Background: In order to increase the detection rate of pathogenic microorganisms in CSF, an improved specimen handling procedure (ISHP) was created. Methods: This study enrolled encephalitis and control groups, both groups were handled with traditional specimen handling procedure (TSHP) and ISHP. Glutaraldehyde was added to the ISHP. Observed items included: total protein, glucose, chloride, adenosine deaminase, lactate dehydrogenase, sediment, cell and pathogen, Pandy's test. Results: Sediment test of CSF: There was 1 specimen in 10 control specimens tested by TSHP in which Pandy's test was positive; there were 2 specimens tested by ISHP which could see sediment by eye. There was no statistical difference between those two methods (p = 1.000, Table 1). Ten specimens in 23 of the encephalitis group processed by TSHP were positive with Pandy's test; 23 specimens processed by ISHP could all see sediments by eye (Figure 1). There was a statistical difference between the two methods (p = 0.000, Table 1). Pathogen test of CSF: no pathogen was found in the control group processed by TSHP and ISHP. No pathogen was found in the encephalitis group specimens processed by TSHP. Pathogen tests were positive in 7 encephalitis specimens processed by ISHP (p = 0.009, Table 1), which were confirmed as Rickettsia spp. by Gimenze stain (Figure 1B), IFA (Figure 2). Conclusions: The results revealed that ISHP contributes to the separation of cells, pathogens (such as Rickettsia), and proteins.