Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis.
A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass.
The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which
this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology.
Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further validated using varying ratios and mixtures of cells to ensure homogenous staining compared to that of individual cells, and were utilized for flow analyzer and FACS labeling.
This technology focuses on the purification and concentration of cells from low-biomass spacecraft assembly facility samples. Currently, purification and concentration of low-biomass samples plague planetary protection downstream analyses. Having a capability to use flow cytometry to concentrate cells out of low-biomass, high-volume spacecraft/ facility sample extracts will be of extreme benefit to the fields of planetary protection and astrobiology.
Successful research and development of this novel methodology will significantly increase the knowledge base for designing more effective cleaning protocols, and ultimately lead to a more empirical and “true” account of the microbial diversity present on spacecraft surfaces. Refined cleaning and an enhanced ability to resolve microbial diversity may decrease the overall cost of spacecraft assembly and/or provide a means to begin to assess challenging planetary protection missions.
This work was done by James N. Benardini, Myron T. La Duc, and Rochelle Diamond of Caltech for NASA’s Jet Propulsion Laboratory. NPO-48086
This Brief includes a Technical Support Package (TSP).
Purifying, Separating, and Concentrating Cells From a Sample Low in Biomass
(reference NPO-48086) is currently available for download from the TSP library.
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Overview
The document is a Technical Support Package from NASA's Jet Propulsion Laboratory (JPL) titled "Purifying, Separating, and Concentrating Cells From a Sample Low in Biomass" (NPO-48086). It outlines innovative methodologies developed for the efficient purification, separation, and concentration of cells, particularly in samples that contain low biomass. This is crucial for various applications in scientific research, biotechnology, and space exploration, where obtaining viable cell samples can be challenging.
The document includes several figures illustrating the use of flow analyzers to differentiate between various cell types, specifically focusing on Bacillus pumilus SAFR-032 spores and cells, Deinococcus radiodurans R1, and Aerobasidium pullans 28v1. These figures demonstrate the proposed gating strategies that can be employed to discern between these cell types during analysis. For instance, different mixtures of cells and reference beads are tested to optimize the detection and separation process.
The flow analyzer runs depicted in the figures show how different concentrations and combinations of the cell types can be analyzed, providing insights into the effectiveness of the proposed methods. The use of internal reference beads is highlighted, which aids in calibrating the flow analyzer and improving the accuracy of the results.
The document emphasizes the importance of these techniques in the context of NASA's research and technology initiatives, particularly in the exploration of extraterrestrial environments where low biomass samples may be encountered. The methodologies discussed are not only applicable to space missions but also have broader implications for environmental monitoring, medical diagnostics, and industrial applications.
Additionally, the document serves as a resource for those interested in the commercial applications of these technologies, as it is part of NASA's Commercial Technology Program aimed at disseminating aerospace-related developments with wider technological relevance.
Overall, this Technical Support Package provides a comprehensive overview of advanced techniques for cell purification and separation, showcasing NASA's commitment to innovation and collaboration in scientific research and technology development. For further inquiries or assistance, the document provides contact information for the Innovative Technology Assets Management at JPL.
