RESUMEN
The control of microbes in manned spaceflight is essential to reducing the risk of infection and maintaining crew health. The primary issue is ensuring the safety of a potable water system, where simultaneous monitoring of microbial abundance and community structure is needed. In this paper, we develop a flow cytometry-based counting protocol targeting cellular flavin autofluorescence as a tool for rapid monitoring of bacterial cells in water. This was successfully applied to estimate the bacterial bioburden in the potable water collected from the International Space Station. We also demonstrate the efficacy of the MinION nanopore sequencer in rapidly characterizing bacterial community structure and identifying the dominant species. These monitoring protocols' rapidity and cost effectiveness would contribute to developing sustainable real-time surveillance of potable water in spaceflight.
Asunto(s)
Agua Potable , Vuelo Espacial , Etnicidad , Flavinas , Citometría de Flujo , HumanosRESUMEN
Bacteria tests are conducted for quality control in many different industries. However, the cultivation method takes a long period of time to obtain results and there are more than a few bacteria that are difficult to cultivate. We have focused on the autofluorescence substance in the bacteria to detect them, and developed a sensor to measure the bacteria in real-time, without any pretreatments or addition of any reagents. This system uses a 405nm laser focused on the sample flowing through the flow-cell in order to detect the fluorescent light from the bacteria as well as scattered light. Fluorescent light and scattered light are separated by a dichroic mirror, and the number of viable particles (bacteria) and that of non-viable particles are obtained. We tested this system using fluorescent polystyrene latex particles and several bacterial strains, and confirmed that it had good detection capability. We believe that this system will become a next-generation bacteria detection system and help the introduction of PAT (process analytical technology) to all areas where real time and on-site detection is needed.
Asunto(s)
Bacterias/aislamiento & purificación , Citofotometría/métodos , Agua Dulce/microbiología , Bacterias/química , Sistemas de Computación , Citofotometría/instrumentación , Ecosistema , Diseño de Equipo , FluorescenciaRESUMEN
We proposed a new method of detecting the onset of thrombus formation based upon the backscattered light intensity changes caused by the alteration of blood flow behavior in the cardiopulmonary devices. In an optical senor based upon the First Order Scattering theory, the relationship between the backscattered light intensity and hematocrit exhibited a monotonically decaying curve for the hematocrit level greater than 45%. To distinguish the effect either by thrombus formation or erythrocyte aggregation on the back-scattered light intensity with a flow chamber connected to a rabbit arterial-venous bypass model, we generated an oscillatory flow using a roller pump and analyzed the results using a Fast Fourier Transform (FFT) method. Our hypothesis was that the nonclotting blood flow would yield an unchanged fundamental spectral power density of the oscillation frequency generated by a roller pump, whereas the thrombus formation would attenuate its power. We measured the back-scattered light intensity in the flow chamber of high shear region and low shear region (n = 5). The blood flow rate was 40 ml/min with the roller pump. The activated clotting time and the hematocrit level were adjusted to 170 +/- 10 s with heparin and 35 +/- 5% with a phosphate buffered sulfate solution, respectively. As a result, the backscattered light intensity from the low shear rate region gradually diminished with time (p < 0.05; 0-5 minutes vs. more than 10-15 minutes), whereas that from the high shear rate region remained fairly constant. The experimental finding supported the hypothesis that the decrease of the backscattered light intensity and diminished spectral power density were caused by the aggregation or clotting erythrocytes. In conclusion, we found that the measurement of the average backscattered light intensity level together with FFT analysis of the backscattered light from the flowing blood is a valuable approach in detecting the onset of thrombus formation.