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.

中文翻译：

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计数秀丽隐杆线虫：液体培养基中种群增长和毒性研究的方案优化和应用。

线虫秀丽隐杆线虫广泛用于分子，毒理学和遗传学研究。然而，尽管线虫的广泛使用并且需要精确的测量，但在液体培养中用于计数线虫的标准化方法尚不存在。本文中，我们提供了一种简单且价格合理的计数方案，旨在最大程度地提高计数准确性并最大程度减少液体线虫培养中的变异性。确定了计数过程中变异性的来源，并在14个单独的实验中进行了测试。三个变量对线虫数量产生了重大影响：培养物的摇动，吸液管吸头的灌注以及微量离心管内的采样位置。运营商之间的差异对计数没有统计学意义的影响，即使是技能不同的运营商也是如此。该协议用于评估线虫在两种不同但常见的液体生长培养基中的线虫种群生长速率：改良的线虫秀丽隐杆线虫栖息和繁殖培养基（mCeHR）和S-基础完全培养基。在mCeHR中，线虫种群每天增加一倍，持续10天。S基础完全种群最初每12小时增加一倍，但在7天之内减慢。我们还检测到mCeHR中胚胎到孵化潜伏期5 d与S基底完全潜伏期4 d的统计学差异。发达的秀丽隐杆线虫计数方法减少了变异性，并允许进行严格而可靠的实验。线虫种群每天增加一倍，持续10天。S基础完全种群最初每12小时增加一倍，但在7天之内减慢。我们还检测到在mCeHR中胚胎孵化潜伏期5 d与在S基底完全孵化4 d之间的差异具有统计学意义。发达的秀丽隐杆线虫计数方法减少了变异性，并允许进行严格而可靠的实验。线虫种群每天增加一倍，持续10天。S基础完全种群最初每12小时增加一倍，但在7天之内减慢。我们还检测到mCeHR中胚胎到孵化潜伏期5 d与S基底完全潜伏期4 d的统计学差异。发达的秀丽隐杆线虫计数方法减少了变异性，并允许进行严格而可靠的实验。