Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometry
Randall, Carly J, Speaks, Justin E., Lager, Claire, Hagedorn, Mary, Llewellyn, Lyndon, Pulak, Rock, Thompson, Julia, Bay, Line K., Mead, David, Heyward, Andrew J., and Negri, Andrew P. (2020) Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometry. Scientific Reports, 10. 12919.
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Abstract
Research with coral embryos and larvae often requires laborious manual counting and sorting of individual specimens, usually via microscopy. Because many coral species spawn only once per year during a narrow temporal window, sample processing is a time-limiting step for research on the early life-history stages of corals. Flow cytometry, an automated technique for measuring and sorting particles, cells, and cell-clusters, is a potential solution to this bottleneck. Yet most flow cytometers do not accommodate live organisms of the size of most coral embryos (> 250 µm), and sample processing is often destructive. Here we tested the ability of a large-particle flow cytometer with a gentle pneumatic sorting mechanism to process and spectrally sort live and preserved Montipora capitata coral embryos and larvae. Average survival rates of mechanically-sorted larvae were over 90% and were comparable to those achieved by careful hand-sorting. Preserved eggs and embryos remained intact throughout the sorting process and were successfully sorted based on real-time size and fluorescence detection. In-line bright-field microscopy images were captured for each sample object as it passed through the flow-cell, enabling the identification of early-stage embryos (2-cell to morula stage). Samples were counted and sorted at an average rate of 4 s larva−1 and as high as 0.2 s larva−1 for high-density samples. Results presented here suggest that large-particle flow cytometry has the potential to significantly increase efficiency and accuracy of data collection and sample processing during time-limited coral spawning events, facilitating larger-scale and higher-replication studies with an expanded number of species.
| Item ID: | 64859 |
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| Item Type: | Article (Research - C1) |
| ISSN: | 2045-2322 |
| Keywords: | Animal physiology, Biological techniques, Developmental biology, Ocean sciences, Zoology |
| Copyright Information: | © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
| Funders: | Australian Institute of Marine Science (AIMS), Reef Restoration and Adaptation Program (RRAP) |
| Date Deposited: | 21 Feb 2021 22:19 |
| FoR Codes: | 41 ENVIRONMENTAL SCIENCES > 4103 Environmental biotechnology > 410304 Environmental biotechnology diagnostics (incl. biosensors) @ 100% |
| SEO Codes: | 97 EXPANDING KNOWLEDGE > 970110 Expanding Knowledge in Technology @ 50% 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 50% |
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