Optimized preparation pipeline for emergency phage therapy against Pseudomonas aeruginosa at Yale University耶鲁大学针对铜绿假单胞菌的紧急噬菌体疗法的优化制备流水线

Quantification of the bacterial products pyocyanin and elastase
细菌产物焦花青素和弹性蛋白酶的定量分析

The blue-colored phenazine pyocyanin was measured by spectrophotometry⁴⁶. Overnight cultures of test bacteria were standardized to OD₆₀₀ = 1.0 before inoculating in 6 mL of LB with 1:100 dilution in 15 mL tubes. After 48 h, cultures were normalized (OD₆₀₀) reflecting about 1e8 CFU/mL and supernatants were collected by centrifugation at 4000 rpm for 15 min. Subsequently, 2.5 mL of chloroform (AmericanBio, Canton, MA, USA) was added to 5 mL supernatants, vortexed 5 × 5 s, and centrifuged for 15 min at 4000 rpm. The bottom (blue) layer was mixed with 500 µL of 0.2 M HCl, vortexed 5 × 5 s and centrifuged for 5 min at 4000 rpm. 100 µL of the upper (pink) layer were transferred to a 96 well plate for spectrophotometric measurements at OD₅₂₀. Pyocyanin concentration (µL/mL) was calculated by subtracting the blank value (0.2 M HCl) from the sample values then multiplying the values by 17.072^47,48.
通过分光光度法 ⁴⁶ 测量了蓝色的酚嗪酞菁。将过夜培养的试验细菌标准化至 OD ₆₀₀ = 1.0，然后接种到 6 mL LB 中，并以 1:100 稀释到 15 mL 试管中。48 小时后，培养物标准化（OD ₆₀₀ ）反映出约 1e8 CFU/mL，并以 4000 rpm 离心 15 分钟收集上清。随后，在 5 mL 上清液中加入 2.5 mL 氯仿（AmericanBio，Canton，MA，USA），涡旋 5 × 5 s，4000 rpm 离心 15 分钟。底层（蓝色）与 500 µL 0.2 M HCl 混合，涡旋 5 × 5 s，4000 rpm 离心 5 分钟。将 100 µL 上层（粉红色）转移到 96 孔板中，在 OD ₅₂₀ 下进行分光光度测量。从样品值中减去空白值（0.2 M HCl），再乘以 17.072 ^47,48 计算出 Pyocyanin 的浓度（µL/mL）。

Pseudomonas aeruginosa elastase was measured with a fluorometric assay as indicated by the manufacturer’s protocol (EnzChek Elastase Assay Kit, Thermo Scientific, Waltham, MA, USA).
铜绿假单胞菌弹性蛋白酶的测定采用荧光测定法，该方法由生产商提供（EnzChek Elastase Assay Kit，Thermo Scientific，Waltham，MA，USA）。