440 MICROBIOLOGICAL TECHNIQUES 



Lowry and Bessey Method for Riboflavin 



SPECIAL REAGENTS AND MATERIALS 



Lactobacillus casei Culture and Inoculum. The procedure for 

 transfer of cultures is shown in the following diagram: 



stock culture h^ 



Stock culture a 



^culture d, etc. 

 culture c 



inoculum e —*■ inoculum / — *■ inoculum g, etc. 



assay tubes h 



Make the stab transfers h, c, d, etc. into yeast-water agar, con- 

 taining 1% glucose and 0.15 M potassium acetate, and, after 24 

 hr. incubation at 37°, store in a refrigerator. Reserve at least one 

 tube, h, as a stock culture. When assays are made on successive 

 days, do not grow inoculum from stock cultures c and d each day, 

 but transfer a drop of inoculum e to a similar tube, /, which is 

 incubated for use the next day. Do not use inoculum cultures 

 after they are more than 36 hr. old. Return to a stock culture about 

 every 5 days to minimize the possibility of contamination and 

 variation. Each month prepare new stock cultures corresponding 

 to h, c, and d, from a tube such as h of the preceding month. For 

 inoculum, make a stab from a stock culture into a sterile tube of 

 basal medium containing 0.5-1.0 ju,g. added riboflavin per 10 ml. 

 Incubate for 24 hr. at 37°, centrifuge out the cells, and resuspend 

 in an equal vol. of sterile 0.9% sodium chloride soln., observing 

 aseptic technique throughout. The most consistent results are 

 obtained from a culture made from a work stab not over 

 a week old. 

 Basal Medium. Consists of the following: photolyzed sodium 

 hydroxide-treated peptone, 0.5%; glucose, 1%; sodium acetate, 

 0.6%; cystine, 0.01%; inorganic salts; and riboflavin-free yeast 

 supplement equivalent to 0.1% yeast extract. 



