Total containment of a continuous-flow zonal centrifuge

A continuous-flow, high throughput, turbine-driven zonal centrifuge has been contained for use with biologically hazardous materials. The centrifuge and all ancillary equipment are enclosed in three sealed cabinets that are maintained at negative pressure, decontaminated with ethylene oxide, and provide cooling for the process fluids. Sample handling is semiautomated and remotely controlled for ease of operation and includes an automated decontamination system. The centrifuge subsystems can be decontaminated prior to an engineer servicing the machine, and apparatus is provided for absolute filtration of the centrifuge turbine exhaust.     

Evaluation of a class III biological safety cabinet for enclosure of an ultracentrifuge

An evaluation of a special safety cabinet housing a high-speed centrifuge was made. The cabinet enclosed both the top access port and the drive and pumping machinery of the centrifuge. A titanium rotor was loaded with tubes containing a bacterial culture, weakened, and driven until rotor rupture occurred. There were several bent and broken components in the centrifuge, and bacteria leaked from the vacuum chamber. Although the forces were sufficient to displace the cabinet, none of the test bacteria were found outside the cabinet.     

Zonal centrifuge profiles of rat brain homogenates: Instability in sucrose,stability in iso-osmotic Ficoll-sucrose

By substituting iso-osmotic Ficoll-sucrose for hyperosmotic sucrose between the densities of 1.043 and 1.088 in sucrose density gradients in the B-XV rotor of an Anderson-NIH-AEC zonal centrifuge system, it was possible to stabilize the zonal centrifuge absorbancy profiles of adult rat brain homogenates. The reason for the instability in ordinary sucrose gradients was found to be the interaction of myelin with other brain structures in hyperosmotic sucrose. No such interaction occurred in isoosmotic sucrose (0.32 M) with or without Ficoll. In Ficoll-sucrose, myelin was separated at three reproducible densities of 1.054, 1.060, and 1.066 gm/ml. No myelin appeared at a density if 1.094 gm/ml, which represented the main collection point in ordinary sucrose. Synaptosomes were separated at peak densities of 1.072 and 1.152 gm/ml. Mitochondria were obtained at a density of 1.176 gm/ml. Areas under zonal centrifuge absorbancy profiles of rat brain homogenates were found to be constant regardless of the values of ω²t that were reached.     

Biohazards Assessment in Large-Scale Zonal Centrifugation

A study was conducted to determine the biohazards associated with use of the large-scale zonal centrifuge for purification of moderate risk oncogenic viruses. To safely and conveniently assess the hazard, coliphage T₃ was substituted for the virus in a typical processing procedure performed in a National Cancer Institute contract laboratory. Risk of personnel exposure was found to be minimal during optimal operation but definite potential for virus release from a number of centrifuge components during mechanical malfunction was shown by assay of surface, liquid, and air samples collected during the processing. High concentration of phage was detected in the turbine air exhaust and the seal coolant system when faulty seals were employed. The simulant virus was also found on both centrifuge chamber interior and rotor surfaces.     

Purification of the protein crystal from Bacillus thuringiensis by zonal gradient centrifugation.

A method is described for the large-scale purification of the Bacillus thuringiensis protein crystal by zonal gradient centrifugation. NaBr gradients are employed in a Beckman J21-B centrifuge equipped with a JCF-Z rotor.     

THE SURFACE DECONTAMINATION OF SEEDS TO PRODUCE AXENIC SEEDLINGS

A variety of sterilizing agents were tested to develop a standard procedure for surface decontaminating seeds to produce axenic seedlings. The use of calcium hypochlorite (0.5% phosphate buffer, pH 6) for 10 min followed by three sterile water rinses was among the most effective agents, and it did not injure some species as did sodium hypochlorite, formaldehyde, ethylene oxide and mercuric chloride. Some species contained internal microbes requiring severe treatments which killed or injured the seedling, while other species were “decontaminated” with a sterile water rinse. The percentage of seeds with internal microbes varied considerably among plant species, seed lot, and the length of seed storage. Thus, with seeds not easily decontaminated, screening of additional seed lots would be more profitable than testing additional decontamination agents. Release of microbes from the seed's interior is associated with germination, and microbial testing must last at least 11 days. Nutrient agar permitted growth, although the seedlings outgrew petri plates too quickly for adequate certification. These seedlings were transferred to nutrient agar in quart jars in which an internal pool of broth was periodically agitated to permit microbial sampling of the leaves while the plant grew.     

Transport Phenomena in Zonal Centrifuge Rotors: I. Velocity and Shear-Stress Distributions of Fluid During Acceleration

An analysis is presented for the evaluation of velocity components and shear-stress distributions of fluid in zonal centrifuge rotors during acceleration. Analytical expressions for the distribution of tangential and radial velocity components and the tangential shear-stress and the radial shear-stress distributions of fluid are obtained for the transient case. Characteristics of each distribution for a typical density gradient liquid in a zonal centrifuge rotor are computed from the relations derived, and are presented as figures. An unusual phenomenon—the tangential velocity of the gradient exceeding the velocity of the rotor during a particular period of acceleration—is demonstrated.     

Respiratory Syncytial Virus Isolation by Combined Continuous Flow-Isopycnic Banding Centrifugation

A new zonal centrifuge rotor (B-IX) which combines continuous sample flow centrifugation with isopycnic banding has been used to isolate and concentrate respiratory syncytial virus from liter volumes of culture fluid. This isolation technique utilizes a sucrose density gradient to trap and isopycnically band the virus particles, and permits recovery of the particles from the rotor in an unaggregated condition.     

Rapid screening for plasmid DNA

Summary A procedure is described for demonstrating plasmid DNA and its molecular weight, based on rate zonal centrifugation of unlabelled DNA in neutral sucrose gradients containing a low concentration of ethidium bromide. Each DNA species is then visualized as a discrete fluorescent band when the centrifuge tube is illuminated with ultra-violet light. Plasmids exist as closed circular and as relaxed circular molecules, which sediment separately, but during preparation of lysates, closed circular molecules are nicked so that each plasmid forms only a single band of relaxed circles within the gradient.     

Herbicide-resistant, transgenic zonal pelargoniums produced by step-down glyphosate selection and plantlet recovery in the presence of aromatic amino acids

A genetic transformation protocol was developed using the transfer of a synthetic CP4 EPSPS transgene, as a conditional positive selectable marker, into commercially relevant zonal pelargoniums using an Agrobacterium tumefaciens strain in combination with a novel step-down glyphosate selection system. The transformation efficiency based on independent T-DNA integration events averaged 1.9?% over 10 experiments. Some 273 independent transformants were produced within an average time of 6?mo from explant inoculation with Agrobacterium to plantlet recovery. For plantlet recovery, three aromatic amino acids were incorporated into the rooting medium to ameliorate the accumulative effects of glyphosate selection. The T-DNA also contained a mutant ethylene receptor (etr1-1) cDNA from Arabidopsis thaliana, under control of the petunia flower-specific, floral-binding protein promoter, to confer ethylene insensitivity. However, delayed flower senescence was not obtained. The transformation protocol provides a reliable method to add herbicide resistance and other traits to zonal pelargonium.     

The fractionation of nuclei from mammalian cells by zonal centrifugation

1. Purified liver nuclei from adult rats separate into two main zones when centrifuged in the slow-speed zonal rotor. One zone contains diploid nuclei, the other tetraploid. 2. The effect of age on the pattern of rat liver ploidy was examined. Tetraploid nuclei are virtually absent from young animals. They increase in proportion steadily with age. Partial hepatectomy disturbs the pattern of ploidy. 3. The zonal centrifuge permits the separation of diploid, tetraploid, octaploid and hexadecaploid nuclei from mouse liver. 4. Rat liver nuclei are isopycnic with sucrose solutions of density 1.35 at 5 degrees .     

Affinity partitioning of membranes. Cholinergic receptor-containing membranes from Torpedo californica.

Affinity partitioning has been employed in the purification of membranes rich in cholinergic receptor from Torpedo californica electric organs. The procedure involves a modification of poly(ethylene oxide)-dextran aqueous phase partitioning systems where a ligand selective for the receptor is conjugated to the poly(ethylene oxide). Specific partitioning of the receptor-containing membranes into the poly(ethylene oxide)-rich phase occurs when bis-alpha,omega-trimethylamino poly(ethylene oxide) or bis-rho-tri-methylammonium phenylamino poly(ethylene oxide) was added to the phase system in low mole ratio. bis-alpha,omega-Methylamino poly(ethylene oxide), which should impart equivalent interfacial electromotive potential to the system but bind poorly to the receptor sites, was much less effective in producing phase distribution changes. The ligand-polymer-dependent phase distribution shifts were blocked by bisquaternary methonium ligands at concentrations consistent with their relative affinities for the cholinergic receptor. Titration or receptor sites with cobra alpha-toxin decreased the phase distribution changes in a linear fashion up to the point of stoichiometry. These observations are consistent with the phase distribution changes being consequent to ligand-polymer association with the pharmacologically important site on the receptor. The affinity partitioning procedure, when employed following an initial purification of the membranes by differential and density gradient centrifugation, yields membrane preparations with a high degree of morphological uniformity and a specific activity between 2.9 and 4.6 nmol of bound cobra alpha-toxin/mg of protein.     

Vapor-phase hydrogen peroxide as a surface decontaminant and sterilant.

The feasibility of utilizing vapor-phase hydrogen peroxide (VPHP) as a surface decontaminant and sterilant was evaluated in a centrifuge application. The prototype VPHP decontamination system, retrofitted into a Beckman L8-M ultracentrifuge, was designed to vaporize a 30% (wt/wt) solution of aqueous hydrogen peroxide continuously injecting and withdrawing VPHP in a deep-vacuum flow-through system. VPHP cycles of 4, 8, 16, and 32 min were examined for cidal activity against spores of Bacillus subtilis subsp. globigii and Bacillus stearothermophilus. Spore inocula (approximately 10(6)/coupon) were dried onto 0.5-in. (1.27-cm)-square stainless-steel coupons, and coupons were suspended in the centrifuge chamber, the space between the refrigeration can and the barrier ring (inner gap), and the space between the barrier ring and the vacuum ring (outer gap). At a chamber temperature of 4 degrees C, B. subtilis subsp. globigii spores were inactivated within 8 min, while inactivation of spores located in the outer gap at 27 degrees C required 32 min. The elevated temperature and high surface area/volume ratios in the outer gap may serve to decompose the gas more rapidly, thus reducing cidal efficacy. Of the two test spores, B. stearothermophilus was more resistant to VPHP. Nonetheless, VPHP was shown to possess significant sporicidal capability. For practical decontamination applications of the type described, VPHP shows promise as an effective and safer alternative to currently used ethylene oxide or formaldehyde vapors.     

Vapor-phase hydrogen peroxide as a surface decontaminant and sterilant

The feasibility of utilizing vapor-phase hydrogen peroxide (VPHP) as a surface decontaminant and sterilant was evaluated in a centrifuge application. The prototype VPHP decontamination system, retrofitted into a Beckman L8-M ultracentrifuge, was designed to vaporize a 30% (wt/wt) solution of aqueous hydrogen peroxide continuously injecting and withdrawing VPHP in a deep-vacuum flow-through system. VPHP cycles of 4, 8, 16, and 32 min were examined for cidal activity against spores of Bacillus subtilis subsp. globigii and Bacillus stearothermophilus. Spore inocula (approximately 10(6)/coupon) were dried onto 0.5-in. (1.27-cm)-square stainless-steel coupons, and coupons were suspended in the centrifuge chamber, the space between the refrigeration can and the barrier ring (inner gap), and the space between the barrier ring and the vacuum ring (outer gap). At a chamber temperature of 4 degrees C, B. subtilis subsp. globigii spores were inactivated within 8 min, while inactivation of spores located in the outer gap at 27 degrees C required 32 min. The elevated temperature and high surface area/volume ratios in the outer gap may serve to decompose the gas more rapidly, thus reducing cidal efficacy. Of the two test spores, B. stearothermophilus was more resistant to VPHP. Nonetheless, VPHP was shown to possess significant sporicidal capability. For practical decontamination applications of the type described, VPHP shows promise as an effective and safer alternative to currently used ethylene oxide or formaldehyde vapors.     

Ethylene Oxide Gaseous Sterilization: I. Concentration and Temperature Effects

The relationships of reaction temperature and concentration of gaseous ethylene oxide to the time required for inactivation of air-dried Bacillus subtilis var. niger spores are more complex than previously reported. A plot of temperature vs. the logarithm of “thermochemical death time” (TCDT) resulted in a straight line between 18 and 57 C for systems of “high” ethylene oxide concentration. The TCDT values were independent of ethylene oxide concentrations above certain temperature-dependent limits. A given ethylene oxide concentration produced a TCDT curve identical in the upper temperature regions with that for higher concentrations. As the temperature was lowered beyond a critical point, this curve diverged from that for higher concentrations, as a straight line of lesser slope. Thus, a series of curves exists for a range of ethylene oxide concentrations. They are characterized by two segments, both logarithmic, intersecting at a critical temperature for each concentration. The intersecting point is at a temperature inversely related to the ethylene oxide gas concentration. The temperature quotient for the high temperature segments of all systems was 1.8. This value was characteristic for ethylene oxide concentrations of 440 and 880 mg/liter at temperatures above 40.6 and 33.4 C, respectively. Below these critical temperatures, the Q₁₀ values for the respective systems were 3.2 and 2.3.     

The zonal centrifuge applied to the purification of a low-yield virus in human leukemia cells

The herpes-type virus found in certain cell cultures derived from Burkitt's lymphoma, other human leukemias, and normal human leukocytes, was concentrated and partially purified by large-volume density gradient centrifugation using zonal centrifuge systems. Using the Jiyoye (P-3) cell line as a model, rate-zonal runs on disrupted cell suspensions in sucrose gradients yielded concentrates with high virus particle counts when 10–15 ml of packed cells were processed per liter of gradient. Isolation and removal of cell nuclei or fluorocarbon treatment of cell sonicates permitted virus recovery from larger volumes of cells per experiment. Zonal centrifugation of concentrated cell-free spent media from highly infected cell cultures yielded more purified virus than obtained from cells. Viral concentrates were prepared with particle counts of 10¹⁰–10¹¹/ml and total protein concentrations of 0.2–0.5 mg/ml. Subsequent isopyenie-zonal centrifugation of the various high-count virus fractions from the zonal centrifuge showed a heterogeneity in buoyant virus density ranging from 1.18 to 1.27 in potassium tart rate. The spread in virus density was attributed to the different morphological forms of the virus observed by electron microscopy.     

Continuous-Flow Ultracentrifugation of Canine Distemper Virus and Infectious Canine Hepatitis Virus

The use of a Spinco L-4 zonal centrifuge with the B-XVI continuous-flow rotor for the purification of canine distemper and infectious canine hepatitis viruses is described. Up to 68 liters of virus was processed at one time. Infectious canine hepatitis virus was found to band at 39% sucrose and canine distemper virus banded between 32 and 48% sucrose. The virus was concentrated 10-fold, and the purity of the virus, as measured by protein concentration, was increased by 99%.     

Microbiological Aspects of Ethylene Oxide Sterilization: I. Experimental Apparatus and Methods

A specially built thermochemical death-rate apparatus is described which can be used to determine the resistance of microorganisms to ethylene oxide under controlled conditions. The apparatus was designed to provide instantaneous exposure of microorganisms to ethylene oxide and to eliminate variables that could result in errors when death kinetic reaction rates are calculated. The apparatus is used to obtain ethylene oxide resistance data which are useful in evaluating and developing sterilizing cycles for materials with known bacterial concentrations, as well as for calculating probability factors on which a given test condition can be expected to provide sterilization.     

Microbiological Aspects of Ethylene Oxide Sterilization: III. Effects of Humidity and Water Activity on the Sporicidal Activity of Ethylene Oxide

An investigation determined the effects of environmental moisture content or water activity (Aw), exposure humidity, and sterilant concentration on the resistance of microbial spores. Decimal reduction values [expressed as D values at 54.4 C-specified concentration (milligrams per liter) of ethylene oxide] were determined from spore destruction curves of Bacillus subtilis var. niger dried on hygroscopic and nonhygroscopic surfaces. Four groups of spore preparations were preconditioned in one of four Aw environments (<0.1, 0.1, 0.5, 0.95) for 2 weeks or longer and were exposed to 500 mg of ethylene oxide per liter at 54.4 +/- 3 C and 10, 50, and 95% relative humidity in a specially designed thermochemical death rate apparatus. A fifth group did not receive any preconditioning treatment and was exposed immediately after preparation, in the same apparatus at the same temperature, to ethylene oxide concentrations of 200, 400, 600, 800, and 1,200 mg/liter and relative humidities of 15, 30, 50, 60, and 90%. The resistance of the spores on both types of surfaces to ethylene oxide increased proportionately with the Aw of the conditioning environment. The study also showed that moisture in the exposure system was not as critical a variable as the ethylene oxide concentration. The spore destruction rates, irrespective of the carrier types at all concentrations and at different humidities, varied little from one another. The decimal reduction values were reduced as the ethylene oxide concentration increased, and no optimal exposure humidity concentration was observed.     

Synthesis and characterization of poly(ethylene oxide)-block-poly(methylidene malonate 2.1.2) block copolymers bearing a mannose group at the PEO chain end

A poly(ethylene oxide)-block-poly(methylidene malonate 2.1.2) block copolymer (PEO-b-PMM 2.1.2) bearing a mannose moiety at the poly(ethylene oxide) chain end was synthesized by sequential anionic polymerization of ethylene oxide (EO) and methylidene malonate 2.1.2 (MM 2.1.2), followed by a coupling reaction between its poly(ethylene oxide) amino- or aldehyde-end group and a sugar derivative. Different coupling procedures, either in organic media or in aqueous micellar solutions, were examined in order to optimize the poly(ethylene oxide) end-glycosylation yield. The micellar size of the functionalized block copolymers was determined by dynamic light scattering.