In recent years, the role of intestinal microorganisms in human health and diseases has been widely researched. The application of a germ-free animal model in intestinal microecology is extensive and this model provides a very effective research tool for human physiological and pathological research. To ensure the quality of experimental animals, aseptic tests must be frequently performed. However, sterility tests are affected by many factors. At present, the traditional detection method are mainly bacterial and fungal culture and Gram staining. With the development of high- throughput sequencing, polymerase chain reaction method have been used to identify bacteria using 16S rRNA. However, these method have some limitations. In this paper, we discuss the advantages and disadvantages of these method and the influencing factors.