Equipment and a method for rapidly assaying solid surfaces for contamination by bacterial spores are undergoing development. The method would yield a total (nonviable plus viable) spore count of a surface within minutes and a viable-spore count in about one hour. In this method, spores would be collected from a surface by use of a transparent polymeric tape coated on one side with a polymeric adhesive that would be permeated with one or more reagent(s) for detection of spores by use of visible luminescence. The sticky side of the tape would be pressed against a surface to be assayed, then the tape with captured spores would be placed in a reader that illuminates the sample with ultraviolet light and counts the green luminescence spots under a microscope to quantify the number of bacterial spores per unit area. The visible luminescence spots seen through the microscope would be counted to determine the concentration of spores on the surface.
This method is based on the chemical and physical principles of methods described in several prior NASA Tech Briefs articles, including “Live/Dead Spore Assay Using DPA-Triggered Tb Luminescence” (NPO-30444), Vol. 27, No. 3 (March 2003), page 7a. To recapitulate: The basic idea is to exploit the observations that (1) dipicolinic acid (DPA) is present naturally only in bacterial spores; and (2) when bound to Tb3+ ions, DPA triggers intense green luminescence of the ions under ultraviolet excitation; (3) DPA can be released from the viable spores by using L-alanine to make them germinate; and (4) by autoclaving, microwaving, or sonicating the sample, one can cause all the spores (non-viable as well as viable) to release their DPA.
One candidate material for use as the adhesive in the present method is polydimethysiloxane (PDMS). In one variant of the method — for obtaining counts of all (viable and nonviable) spores — the PDMS would be doped with TbCl3. After collection of a sample, the spores immobilized on the sticky tape surface would be lysed by heating or microwaving to release their DPA. Tb3+ ions from the TbCl3 would become bound to the released DPA. The tape would then be irradiated with ultraviolet and examined as described above. In another variant of the method — for obtaining counts of viable spores only — the PDMS would be doped with L-alanine in addition to TbCl3.
As now envisioned, a fully developed apparatus for implementing this method would include a pulsed source of ultraviolet light and a time-gated electronic camera to record the images seen through the microscope during a prescribed exposure interval at a prescribed short time after an ultraviolet pulse. As in the method of the second mentioned prior article, the pulsing and time-gating would be used to discriminate between the longer-lived Tb3+/DPA luminescence and the shorter-lived background luminescence in the same wavelength range. In a time-gated image, the bright luminescence from bacterial spores could easily be seen against a dark background.
This work was done by Adrian Ponce of Caltech for NASA’s Jet Propulsion Laboratory. For further information, access the Technical Support Package (TSP) free online at www.techbriefs.com/tsp under the Bio-Medical category.
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-40646, volume and number of this NASA Tech Briefs issue, and the page number.
This Brief includes a Technical Support Package (TSP).
Surface Bacterial-Spore Assay Using Tb3+/DPA Luminescence
(reference NPO-40646) is currently available for download from the TSP library.
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Overview
The document outlines a novel method developed by NASA's Jet Propulsion Laboratory (JPL) for detecting bacterial spores on surfaces, which is crucial for ensuring sterility in various industries, including pharmaceuticals, healthcare, and food preparation. Traditional bioburden testing methods, which rely on culture-dependent techniques, can take 3-5 days to yield results and often underestimate the presence of bacterial spores due to aggregation and the limitations of colony-counting methods. These methods primarily account for cultivable spore-forming species, which represent less than 1% of environmental samples.
The new approach utilizes a surface bacterial-spore assay that combines the detection of dipicolinic acid (DPA), a unique chemical marker found in bacterial spores, with an optically transparent adhesive polymer, specifically polydimethylsiloxane (PDMS). This polymer is doped with terbium chloride (TbCl₃) and L-alanine, which facilitates the germination of spores and the release of DPA. When exposed to UV light, the Tb³⁺ binds to the DPA, triggering green luminescence that can be quantified using a photodetector. This method allows for rapid assessment of both total and viable bacterial spore counts, significantly reducing the time required for sterility testing.
The document also highlights the importance of bioburden testing in the context of NASA's planetary protection provisions for robotic extraterrestrial missions. For missions categorized as Class IVa, which do not involve life-detection experiments, a bioburden limit of 3×10⁵ spores per vehicle and fewer than 300 spores per square meter is mandated. Missions with life-detection experiments face stricter requirements, necessitating additional procedures to limit the total bioload to 30 spores.
Overall, the new bacterial spore detection technology represents a significant advancement in sterility validation, providing faster and more accurate results compared to traditional methods. This innovation not only supports compliance with bioburden standards set by regulatory bodies like the USP, FDA, PDA, and AAMI but also enhances the safety and reliability of space missions by ensuring that spacecraft are free from excessive microbial contamination prior to launch. The document serves as a technical support package, detailing the methodology and potential applications of this technology in both aerospace and commercial sectors.
