An autoclavable stainless steel isolator for small scale gnotobiotic experiments (author's transl)

A lightweight stainless steel autoclavable pentagon isolator was designed for experiments using gnotobiotic mice. The chamber, 400 x 450 x 350 mm, has an entry port 200 mm in diameter at the back and a window at the ceiling. The globes and two filters were equipped at the front and each side, respectively. An inner stainless steel cap of the entry lock was sealed by a seamless silicone band. It was possible to ventilate this isolator by either free-flow or blower operation. After autoclaving the isolators 10 to 16 times for a year, none of them was repaired. Five mice can be kept in this isolator for about 1 month without supply of diet, water and wood shaving after the first setting.     

An autoclavable intensive care isolator for the adult gnotobiotic dog.

A lightweight stainless steel isolator which allows 3 investigators simultaneous access to an adult gnotobiotic dog for the purpose of rendering intensive care was designed and constructed. This isolator is 54' high X 60' long X 42' wide. It has an 18' diam entry port at 1 end and a pair of gloves mounted beneath a window on each of the other 3 sides. The chamber rests on a 12' high pallet and is moved about the laboratory by a portable hydraulic platform lift. It can be rapidly cleansed with soap and water and sterilized in a large autoclave. Over the past 2 yr. this intensive care isolator has enabled us to perform a variety of manipulations on the adult gnotobiotic dog in a safe, convenient, and efficient manner.     

Permeability and Self-Induction as Factors in Water Transport through Bean Roots

Water uptake of root systems of Phaseolus vulgaris was measured in a Scholander pressure cylinder at a constant pressure. Water uptake rises gradually till a steady state is reached after 15 to 60 minutes. This time course can be described as a transport process with the property of a self-induction. The latter was not affected by temperature. It is concluded that the property of self-induction is located in the cytoplasm or at the interface between cytoplasm and plasmalemma of the endodermal cells. It is suggested that cytoplasmic streaming controls the time course of water transport.     

Negative-pressure plastic isolator for patients with dangerous infections.

A negative-pressure plastic isolator is effective for dealing with patients suffering from dangerous infections. So far it has been used to treat seven patients suspected of having infections due to Lassa, Marburg, or Ebola viruses. One patient spent 32 days in the isolator. The isolator was proved comfortable and acceptable to patients, and it gives the nursing and medical attendants a high degree of protection. All routine nursing and medical procedures can be carried out with minimal interference by the physical barrier, though it is not practicable to undertake artificial respiration or haemodialysis.     

Lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs

Amid growing evidence that numerous viral infections can produce immunopathology, including nonspecific polyclonal lymphocyte activation, the need to test the direct impact of an infecting virus on the immune system of the host is crucial. This can best be tested in the isolator piglet model in which maternal and other extrinsic influences can be excluded. Therefore, neonatal isolator piglets were colonized with a benign Escherichia coli, or kept germfree, and then inoculated with wild-type porcine reproductive and respiratory syndrome virus (PRRSV) or sham medium. Two weeks after inoculation, serum IgM, IgG, and IgA levels were 30- to 50-, 20- to 80-, and 10- to 20-fold higher, respectively, in animals receiving virus vs sham controls, although <1% was virus specific. PRRSV-infected piglets also had bronchial tree-associated lymph nodes and submandibular lymph nodes that were 5-10 times larger than colonized, sham-inoculated animals. Size-exclusion fast performance liquid chromatography revealed that PRRSV-infected sera contained high-molecular-mass fractions that contained IgG, suggesting the presence of immune complexes. Lesions, inflammatory cell infiltration, glomerular deposits of IgG, IgM, and IgA, and Abs of all three isotypes to basement membrane and vascular endothelium were observed in the kidneys of PRRSV-infected piglets. Furthermore, autoantibodies specific for Golgi Ags and dsDNA could be detected 3-4 wk after viral inoculation. These data demonstrate that PRRSV induces B cell hyperplasia in isolator piglets that leads to immunologic injury and suggests that the isolator piglet model could serve as a useful model to determine the mechanisms of virus-induced immunopathology in this species.     

Biomechanical efficiency of wrist guards as a shock isolator

Despite the use of wrist guards during skate- and snowboard activities, fractures still occur at the wrist or at further proximal locations of the forearm. The main objectives of this study were to conduct a human subject testing under simulated falling conditions for measurement of the impact force on the hand, to model wrist guards as a shock isolator, to construct a linear mass-spring-damper model for quantification of the impact force attenuation (Q-ratio) and energy absorption (S-ratio), and to determine whether wrist guards play a role of an efficient shock isolator. While the falling direction (forward and backward) significantly influenced the impact responses, use of wrist guards provided minimal improvements in the Q- and S-ratios. It was suggested based on the results under the submaximal loading conditions that protective functions of the common wrist guard design could be enhanced with substantial increase in the damping ratio so as to maximize the energy absorption. This would bring forth minor deterioration in the impact force attenuation but significant increase in the energy absorption by 19%, which would help better protection against fall-related injuries of the upper extremity.     

Vacuum-cleaning System for Isolation Chambers

To encourage the utilization of the isolation chamber as a research tool, the cost of its use should be lowered. Methods and devices must be developed which make more efficient use of the space within the isolator and allow the operator to work more effectively in this confined area. A simple vacuum-cleaning system is described; it consists of a nozzle and flexible hose which connect through the isolator wall to an externally placed waste tank, attached by way of its outlet filter to a source of vacuum. The cylindrical waste tank [48 inches (1.219 m) high and 36 inches (0.914 m) in diameter] was sterilized in a large autoclave. During a 9-month test period, the system was used to remove soiled corncob bedding from a large isolator containing 90 adult monocontaminated rats. During this period, the microbial flora of the isolator was unchanged, and the time required to clean the cages was reduced by 50%. This vacuum-cleaning system is a safe, convenient, and economical means of increasing the efficiency of an isolation chamber.     

Sterile microenvironment in prevention of wound infection.

A prospective controlled trial was carried out to assess the effect of using a wound isolator on reducing postoperative infection. A total of 291 patients undergoing hip pinning for fractures of the neck of femur entered the trial and were allocated at random to have their wound contained in a wound isolator (study group) or dressed with a standard gamma-irradiated adhesive dressing (control group). The bacteriological flora of the patient was monitored before, during, and after operation and that of the ward before and after. No significant difference was found in the flora of the wards in which the patients were nursed. On several occasions the source of the infective organism was traced to the ward but never to the theatre. The isolator prevented direct contamination and airborne cross-infection of the wound and appreciably reduced the rate of infection.     

隔离器饲育SPF鸡微生物质量控制方法的建立

目的建立隔离器饲育SPF鸡生产管理方式,探讨并建立其微生物质量控制方法。方法对引进的SPF鸡种蛋进行孵化后,将种鸡置于隔离器中进行饲养,在种鸡10、20、40周龄时采取鸡血清进行微生物质量监测,检验当前SPF鸡生产管理和微生物质量控制方法的有效性。结果隔离器饲育SPF鸡初期由于对孵化环节控制不严出现了微生物污染现象。在对孵化环节操作管理改进后,隔离器饲育SPF鸡的微生物状况得到控制。结论通过加强各个环节消毒,隔离器饲育环境能够保证SPF鸡的微生物质量。适当控制隔离器内SPF鸡的密度不仅有利于SPF鸡的繁育和种蛋回收,而且有利于环境微生物的控制。     

Equipment for germ-free caesarean section and baby care

A germ-free isolator system was designed and constructed, It permits a sterile caesarean section and rearing of an infant under germ-free conditions. The system includes a plastic surgical and rearing isolator and two supply isolators; one of the supply isolators served as a transport mobile unit for the transfer of the newborn from the operation room to the neonatal intensive care unit. All members of staff participating in the sterile caesarean section and in the postnatal care of the germ-free newborn underwent to a special training and all of them were able to meet the high requirements of this work. The surgery itself was without complications, the newborn was absolutely sterile up to the age of 1 month; afterwards the infant was gradually colonized with selected strains of bacteria and thus prepared for conventialization. In the course of this work further possibilities of application of gnotobiological techniques in pediatrics were proposed (e.g. care of premature, high-risk neonates).     

Creation of an experimental rearing environment for microbiome animal research using an individually ventilated cage system and bioBUBBLE enclosure

To avoid microbial contamination risk, vinyl film isolators are generally used in animal microbiome experiments involving germ-free (GF) mice and/or gnotobiotic (GB) mice. However, it can take several months to gain expertise in operating the isolator competently. Furthermore, sterilization and sterility testing, which are essential for isolator preparation, can take more than 20 days. Hence, we built an experimental rearing environment that combines an individual ventilation cage system and a bioBUBBLE clean room enclosure to easily set up an experimental animal microbiome environment for animal facilities. In this work, a three-step evaluation was conducted. First, we examined whether GF mice can be maintained in this rearing environment without bacterial contamination. Next, we examined whether GF and GB mice can be maintained without cross-contamination in one individual ventilation cage rack. Finally, we tested whether GF mice can be maintained in a biological safety cabinet controlled by negative pressure. In our series of experiments, no microbial contamination occurred over more than 3 months. These results indicated that our rearing system that combines the individual ventilation cage and bioBUBBLE systems can be used not only for experiments with GF mice but also for Biosafety Level 2 experiments that handle bacteria. Our system can mitigate various disadvantages of using vinyl film isolators. In conclusion, we established an experimental method with improved working time and efficiency compared with those of the previous vinyl isolator method.     

The Surgical Isolator

A plastic film isolator has been developed which is intermediate between a half-suit and an arm isolator. Used for orthopaedic surgery in an aseptic environment few operating difficulties have been found, and an extended trial of its use should be carried out to assess its effect on the incidence of postoperative wound infection.     

Anaerobic transfer of antibiotic resistance from Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen that often initiates infections from a reservoir in the intestinal tract, may donate or acquire antibiotic resistance in an anaerobic environment. Only by including nitrate and nitrite in media could antibiotic-resistant and -sensitive strains of P. aeruginosa be cultured in a glove box isolator. These anaerobically grown cells remained sensitive to lytic phage isolated from sewage. After incubation with a phage lysate derived from P. aeruginosa 1822, anaerobic transfer of antibiotic resistance to recipients P. aeruginosa PS8EtBr and PS8EtBrR occurred at frequencies of 6.2 x 10(-9) and 5.0 x 10(-8) cells per plaque-forming unit, respectively. In experiments performed outside the isolator, transfer frequencies to PS8EtBr and PS8EtBrR were higher, 1.3 x 10(-7) and 6.5 x 10(-8) cells per plaque-forming unit, respectively. When P. aeruginosa 1822 was incubated aerobically with Escherichia coli B in medium containing nitrate and nitrite, the maximum concentration of carbenicillin-resistant E. coli B reached 25% of the total E. coli B population. This percentage declined to 0.01% of the total E. coli B population when anaerobically grown P. aeruginosa 1822 and E. coli B were combined and incubated in the glove box isolator. The highest concentration of the recipient population converted to antibiotic resistance occurred after 24 h of aerobic incubation, when an initially high donor/recipient ratio (>15) of cells was mixed. These data indicate that transfer of antibiotic resistance either by transduction between Pseudomonas spp. or by conjugation between Pseudomonas sp. and E. coli occurs under strict anaerobic conditions, although at lower frequencies than under aerobic conditions.     

Germfree status of mice obtained by embryo transfer in an isolator environment

The technique of embryo transfer has been evaluated for the purpose of changing the mouse stocks to a germfree (GF) status. Our results show reproducible and quality-assured conversion of animals to those which are negative for the presence of microorganisms. Rapid and easy access to GF mice is advantageous for studies of selected microflora and their cross-talks with the host, when applying, e.g. genomic, proteomic and metabolic methodology.The study involved embryo transfer in an isolator environment, thereby allowing implantation of cleansed embryos into GF recipients under well-controlled conditions. The recipient females gave birth normally and took care of the offspring as if they were their own pups, thus enhancing the survival rate. Access to full technical resources required to maintain GF isolators are, however, a prerequisite. In this study, we used stainless steel isolators designed by Gustafsson (1959), on which a stereomicroscope was mounted to facilitate embryo transfer inside the isolator.The use of embryo transfer and isolator techniques will facilitate the availability of various mouse mutant models under different gnotobiotic conditions, GF, monoxenic or polyxenic animals, to enable comparison with conventional animals for physiological and pathophysiological studies.     

Stability of elliptical cylinders in two-dimensional channel flow

The flow around rigid cylinders of elliptical cross section placed transverse to Poiseuille flow between parallel plates was simulated to investigate issues related to the tumbling of red blood cells and other particles of moderate aspect ratio in the similar flow in a Field Flow Fractionation (FFF) channel. The torque and transverse force on the cylinder were calculated with the cylinder freely translating, but prevented from rotating, in the flow. The aspect ratios (long axis to short axis) of the elliptical cylinders were 2, 3, 4, and 5. The cylinder was placed transversely at locations of y0/H = 0.1, 0.2, 0.3, and 0.4, where y0 is the distance from the bottom of the channel and H is the height of the channel, and the orientation of the cylinder was varied from 0 to 10 deg with respect to the axis of the channel for a channel Reynolds number of 20. The results showed that equilibrium orientations (indicated by a zero net torque on the cylinder) were possible for high-aspect-ratio cylinders at transverse locations y0/H < 0.2. Otherwise, the net torque on the cylinder was positive, indicating that the cylinder would rotate. For cylinders with a stable orientation, however, a transverse lift forced existed up to about y0/H = 0.25. Thus, a cylinder of neutral or low buoyancy might be lifted with a stable orientation from an initial position near the wall until it reached y0/H < 0.2, whereupon it would begin to tumble or oscillate. The dependence of lift and torque on cylinder orientation suggested that neutral or low-buoyancy cylinders may oscillate in both transverse location and angular velocity. Cylinders more dense than the carrier fluid could be in equilibrium both in terms of orientation and transverse location if their sedimentation force matched their lift force for a location y0/H < 0.2.     

A technique for rearing germfree piglets obtained without surgery

A relatively simple procedure is described for obtaining germfree piglets which does not involve hysterectomy or hysterotomy. Newborn pigs were delivered into an isolator and their freedom from microbial contamination was ensured by applying bactericidal solutions externally and a combination of antibiotics in solution per os. 40 piglets so derived have been maintained free from detectable micro-organisms, some for up to 140 days. Equipment is described which allowed the long-term holding of these animals so that nutritional balance studies could be completed.     

EEG potential theory in a model with thin brain integuments. I. Multilayer spherical cable model]

The problem of finding EEG potentials is solved assuming that integuments of the brain are regarded as infinitely thin spheres. The potential is not changed along the radius in the layers with high conductivity, and the current in the resistive layers is only radial. It is shown that the EEG potentials could be described exactly enough supposing the lower bone plate to be an ideal isolator. The potentials of EEG are obtainable from ECoG by means of a comparatively simple algebraic procedure.     

A new technique to determine hydrogen excreted by gnotobiotic rats

A new system, that allowed the monitoring of hydrogen (H2) excretion by gnotobiotic rats without affecting their defined microbial status, was developed. The system consists of an isolator containing a chamber for an experimental animal, and a life-support system (LSS), with a sampling port outside the isolator connected to it. H2 accumulation in the system was measured by analysing a defined volume of gas after removal. H2 concentrations were determined with an electrochemical cell or by gas chromatography. To validate this technique, H2 excretion by germ-free (GF) and mono-associated rats fed a chemically defined diet was measured after oral application of lactulose. Mono-associated rats had been obtained by colonizing GF rats with a H2-producing Clostridium perfringens type A strain isolated from human faeces of a healthy volunteer. Application of 50 mg lactulose to the mono-associated rats resulted in a significant increase in H2 excretion. The net H2 excretion was 7.82+/-1.28 ml H2 in 12 h corresponding to a net maximal rate of 1.1+/-0.3 ml H2/h. In contrast, in experiments with GF rats, less than 0.13 ml H2 were detectable within 12 h. The technique presented is a useful tool for studying bacterial H2 metabolism in vivo under gnotobiotic conditions.     

Radial histophysiologic gradient culture chamber: Rationale and preparation

Summary Histophysiologic gradient culture methods reconstitute important spatial relationships that occur in nature between a parenchyma and its supporting stroma. At the epithelial-stromal interface, epithelia are firmly attached to the stromal substrate, initiation of renewal takes place, and metabolites are exchanged by a process of diffusion between epithelium and substrate. Other spatial imperatives characteristic of stratified epithelium are high density of cells, gradients of maturation, and continuity of epithelia along the entire course of the stromal-parenchymal interface. In radial gradient culture these relationships of epithelial cells, and supporting substrates are reconstituted. The culture chamber consists of a thin-walled cylinder, 2 to 3 mm in diameter and 3 cm long. The wall is a transparent collagen membrane in whose substance is embedded a reinforcing nylon mesh. To prepare a culture, one end of the cylinder is ligated, 1 or 2 particulate inocula are inserted in the open end of the cylinder, guided toward the ligature, and the open end is ligated. Subsequently, during incubation in a container with medium, the explants attach and proliferate. Proliferation and migration result in the cylinder being completely lined by a complex organoid tissue with structural characteristics of the original tissue. The tissue patterns in radial gradient culture of two human cell lines, RT-4, a bladder cancer, and 87×50, and ovarian cancer, are illustrated.