Assessment of a disc stack centrifuge for use in mammalian cell separation

A prototype disc stack centrifuge was tested for the separation of mammalian cell cultures from 80- and 2000-L fermentations. The clarification capacity for mammalian cells was excellent, but some smaller particles remained in the supernatant and reduced its usefulness for downstream processing. In order to identify the source of such particle formation, several parameters were assessed and minimum particle size for separation was calculated. An analysis of particle distribution was performed. Temperature and pressure effects inside the centrifuge bowl were measured. Some modifications of mechanical engineering can be suggested for the improvement of the use of standard disc stack centrifuges for mammalian cells. (c) 1995 John Wiley & Sons, Inc.     

High-resolution particle size analysis in biotechnology process control.

Many industrially important proteins can now be expressed intracellularly as insoluble protein inclusion bodies. In production, large-scale centrifugation is commonly used to separate and recover the inclusion bodies. Recovery efficiency depends critically on the centrifuge feed rate, which must be optimized to minimize production costs. We have used a disc centrifuge photosedimentometer to make high-resolution measurements of the particle size distribution (PSD) of the supernatant during the production of porcine somatotropin (pST) inclusion bodies. These measurements readily monitor the breakthrough of inclusion bodies into the supernatant and allow the centrifugation operation to be optimized.     

The performance of a scaled down industrial disc stack centrifuge with a reduced feed material requirement

A method is described for the scale-down of a disc stack centrifuge which reduces the number of separating discs and also the liquid and solid hold-up of the centrifuge bowl. This is to enable a reduced volume of process material to be used for study of clarification. Scale-down is achieved in stages using a series of interlocking inserts to suit particular applications. Maximum scale-down gives a 76% reduction in the separation area and a bowl volume reduction of 70%. Separation performance of the full stack machine and scale-down variants is compared using the grade efficiency concept. Polyvinyl acetate and bakers' yeast homogenate particle suspensions are used for the comparison. The grade efficiency curves produced by the scale-down variants closely follow the curves for the full stack machine. This resulted in supernatants of the same volume size distribution and concentration when using scale-down methodology to mimic the full scale operation.

     

Performance prediction of industrial centrifuges using scale-down models

Computational fluid dynamics was used to model the high flow forces found in the feed zone of a multichamber-bowl centrifuge and reproduce these in a small, high-speed rotating disc device. Linking the device to scale-down centrifugation, permitted good estimation of the performance of various continuous-flow centrifuges (disc stack, multichamber bowl, CARR Powerfuge) for shear-sensitive protein precipitates. Critically, the ultra scale-down centrifugation process proved to be a much more accurate predictor of production multichamber-bowl performance than was the pilot centrifuge.     

Aerial Dispersal and Epiphytic Survival of Pseudomonas syringae during a Pretest for the Release of Genetically Engineered Strains into the Environment

Prospective experimental field evaluation of genetically engineered microorganisms, such as microbial pest control agents, raises issues of how to properly ascertain their fate and survival in the environment. Field trials with recombinant organisms must reflect requirements for sampling and monitoring. Field trials were conducted at Tulelake, Calif., to monitor the numbers of viable cells of a nonrecombinant strain of Pseudomonas syringae that entered the atmosphere and landed on plants and soil during and after an aerosol spray application. An exponential decrease in numbers of viable cells deposited at increasing distances from three sprayed plots was observed. The relative rate of survival of cells sprayed directly on plants was more than 10 times higher than that of cells dispersed through the air to similar adjacent plants. Results are being used to gain experience with the characteristics of a release site that influence containment or dispersal and to develop appropriate sampling methodologies for evaluating survival and dispersal characteristics of genetically engineered bacteria released into the environment. The ability to make predictions about microbial dispersal and survival will reduce the uncertainties associated with environmental releases of recombinant organisms.     

The potential spread of infection caused by aerosol contamination of surfaces after flushing a domestic toilet

AIMS: To determine the level of aerosol formation and fallout within a toilet cubicle after flushing a toilet contaminated with indicator organisms at levels required to mimic pathogen shedding during infectious diarrhoea. METHODS AND RESULTS: A semisolid agar carrier containing either Serratia marcesens or MS2 bacteriophage was used to contaminate the sidewalls and bowl water of a domestic toilet to mimic the effects of soiling after an episode of acute diarrhoea. Viable counts were used to compare the numbers of Serratia adhering to the porcelain surfaces and those present in the bowl water before and after flushing the toilet. Air sampling and settle plates were used to determine the presence of bacteria or virus-laden aerosols within the toilet cubicle. After seeding there was a high level of contamination on the porcelain surfaces both under the rim and on the sides of the bowl. After a single flush there was a reduction of 2.0-3.0 log cycles cm(-2) for surface attached organisms. The number of micro-organisms in the bowl water was reduced by 2.0-3.0 log cycles ml(-1) after the first flush and following a second flush, a further reduction of c. 2.0 log cycles ml(-1) was achieved. Micro-organisms in the air were at the highest level immediately after the first flush (mean values, 1370 CFU m(-3) for Serratia and 2420 PFU m(-3) for MS2 page). Sequential flushing resulted in further distribution of micro-organisms into the air although the numbers declined after each flush. Serratia adhering to the sidewalls, as well as free-floating organisms in the toilet water, were responsible for the formation of bacterial aerosols. CONCLUSIONS: Although a single flush reduced the level of micro-organisms in the toilet bowl water when contaminated at concentrations reflecting pathogen shedding, large numbers of micro-organisms persisted on the toilet bowl surface and in the bowl water which were disseminated into the air by further flushes. SIGNIFICANCE AND IMPACT OF THE STUDY: Many individuals may be unaware of the risk of air-borne dissemination of microbes when flushing the toilet and the consequent surface contamination that may spread infection within the household, via direct surface-to-hand-to mouth contact. Some enteric viruses could persist in the air after toilet flushing and infection may be acquired after inhalation and swallowing.     

Analytical techniques for cell fractions. XVI. Preparation of protein-free supernatants with a "Z"-path rotor

A new “Z”-path centrifuge rotor is described for the separation of pellets from supernatants in up to 15 samples simultaneously. The samples and precipitating solutions are stored in separate chambers at rest, and are transferred to a sedimentation chamber by centrifugal force during rotation, where mixing occurs during rapid changes in speed, followed by sedimentation of the precipitate. The supernatant decants itself at rest into a third series of chambers, where it may be transferred centrifugally into radially disposed measuring devices, transferred with suitable pipettes, or used as a sample ring for conventional analyzers.     

A vacuum benthos sampler suitable for diverse habitats

The vacuum benthos sampler consists of a vacuum chamber equipped with a collecting net connected to the intake of a 12 volt pump, a battery, and a standpipe. Contents of the standpipe are vacuumed while substrate is removed and washed with the exhaust hose. The vacuum chamber is designed for rapid changing of nets during replicate sampling. This sampler is equally efficient in flowing and standing water. It was more effective than a modified Hess sampler for collecting a large variety of benthos from flowing (ca 0.25–0.75 m/s) riffles. Required operation time is variable, but 93% of invertebrates caught in 10 min were captured in the first 5 min during our tests, and there was a 94% mean recovery of released organisms during 10 min of subsequent operation. Advantages over previous suction samplers include interception of organisms before they pass through a pump, return of outlet water to the standpipe, capability of sampling in shallow (20–30 mm) water, and that it can be carried and operated by one person.     

Evaluation of four sampling devices for Burkholderia pseudomallei laboratory aerosol studies

Previous field and laboratory studies investigating airborne Burkholderia pseudomallei have used a variety of different aerosol samplers to detect and quantify concentrations of the bacteria in aerosols. However, the performance of aerosol samplers can vary in their ability to preserve the viability of collected microorganisms, depending on the resistance of the organisms to impaction, desiccation, or other stresses associated with the sampling process. Consequently, sampler selection is critical to maximizing the probability of detecting viable microorganisms in collected air samples in field studies and for accurate determination of aerosol concentrations in laboratory studies. To inform such decisions, the present study assessed the performance of four laboratory aerosol samplers, specifically the all-glass impinger (AGI), gelatin filter, midget impinger, and Mercer cascade impactor, for collecting aerosols containing B. pseudomallei generated from suspensions in two types of culture media. The results suggest that the relative performance of the sampling devices is dependent on the suspension medium utilized for aerosolization. Performance across the four samplers was similar for aerosols generated from suspensions supplemented with 4% glycerol. However, for aerosols generated from suspensions without glycerol, use of the filter sampler or an impactor resulted in significantly lower estimates of the viable aerosol concentration than those obtained with either the AGI or midget impinger. These results demonstrate that sampler selection has the potential to affect estimation of doses in inhalational animal models of melioidosis, as well as the likelihood of detection of viable B. pseudomallei in the environment, and will be useful to inform design of future laboratory and field studies.     

Lysis-free separation of hybridoma cells by continuous disc stack centrifugation

The non-destructive removal of hybridoma cells from fermentation broth with an improved disc stack centrifuge (CSA1, Westfalia Separator AG, Oelde, Germany) was investigated. The centrifuge was equipped with a hydrohermetic feed system, which allowed a gentle, shearless acceleration of the cells inside the bowl. No significant cell damage was observed during the separation of hybridoma cells from repeated batch fermentation in 100 liter scale. In the clarified liquid phase there was no increase in Lactate-Dehydrogenase (LDH) activity. Consequently, there was no increased exposure of the product to intracellular components.Due to continuous operation with a periodic and automatic discharge of sediment, a high throughput was achieved without any considerable loss of product. The clarification for mammalian cells was in the range of 99% to 99.9%, depending on the operating conditions. The content of cell debris and other small particles decreased about 30 to 50%, depending on the particle load in the feed stream. The centrifuge was fully contained; cleaning and sterilizing in place possible. Therefore, the decice could be integrated easily into the fermentation process.     

Inactivation of viruses in bubbling processes utilized for personal bioaerosol monitoring

A new personal bioaerosol sampler has recently been developed and evaluated for sampling of viable airborne bacteria and fungi under controlled laboratory conditions and in the field. The operational principle of the device is based on the passage of air through porous medium immersed in liquid. This process leads to the formation of bubbles within the filter as the carrier gas passes through and thus provides effective mechanisms for aerosol removal. As demonstrated in previous studies, the culturability of sampled bacterium and fungi remained high for the entire 8-h sampling period. The present study is the first step of the evaluation of the new sampler for monitoring of viable airborne viruses. It focuses on the investigation of the inactivation rate of viruses in the bubbling process during 4 h of continuous operation. Four microbes were used in this study, influenza, measles, mumps, and vaccinia viruses. It was found that the use of distilled water as the collection fluid was associated with a relatively high decay rate. A significant improvement was achieved by utilizing virus maintenance fluid prepared by using Hank's solution with appropriate additives. The survival rates of the influenza, measles, and mumps viruses were increased by 1.4 log, 0.83 log, and 0.82 log, respectively, after the first hour of operation compared to bubbling through the sterile water. The same trend was observed throughout the entire 4-h experiment. There was no significant difference observed only for the robust vaccinia virus.     

Inactivation of Viruses in Bubbling Processes Utilized for Personal Bioaerosol Monitoring

A new personal bioaerosol sampler has recently been developed and evaluated for sampling of viable airborne bacteria and fungi under controlled laboratory conditions and in the field. The operational principle of the device is based on the passage of air through porous medium immersed in liquid. This process leads to the formation of bubbles within the filter as the carrier gas passes through and thus provides effective mechanisms for aerosol removal. As demonstrated in previous studies, the culturability of sampled bacterium and fungi remained high for the entire 8-h sampling period. The present study is the first step of the evaluation of the new sampler for monitoring of viable airborne viruses. It focuses on the investigation of the inactivation rate of viruses in the bubbling process during 4 h of continuous operation. Four microbes were used in this study, influenza, measles, mumps, and vaccinia viruses. It was found that the use of distilled water as the collection fluid was associated with a relatively high decay rate. A significant improvement was achieved by utilizing virus maintenance fluid prepared by using Hank's solution with appropriate additives. The survival rates of the influenza, measles, and mumps viruses were increased by 1.4 log, 0.83 log, and 0.82 log, respectively, after the first hour of operation compared to bubbling through the sterile water. The same trend was observed throughout the entire 4-h experiment. There was no significant difference observed only for the robust vaccinia virus.     

Model to predict aerial dispersal of bacteria during environmental release

Risk assessment for genetically engineered bacteria sprayed onto crops includes determination of off-site dispersal and deposition. The ability to predict microbial dispersal patterns is essential to characterize the uncertainty (risk) associated with environmental release of recombinant organisms. Toward this end, a particle dispersal model was developed to predict recovery of bacteria on fallout plates at various distances and directions about a test site. The microcomputer simulation incorporates particle size distribution, wind speed and direction, turbulence, evaporation, sedimentation, and mortality, with a time step of 0.5 s. The model was tested against data reported from three field applications of nonrecombinant bacteria and two applications of recombinant bacteria. Simulated dispersal of 10(5) particles was compared with reported deposition measurements. The model may be useful in defining appropriate populations of organisms for release, methods of release or application, characteristics of a release site that influence containment or dispersal, and in developing an appropriate sampling methodology for monitoring the dispersal of organisms such as genetically engineered bacteria.     

Model to predict aerial dispersal of bacteria during environmental release.

Risk assessment for genetically engineered bacteria sprayed onto crops includes determination of off-site dispersal and deposition. The ability to predict microbial dispersal patterns is essential to characterize the uncertainty (risk) associated with environmental release of recombinant organisms. Toward this end, a particle dispersal model was developed to predict recovery of bacteria on fallout plates at various distances and directions about a test site. The microcomputer simulation incorporates particle size distribution, wind speed and direction, turbulence, evaporation, sedimentation, and mortality, with a time step of 0.5 s. The model was tested against data reported from three field applications of nonrecombinant bacteria and two applications of recombinant bacteria. Simulated dispersal of 10(5) particles was compared with reported deposition measurements. The model may be useful in defining appropriate populations of organisms for release, methods of release or application, characteristics of a release site that influence containment or dispersal, and in developing an appropriate sampling methodology for monitoring the dispersal of organisms such as genetically engineered bacteria.     

Release and containment of microorganisms from applied genetics activities

In the normal course of manipulating microorganisms in laboratories and commercial facilities, some organisms inevitably are dispersed through aerosol formation, drips, etc. Accidents can increase this dispersal. The objective of a recent study was to assess the release of microorganisms to the environment and exposure to technicians during applied genetics protocols. Towards this end, a computer simulation model that incorporates the complex interactions of microorganism dispersal, organism characteristics, facility design, containment equipment and facility practices was developed. The model uses the concepts of mass balance and compartments. The results of the simulations suggest the effects of different levels of containment on technician exposure and environment release.     

Centrifugal dewatering of acid casein curd: effect of casein manufacturing and centrifugation variables on curd compression in a laboratory centrifuge

Data relevant to curd compression in a horizontal, solid bowl decanter centrifuge have been obtained by studying the dewatering of acid casein curd in a batch laboratory centrifuge. Analysis of curd compression under centrifugal force predicts a moisture content gradient in the dewatered curd from a maximum at the curd-liquid interface to a minimum at the centrifuge bowl wall. This moisture content gradient was also measured experimentally, and its practical implications are discussed. Increases in centrifugal force, centrifugation time, and centrifugation temperature all caused a marked de crease in dewatered curd moisture content, whereas in creases in precipitation pH and maximum washing temperature caused a smaller decrease in dewatered curd moisture content.     

Statistical evaluation of a newly modified Robbins device using a bioluminescent pseudomonad to quantify adhesion to plastic

A modified Robbins device (MRD) has frequently been used as a model system to study adhesion and biofilm formation. This study investigates the reproducibility of attachment and whether a statistically significant gradient of adhesion exists along the 25 sampling ports of a MRD. A simple, quantitative, non‐destructive, bioluminescence assay was developed in order to measure attachment of bioluminescent P. veronii BL146bio cells to plastic discs of Thermanox? in newly modified Robbins devices (nMRD). No statistically significant difference in mean bioluminescence values occurred between pairs of nMRDs run in parallel, but there was a significant difference in bioluminescence values between different batches of bacteria (p < 0.05). Generalised Linear Modelling showed that the position of the sample disc influenced the numbers attaching. In 50% of devices a significant positive gradient of attachment occurred and bioluminescence values varied from disc 1 to disc 25 by 29.6–58.0%. In the other 50% of nMRDs there was a smaller, non‐significant gradient. A disc sampling regime was devised to take this gradient into account and used to prove a positive correlation between bioluminescence and numbers of viable P. veronii BL146bio cells during a 6h biofilm accumulation period.     

Comparison of methods for quantitative determinations of airborne bacteria and evaluation of total viable counts.

Three different methods of estimating airborne bacteria were compared. An Anderson sampler, a slit sampler, an impinger, and filter samplers with gelatine filters or membrane filters were tested for collection efficiency. The comparisons were made in laboratory experiments with an aerosol of Staphylococcus epidermidis or Serratia marcescens, in field experiments in two different industries, i.e., cotton mill and sewage plant, and in experiments with skin fragment sampling. Experiments were also performed estimating the total number of viable microorganisms on the airborne particles. The Andersen sampler gave the highest bacterial counts in all environments tested. The slit sampler gave statistically lower counts only in the aerosol experiments and cotton mill experiments, where the size of the majority of the particles carrying visible bacteria was 2 to 6 micrometers or smaller. In the sewage plant and skin fragment experiments, where the particles were mainly 5 micrometers or larger, the difference was not significant. The filters were efficient in sampling in skin fragment experiments only. With the agar impingement method, the total viable cell count showed a rising index value with increasing particle size. A mean of 13 bacteria was found per particle in the cotton mill, a mean of 24 in the sewage plant, and a mean of 147 in skin fragment experiments.     

Design of a large-scale mammalian cell,suspension culture facility

The design of a suspension culture facility capable of producing approximately 10¹² cells per week has been developed on a small-scale system which has evolved from various architectural, engineering, biological, and biohazard considerations. The smaller system is composed of spinner flasks (50 ml to 8 liters) modified for semicontinuous culture conditions, metal reservoirs, a continuous flow centrifuge, and supportive equipment. The large system which is under construction is composed of metallic vessels of up to 500 liter working volume with hard plumbing, monitors, controllers, recorders, continuous flow centrifuge and other ancillary equipment. This system begins with medium preparation and ends with harvesting of cells and disposition of supernatant. The design of this turn-key operation was developed over a two and one-half year period through the cooperation of private industry, the federal government, and the academic community.     

Microbiology of Anaerobic Sludge Fermentation: I. Enumeration of the Nonmethanogenic Anaerobic Bacteria1

An anaerobic medium containing sludge supernatant fluid and glucose was used for enumeration of bacteria from the sludge fermentation. Comparison of viable counts from several separate samples consistently showed 10 to 100 times more anaerobic than aerobic bacteria. However, viable counts of the various samples differed by as much as 10 times; this variation probably reflects a change in the natural environment or sampling errors, or a combination of the two. Direct microscopic counts yielded values of about 10(10)/ml. The discrepancy between viable (10(8) to 10(9)/ml) and direct counts may be due to large numbers of dead cells. Random isolates of representative colonies from high dilutions exhibited the ability to ferment sugars and are not likely to be methane bacteria.