Bx51f

Microbial cell morphology was examined under a fluorescence microscope (Olympus BX51F, Tokyo, Japan) equipped with a color CCD camera system (Olympus DP72). For scanning electron microscopic (SEM) images, lignite particle and sponge samples were prefixed in 2.5% (w/v) glutaraldehyde in the anaerobic medium used for the DHS reactor cultivation without organic compounds. After the samples were rinsed with distilled water, the conductive staining was performed by incubation in 0.2% aqueous tannic acid (pH 6.8) for 30 min, the samples were then washed with distilled water and treated with 1% aqueous osmium tetraoxide for 1 hour. Subsequently, the samples were dehydrated in a graded ethanol series and critical point dried in a JEOL JCPD-5 (Tokyo, Japan). The samples were then coated with osmium using an osmium plasma coater (POC-3; MEIWAFOSIS Co., Ltd., Tokyo, Japan) and observed with a JEOL JSM-6700F field emission SEM operated at 5 kV.

Lung and kidney tissue was fixed in 4% neutral-buffered formalin and subsequently embedded in paraffin. Three-mm thick sections were stained with hematoxylin and eosin (H&E) using a standard protocol [19 (link)]. The sections were analyzed by light microscopy and images were taken with an Olympus DP70 digital camera system in an Olympus BX51F microscope. Histological changes were evaluated by a pathologist blind to the treatment regimen. A scoring system to grade the degree of lung and kidney injury was employed [20 (link),21 (link)] based on the histological features specified in Tables 1 and 2. Each feature was graded as absent, mild, moderate, or severe, with a score of 0–3. A total score was calculated for each animal.