Preculture preparation

A. Preculture preparation. A 250-mL Erlenmeyer flask containing 50 mL of mineral salt broth (MSB) (Note 1) with 0.2% L-arginine hydrochloride (L-arginine-HCI) and fitted with a cotton plug is sterilized (Note 2). In a laminar flow hood, a single colony of Pseudomonas putida 39/D (Pp 39/D) (Note 3) is selected from a fully grown plate (Notes 4, 5, 6) and transferred to the solution with a sterile loop taking care not to place the hand over the plate or flask. The flask is then placed in a benchtop orbital incubator shaker at 30°C and 200 rpm for 24 hr. 

Plug these flasks with cotton plugs and autoclave at 15 psi pressure for 20 min. Transfer 2 mL of preculture as prepared above into each of the flasks. Add 0.2 mL of filter sterile propionitrile (filter 5 mL of propionitrile using 0.22 pm filter into a sterile tube) into each flask as inducer for the induction of nitrilase in G. terrae NDB 1165 cells. Incubate these at 30 °C in a gyratory shaker (180 rpm) for 24 h. 

Preculture (100 ruL) was prepared in the liquid medium containing an inoculum of H402 X pTKLl yeast,that was stored at —80 °C, and was grown for 48 h at 30 °C in an Erlenmeyer flask (500 mL) under rotatory shaking (200 rpm). 

The inocula for these experiments were prepared by preculturing green hairy roots in MS liquid medium for 14 days at incident light intensity, I, of 11.1 W m 2 under the same conditions as mentioned above. 

In the dryness tolerance experiment, the precultured alga was harvested by centrifugation at 3,000 rpm for 10 min and washed twice with distilled water. The pellet was resuspended in sterilized water so that the optical density was 1.0 at 660 nm. One ml of the suspension was filtered using a filter paper of 2.5 cm in diameter. Two filter papers were prepared. One was dried at 25°C for 7 days, the other was dried at 45°C for 2 days. Each of the dry filter papers was transferred into 40 ml medium in a 100-ml Erlenmeyer flask and cultured at 25°C on a rotary shaker (130 rpm). 

For cultivation by the submerged culture technique, a preculture is first prepared as follows. The medium used is a 4.5% aqueous malt extract solution of pH 5. One litre of this solution is sterilised for 20 minutes at 110° C. in a 2-litre conical flask, then inoculated with 6.10 to the) 8 th) conidia of a 15-days old agar culture and incubated for 3 days on a rotating shaking machine at 24° C. A dense culture of fine mycelial flocks forms, each flock consisting of a loose bunch of hyphae of diameter 2 to 4 mm. No alkaloids can be identified. 

Streptococcus sobrinus (5. sobrinus) 6715 and Streptococcus mutans (S. mutans) MTS 148 were obtained from the Institute of Physical and Chemical Research (RIKEN), Saitama, Japan. S. sobrinus 6715, 5. mutans MTS 148, and S. mutans IFO 13955 were precultured in BHI broth overnight to prepare the seeded solution. The precultured mutans streptococci was adjusted to 10 cfii/m/ with BHI broth by optical density (660 nm). Each isothiocyanate, in an 80% aqueous methanol solution (50 pL), was added to the test tube containing five mL of each mutans streptococci BHI broth (10 cfii/mL). The test tube was incubated for 24 h at 37 C and the minimum inhibitory concentration (MIC) value was measured. All tests were run in triplicate and averaged. 

Prepare a preculture of wild-type strain in YPD medium incubated at 25 °C. Inoculate cells into 96 wells of a microtiter plate containing fresh YPD medium and various inhibitory concentrations of the drug or the solvent alone (r Note 8). 

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