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.     

An autoclavable electronic interface for germfree isolators

An electronic interface was designed and fabricated for use with germfree isolators. It allows a variety of sensors or electrical instruments inside an isolator to be readily connected to monitoring or activating devices on the outside of an isolator. It was built into a 25 cm diameter cylinder whose open ends were loosely covered by an inner and outer connector panel. These two panels were wired together through an airtight dividing wall placed transversely across the middle of the cylinder. The interface was mounted in an entry port of a stainless steel isolator and steam sterilized with the isolator. It has proven to be a useful, contamination-free, durable device.     

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.     

Transfer isolator to protect personnel from exposure to peracetic acid.

A transfer isolator is described which confines peracetic acid fumes used in the gnotobiotic operation of isolators. The use of this isolator protects both personnel and animals from contact with peracetic acid, provides additional isolator space and reduces wear on the gloves and sleeves of the main isolator.     

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。     

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.     

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.     

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.     

Rationalisation and modification of a germ-free isolator design

A modified design of polyvinyl chloride isolator, suitable for small laboratory animals utilises room space more effectively than previous models and is particularly useful where accommodation is restricted. The standard attachments and ancillary equipment used with other flexible isolators are compatible with the modified isolator.     

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.     

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.     

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.     

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.     

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.     

A case of Ebola virus infection.

In November 1976 an investigator at the Microbiological Research Establishment accidentally inoculated himself while processing material from patients in Africa who had been suffering from a haemorrhagic fever of unknown cause. He developed an illness closely resembling Marburg disease, and a virus was isolated from his blood that resembled Marburg virus but was distinct serologically. The course of the illness was mild and may have been modified by treatment with human interferon and convalescent serum. Convalescence was protracted; there was evidence of bone-marrow depression and virus was excreted in low titre for some weeks. Recovery was complete. Infection was contained by barrier-nursing techniques using a negative-pressure plastic isolator and infection did not spread to attendant staff or to the community.     

Evaluation of countermeasures for reduction of mouse airborne allergens

Three kinds of countermeasures for reduction of mouse airborne allergens were evaluated with use of an air sampler and immunochemical methods. Mouse cages and the sampler were placed inside a flexible-film isolator, and concentrations of mouse major allergens in the air were measured. The levels of the airborne allergens, prealbumin and albumin, generated by 10 males, were 3,050 and 492 pg/m3, respectively. Those by 10 females were lower, 317 and 270 pg/m3, respectively. When mouse cages were covered with a filter cap, the airborne allergens inside the isolator were decreased by 90%. When corncob was used as bedding in place of wood shavings, the airborne allergens were decreased by 57 and 77%, respectively. Therefore, for reduction of mouse airborne allergens, we recommend using female mice, covering the cages with filter caps, and using corncob bedding.     

Good design practices for an integrated containment and production system for advanced therapies

Advances in molecular biology and the possibility of differentiating stem cells have opened up new scenarios in therapies that use progenitor or variously differentiated cells. Regardless of the choice of the system, designing a plant for producing advanced therapies requires a clear understanding of the final objective (the product), taking into account all the regulatory, environment, process, risk assessment, asepsis, and validation aspects involved until its implementation. Good Manufacturing Practice (GMP) compliant procedures are a prerequisite for cell production in clinical application, and clean rooms are zones for producing cell therapies. Clean rooms for clinical application require high running and maintenance costs and need trained operators and strict procedures to prepare the rooms and the people involved in the processes. While today production mainly occurs in open systems (clean rooms), there is evidence of processes in closed systems (isolators). The isolator is a Grade A aseptic closed system that requires a controlled environment and at least a Grade D environment in the case of sterile productions (A in D closed system). The use of isolators can ensure a very high level of protection against the risk of product contamination and, at the same time, provide the operators with a very safe working environment. Furthermore, working with closed systems can optimize and facilitate the production of Advanced Therapy Medical Products in GMP environments, by providing an easily reproducible working tool even for large-scale production, with generally lower costs compared to a classical clean room approach. In conclusion, the isolator workstation as a possible alternative to the classic clean room, due to its small size and the simplification of the working and maintenance operational procedures, may represent an interesting solution in the perspective of the increasingly more stringent requests for cost reductions of GMP in clinical application.     

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).     

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.     

Use of the flexible film isolator as a single circuit hypoxic chamber for small animals

PVC isolators are now widely used for housing animals and provide a readily available pretested air-tight chamber (Pendry, 1984; Trexler, 1984). We have adapted a flexible film isolator for use as a hypoxic chamber for small animals. The environment within the chamber can be easily and continuously monitored with indwelling probes, obviating the need for a separate circuit for gas analysis. This design has been used for long-term studies of chronic hypoxia.