Autoclave Method Research Articles

Autoclave method for rapid preparation of bacterial PCR-template DNA

An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost.

SYNTHESIS AND CHARACTERIZATION ON THE LUMINESCENCE PROPERTIES OF MANGANESE DOPED ZnS NANOCRYSTALS

ZnS and manganese doped ZnS (ZnS:Mn2+) nanocrystals were prepared by adding Na2S to the solutions of zinc oleate and zinc oleate mixed with Mn(NO3)2. It was done by thermal decomposition using auto-clave method. The aging of the complexes in auto-clave resulted in ZnS and Mn2+ doped ZnS nanoparticles. The diameters of both nanoparticles were determined as approximately 10.3±2.1 nm from XRD line widths and TEM images. The ZnS:Mn2+ nanoparticles emitted at 500 nm∼650 nm (λmax=575 nm) in contrast to ZnS nanoparticles. The luminescence Intensity of ZnS:Mn2+ highly depend on the amount of Mn2+ added.

SYNTHESIS OF ZnS:Mn2+ NANOCRYSTALS AND LUMINESCENCE PROPERTIES OF LDPE FILM CONTAINING ZnS:Mn2+

The manganese-doped ZnS ( ZnS:Mn 2+) nanocrystals were prepared by addition of Na 2 S to an Zinc Oleate and Mn ( NO 3)2 solution. It was done using auto-clave method. The aging in auto-clave resulted in Mn 2+-doped ZnS particle. The emitting band ZnS:Mn 2+ showed red-shift from that of ZnS and results in the emission band at 500 nm ~ 650 nm (λ max =575 nm ). ZnS:Mn 2+ particles dispersed in polymer for production of the green house films. So this film will be used as light wavelength modification materials for the utilization of plant growth acceleration. Luminescence properties of the film were measured by luminescence spectrometer.

A STUDY ON A NEW SYNTHETIC METHOD OF CdS AND CdSe NANOPARTICLES AND THEIR ORGANIC/INORGANIC NANOCOMPOSITE

Materials such as CdS and CdSe inorganic nanoparticles have photoluminescence. Sodium oleate has been used as effective stabilizers for the synthesis of CdS and CdSe nanoparticles in water by autoclave method. Photoluminescence of CdS and CdSe with particle size of 5–14 nm showed λ max at 520 nm and 600 nm, respectively, when were excited at 365 nm. These nanoparticles doped into the PVA resulted in the organic/inorganic films ( PVA/CdS , CdSe ). Photoluminescence, X-ray diffraction and transmission electron microscopy were employed for their characterization.

Diclofenac Sodium Injection Sterilized by Autoclave and the Occurrence of Cyclic Reaction Producing a Small Amount of Impurity

A known impurity is formed in the production of a parenteral dosage form of diclofenac sodium if terminally sterilized by autoclave. This impurity has been detected as 1‐(2,6‐diclorophenyl) indolin‐2qone, which is also an intermediate from which diclofenac sodium is generally synthesized. It is only the condition of the autoclave method (i.e., 123 ± 2°C) that enforces the intramolecular cyclic reaction of diclofenac sodium forming the indolinone derivative and sodium hydroxide. The formation of this impurity has been found to depend on the initial pH of the formulation. The reaction follows first‐order kinetics, and the energy of activation is 5.34 kcal/mol. The other excipients in the formulation do not have a role in this reaction. The concentration of the impurity in the resultant product in the ampule goes beyond the limit of the raw materials in the pharmacopoeias. It is thus preferable to use an alternative sterilization method; that is, an aseptic filtration method in which the formation of this impurity can be avoided. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:541–544, 2001

Fish soup versus fish mince: a rapid autoclave method for preparing fish flesh for chemical analysis

Compared to the labour-intensive mincing method, autoclaving (pressurised steam cooking) is a fast way of preparing samples for chemical analysis. In addition, autoclaving dissolves fish scales and so enables more homogenous samples to be prepared. Initial method development work has shown that autoclaving at 150 kPa for 4 h is suitable for preparing samples for proximate and fatty acid analyses. However, the effect of autoclaving period and pressure on amino acid recovery has not been measured. A comparative study was made between autoclaving and mincing. The proximate chemical, fatty acid and amino acid composition of silver perch (Bidyanus bidyanus) was measured. The control samples (n = 3) were minced but not autoclaved. Each autoclaved sample (n = 36) was subjected to one of four autoclave periods (1, 2, 3 or 4 h) and one of three pressures (103, 155 or 207 kPa). Compared with mincing, autoclaving decreased nitrogen, increased energy and fat but had a variable effect on fatty acid recovery. Compared with mincing, autoclaving either had no effect on or increased the recovery of all amino acids, except cystine which was marginally reduced. Thus autoclaving appears to offer a superior method for preparing fish samples for amino acid analysis compared with mincing alone. Increasing autoclave pressure from 103 to 207 kPa had no measured effect on flesh composition. Sample processing time in the autoclave can be reduced from 4 to 1 h with silver perch flesh. © 2000 Society of Chemical Industry

Surface Geometric Structure of Chemically Modified Silica Studied by Direct Atomic Force Microscopy (AFM) Imaging and Adsorption Method

The relationship between wettability and surface geometric structure of modified silica particles and glass plates was investigated. Surface modification was performed by the autoclave method with 1-dodecanol to control the surface wettability. The preferential dispersion test proved that wettability varied at a surface modification ratio of 20%, which coincided with the changing point of the geometric structure of modifier chains determined by the adsorption method. The geometric structure was also evaluated by atomic force microscopy (AFM), and we could obtain the hexagonal packing of chains of the modifiers both in water and air at high surface modification ratio. Imaging of the surface of the nanosized particles on a glass plate was accomplished in water by taking advantage of the hydrophobic attractive force, which was proved by adhesion force measurements.

Study of Acidity of Aerogels ZrO2-SO42− by Isopropanol Dehydration Reaction, Surface Potential and X-Ray Photoelectron Spectroscopy

Sulphated zirconia aerogels, with definite atomic ratio S/Zr and hydrolysis ratio (H = H2O/Zr) were prepared by the autoclave method. The addition of sulphate ions causes a decrease of the cristallinity of zirconia. XPS results show the O1s photoelectronpeak which could be decomposed in two components for the reticular oxygen of the zirconia framework and for oxygen attributed to the OH groups and/or sulphates groups, and the S2p photopeak characteristic of sulphates species. The Kelvin probe shows that the value of pure zirconia is around 200 mV. This value grows up to 1200 mV for sulphate doped catalysts. The modification of the work function is probably due to the charge transfer from the zirconium to an oxygen species, responsible for the increase of Lewis acidity. The catalysts prepared with hydrolysis ratio of H = 4 exhibit higher activities in the isopropanol dehydration reaction than those with H = 2 in the temperature range 373 K–423 K.

Fish soup versus fish mince: a rapid autoclave method for preparing fish flesh for chemical analysis

Compared to the labour-intensive mincing method, autoclaving (pressurised steam cooking) is a fast way of preparing samples for chemical analysis. In addition, autoclaving dissolves fish scales and so enables more homogenous samples to be prepared. Initial method development work has shown that autoclaving at 150 kPa for 4 h is suitable for preparing samples for proximate and fatty acid analyses. However, the effect of autoclaving period and pressure on amino acid recovery has not been measured. A comparative study was made between autoclaving and mincing. The proximate chemical, fatty acid and amino acid composition of silver perch (Bidyanus bidyanus) was measured. The control samples (n = 3) were minced but not autoclaved. Each autoclaved sample (n = 36) was subjected to one of four autoclave periods (1, 2, 3 or 4 h) and one of three pressures (103, 155 or 207 kPa). Compared with mincing, autoclaving decreased nitrogen, increased energy and fat but had a variable effect on fatty acid recovery. Compared with mincing, autoclaving either had no effect on or increased the recovery of all amino acids, except cystine which was marginally reduced. Thus autoclaving appears to offer a superior method for preparing fish samples for amino acid analysis compared with mincing alone. Increasing autoclave pressure from 103 to 207 kPa had no measured effect on flesh composition. Sample processing time in the autoclave can be reduced from 4 to 1 h with silver perch flesh. © 2000 Society of Chemical Industry

Interaction of Aluminum with PHFτ in Alzheimer's Disease Neurofibrillary Degeneration Evidenced by Desferrioxamine-Assisted Chelating Autoclave Method

To demonstrate that aluminum III (Al) interacts with PHFtau in neurofibrillary degeneration (NFD) of Alzheimer's disease (AD) brain, we developed a "chelating autoclave method" that allows Al chelation by using trivalent-cationic chelator desferrioxamine. Its application to AD brain sections before Morin histochemistry for Al attenuated the positive fluorescence of neurofibrillary tangles, indicating Al removal from them. This method, applied for immunostaining with phosphorylation-dependent anti-tau antibodies, significantly enhanced the PHFtau immunoreactivity of the NFD. These results suggest that each of the phosphorylated epitopes in PHFtau are partially masked by Al binding. Incubation of AD sections with AlCl(3) before Morin staining revealed Al accumulation with association to neurofibrillary tangles. Such incubation before immunostaining with the phosphorylation-dependent anti-tau antibodies abolished the immunolabeling of the NFD and this abolition was reversed by the Al chelation. These findings indicate cumulative Al binding to and thereby antigenic masking of the phosphorylated epitopes of PHFtau. Al binding was further documented for electrophoretically-resolved PHFtau on immunoblots, indicating direct Al binding to PHFtau. In vitro aggregation by AlCl(3) was observed for PHFtau but was lost on dephosphorylation of PHFtau. Taken together, phosphorylation-dependent and direct PHFtau-Al interaction occurs in the NFD of the AD brain.

The effect of different synthesis methods on properties of GaP nanocrystals

GaP nanocrystals were synthesized by the benzene-thermal synthesis method in autoclave at 260°C for 6 h. The particle size distribution and optical absorption property were studied and were compared with that of the reflux method in organic solvent. The advantages of the benzene-thermal synthesis method are the lower temperature and shorter time required than in the reflux methods in organic solvent.

The expression of P-selectin in inflammatory and non-inflammatory lung tissue.

An initial attachment of leucocytes to blood vessel walls is mediated by selectins. A feature of adhesion mediated by P-selectin is the "rolling" of leucocytes on the endothelium. The time dependent expression of p-selectin in lung tissue was investigated in five groups of cases with different causes of death: carbon-monoxide and cyanide intoxication (n = 11), drowning (n = 5), hanging (n = 9), pneumonia (n = 13) and polytrauma with blunt thorax trauma (n = 14). In paraffin-embedded archival specimens immunostaining was achieved using an adapted APAAP-immunoperoxidase technique together with a wet autoclave method. P-selectin detection was scored by a semiquantitative method evaluating the intensity and incidence of positively stained endothelial cells. The distribution pattern of endothelial P-selectin of blood vessels in cases of pneumonia and septic shock were heterogenius and weak. In one case with lung contusion (survival time 3 h) moderate infiltrates of granulocytes were found near to septal and subpleural hemorrhages. In these inflammatory areas the positive endothelial immunostaining of small vessels was often weaker than in other lung segments or compared to the intensely stained platelets in corresponding vessels.

A case study of two airport runways affected by alkali-carbonate reaction. Part one: Evidence of deterioration and evaluation of aggregates

Through the inspection of field concrete and detailed petrographic examinations of concrete cores collected from two airport runways, it was found that the main reason of the damages in the concrete structures was alkali-carbonate reaction. The test results of the newly developed autoclave method agreed well with the field performance of the aggregates used. It is suggested that the autoclave method is an effective method to evaluate the reactivity of carbonate aggregates though its general validity should be widely inspected.

The use of extracted teeth for in vitro bonding studies: A review of infection control considerations

Objective. Infection control concerns regarding the handling of teeth for research purposes have prompted investigators to evaluate the effects of disinfection/sterilization on extracted teeth. The objectives of this literature review were to assess the reported findings of the effect of disinfection or sterilization on teeth used for in vitro bonding studies and make recommendations on their use. Methods. A search of the literature was performed to obtain background information on infection control guidelines and current findings regarding disinfection or sterilization of extracted teeth. Published articles addressing the effects of different disinfection and sterilizing procedures on the tooth, such as structural changes of dentin and dentin permeability or effect on bond strengths, were examined and compared for agreement or disagreement of findings. The review was organized by the type of disinfection or sterilization method utilized by the investigators. These methods were evaluated in an attempt to address whether disinfection/sterilization of extracted teeth can be recommended or if the procedure simply produces another variable. Results. Formalin, chemical heat sterilization (Chemiclave), autoclave, ethylene oxide and gamma radiation methods of disinfection/sterilization have been investigated for their effects on extracted teeth. When the effect of formalin storage on dentin bond strengths was examined, investigator results were extremely varied. Autoclave and chemical sterilization were found to produce comparable dentin bond results when compared to controls. Gamma radiation did not produce structural changes in the dentin but it currently has not been investigated for its effect on dentin bond strength. And while ethylene oxide produced similar dentin bond strength results when compared to controls, its use as a sterilant was determined to be ineffective. Significance. Investigators have found formalin storage to be effective for infection control purposes. It cannot, however, be recommended as a storage medium for dentin bonding studies due to the variability in dentin bond strengths resulting from its use. Ethylene oxide is also not recommended due to its inability to effectively sterilize teeth. Chemical heat and autoclave sterilization methods are recommended for preventing cross-contamination during in vitro dentin bonding research. This recommendation is based on current research findings which examined the effect of these sterilization methods on dentin and dentin bond strengths.

A critical review of ultra-accelerated tests for alkali-silica reactivity

A large number of ultra-accelerated test procedures, for determining the potential alkali reactivity of aggregates, have been developed, particularly in the past 15 years. An ultra-accelerated test method is defined as one which yields results within a few days or, at most, a few weeks. A number of ultra-accelerated test methods have been adopted as ‘standard tests’, but few have been adequately evaluated. The rapid globalization of the construction industry will require the harmonization of National Standard Test Methods. The major requirement of ultra-accelerated test methods is that they should correctly predict the potential reactivity of aggregates in greater than 95% of the cases. Due to the complexity and variability in the composition and grain size of aggregates, it is improbable that a single test method will be developed which would be appropriate for evaluating all types of aggregates. Another major requirement for ultra-accelerated test methods is that the interlaboratory coefficient of variation should be low, preferably less than 12%. At present, only the NBRI accelerated mortar bar method has been subject to adequate inter-laboratory evaluation. However, a more limited inter-laboratory investigation showed that the autoclave mortar bar test also shows considerable potential, as a satisfactory ultra-accelerated test method. Further refinement of the NBRI and autoclave methods is required to improve their performance with a wide variety of aggregates.

Demonstration of silver-stained nucleolar organizer region associated proteins (AgNORs) after wet autoclave pretreatment in breast carcinoma: correlation to tumor stage and long-term survival.

Argyrophilic nucleolar organizer region associated proteins (AgNORs) are known to reflect cellular and nucleolar activity. Due to a novel staining procedure, which substantially improves visualisation of AgNORs on formalin-fixed and paraffin-embedded material, AgNORs can be reliably demonstrated as true substructures of the nucleoli. The aim of the present study was to apply a standardized morphometric AgNOR quantification on a large series of breast carcinomas with regard to its prognostic relevance. AgNOR quantity was evaluated on archival tumor tissues of 115 adenocarcinomas of the breast treated with the wet autoclave method prior to standardized silver-staining and morphometric analysis. AgNOR parameters were correlated to prognostic features (steroid hormonal receptor status, tumor type, tumor size, histological grading, pTNM, and UICC stage) carrying out both univariate and multivariate survival analyses. AgNOR number and area were proven to be statistically significantly related (Pearson correlation coefficient: 0.67, Bonferroni adjusted P = 0.0001). Almost all AgNOR parameters, in particular CV (coefficient of variation) of corrected area (delta-area) and CV of number, were statistically significantly correlated to estrogen and progesterone receptor status as well as histological grading of tumors. Increased AgNOR parameters were statistically significantly associated with early tumor relapse and cancer related death. Univariate and multivariate analysis by means of Cox regression revealed independent prognostic significance for CV of delta-area and number of AgNORs. Various AgNOR parameters (CV of number, CV of delta-area, CV of area, mean delta-area, and mean area of AgNORs per nucleus) determined on wet autoclave pre-treated formalin-fixed and paraffin-embedded breast cancer tissues are statistically highly significantly associated with the prognostic outcome, independently predicting tumor-free and overall survival.

Manufacturing of Adaptive Graphite/Epoxy Structures with Embedded Nitinol Wires

Shape memory alloy (SMA) composites are a class of smart materials in which embedded nitinol wires are used as actuators. Previous research in SMA composites has been limited to wires using one-way shape memory behavior. The objective of this paper is to assess the feasibility of using two-way shape memory (TWSM) training of nitinol, a nickel-titanium SMA, wires for their use as embedded actuators in SMA composites and to investigate the effects of processing conditions on SMA composite quality and actuation ability. Training of the TWSM behavior in nitinol was investigated for a range of initial strains and training cycles. In each case, over 50% of the initial strain can be recovered after training and trained response is generally stable after about 30 training cycles. SMA composite specimens were manufactured using either hot press or autoclave methods and several different types of cure cycles. Voids were found to congregate near the embedded wire surface and can typically be as high as 10% in these regions. Using a staged-cure cycle, the average void content was reduced to 1.3-1.5%. The effect of the composite cure cycle was found to have no significant effect on trained nitinol wire behavior. Finally, the structural behavior of an SMA composite beam was investigated by actuating wires through electrical resistance heating. The tip deflection of a cantilevered beam was found to be primarily composed of SMA transformational effects, however, some localized heating was evident.

Wet autoclave pretreatment for immunohistochemical demonstration of oestrogen receptors in routinely processed breast carcinoma tissue.

The immunohistochemical demonstration of oestrogen receptor (OR) was performed on 32 randomly selected and routinely processed breast carcinomas after wet autoclave pretreatment of sections. The autoclave method was compared to the OR status found on frozen sections as well as to alternative pretreatment methods such as enzymatic predigestion and microwave irradiation. Using four different monoclonal antibody clones (H222, LH1, CC4-5, 1D5.26), the OR status was evaluated for each of the various pretreatment methods applied. All cases with a high OR content on frozen sections (n = 11) also showed a high OR status on wet autoclave-pretreated paraffin tissues using antibody clones 1D5.26 and CC4-5; in cases with low OR content on frozen sections, no false-negative cases were recorded using only the antibody 1D5.26 neither after wet autoclave nor microwave pretreatment. In addition, with this antibody, OR was detectable after autoclave pretreatment in two cases which were considered to be OR-negative even on frozen sections. When the primary antibody was omitted, no false-positive cases were observed after wet autoclave pretreatment. Thus, in our hands, wet autoclave pretreatment, in combination with the antibody 1D5.26, offers a highly sensitive method for the immunohistochemical demonstration of OR in routinely formalin-fixed, paraffin-embedded sections of breast carcinomas.

A procedure for the simultaneous oxidation of total soluble nitrogen and phosphorus in extracts of fresh and fumigated soils and litters

Abstract An autoclave method based on the oxidation of dissolved organic matter with alkaline persulphate for the analysis of total soluble nitrogen (N) and phosphorus (P) in 0.01M calcium chloride (CaCl2), 0.5M potassium sulfate (K2SO4), or 0.2M sulfuric acid (H2SO4) extracts of fresh and fumigated soils is described. Autoclaving at 110°C for 30 min oxidized N at 50 mg N/dm3 from urea, glucosamine, and the sodium salt of ethylenediaminetetra‐acetic acid (EDTA) to nitrate (NO3). Recoveries decreased with increasing concentrations of total organic carbon (TOC), hence glucosamine and EDTA required an increased concentration of persulphate. Tests with added glucose showed that recovery of urea N was complete up to a carbon (C) concentration of 100 mg/dm3. For P, it was important that solutions of 0.01M CaCl2 were acidic after oxidation to avoid the precipitation of appatite. Recoveries of P from orthphosphate and inositol hexaphosphate dodeca sodium salt in water or in 0.2M H2SO4 were complete after oxidatio...

Wet autoclave pretreatment for antigen retrieval in diagnostic immunohistochemistry

Interest has recently been shown in adapting the microwave oven heating technique for antigen retrieval to routine diagnostic immunocytochemical practice. Although it has proved effective as a specialist method for individual antigen localization in many laboratories, it has certain drawbacks which have hampered its wider routine application. These include the need to monitor the sections during the microwave treatment to prevent damage or drying, the limited number of sections that can be accommodated in the microwave oven, and the inevitable alteration in nuclear morphology induced by the microwaves. In order to obviate these difficulties, we have modified the wet autoclave method of Shin et al. (Lab Invest 1991; 64: 693-702) as a routine technique for retrieval of a variety of cell surface, cytoplasmic, and nuclear antigens in formalin-fixed and paraffin-embedded tissue. The technique produces even enhancement of several refractory antigens in anatomically different sites and has the potential to handle reliably up to 200 sections at a time without significant damage to the section or to nuclear morphology.