National Institute of Standards and Technology, Material Measurement Laboratory - Biomolecular Measurements Division, 100 Bureau Drive, Gaithersburg, MD, 20899, United States.
California EPA Department of Pesticide Regulation, Sacramento, CA, 95814, United States.
Sci Rep. 2018 Jan 17;8(1):904. doi: 10.1038/s41598-018-19187-3.
The nematode Caenorhabditis elegans is used extensively in molecular, toxicological and genetics research. However, standardized methods for counting nematodes in liquid culture do not exist despite the wide use of nematodes and need for accurate measurements. Herein, we provide a simple and affordable counting protocol developed to maximize count accuracy and minimize variability in liquid nematode culture. Sources of variability in the counting process were identified and tested in 14 separate experiments. Three variables resulted in significant effects on nematode count: shaking of the culture, priming of pipette tips, and sampling location within a microcentrifuge tube. Between-operator variability did not have a statistically significant effect on counts, even among differently-skilled operators. The protocol was used to assess population growth rates of nematodes in two different but common liquid growth media: axenic modified Caenorhabditis elegans Habitation and Reproduction medium (mCeHR) and S-basal complete. In mCeHR, nematode populations doubled daily for 10 d. S-basal complete populations initially doubled every 12 h, but slowed within 7 d. We also detected a statistically significant difference between embryo-to-hatchling incubation period of 5 d in mCeHR compared to 4 d in S-basal complete. The developed counting method for Caenorhabditis elegans reduces variability and allows for rigorous and reliable experimentation.
秀丽隐杆线虫被广泛应用于分子、毒理学和遗传学研究。然而，尽管线虫的应用广泛且需要进行精确的测量，但目前仍没有标准化的液体培养线虫计数方法。本文提供了一种简单且经济的线虫计数方案，旨在最大限度地提高计数的准确性和减少液体培养线虫计数过程中的变异性。在 14 项独立实验中确定并测试了计数过程中的变异性来源。有三个变量对线虫计数有显著影响：培养物的晃动、移液管尖端的预润湿以及微离心管内的取样位置。即使在不同技能水平的操作人员之间，操作人员之间的变异性也没有对计数产生统计学上的显著影响。该方案用于评估两种不同但常见的液体生长培养基中秀丽隐杆线虫的种群增长率：无菌改良秀丽隐杆线虫栖息地和繁殖培养基（mCeHR）和 S-基础完全培养基。在 mCeHR 中，线虫种群在 10 天内每天翻倍。S-基础完全培养基中的线虫种群最初每 12 小时翻倍一次，但在 7 天内减缓。我们还检测到 mCeHR 中的胚胎孵化期为 5 天与 S-基础完全培养基中的 4 天之间存在统计学上的显著差异。开发的秀丽隐杆线虫计数方法减少了变异性，允许进行严格可靠的实验。
