Acetone

The combinations with the lowest FICI values were examined using scanning electron microscopy (SEM). Leptospira cultures (2 × 10⁶ CFU/mL) were incubated at 30 °C and 40 rpm for seven days and treated with the MICs of the extracts and antibiotic solutions separately and combined for 18 h. A negative control (untreated Leptospira) sample was prepared. The samples were centrifuged (4560 rpm at 2000 g for 10 min) (Hettich, Westphalia Germany) and the pellets were fixed using McDowell-Trump’s fixative (Electron Microscopy Sciences, Hatfield, PA, USA) at 4 °C for two hours, centrifuged again, washed with 0.1 M PBS (Electron Microscopy Sciences, Hatfield, PA, USA), and fixed using 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA) at 4 °C for one hour. The pellets were then washed with 0.1 M PBS and dehydrated using 50%, 75%, 95%, and 100% acetone (Electron Microscopy Sciences, Hatfield, PA, USA) for ten minutes each. Then, 100% hexamethyldisilazane (Electron Microscopy Sciences, Hatfield, PA, USA) was added to the pellets for 10 min and air-dried overnight. Specimens were then mounted on sample stubs and coated with gold (Electron Microscopy Sciences, Hatfield, PA, USA) for examination with a high-resolution versatile SEM instrument (FEI, Brno, Czech Republic).

The precursor compound of Al-MOF-TCPPH₂ was tetrakis(4-carboxyphenyl)porphyrin (TCPPH₂) of purity ≥ 97.0% from TCI Chemicals (Portland, OR, USA). Aluminum chloride AlCl₃·6H₂O of 99% purity was from Alfa Aesar (Tewksbury, MA, USA). Acetone (of reagent purity) was from Electron Microscopy Sciences (Hatfield, PA, USA). N,N-dimethylformamide (DMF) of ≥99.5% purity and n-pentane of >97% purity were from TCI Chemicals.

Frozen samples were collected in liquid nitrogen and transferred into a freeze-substitution chamber (Leica EM AFS; Leica, Germany), where they were freeze substituted for 12–14 hours at −90 °C in acetone containing 1% osmium tetroxide (OsO₄; Electron Microscopy Sciences, Germany) to enhance contrast and simultaneously stabilize the glycocalyx. Subsequently, the temperature was gradually increased (5 °C/1 h) to 20 °C. Afterwards, the cells were centrifuged (1000 rpm, 3 min), washed with acetone (Roche, Germany) and centrifuged again. After rinsing several times with acetone, the dehydrated samples were infiltrated first with HPMA (2-hydroxypropyl methacrylamide; Sigma-Aldrich, Germany), centrifuged at 3000 rpm for 5 min and finally infiltrated overnight with HPMA-Epon mix (1:1). Suspension cells were further centrifuged at 5000 rpm for 5 min and infiltrated with pure freshly prepared Epon for several hours. Finally, the samples were embedded in Epon and polymerized overnight at 60 °C.