Add purple broth base to distilled water. Dispense 9 ml into 16 x 125 mm tubes containing Durham tubes. Autoclave at 121°C for 15 min. Prepare carbohydrates, except esculin, as sterile 5% solutions.

Filter-sterilize or autoclave, as appropriate. Add 1 ml carbohydrate solution to 9 ml broth base to yield 0.5% carbohydrate in broth.

Add esculin directly into base broth to make a 0.5% solution and autoclave 15 min at 115°C. At room temperature, a 5% solution of esculin is a gel that cannot be pipetted.

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