A method of measuring the fraction of bacterial spores in a sample that remain viable exploits DPA-triggered luminescence of Tb3+ and is based partly on the same principles as those described in the immediately preceding article. Unlike prior methods for performing such live/dead assays of bacterial spores, this method does not involve counting colonies formed by cultivation (which can take days), or counting of spores under a microscope, and works whether or not bacterial spores are attached to other small particles (i.e., dust), and can be implemented on a time scale of about 20 minutes.
Like the method of the preceding article, this method exploits the facts that (1) DPA is present naturally only in bacterial spores; (2) when bound to Tb3+ions, DPA triggers intense green luminescence of the ions under ultraviolet excitation; and (3) the intensity of the luminescence can be correlated with the concentration of DPA released from spores and, thus, with the number density of the spores. It has been found that in the case of a sample comprising bacterial spores suspended in a solution, the DPA can be released from the viable spores into the solution by using L-alanine to make them germinate. It has also been found that by autoclaving, microwaving, or sonicating the sample, one can cause all the spores (non-viable as well as viable) to release their DPA into the solution. When the released DPA binds Tb3+ ions in the solution and the sample is exposed to ultraviolet light, the solution luminesces, as described in the preceding article.
Therefore, in this method, one divides a sample into two parts. For the first part, germination is used to release the DPA from the viable spores; for the second part, one of the three other techniques is used to release DPA from all the spores. The intensities of the DPA-triggered luminescence of both parts of the sample are measured. Then the fraction of viable spores is calculated as the ratio between the measured luminescence intensities of the first and second parts of the sample.
This work was done by Adrian Ponce of Caltech for NASA's Jet Propulsion Laboratory.
In accordance with Public Law 96-517, the contractor has elected to retain title to this invention. Inquiries concerning rights for its commercial use should be addressed to
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Refer to NPO-30444.
This Brief includes a Technical Support Package (TSP).
Live/Dead Bacterial Spore Assay Using DPA-Triggered Tb Luminescence
(reference NPO30444) is currently available for download from the TSP library.
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Overview
The document is a technical support package prepared under the sponsorship of the National Aeronautics and Space Administration (NASA), specifically detailing a novel assay for detecting live and dead bacterial spores using DPA-triggered terbium (Tb) luminescence. This work is documented in the NASA Tech Brief Vol. 27, No. 3, and is associated with Jet Propulsion Laboratory (JPL) New Technology Report NPO-30444.
The primary focus of the document is on the development and application of a sensitive and rapid assay that leverages the unique properties of terbium luminescence to differentiate between viable and non-viable bacterial spores. The assay utilizes dipicolinic acid (DPA), a compound found in bacterial spores, which, when triggered, emits luminescence that can be measured. This method offers a significant advancement over traditional techniques for spore detection, which may be time-consuming or less sensitive.
The document outlines the methodology, potential applications, and implications of this technology in various fields, including space exploration, where the detection of microbial life is crucial for planetary protection and ensuring the safety of astronauts. The ability to quickly and accurately assess the viability of bacterial spores can also have applications in environmental monitoring, food safety, and healthcare.
The report emphasizes that the work was conducted at JPL under a contract with NASA, highlighting the collaborative nature of the research and the expertise of the team involved. It also includes a disclaimer stating that references to specific commercial products or manufacturers do not imply endorsement by the U.S. Government or JPL.
Overall, this technical support package presents a significant advancement in microbial detection technology, showcasing the innovative research being conducted at NASA and JPL. The DPA-triggered Tb luminescence assay represents a promising tool for various applications, enhancing our ability to monitor and understand microbial life in diverse environments.
