The total containment of a batch-type zonal centrifuge.

A batch-type zonal centrifuge has been modified and totally contained for use with biologically hazardous materials. A sealed cabinet encloses the centrifuge and the ancilliary equipment. It is operated with a flow of filtered air when the zonal system is on, decontaminated with ethylene oxide, and maintained at a negative pressure throughout. The centrifuge subsystems can be drained, flushed, and decontaminated with ethylene oxide before an engineer services the machine. The sample handling system within the cabinet is remotely controlled.     

Tube inserts as aids in preparative ultracentrifugation

Conventional preparative ultracentrifugation was modified by using inserts in the centrifuge tubes, causing a reduction of the time necessary to centrifuge a protein at reduced rotor velocity from suspension.     

Impact of process conditions on the centrifugal recovery of a disabled herpes simplex virus

Despite continuous improvements in culturing and recovery techniques, high-titer stocks of purified disabled herpes simplex virus type-1 (HSV-1 DIS) vector for drug discovery and use in preclinical and clinical trials are currently difficult to achieve. Efforts to improve their centrifugal recovery have been addressed in this paper. The operation of a swing-out centrifuge rotor was assessed, and its operational conditions were defined for the recovery of viable HSV-1 DIS. 80% virus recovery was achieved after 90 min at 26000g. The 20% loss of virus was attributed to damage to the viral envelope by overcompaction of the pellet and impaction with the base of the centrifuge tube. Virus recovery was increased by a further 10% by using a fixed-angle centrifuge rotor operating at 26000g. Plaque assays of recovered HSV-1 DIS gave values on the order of 10(6) pfu/mL, compared to values typically above 10(9) pfu/mL obtained for the replication-competent HSV-1 viron.     

8. Tube Inserts as Aids in Preparative Ultracentrifugation

ABSTRACT

Conventional preparative ultracentrifugation was modified by using inserts in the centrifuge tubes, causing a reduction of the time necessary to centrifuge a protein at reduced rotor velocity from suspension.     

Tube Inserts as Aids in Preparative Ultracentrifugation

Abstract

Conventional preparative ultra centrifugation was modified by using inserts in the centrifuge tubes, causing a reduction of the time necessary to centrifuge a protein at reduced rotor velocity from suspension.     

Purification of human sperm by a discontinuous Percoll density gradient with an innercolumn

Human sperm were highly purified through the use of a discontinuous Percoll density gradient placed in an inner column of a centrifuge tube. Six ml of 80% Percoll solution were poured into a centrifuge tube with an inner column containing successive 1.0-ml layers of 70, 60, and 40% Percoll solutions. Diluted semen was placed on top of the gradient, and the tube was centrifuged at 600 X g for 30 min using a swing-out rotor. After centrifugation, the majority of the progressive motile sperm were isolated in the sediment; they had a mean motility of 93 +/- 4.1% (n = 10). Other cellular components, including bacteria, remaining in the inner column. The level of bacterial contamination in the purified sperm fraction was below detection for most of the species quantified. The purified sperm were found to be more than 92 +/- 3.2% viable, as judged by dye exclusion, and abnormal sperm were reduced to 5.2 +/- 1.4%. Because of the use of the inner column, the contamination by seminal plasma was negligible in the purified sperm, as estimated by residual protein, fructose, and acid phosphatase activity.     

Descriptions of melissopalynological methods involving centrifugation should include data for calculating Relative Centrifugal Force (RCF) or should express data in units of RCF or gravities (g)

Techniques of pollen recovery involving centrifugation of honey pollen (melissopalynology) described in certain publications are difficult to 1) reproduce and 2) compare to other melissopalynological methods because dimensions of the centrifuge rotor were not described. Centrifugation data should be stated either in units of Relative Centrifugal Force (RCF) or gravities (g), or the data should be stated so RCF can be calculated using revolutions per minute (RPM) and the dimensions of the centrifuge rotor.     

Temperature regulation during centrifugal elutriation and its effect on cell separation

Centrifugal elutriation appears to be a promising method for cell separation. The quality of the separation may be limited by the control of temperature within the separation chamber, which affects the fluid viscosity and rotor speed. The factors affecting the temperature regulations have been re-examined. At flow rates between 10 and 40 mL/min the temperature within the chamber was primarily dependent on the temperature of the fluid flowing into the rotor. Increases in the temperature of the fluid while it flowed through the rotor were observed and were greater at higher rotor speeds and lower flow rates. This heating, caused by friction at the rotating seal, could raise the fluid temperature within the chamber by as much as 6 degrees C. Fluctuations in the temperature of the centrifuge produced temperature variations of only 0.3 degrees C in the fluid in the elutriation chamber. Small increases in the rate of elutriation of cells, concomitant with centrifuge cooling and speed fluctuations, were detected by optical density measurements. However, neither the modal volume nor coefficient of variation of the collected cells were affected.     

Vulnerability curves by centrifugation: is there an open vessel artefact,and are ‘r’ shaped curves necessarily invalid?

Vulnerability curves using the 'Cavitron' centrifuge rotor yield anomalous results when vessels extend from the end of the stem segment to the centre ('open-to-centre' vessels). Curves showing a decline in conductivity at modest xylem pressures ('r' shaped) have been attributed to this artefact. We determined whether the original centrifugal method with its different rotor is influenced by open-to-centre vessels. Increasing the proportion of open-to-centre vessels by shortening stems had no substantial effect in four species. Nor was there more embolism at the segment end versus centre as seen in the Cavitron. The dehydration method yielded an 'r' shaped curve in Quercus gambelii that was similar to centrifuged stems with 86% open-to-centre vessels. Both 'r' and 's' (sigmoidal) curves from Cercocarpus intricatus were consistent with each other, differing only in whether native embolism had been removed. An 'r' shaped centrifuge curve in Olea europaea was indistinguishable from the loss of conductivity caused by forcing air directly across vessel end-walls. We conclude that centrifuge curves on long-vesselled material are not always prone to the open vessel artefact when the original rotor design is used, and 'r' shaped curves are not necessarily artefacts. Nevertheless, confirming curves with native embolism and dehydration data is recommended.     

Temperature regulation during centrifugal elutriation and its effect on cell separation

Centrifugal elutriation appears to be a promising method for cell separation. The quality of the separation may be limited by the control of temperature within the separation chamber, which affects the fluid viscosity and rotor speed. The factors affecting the temperature regulation have been re-examined. At flow rates between 10 and 40 mL/min the temperature within the chamber was primarily dependent on the temperature of the fluid flowing into the rotor. Increases in the temperature of the fluid while it flowed through the rotor were observed and were greater at higher rotor speeds and lower flow rates. This heating, caused by friction at the rotating seal, could raise the fluid temperature within the chamber by as much as 6°C. Fluctuations in the temperature of the centrifuge produced temperature variations of only 0.3°C in the fluid in the elutriation chamber. Small increases in the rate of elutriation of cells, concomitant with centrifuge cooling and speed fluctuations, were detected by optical density measurements. However, neither the modal volume nor coefficient of variation of the collected cells were affected.     

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.     

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.     

Strain differentiation of epidemic typhus rickettsiae (Rickettsia prowazekii) by DNA restriction endonuclease analysis

We have investigated the number of methanogenic bacteria in different samples of fresh feces and effluents from the anaerobic digestion of cattle and swine waste and municipal sludges. We have further compared conventional anaerobic techniques with the rigorous, oxygen-free, anaerobic, experimental conditions obtained in a controlled glove cabinet atmosphere. It was found that the total number of oxygen-intolerant methanogenic bacteria was about 10–100 times higher than that of anaerobic oxygen-tolerant methanogenic bacteria. We have also developed a simple technique for the growth of methanogenic bacteria that permits the realization of rigorous anaerobic conditions without the use of a glove cabinet.     

Vulnerability to cavitation in Olea europaea current‐year shoots: further evidence of an open‐vessel artifact associated with centrifuge and air‐injection techniques

Different methods have been devised to analyze vulnerability to cavitation of plants. Although a good agreement between them is usually found, some discrepancies have been reported when measuring samples from long‐vesseled species. The aim of this study was to evaluate possible artifacts derived from different methods and sample sizes. Current‐year shoot segments of mature olive trees (Olea europaea), a long‐vesseled species, were used to generate vulnerability curves (VCs) by bench dehydration, pressure collar and both static‐ and flow‐centrifuge methods. For the latter, two different rotors were used to test possible effects of the rotor design on the curves. Indeed, high‐resolution computed tomography (HRCT) images were used to evaluate the functional status of xylem at different water potentials. Measurements of native embolism were used to validate the methods used. The pressure collar and the two centrifugal methods showed greater vulnerability to cavitation than the dehydration method. The shift in vulnerability thresholds in centrifuge methods was more pronounced in shorter samples, supporting the open‐vessel artifact hypothesis as a higher proportion of vessels were open in short samples. The two different rotor designs used for the flow‐centrifuge method revealed similar vulnerability to cavitation. Only the bench dehydration or HRCT methods produced VCs that agreed with native levels of embolism and water potential values measured in the field.     

10. Purification of Viruses by Cenlrifugation in Gradients of Inert Substances

ABSTRACT

By using a reorienting gradient centrifuge rotor cut from a block of Nylon and fitted with eight septae, it was possible to separate the components of the haemolymph of the mollusc Turbo sarmaticus into three fractions in a sucrose gradient held in the bowl of the rotor. The fractions were (108 and 98)S, 44S and 16-22S. The success of the experiment was due to the large differences in the sedimentation coefficients of the components. When the rotor was applied to the natural mixture of the five viruses of the caterpillars of Nudaurelia cytheria only the main component could be isolated in a pure state. The viruses were separated by isopycnic centrifugation in “self formed” caesium chloride gradients, using a Beckman Model E analytical centrifuge in which a separation cell fitted with a centerpiece with two perforated partitions was used.

Centrifugation in gradients of inert substances is useful for the separation of components in a mixture¹. There are two principles involved in this type of separation. One, termed reorienting gradient centrifugation (reograd) relies on the differences in masses or, better still on the sedimentation coefficients of the different components in the mixture and the second, termed isopycnic centrifugation², on the densities or specific gravities of the different entities.     

High-resolution flow-zonal centrifuge system.

A modified CF-32 Beckman flow centrifuge rotor has been developed that provides a long sedimentation path length with high gravitational force at the gradient sample interface. The modified rotor exhibits excellent separative capability and extraction efficiency when applied to purification of human influenza B and herpes simplex viruses.     

Mass Airflow Cabinet for Control of Airborne Infection of Laboratory Rodents

A mass airflow cabinet for handling and housing of laboratory rodents has been developed and tested. The unit consists of a high-efficiency particulate air filter and uniform distribution of air at a vertical velocity of 19 cm per s. Animals are maintained without bedding in mesh-bottomed cages that rest on rollers for rotation inside the cabinet. There is an air barrier of 90 cm per s separating the cabinet air from room air. Sampling for airborne bacteria yielded an average of 0.03 colony-forming units (CFU) per ft(3) of air inside the cabinet, whereas 28.8 CFU per ft(3) was simultaneously detected outside the cabinet during housekeeping, a reduction of almost three logs. The efficiency of the air barrier was tested by aerosolization of T3 phage. When phage was aerosolized 5 cm outside the cabinet, no phage could be detected 5 cm inside when the fans were operating; with the fans off an average of 1.6 x 10(4) plaque-forming units (PFU) per ft(3) was detected in six tests. Aerosolization of phage inside the cabinet yielded an average of 9 x 10 PFU per ft(3) outside; an average of 4.1 x 10(6) PFU per ft(3) were detected with the fans not in operation, a reduction of more than four logs. In-use studies on effectiveness showed that the cabinet significantly reduced the incidence of mice originally titer-free to Reo-3 virus. Hemagglutination inhibition antibodies to Reo-3 were detected in 9/22 (42%) mice housed in a conventionally ventilated animal laboratory while no seroconversion was detected in any of 22 mice housed in the mass air flow cabinet in the same laboratory.     

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.     

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.     

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.