Objective To investigate the effects of different culture conditions (culture medium, culture temperature, and culture time) on the identification of filamentous fungi by matrix⁃assisted laser desorption/ionization time⁃of⁃flight mass spectrometry (MALDI⁃TOF MS). Methods Eighty⁃one strains of Aspergillus filamentous fungi were included, and all strains were identified by conventional morphology combined with the “gold standard” molecular biology method. The 81 strains of filamentous fungi were inoculated onto planes of Sabouraud dextrose agar (SDA) medium, potato dextrose agar (PDA) medium, and novel Pan⁃fungal (PF) medium, and then various medium planes were cultured at two different temperatures of 28 ℃ and 35 ℃ for 2 days, respectively, followed by MALDI⁃TOF MS identification. The identification results were analyzed to screen the optimal medium and culture temperature suitable for mass spectrometry identification. After determining the optimal medium and culture temperature, the 81 strains of filamentous fungi were cultured for 1 to 6 days, respectively, and identified by MALDI⁃TOF MS. The identification results on days 2, 3, 4, 5, and 6 of culture were analyzed to screen the optimal culture time suitable for mass spectrometry identification. Results There were statistically significant differences in the detection rates of the 81 strains of filamentous fungi among different culture media and culture temperatures (P<0.05). The detection rate of filamentous fungi under the SDA medium+28 ℃ condition was 83.95%, which was higher than that under the SDA medium+35 ℃, PDA medium+35 ℃, PF medium+28 ℃, and PF medium+35 ℃ conditions (P<0.05). Under the SDA medium+28 ℃ condition, there were statistically significant differences in the detection rates of filamentous fungi among different culture times (P<0.05), showing an overall gradual downward trend, and the detection rate on day 2 was higher than that at the remaining culture times (P<0.05). There was no statistically significant difference in the detection rate between day 3 and day 4 (P>0.05), but both were higher than those on day 5 and day 6 (P<0.05). Conclusion Culture on SDA medium at 28 ℃ for 2 to 4 days is the optimal protocol for MALDI⁃TOF MS identification of filamentous fungi. However, different strains differ in their sensitivity to culture time, and culture strategies should be flexibly adjusted in clinical practice.

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