2.2. Ellinghausen McCullough Johnson Harris Broth Preparation

Ellinghausen McCullough Johnson Harris (EMJH) broth was prepared by dissolving Difco^TM Leptospira Medium Base EMJH (Becton, Dickinson and Company, Sparks, MD, USA) in distilled water (2.3 g/L), which was autoclaved (Tomy Kogyo Co., Ltd., Tokyo, Japan) and left to cool. Difco^TM Leptospira Enrichment EMJH (Becton, Dickinson, and Company, Sparks, MD, USA) (100 mL/L) and filtered (0.22-µm syringe filter; Bioflow, Kuala Lumpur, Malaysia), and 5-fluorouracil (5-FU; Intas Pharmaceuticals Ltd., Ahmedabad, India) (200 µg/mL) was added. The broth was transferred to 15-mL centrifuge tubes (Nest Biotech Co., Ltd., Wuxi, Jiangsu, China).

EMJH agar was prepared by dissolving Difco^TM Leptospira Medium Base EMJH (2.3 g/L) and Bacto^TM Agar (Becton, Dickinson and Company, Sparks, Maryland, USA) (9 g/mL) in distilled water, followed by autoclaving, leaving to cool, and then adding Difco^TM Leptospira Enrichment EMJH (100 mL/L) supplemented with filtered 5-FU (200 µg/mL). The solution was poured into plastic petri dishes. Both EMJH broth and agar were incubated at 30–35 °C for 24 h for sterility testing and stored in a cold room prior to use.