Abstract: With the progressive shift of space biological experiments from post-flight observations following short-term missions to long-term in-orbit observations, coupled with the increasing frequency of extravehicular activities by astronauts, the study of biological damage induced by the external space environment has emerged as a pressing and pivotal direction in the field of space life sciences. To achieve long-term in-orbit observation of individual nematode development in the extravehicular environment, it is necessary to prepare samples that meet the requirements of the microfluidic chip system used for nematode encapsulation, ensuring compatibility with the chip's loading specifications. The nematode chip regulates the entry of individual nematodes into the cultivation chambers through the precise dimensions of its microchannels. Consequently, the developmental stage of the samples must meet exacting criteria, which are directly correlated with the nematode's body width (requiring a body width range of 24~29 μm). To analyze the loading and developmental conditions of nematodes responsive to radiation and microgravity, thereby enhancing the sample loading efficiency of various nematode strains in microfluidic chips on the future Chinese Space Station, this study establishes a standardized operational protocol and verification method for the preparation, propagation, and post-synchronization developmental timing confirmation of nematode samples for microfluidic chip applications. The incineration method was employed to measure the body width of various nematode strains under different propagation and development periods, aiming to ascertain the optimal propagation time and the most suitable developmental stage for each nematode strain. The experimental results revealed the following findings: the wild-type strain exhibited a body width ranging from 25.41~26.41 μm after 3 weeks of propagation and 104-110 hours of development; the AM141 strain displayed a body width of 20.26 μm after 3 weeks of propagation and 96 hours of development; the SSM264 strain showed a body width of 23.51 μm after 4 weeks of propagation and 144 hours of development; and the TG11 strain demonstrated a body width of 26.16 μm under the same conditions of 4 weeks of propagation and 144 hours of development. These measurements meet the requirements for chip loading. By confirming the sample conditions before and after loading into the microfluidic chip, it was determined that the body width range of the samples from the four strains added to the chip was 27.71~28.02 μm, thereby verifying the validity of the samples.