100 μl of the suspension was spread on CSPYME agar consisting of casein (0.1%), corn starch (1.0%), K2HPO4, (0.05%), peptone (0.1%), yeast extract (0.1%), malt extract (1.0%), agar agar (1.5%), hydroxylamine (0.01%) and amphotericin-B (0.002%) with initial pH value of 7.0.
One of them was purified on MRSC agar, consisting of MRS ([ 19] Biolife, Milan, Italy) supplemented with 0.05 % L- cysteine hydrochloride and 1.5 % agar, and propagated in MRSC broth.
Agar consists of a mixture of agarose and agaropectin.
The first was water agar (1.0 L of agar consisted of 20 g of agar and 1000 mL of water) and the second was nutrient agar (1.0 mL of the nutrient agar contained 100 mL of a basal salt solution, 5.0 g of glucose [Difco], 2.5 g of yeast extract [Difco], 20.0 g of agar, and 900 mL of distilled water adjusted to pH 5.5).
Briefly, 5000 (HEK293T cells), 10 000 (PC3, MCF-7 and UXF1138L cells) or 50 000 (UXF1138L tumour tissue) vital cells were added to 0.2 ml Iscove's medium/20% fetal bovine serum/0.05% gentamycin containing 0.4% agar and plated on top of a base layer consisting of 0.2 ml medium with 0.75% agar.
ZR-75-1 cells, MCF-7 cells, or derivative cell lines were cultured in 12-well plates containing a bottom agar layer consisting of culture medium plus 0.7% agar and 2 ng/mL puromycin.
For fen treatment, seedlings were grown on agar plates consisting of the above medium containing 0.8% plant agar (Duchefa) and 200 μg/ml fen for 5 days.
The culture was maintained at 4 °C on YPD agar plate consisting of 10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose and 25 g/l agar.
The strain was maintained on MGYP agar medium consisting of 0.3 % malt extract, 1 % glucose, 0.3 % yeast extract, 0.5 % peptone and 2%% agar and it was sub-cultured once in every 15 days.
The soft top agar of the test agar plate consisted of 10 gL-1 tryptone extract, 5 gL-1 yeast extract, 5 gL-1 NaCl, and 16 gL-1 agar, and the indicator cells were added into the soft agar at a final concentration of 10 cfu/ml.
