What Was Observed? (Introduction)

• The goal of this research is to establish and maintain healthy colonies of planarians (a type of flatworm) for experimentation.
• Planarians are important in biological research due to their ability to regenerate lost body parts, making them useful for studies on regeneration, stem cells, and behavior.
• Researchers need a stable, healthy population of planarians to avoid variability that could interfere with experimental results.
• This protocol outlines how to maintain colonies of three common planarian species: Dugesia japonica, Schmidtea mediterranea, and Girardia tigrina.

Obtaining Planarians

• Planarians can be found in the wild (ponds, streams) or purchased from commercial suppliers. However, some species are not commercially available and may need to be obtained from research labs.
• Planarians should be shipped carefully in water-filled containers to avoid harm, with temperature carefully monitored (between freezing and 25°C).
• Upon arrival, the water in which they were shipped should be changed to fresh, oxygenated water to remove harmful byproducts.

Culture Conditions

• The type of water used for planarian culture is critical. The water must be fresh and carefully prepared because the pH can change over time.
• For G. tigrina, the water should have a pH range between 7.5 and 9.5. For other species like D. japonica, Poland Spring water is a good option.
• Containers for the planarians should be large, clean, and not overcrowded to prevent stress. Typically, about 400-700 worms are kept per 2000 mL of water.
• The ideal temperature for planarian colonies is between 17°C and 20°C. Higher temperatures may lead to bacterial growth and stress, especially for regenerating worms.

Light and Oxygen Conditions

• Planarians are nocturnal creatures and should be kept in dark environments. They can be exposed to light during feeding and cleaning.
• A proper light/dark cycle is necessary for experiments. A 12-hour light/12-hour dark cycle is recommended to synchronize their behavior.
• Oxygen levels in the water are important for worm health. If the environment is well-maintained, the oxygen should be sufficient without additional aeration.

Food Preparation and Allocation

• Planarians are fed beef liver paste. Here’s how to prepare it:
• Cut fresh organic beef liver into small cubes and remove any veins or fatty tissue.
• Blend the liver into a smooth puree, then strain to remove any remaining connective tissue.
• Centrifuge the liver paste to remove air bubbles, then aliquot into Petri dishes and freeze.
• Feed the planarians once a week. Before experiments, they should be starved for 7-15 days to ensure a consistent metabolic state.
• When feeding, add the liver paste to the water and ensure it sinks to the bottom for the worms to consume.
• After 1-2 hours, remove any uneaten food to avoid contamination and water quality issues.

Cleaning the Culture Containers

• Cleaning planarian containers is critical to maintaining healthy conditions. Cleaning should occur after every feeding and again 2 days later to remove metabolic waste.
• Use a pipette to gently move worms from the surface to the bottom of the container, then pour off the old water.
• Rinse the sides of the container, then wipe away any debris using paper towels.
• Refill the container with fresh, properly prepared medium for the worms.

Reproduction of Planarians

• Planarians reproduce by fissioning. A single worm can generate up to 40 offspring in 6 months with proper feeding.
• If clonal colonies are desired, you can cut worms to induce faster reproduction, ensuring all worms are genetically identical.
• Both D. japonica and S. mediterranea reproduce faster than G. tigrina, doubling their population every 2-3 weeks.

Troubleshooting

• If planarians appear stressed, lying limp or curled up, check water quality. High ammonia levels, low oxygen, or incorrect pH can harm the worms.
• If the colony becomes overcrowded, split them into multiple containers to reduce stress and prevent infections.
• For protozoan infections, remove sick animals, and treat the colony by chilling it to 10°C overnight, then slowly warming it up to 18°C the next day.
• Infected colonies can also be treated with antibiotics, but chilling the worms is the most effective method.

观察到了什么？ (引言)

• 这项研究的目的是为了建立并维护健康的计划虫（扁形虫）殖民地，以供实验使用。
• 计划虫因其再生失去的身体部位的能力而在生物学研究中非常重要，因此它们适用于再生、干细胞和行为等研究。
• 研究人员需要一个稳定、健康的计划虫群体，以避免干扰实验结果的变异性。
• 本协议概述了如何维持三种常见计划虫物种的殖民地：Dugesia japonica、Schmidtea mediterranea 和 Girardia tigrina。

如何获取计划虫

• 计划虫可以在野外（池塘、溪流）找到，或者从商业供应商那里购买。然而，某些物种无法商业购买，可能需要从研究实验室获取。
• 计划虫应谨慎运输，确保水满容器以避免伤害，同时仔细监控温度（在冰点和25°C之间）。
• 到达后，应立即更换运输水中的部分水，以去除有害副产品。

培养条件

• 用于计划虫培养的水类型非常关键。水必须是新鲜且仔细准备的，因为pH可能随时间变化。
• 对于G. tigrina，水的pH范围应在7.5到9.5之间。对于其他物种，如D. japonica，Poland Spring水是一个不错的选择。
• 计划虫容器应大而干净，避免过度拥挤，以防止压力。通常，每2000 mL水中保持400至700只虫。
• 计划虫群体的理想温度在17°C至20°C之间。较高的温度可能导致细菌生长和压力，尤其是对再生的虫子来说。

光照与氧气条件

• 计划虫是夜行性生物，应保持在黑暗环境中。它们在喂食和清洁时会暴露在光线下。
• 适当的光/暗周期对于实验至关重要。推荐12小时光照/12小时黑暗周期，以同步它们的行为。
• 水中的氧气水平对虫子健康非常重要。如果环境得到良好维护，氧气应充足，不需要额外的空气循环。

食物准备和分配

• 计划虫的食物是牛肝糊。以下是准备方法：
• 将新鲜有机牛肝切成小块，去除所有的静脉和脂肪组织。
• 将肝脏搅拌成光滑的泥状物，然后过滤以去除任何残余的结缔组织。
• 将肝脏糊液离心，以去除空气泡，然后分装到培养皿中并冷冻。
• 每周喂食计划虫。在实验前，应禁食7至15天，以确保它们的代谢状态一致。
• 喂食时，将牛肝糊加入水中，确保它沉到底部供虫食用。
• 1至2小时后，移除任何未吃完的食物，以避免污染和水质问题。

清洁培养容器

• 清洁计划虫容器对于维持健康的环境至关重要。每次喂食后都要清洁容器，并在2天后再清洁一次以去除代谢废物。
• 使用移液管轻轻将虫子从容器表面移到底部，然后倒掉旧水。
• 用水冲洗容器的侧面，然后用纸巾擦拭去除残留物。
• 使用新鲜、正确准备的培养基来填充容器。

计划虫的繁殖

• 计划虫通过分裂繁殖。一只虫子在6个月内可以生成最多40只后代。
• 如果需要无性繁殖的虫群，可以通过切割虫子来加速繁殖过程，确保所有虫子基因相同。
• D. japonica和S. mediterranea繁殖速度比G. tigrina更快，每2到3周就能繁殖一倍。

故障排除

• 如果计划虫表现出压力反应，如蜷曲或无力地躺在容器底部，请检查水质。氨水平过高、氧气不足或pH不正确可能会伤害虫子。
• 如果容器过于拥挤，请将虫子分开。
• 对于原生动物感染，移除病虫，使用冷却处理来恢复群体。
• 使用抗生素治疗感染，但冷却虫群是更有效的方法。