The culturing techniques are important and widely used in microbiological studies. However, it has been revealed in 1980's that many microbes in the environment cannot be detected by conventional culturing techniques with long history of more than 100 years. Culturing processes are time-consuming and increase the biomass of microbial cells, which shall result to increase the risk of biohazard. We regards microbes as “particles with genetic information and physiological activity”, and develop culture-independent techniques to analyze microbial cells at the single level, a cell itself, and also community level, by using fluorescent staining methods and molecular microbial ecological approaches. By using these techniques, microbial cells can be detected and enumerated rapidly and their physiological status can be estimated at the same time. Targeted cells are detected and their dynamics can be monitored accurately based on their genetic information. Culture-independent techniques are rapid and rather simple. Results can be obtained within one hour to half a day, while conventional culturing techniques require more than a day to obtain reliable results. In order to popularize these techniques, validation and automatization is important. We applied image analysis and flow cytometry for rapid analysis. We also designed a semi-automated system for enumeration of microcolony-forming cells. Compare to conventional fluorescent microscopy, applying these new approaches is more likely to bring us more rapid and quantitative results. We now apply microfluidic devices (on-chip flow cytometry) to automatize enumeration of microbial cells, which enable us to acquire data rapidly with high reproducibility and reduce the risk of biohazard by rather simple operation. These techniques should be useful in microbiological quality assurance of freshwater.
Regenerative medicine, which involves advanced techniques using ex vivo processed tissue-engineering products (e.g., viable cells, tissues), is one of the newest and most promising methods for treating various intractable diseases and damaged organs. For clinical use, tissue-engineered products require sterilization, so they are usually prepared manually, using aseptic processing procedures in a biological clean room (BCR) to prevent contamination with microbiological and/or hazardous materials. However, these procedures carry the unavoidable risk of contamination from the people performing them. To reduce this risk, personnel are required to wear sterilized gowns and follow written standard operating procedures (SOPs) for every process. These measures make procedures performed in the BCR laborious and expensive. In this study, to develop a system for producing tissue engineering products that is safer, less laborious, and less expensive than those used in the BCR, we examined the applicability of an isolator system for human cell cultures and developed a new system, the Advanced Isolator System for Tissue Engineering (AIST). The AIST is a compact, closed isolator system intended for aseptic human cell cultures. It has a built-in CO2 incubator and the equipment needed for cell culture (e.g., microscope, centrifuge) is set up inside it. It is decontaminated in its entirety by vaporized hydrogen peroxide (VHP), and it can be maintained in a cleaner condition with less effort than equipment in a BCR. We have been able to culture various human cells in the microbiologically closed AIST using a half-suit unit as the operator interface, so the potential risk of contamination from personnel is as low as possible. Cell processing in the AIST will maintain the quality of human cells for clinical use at a higher level than can be attained in a BCR, and we expect it to contribute greatly to the early realization of regenerative medicine using ex vivo processed human cells.
Bowie-Dick test is used to detect inefficient vacuum pump function, inward air leak into the chamber and non-condensable gases in the supplied steam. Health Technical Memorandum 2010 recommends to perform vacuum leak test when prevacuum autoclave fails to pass Bowie-Dick test, because the state of vacuum pump function and the degree of inward air leak into the chamber are evaluated, respectively, using this test. However, the microprocessor-controlled prevacuum autoclave installed in our surgical center is not equipped with the automatic vacuum leak test mode. The authors devised a simple method to perform vacuum leak test in our prevacuum autoclave: the microprocessor boards were operated manually when the test was performed. In the present study, vacuum leak test was performed twice in the prevacuum autoclave when it failed to pass Bowie-Dick test: before and after it underwent the mechanical repair. Vacuum leak test detected the impaired function of the vacuum pump, which had been replaced four years previously. After this impaired vacuum pump was replaced with a new one, the prevacuum autoclave passed Bowie-Dick test. Moreover, this impaired vacuum pump was disassembled to clarify the cause of its impaired function. The main parts underwent rusty corrosion. The present study suggested that vacuum leak test is performed easily using our method in the prevacuum autoclave without the automatic test mode. It is also suggested that vacuum leak test is useful in the repair of the prevacuum autoclave failing Bowie-Dick test.