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.
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