Ammonium acetate

Clostridium difficile strain CD105HE1 ribotype 076 was used to propagate the phage as per the method of Nale et al. [28 (link)] Bacteriophage CDKM9 is a myovirus previously isolated from an environmental water sample as described by Rashid et al. [48 ]
The identity and purity of the phages used for encapsulation was confirmed using transmission electron microscopy (TEM) analysis. 1 ml of phage stock was centrifuged at 10,000 rpm for 1 hour at 4°C and the pellet was washed two times with 0.1 M ammonium acetate (Fisher, UK) and re-suspended in 100 μl of ammonium acetate. 4 μl of sample suspension was placed on glow discharged pioloform/ carbon coated copper grids which were allowed to stand for 5 min for bacteriophage to bind, 1% w/v uranyl acetate was added to stain the sample. 10μl of deionised water was used to rinse the sample and the prepared sample was left to dry for 24 h. The grids were examined using a JEOL 1220 electron microscope with an accelerating voltage of 80 kV. Digital images were captured using SIS Megaview III digital camera with associated analysis software.

Chitosan (CH, MW 230 KDa and 80.6% N-deacetylation) was acquired from Aldrich Chemical Co. (Milwaukee, WI, USA) and hydroxypropyl methylcellulose (HPMC, MW 250 kDa, hydroxypropyl 7–12%, methoxyl 19–24%) was obtained from Eigenmann & Veronelli (Milan, Italy). Gelatine (GEL, type A from pork skin, 125 Bloom value) and sodium alginate (ALG, Sigma-Aldrich Co. Buenos Aires, Argentina) were also used. FMC BioPolymer (Philadelphia, PA, USA) donated samples of food-grade λ and κ-carrageenan (λ-c and κ-C), and sodium lauryl sulphate (SLS) was acquired from Biopack (Buenos Aires, Argentina). Ammonium acetate (HPLC grade) was acquired from Thermo Fisher Scientific (Cleveland, OH, USA), acetonitrile (HPLC grade) and sorbitol (70%) were obtained from Cicarelli (Buenos Aires, Argentina), while lidocaine hydrochloride (LDCH) and miconazole nitrate (MN), both of pharmaceutical grade, were obtained from Parafarm (Buenos Aires, Argentina). All other reagents were of analytical grade.

Shuanghuanglian for injection (lyophilized) (600 mg) was purchased from Harbin Pharmaceutical Group No. Two Traditional Chinese medicine factory (Harbin, China) (Batch number: 1904002). Shenmai injection (10 mL) was purchased from Chiatai Qingchunbao Pharmaceutical Co., Ltd. (Hangzhou, China) (Batch number: 1912094). Compound 48/80 (C48/80) was purchased from Sigma Aldrich (Saint Louis, MO, USA) (Batch number: 088M4120V). Mouse Histamine ELISA Kit was purchased from Elabscience Biotechnology (Wuhan, China) (Batch number: DPM78G9DWG). Mouse tryptase Kit was purchased from Jianglai biology Co., Ltd. (Shanghai, China) (Batch number: April 2020). Chromatographic pure methanol, formic acid, ammonium acetate was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Normal saline (NS) for injection was purchased from Hunan Kelun Pharmaceutical Co., Ltd. (Yueyang, Hunan, China) (Batch number: 088M4120V). Evans Blue (EB) was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China).

Methanol, acetonitrile, isopropanol, formic acid, and ammonium acetate were purchased from Thermo Fisher Scientific (Fair Lawn, NJ). Ammonium bicarbonate (LC-MS grade) and methyl tert-butyl ether (MTBE) were purchased from Sigma-Aldrich (St. Louis, MO). Ultra-pure water (18.2 m ω cm) was prepared in house by a Milli-Q purified water system (Merck KGaA, Darmstadt, Germany).

Samples containing Mtb were heat-treated on a thermomixer (95°C, 30 minutes, 1400 rpm). Subsequently, in order to concentrate and purify the extracted DNA, samples were vortexed in the presence of ethanol (81% [v/v], VWR International, Radnor, USA) ammonium acetate (9% [v/v], Thermo Fisher Scientific, Waltham, USA) and 60 μg glycogen (Roche, Basel, Switzerland) and incubated overnight at -20°C. Following centrifugation (30 minutes, 16.200xg), the supernatant was discarded. This step was repeated. Subsequently, the pellet was washed with ethanol (75% [v/v] and sample centrifuged (10 minutes, 16.200xg). Finally, the pellet was air dried for 15 minutes and dissolved in 5μl H₂O before proceeding further.