subjected to different grinding methods for 15, 30, 45, and 60 min: Method A, grinding at 20°C under air atmosphere; Method B, grinding in presence of liquid nitrogen under air atmosphere; Method C, grinding at 20°C under nitrogen atmosphere; and Method D, grinding in presence of liquid nitrogen under nitrogen atmosphere. Scanning electron microscopy, differential scanning calorimetry, X-ray powder diffractometry, thermogravimetry, and infrared spectroscopy were used to follow changes in the particle size and in crystalline structures. Batches from Methods A and C underwent partial amorphization immediately after grinding; Form II was obtained by heating these partially amorphous forms or after spontaneous crystallization after 1 and 5 months storage. Method B promoted the hydration of nicergoline to a monohydrate form. Batch D was stable under grinding and neither amorphization nor hydration were observed. The best intrinsic dissolution rate was that of metastable Form II, followed by Form I, while the worst was that of the Method B monohydrate form. The slowest particle dissolution was observed for hydrated particles, because of the lowest IDR, while the most rapid was exhibited by batch D, because of the very small particle size.

Effect of cryomilling on matrix/reinforcement interfaces and properties of Al-TiB2 composites

The present study reports the effects of cryomilling on the microstructural and mechanical properties of pressurelessly sintered Al-xTiB2 (x = 5, 10, or 15 wt%) composites. Composite powders milled under cryogenic conditions in the presence of liquid nitrogen were compacted in a hydraulic press, and the compacts were pressurelessly sintered at 650 °C for 5 h under Ar/H2 gas. The effects of cryomilling time (10 or 20 min) and TiB2 content (5, 10, or 15 wt%) on the microstructures, densities, and mechanical properties (wear characteristics, hardness, elastic modulus, indentation response) of the sintered products were investigated. Because the milling process was under cryogenic conditions, the formation of intermetallic phases between the matrix and reinforcement particles, such as Al3Ti, was not observed at the interfaces of the sintered Al-TiB2 composites, even when 15 wt% TiB2 was used. Cryomilling for 10 min followed by pressureless sintering of Al matrix composites reinforced with 5–15 wt% TiB2 particles resulted in bulk samples with relative densities of 97.38–98.43%, hardness values of 0.75–1.35 GPa, wear volume losses of 0.276–1.265 mm3, and elastic moduli of 283–433.7 GPa.

Effect of liquid nitrogen pre-treatment on various types of wool waste fibres for biogas production

This study investigated the role of liquid nitrogen (LN2) in increasing microbial accessibility of wool proteins for biogas production. It involves a mechanical size reduction of four different types of raw wool fibres, namely, Blackface, Bluefaced Leicester, Texel and Scotch Mule, in presence of liquid nitrogen, followed by the determination of the methane production potential of the pre-treated wool fibres. The highest methane yield, 157.3 cm3 g−1 VS, was obtained from pre-treated Scotch mule wool fibre culture, and represented more than 80% increase when compared to the yield obtained from its raw equivalent culture. The increase in biogas yield was attributed to the effectiveness of LN2 in enhancing particle size reduction and the consequent increase in wool solubility and bioavailability. Results also showed that LN2 pre-treatment can enhance size reduction but has limited effect on the molecular structure. The study also showed that the biogas potential of waste wool fibres varies with the type and source of wool.

A HIGH RETURN LOSS OF MICROWAVE BANDPASS FILTER USING SUPERCONDUCTING ELECTROSPUN YBCO NANOSTRUCTURES

A high return loss (-30 dB), small size (100 mm2) and broad bandwidth (1.5 GHz) microwave bandpass filter has been designed using finite element modelling and developed using the superconducting YBa2Cu3O7-δ (YBCO) thin films deposited on a (10 × 10 mm2) LaAlO3 substrate by spin coating. The thin films have been prepared by electrospinning and solid-state techniques. The microwave properties of filter circuits were experimentally determined using the vector network analyser (VNA) at room temperature (300 K) and in the presence of liquid nitrogen (77 K). The solid-state filter showed high return loss (i.e. -22 dB) at operating frequency of 9.7 GHz and broad bandwidth of 1.5 GHz, which is consistent with the simulation results. The insertion losses for YBCO filters are ~-2, ~-1.5 and ~-3 dB for the normal, nanoparticle and nanorod respectively. However, the electrospun filters exhibited lower performance due to the nano-structural properties of YBCO samples at nanoscale which make these sample have a large band gap compared to solid-state sample. The results indicate that the filter design and simulation result are reliable. Hence, HTS YBCO could be a potential microwave bandpass filter in industry.

Effect of Grinding on the Solid-State Stability and Particle Dissolution of Acyclovir Polymorphs

The present work investigated the solid state change of 4 acyclovir polymorphs when ground at room temperature (Method A) and under cryo-grinding in the presence of liquid nitrogen (Method B). Modifications in particle size and shape (evaluated by scanning electron microscopy) and in the water content (evaluated by thermal analysis) were related to transitions at the solid state, as confirmed by X-ray powder diffractometry. Anhydrous Form I was stable under grinding by both Methods A and B. The anhydrous Form II was stable during grinding under Method A, whereas it was progressively converted to the hydrate Form V during grinding under Method B. The hydrate Form V was stable under Method A, whereas it was converted to the anhydrous Form I after 15 min and then to the hydrate Form VI after 45 min of grinding. The hydrate Form VI proved to be stable under grinding by both Methods A and B. Thus, Form I and VI were the only forms that yielded a sizeable decrease in particle size under grinding, with a consequent increase in particle dissolution rate, while maintaining solid state physicochemical stability. Form I treated under Method B grinding gave the best dissolution rate.

Ultrasound assisted methods for enhanced extraction of phycobiliproteins from marine macro-algae, Gelidium pusillum (Rhodophyta)

Extraction of phycobiliproteins (R-phycoerythrin, R-PE and R-phycocyanin, R-PC) from macro-algae is difficult due to the presence of large polysaccharides (agar, cellulose etc.) present in the cell wall which offer major hindrance for cell disruption. The present study is aimed at developing most suitable methodology for the primary extraction of R-PE and R-PC from marine macro-algae, Gelidium pusillum(Stackhouse) Le Jolis. Such extraction of phycobiliproteins by using ultrasonication and other conventional methods such as maceration, maceration in presence of liquid nitrogen, homogenization, and freezing and thawing (alone and in combinations) is reported for the first time. Standardization of ultrasonication for different parameters such as ultrasonication amplitude (60, 90 and 120µm) and ultrasonication time (1, 2, 4, 6, 8 and 10mins) at different temperatures (30, 35 and 40°C) was carried out. Kinetic parameters were estimated for extraction of phycobiliproteins by ultrasonication based on second order mass transfer kinetics. Based on calorimetric measurements, power, ultrasound intensity and acoustic power density were estimated to be 41.97W, 14.81W/cm2 and 0.419W/cm3, respectively. Synergistic effect of ultrasonication was observed when employed in combination with other conventional primary extraction methods. Homogenization in combination with ultrasonication resulted in an enhancement in efficiency by 9.3% over homogenization alone. Similarly, maceration in combination with ultrasonication resulted in an enhancement in efficiency by 31% over maceration alone. Among all the methods employed, maceration in combination with ultrasonication resulted in the highest extraction efficiency of 77 and 93% for R-PE and R-PC, respectively followed by homogenization in combination with ultrasonication (69.6% for R-PE and 74.1% for R-PC). HPLC analysis was carried out in order to ensure that R-PE was present in the extract and remained intact even after processing. Microscopic studies indicated a clear relation between the extraction efficiency of phycobiliproteins and degree of cell disruption in a given primary extraction method. These combination methods were found to be effective for extraction of phycobiliproteins from rigid biomass of Gelidium pusillum macro-algae and can be employed for downstream processing of biomolecules also from other macro-algae.

Changes in the solid state of anhydrous and hydrated forms of sodium naproxen under different grinding and environmental conditions: Evidence of the formation of new hydrated forms

The aim of the present work was to investigate the solid state change of the anhydrous and hydrate solid forms of sodium naproxen under different grinding and environmental conditions. Grinding was carried out manually in a mortar under the following conditions: at room temperature under air atmosphere (Method A), in the presence of liquid nitrogen under air atmosphere (Method B), at room temperature under nitrogen atmosphere (Method C), and in the presence of liquid nitrogen under nitrogen atmosphere (Method D). Among the hydrates, the following forms were used: a dihydrate form (DSN) obtained by exposing the anhydrous form at 55% RH; a dihydrate form (CSN) obtained by crystallizing sodium naproxen from water; the tetrahydrate form (TSN) obtained by exposing the anhydrous form at 75% RH. The metastable monohydrate form (MSN), previously described in the literature, was not used because of its high physical instability.The chemical stability during grinding was firstly assessed and proven by HPLC. Modification of the particle size and shape, and changes in the solid state under different grinding methods were evaluated by scanning electron microscopy, and X-ray powder diffractometry and thermogravimetry, respectively. The study demonstrated the strong influence of starting form, grinding and environmental conditions on particle size, shape and solid state of recovered sodium naproxen forms. In particular, it was demonstrated that in the absence of liquid nitrogen (Methods A and C), either at air or at nitrogen atmosphere, the monohydrate form (MSN) was obtained from any hydrates, meaning that these grinding conditions favored the dehydration of superior hydrates. The grinding process carried out in the presence of liquid nitrogen (Method B) led to further hydration of the starting materials: new hydrate forms were identified as one pentahydrate form and one hexahydrate form. The hydration was caused by the condensation of the atmospheric water on sodium naproxen particles by liquid nitrogen and by the grinding forces that created a close contact between water and drug. The simultaneous disruption of the crystals, occurring during grinding, and their close contact with water molecules promoted the conversion in higher hydrates. Under the Method D, it was possible to highlight a certain tendency to hydration probably due to a rearrangement of water already present into the hydrates, but results were substantially different from Method B. Thus, summarizing, the different SN forms behave differently under different grinding and environmental conditions.

Designing a Cold Plate Used in Rapid Freeze Prototyping Technology

The rapid prototyping with ice is a new manufacturing technology that can generate three-dimensional objects from ice by depositing and rapidly freezing water layer by layer. In order to freeze the water droplets, the plate must meet certain conditions, among those it is worth to be mentioned, the surface temperature, which should not surpass 0°C. The equipment presented in the paper is a part of rapid freeze prototyping equipment, designed and built by the authors. The authors analysed various freezing systems and they discovered that the classical cooling systems are not appropriate to be used in rapid freeze prototyping technology. The unique solution is the utilisation of thermoelectric cells, generic named as Peltier cells. The modern technology can be used in obtaining Peltier cells, with a high degree of reliability, electrical consumption and yield. This can be successfully used as heat pumps in industry. The author developed an original system that permitted to implement the Peltier cells. The cold plate was designed by using CAD software. In addition, the cold plate was produced and tested. The equipment obtained, has a greater power in comparison with the classical ones, even if it has smaller dimensions. The electrical power supply has to be well filtered in order to generate a higher efficiency. Through the main advantages of the Peltier cells can be mentioned: no maintenance have to be performed as this type of cells have no moving parts, thus having a major advantage; no CFC or other consumables parts have to be used, thus proving to be more economical than other systems; the heat regulation can be performed easier compared with the classical systems offering a better control and precision; it can be used in severe environments where conventional cooling systems can fail, being successfully used in the presence of liquid nitrogen; it can perform in various positions and is reversible similar to heat pumps. The main drawback of this system is that it uses a great amount of electrical energy, consequently can have a low yield.

Preparation of glibenclamide nanocrystals by a simple laboratory scale ultra cryo-milling

The objective of this study is to evaluate the ability to reduce the particle size of glibenclamide (GBC) to the nanometric scale through a very simple and well-known laboratory scale method, the laboratory scale ultra cryo-milling. The effect of milling on GBC crystalline properties and dissolution behaviour was deliberately evaluated in the absence of any surfactants as stabilizers. The milling procedure consisted in adding particles to liquid nitrogen and milling them by hand in a mortar with a pestle for different time intervals (15, 30, 40 min). For comparison, the same milling procedure was also applied without liquid nitrogen. The particle size reduction was evaluated for the coarsest samples (>3 μm) by measuring the particle Ferret’s diameter through scanning electron microscopy, while for the smallest one (<3 μm) by dynamic light scattering. A time grinding of 40 min in the presence of liquid nitrogen was revealed highly efficacious to obtain particles of nanodimensions, with a geometric mean particle size of 0.55 ± 0.23 μm and more than the 80 % of particles lower than 1,000 nm. Interestingly, non-agglomerated particles were obtained. Differential scanning calorimetry and X-ray powder diffractometry allowed to assess that under mechanical treatment no polymorphic transitions were observed, while a decrease in crystallinity degree occurred depending on the milling procedure (presence or absence of liquid nitrogen) and the milling time (crystallinity decreases at increasing milling time from 15 to 40 min). A comparison of the intrinsic dissolution rate and the dissolution from particles revealed an interesting improvement of particle dissolution particularly for particles milled in the presence of liquid nitrogen due to an increase in particle surface area and concentration gradient, according to the Noyes–Whitney equation.

Indomethacin nanocrystals prepared by different laboratory scale methods: effect on crystalline form and dissolution behavior

The objective of this study is to select very simple and well-known laboratory scale methods able to reduce particle size of indomethacin until the nanometric scale. The effect on the crystalline form and the dissolution behavior of the different samples was deliberately evaluated in absence of any surfactants as stabilizers. Nanocrystals of indomethacin (native crystals are in the γ form) (IDM) were obtained by three laboratory scale methods: A (Batch A: crystallization by solvent evaporation in a nano-spray dryer), B (Batch B-15 and B-30: wet milling and lyophilization), and C (Batch C-20-N and C-40-N: Cryo-milling in the presence of liquid nitrogen). Nanocrystals obtained by the method A (Batch A) crystallized into a mixture of α and γ polymorphic forms. IDM obtained by the two other methods remained in the γ form and a different attitude to the crystallinity decrease were observed, with a more considerable decrease in crystalline degree for IDM milled for 40 min in the presence of liquid nitrogen. The intrinsic dissolution rate (IDR) revealed a higher dissolution rate for Batches A and C-40-N, due to the higher IDR of α form than γ form for the Batch A, and the lower crystallinity degree for both the Batches A and C-40-N. These factors, as well as the decrease in particle size, influenced the IDM dissolution rate from the particle samples. Modifications in the solid physical state that may occur using different particle size reduction treatments have to be taken into consideration during the scale up and industrial development of new solid dosage forms.

The structural stability of AlPO4-5 zeolite under pressure: Effect of the pressure transmission medium

The structural stability of AlPO4-5 zeolite (AFI) has been studied as a function of pressure up to 34.4 GPa in a diamond anvil cell by using synchrotron x-ray diffraction. It is found that the AFI structural stability can be enhanced significantly when a mixture of silicone oil and liquid nitrogen is used as pressure transmission medium (PTM). In this case, the crystalline-to-amorphous transition pressure for AFI increased to be 15.9 GPa, much higher than that of 8.5 GPa observed in the experiment by using silicone oil as PTM. We found that the average distance of the interplanar crystal spacing along to most planes was expanded obviously when liquid nitrogen is used as one component in the PTM. The presence of liquid nitrogen in the PTM also affects the structural evolution of the AFI channel under pressure. The results demonstrated that nitrogen molecules can be inserted into the channels of porous zeolite AFI single crystals, exerting a supporting effect against the structure collapse of AFI and thus improving their structural stability.

Synthesis and characterization of peptides after high-energy impact on the icy matrix: Preliminary step for further UV-induced formation

In the interstellar medium, the most probable source of organic molecules could be non-equilibrium processes driven by photons, cosmic rays, shock waves and solid bodies’ collisions. The dense cold phase of ISM host icy dust grains—important chemical catalyst during its life cycle. Such particles consist of mineral core composed by silicate or olivine admixed with metal sulfides and oxides, with the water-icy envelope containing organic molecules. Organic molecules in the ISM evolve and become later incorporated in solar system material (comets and meteorites). The formation of polypeptides from single amino acids was traced in simulation experiments representing the inner structure of icy dust grains. Experimental chamber was irradiated at subzero temperatures at the dosage of 2.54 kRad/min. Solid frozen solutions of Gly and Phe were taken as the experimental samples inserted into the metal tube kept at subzero temperatures in the presence of liquid nitrogen. Formation of di- and tri-peptides was demonstrated after applying mass-spectrometry and high performance liquid chromatography (HPLC) techniques. Having polypeptides within the icy matrix, dust grains with ice mantles are transported to warm, dense and active protostellar regions, where ultraviolet irradiation may become important and alter the grain composition. Furthermore UVC radiation may contribute to the formation of additional amounts of polypeptides, since short-wave photons are totally adsorbed by a thin outer layer. This presumption coincides with our previous investigations concerning UV impact on prebiotic formation of the main biological molecules. Combination of two irradiation types in different stages of interstellar flight could compensate the effects of low reagents concentration and temperature. Since the primordial Earth had no atmosphere, the natural carriers could get freely onto its surface and thus raise the concentration of organic molecules.

Dimerization Controls the Activity of Fungal Elicitors That Trigger Systemic Resistance in Plants

The soilborne fungus Trichoderma virens secretes a small protein (Sm1) that induces local and systemic defenses in plants. This protein belongs to the ceratoplatanin protein family and is mainly present as a monomer in culture filtrates. However, Hypocrea atroviride (the telomorph form of Trichoderma atroviride) secretes an Sm1-homologous protein, Epl1, with high levels of dimerization. Nonetheless, the molecular mechanisms involved in recognition and the signaling pathways involved in the induction of systemic resistance in plants are still unclear. In this report, we demonstrate that Sm1 and Epl1 are mainly produced as monomer and a dimer, respectively, in the presence of maize seedlings. The results presented show that the ability to induce plant defenses reside only in the monomeric form of both Sm1 and Epl1, and we demonstrate for the first time that the monomeric form of Epl1, likewise Sm1, induces defenses in maize plants. Biochemical analyses indicate that monomeric Sm1 is produced as a glycoprotein, but the glycosyl moiety is missing from its dimeric form, and Epl1 is produced as a nonglycosylated protein. Moreover, for Sm1 homologues in various fungal strains, there is a negative correlation between the presence of the glycosylation site and their ability to aggregate. We propose a subdivision in the ceratoplatanin protein family according to the presence of the glycosylation site and the ability of the proteins to aggregate. The data presented suggest that the elicitor's aggregation may control the Trichoderma-plant molecular dialogue and block the activation of induced systemic resistance in plants.

Quantitative Measurement of Fungal DNA Extracted by Three Different Methods Using Real-Time Polymerase Chain Reaction

Quantification of fungal populations in the environment is important for gaining a better understanding of various microbial processes. Recently, the development of real-time quantitative PCR (RTQ-PCR) has eliminated the variability associated with conventional quantitative PCR, thereby allowing the routine and reliable quantification of PCR products. Thus, in this present study, RTQ-PCR was used to quantify the fungal target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in the presence of liquid nitrogen, and hot detergent SDS based enzymatic lysis combined with bead beating) to determine the suitability of the quantification of target DNA. For the purpose of this study, pure culture of Aspergillus fumigatus (model organism), sterilized soil seeded with a known amount of A. fumigatus (model soil system), and woodland and grassland soil samples (environmental samples) were chosen to extract DNA by the above three different protocols. The extracted DNA was then quantified by spectroscopy and a RTQ-PCR system. 18S rDNA specific universal fungal primers were used to quantify the target part and then amplification products were verified by agarose gel electrophoresis. Standard curves used for the quantification by RTQ-PCR revealed strong linear relationships ( R 2 =0.9994 for the primer pair NS1 and NS2 and 0.9938 for the primer pair nu-SSU-0817 and nu-SSU-1196) with a higher amplification efficiency, e =0.983 for the primer pair NS1 and NS2 and 0.956 for the primer pair nu-SSU-0817 and nu-SSU-1196. Although for pure culture the hot detergent SDS based enzymatic lysis combined with bead beating method showed the highest target DNA copy number (1.5 × 10 9 copies/μl), for the model soil system and both environmental samples the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (6.16 × 10 8 and 2.7 × 10 8 copies/μl for woodland and grassland, respectively), high DNA yield (6.4 μg/g and 1.8 μg/g of soil for woodland and grassland, respectively), and high recovery on the basis of the target DNA copy number (39.2%), suggesting an overall high extraction efficiency.

Quantitative measurement of fungal DNA extracted by three different methods using real-time polymerase chain reaction

Quantification of fungal populations in the environment is important for gaining a better understanding of various microbial processes. Recently, the development of real-time quantitative PCR (RTQ-PCR) has eliminated the variability associated with conventional quantitative PCR, thereby allowing the routine and reliable quantification of PCR products. Thus, in this present study, RTQ-PCR was used to quantify the fungal target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in the presence of liquid nitrogen, and hot detergent SDS based enzymatic lysis combined with bead beating) to determine the suitability of the quantification of target DNA. For the purpose of this study, pure culture of Aspergillus fumigatus (model organism), sterilized soil seeded with a known amount of A. fumigatus (model soil system), and woodland and grassland soil samples (environmental samples) were chosen to extract DNA by the above three different protocols. The extracted DNA was then quantified by spectroscopy and a RTQ-PCR system. 18S rDNA specific universal fungal primers were used to quantify the target part and then amplification products were verified by agarose gel electrophoresis. Standard curves used for the quantification by RTQ-PCR revealed strong linear relationships ( R 2=0.9994 for the primer pair NS1 and NS2 and 0.9938 for the primer pair nu-SSU-0817 and nu-SSU-1196) with a higher amplification efficiency, e=0.983 for the primer pair NS1 and NS2 and 0.956 for the primer pair nu-SSU-0817 and nu-SSU-1196. Although for pure culture the hot detergent SDS based enzymatic lysis combined with bead beating method showed the highest target DNA copy number (1.5 × 10 9 copies/μl), for the model soil system and both environmental samples the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (6.16 × 10 8 and 2.7 × 10 8 copies/μl for woodland and grassland, respectively), high DNA yield (6.4 μg/g and 1.8 μg/g of soil for woodland and grassland, respectively), and high recovery on the basis of the target DNA copy number (39.2%), suggesting an overall high extraction efficiency.

Preparation of Protein Extracts from Yeast

Three protocols are presented for preparing protein extracts; they differ primarily in the way the cells are broken. In the basic protocol, cells are enzymatically converted to spheroplasts, which are then lysed by a combination of osmotic shock and Dounce homogenization. A support protocol for isolating intact nuclei by differential centrifugation is also presented. An alternate protocol describes mechanical breakage of cells by vortexing in the presence of glass beads. In a second alternate protocol, growing cells are frozen immediately in liquid nitrogen and then lysed by grinding in an industrial-strength blender in the presence of liquid nitrogen.

Chapter 8 Bulk Isolation of Yeast Mitochondria

Publisher Summary Various methods are used for isolating yeast mitochondria. However, most are not suitable for the isolation of mitochondria in gram quantities. Generally, small-scale isolation methods cannot be conveniently scaled up. For example, the treatment of yeast cells with snail gut enzyme yields fragile spheroplasts which can be gently lysed to provide fairly intact mitochondria. Faced with limitations, two methods for the bulk isolation of yeast mitochondria are developed. One involves rupturing the cells in a Waring Blendor in the presence of liquid nitrogen, and the second involves breaking the cells in a commercially available Manton–Gaulin homogenizer. Both methods are successfully used in many laboratories, however are relatively laborious and time-consuming, particularly when large amounts of cells need to be broken. These methods are not always equally reliable with different yeast strains or commercial preparations of yeast. The chapter discusses the Dyno–Mill method which has two major advantages over other large-scale methods of yeast cell breakage. It is a far more convenient and easy procedure; and breakage is very efficient, leading to a high yield of mitochondria. With the Dyno–Mill it is easily possible for one person to process 4–5 kg of yeast per day and to obtain 25 gm or more of mitochondrial protein. As 4 kg can be broken in two hours or less, production is limited mainly by the volume of the yeast suspension that must be subsequently processes.

Unimpaired Infectivity of the Nematode Haemonchus contortus after Freezing for 44 Weeks in the Presence of Liquid Nitrogen

Exsheathed larvae of Haemonchus contortus were frozen over liquid nitrogen, and were kept frozen for 44 weeks. Upon thawing, the larvae showed no loss in viability as judged by motility. Their infectivity to sheep was compared with that of normal larvae (ensheathed and nonfrozen). Sheep given 1,500 frozen exsheathed larvae acquired worm counts (688 to 921, mean 722) similar to those given normal larvae (439 to 852, mean 627). It has been reported from this laboratory that exsheathment of the infective larva of the nematode Haemonchus contortus enables it to withstand freezing in the presence of liquid nitrogen (Campbell et al., 1972). When that report was prepared for publication the longest survival time recorded was 4 weeks; and although the larvae had been shown to be highly infective, it had not then been possible to make a critical comparison of the infectivity of frozen and nonfrozen larvae in worm-free sheep. The present study shows that frozen larvae remain viable (without perceptible loss in numbers) for at least 44 weeks and that their infectivity is unimpaired. MATERIALS AND METHODS Use was made of a strain of H. contortus originating on Branchburg Farm, New Jersey, and maintained by serial passage in sheep for many years. Larvae were harvested from conventional fecal cultures after 7 days' development at 27 C and approximately 90% relative humidity. The larvae of this strain can readily be exsheathed by bubbling carbon dioxide into the suspension of larvae, followed by agitation at 37 C; and this was the method employed for exsheathment. The larvae were concentrated in "Vitro" brand 1-dram screw-cap vials (Wheaton Glass Company, Milville, New Jersey). Each vial contained roughly 150,000 larvae in 1 ml water. They were frozen by transferring the vials directly from air at room temperature to the storage section of a liquid nitrogen container (Union Carbide Corporation, Model Designation Linde LD-40). According to the manufacturer of the unit, the temperature in this sealed space would not be more than 96 C higher than the temperature of the underlying liquid nitrogen (-196 C). The vapor space contained 4 layers of steel storage drawers; the vials were stored in the second layer from the top, but we Received for publication 20 October 1972. have no reason to believe that the exact location was a critical factor. The larvae were thawed by immersing them in water at about 55 C, allowing approximately 300 ml of water per vial, and making no attempt to maintain the initial temperature of the bath. As soon as the contents of the vial were thawed, the larval suspension was diluted several-fold with water at room temperature and allowed to stand at room temperature for 1 hr before the motility was assessed. The lambs used in the infectivity trial had been raised on slatted floors which were cleaned daily, and were kept under these conditions throughout the experiment. They were known to harbor light infections of Capillaria, Strongyloides, and Eimeria spp. at the time of use, but were not passing trichostrongyle eggs in the feces. The lambs were of both sexes and were approximately 3 months old. At necropsy the abomasum was removed and the contents collected on a 200-mesh sieve (pore size 0.074 mm). The contents were stained with iodine and examined in their entirety to determine the total number of worms present. The abomasum was allowed to soak in saline at 37 C for 24 to 48 hr, with the addition of an antibiotic preparation to suppress putrefaction. The resultant material was collected on a 200-mesh screen, stained, and examined in its entirety for worms. EXPERIMENTS AND RESULTS Larvae of H. contortus were exsheathed and frozen on 25 August 1971. A sample of these larvae, henceforth designated "frozen" larvae, was removed from the container on 28 June 1972, that is, after 44 weeks in the frozen state. After thawing, examination of 200 larvae under a stereoscopic microscope revealed that 92% of them were motile. It is possible that an even greater percentage of the larvae in this culture would have been motile prior to freezing; but the motility of 88% of the larvae in the nonfrozen control culture (below) is