• RIBOFLAVIN 441 



(a) Prepare the photolyzed sodium hydroxide-treated peptone 

 by exposing a mixture of 40 g. of Difco Bacto peptone {Difco 

 Laboratories, Inc.) in 250 ml. water and 20 g. sodium hydroxide 

 in 250 ml. water in a 25 cm. crystallizing dish to light from a 100 

 watt lamp, with reflector, at a distance of about 30 cm. for 6-10 

 hr. After standing at room temperature for an additional 14-18 

 hr. the mixture is neutralized with 27.9 ml. glacial acetic acid, 

 7 g. anhydrous sodium acetate is added, and the mixture is diluted 

 to 800 ml. Preserve under toluene. 



(5) Prepare the yeast supplement by adding a soln. of 150 g. 

 basic lead acetate in 500 ml. water to 100 g. Bacto yeast extract 

 (Difco) in 500 ml. water; filter off and discard the precipitate, add 

 ammonium hydroxide to a pH of about 10, filter off and discard 

 the precipitate, add glacial acetic acid to just acidify the filtrate, 

 precipitate excess lead with hydrogen sulfide, and filter off and 

 discard the lead sulfide. Make up the filtrate to 1000 ml. and store 

 in a refrigerator under toluene. One ml. of the preparation is 

 equivalent to 100 mg. of original yeast extract. 



(c) Prepare inorganic salt solns. as follows: Solution A — 

 Dissolve 25 g. potassium monohydrogen phosphate and 25 g. 

 potassium dihydrogen phosphate in 250 ml. water. Solution B — 

 Dissolve 10 g. magnesium sulfate heptahydrate, 0.5 g. sodium 

 chloride, 0.5 g. ferrous sulfate heptahydrate, and 0.5 g. manganese 

 sulfate tetrahydrate in 250 ml. water. 



(d) Combine 50 ml. of the treated peptone soln., 50 ml. 0.1% 

 cystine hydrochloride, 5.0 ml. yeast supplement, 5.0 g. glucose, 

 2.5 ml. inorganic salt soln. A, and 2.5 ml. soln. B. Add potassium 

 acetate to give a concentration of 0.3 M when the mixture is 

 diluted to 250 ml. With a sterile pipette, add 0.5 ml. of 0.4% 

 freshly boiled neutral cysteine soln. to each 10 ml. of sterile basal 

 medium just before inoculation. 



Standard Riboflavin Solutions. Prepare standards containing 0.5, 

 1.0, 1.5, and 2.0 fig. per 100 ml. in 0.002 N hydrochloric acid. Use 

 0.002 N hydrochloric acid for the blank soln. See step 11 imder 

 Procedure. 



Caprylic Alcohol. 



0.1 N Hydrochloric Acid. 



0.002 N Hydrochloric Acid. 



0.3 N Sodium Hydroxide. 



