New Method for <i>Lawsonia intracelullaris</i> Quantification Based on Optical Density by Spectrophotometry
Studies investigating the pathogenesis of <i>Lawsonia intracellularis</i> often require bacterial quantification in suspension. However, due to the organism’s fastidious growth requirements—being both intracellular and microaerophilic—traditional quantification methods, such as colony-fo...
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| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-03-01
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| Series: | Microorganisms |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-2607/13/3/568 |
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| Summary: | Studies investigating the pathogenesis of <i>Lawsonia intracellularis</i> often require bacterial quantification in suspension. However, due to the organism’s fastidious growth requirements—being both intracellular and microaerophilic—traditional quantification methods, such as colony-forming unit counting, are not feasible. Currently, the only widely available method for quantifying <i>L. intracellularis</i> is real-time quantitative PCR (RT-qPCR). Unfortunately, the time required to perform RT-qPCR is incompatible with the bacterium’s limited survival outside its intracellular and microaerophilic environment. As a result, bacterial suspensions are typically quantified subjectively, based on the researcher’s experience for immediate use, with RT-qPCR conducted afterward. Optical density (OD) spectrophotometry is a rapid, although indirect, method of estimating bacterial concentrations in suspension, and it has been applied successfully to fast-growing prokaryotic species. Therefore, the objective of this study was to determine the correlation between RT-qPCR results and the optical density of <i>L. intracellularis</i> suspensions, with the goal of enabling the use of spectrophotometry for immediate bacterial quantification in experimental settings. Optical densities (ODs) were measured at 405 nm and 450 nm, using either a cuvette or microplate, while RT-qPCR was employed to establish a standard curve from samples of known concentration and to quantify the concentration of <i>L. intracellularis</i> in the test suspensions. Four comparison variations between OD and RT-qPCR were evaluated: (1) spectrophotometry at 405 nm using a cuvette vs. RT-qPCR; (2) spectrophotometry at 405 nm using a microplate vs. RT-qPCR; (3) spectrophotometry at 450 nm using a cuvette vs. RT-qPCR; and (4) spectrophotometry at 450 nm using a microplate vs. RT-qPCR. The tests were conducted in two independent replications, with each sample analyzed in duplicate. In all variations, the correlation between the bacterial concentrations determined by RT-qPCR and those estimated by OD was greater than 80%, with a statistical significance of <i>p</i> < 0.05. The following OD conversion equations for determining the number of microorganisms/mL were obtained: (1) f(x) = −7.438 × 10<sup>8</sup> + 1.797 × 10<sup>10</sup>. x; (2) f(x) = 3.255 × 10<sup>8</sup> + 3.003 × 10<sup>9</sup>. x; (3) f(x) = −8.006 × 10<sup>8</sup> + 2.169 × 10<sup>10</sup>. x; (4) f(x) = 3.107 × 10<sup>8</sup> + 3.758 × 10<sup>9</sup>. x. Here, “X” is the Ct value obtained by RT-qPCR. These findings enable researchers to improve the accuracy of their <i>L. intracellularis</i> experiments by utilizing optical spectrometry—a straightforward method that provides immediate results for determining bacterial concentration in suspensions. |
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| ISSN: | 2076-2607 |