Ch20i

Tumor, liver, lungs and kidney tissues were fixed overnight at 4°C in freshly prepared 4% paraformaldehyde and then dehydrated in graded alcohols and embedded in paraffin. Sections of 5μm thickness were cut from representative paraffin blocks. Tumor tissues were cut right through the middle of the tissues to obtain the central core region. The sections were rehydrated and stained with hematoxylin and eosin (H&E). Stained sections were observed under light microscope (Olympus CH20i).

EP in vitro PTC trials followed after above-surface sterilization was routinely observed on a long term (3–4 weeks post in vitro establishment) for any microbial growth emanating from the EP explants and specifically not affecting in vitro response from EP. Any contamination from within 3–4 weeks of in vitro EP establishment or contamination deemed to occur from a source other than the explants was discarded and not considered in the study. When apparent, a loopful of culture or colonies with limited growth around the explant (few colonies) which could be picked using a sterile tooth-pick were streak-plated over a nutrient agar plate (incubated overnight at 37 °C) for colony phenotyping under a light microscope (Olympus CH20i, Haryana, India). Single colonies were then subcultured further onto fresh NA plates and were later used to inoculate 5 mL sterile nutrient broth (NB) for overnight growth (at 37 °C, 150 rpm, dark) for preparation as glycerol stock for culture storage backups and further analyses when needed.

All the stained sections were observed using a binocular light microscope (CH20i, Olympus, made in Noida, India under license from Olympus Corporation, Tokyo, Japan) at 10× magnification, and, with a one-field depth from the basement membrane of the epithelium or from the tumor invasive margins or from the tumor nests, four areas with the highest number of blood vessels (hotspots) were selected. The microvessel density (number of microvessels per optical field) was determined by counting the number of blood vessels in each field at 40× magni-fication, and the average was recorded. During counting, single endothelial cells, clusters of endothelial cells, and endothelial cells lining the lumen which turned a brown or black color with anti-CD34 were considered positively stained blood vessels. Simultaneously, mast cell density (the number of mast cells per optical field) was determined by counting the mast cells in the same four already selected microscopic fields, at 40× magnification, and the average was recorded for each sample. Mast cell granules, appearing violet with bluish background and clearly separated from adjacent clusters, were considered as single mast cells and were counted in each field; the average of the four fields was recorded. All the evaluations were performed by a single investigator.

Under a calibrated compound light microscope (Olympus CH20i) with 10 ×, 40 ×, and 100 × immersion lenses, the main morphological characteristics of the isolated algal strain were examined and digital photomicrographs of the specimens were taken. For molecular identification, the genomic DNA extraction method (CTAB) was used to characterize the algal strain, which was then followed by PCR, gel electrophoresis, and algal identification was done by using rbcL (Ribulose bisphosphate Carboxylase Large subunit) gene sequencing. In addition, that sequence was uploaded to the NCBI database to obtain an accession number.

Bacterial isolates were morphologically characterized based on visual observations of colony growth and behavior over the NA plate, followed by direct close examination under a light microscope (Olympus CH20i) and also using SEM (detailed below). Similarly, plant (wheat and tomato) seeds/seedlings and/or full-grown plants resulting from the various in vitro/ex vitro/field treatments with/without bacterial isolates (discussed below) were also recorded for morphometrics around shoots, roots, their branching, and root hair density using visual observations and/or close examinations under a stereomicroscope (Nikon-745T fitted with a 5 Megapixel digital camera, Nikon, Mumbai, India).