442 MICROBIOLOGICAL TECHNIQUES 



0.030 N Sodium Hydroxide. 

 0.04% Bromotlnjmol Blue. 



PROCEDURE 



1. Add 0.1 ml. 0.1 A^ hydrochloric acid, either with a 0.2 ml. 

 graduated pipette drawn out at the end to a slender tip, or with a 

 constriction pipette (p. 172), to the sample containing 2-8 m/xg. 

 riboflavin in a 0.75 ml. serological tube (6 X 50 mm.). To clean 

 these tubes boil in half-cone, nitric acid for a few min. and then in 

 distilled water several times. 



2. Plug tubes with cotton and autoclave for 15 min. at 15 lb. 

 pressure. Take care to protect from light, particularly while hot. 

 Check weight of several tubes before and after autoclaving to make 

 sure no significant volume change occurred. 



3. After cooling, add exactly 0.3 ml. 0.030 N sodium hydroxide 

 with a slender-tipped 1 ml. graduated pipette, or a constriction 

 pipette, and mix at once by twirling a fine glass rod with a hooked 

 end in the tube. The soln. now has an excess acid concentration of 

 about 0.002 M. 



4. Transfer a 0.1 ml. aliquot to each of three serological tubes, 

 and set up, in triplicate, each of the riboflavin standard solns. and 

 the blank using 0.1 ml. of soln. in each tube. 



5. Plug tubes with cotton, wrap entire tube rack in black cloth, 

 and autoclave for 15 min. at 15 lb. pressure. 



6. When the tubes have cooled, remove black cloth, and with a 

 sterile 1 ml. graduated pipette drawn out at the tip inoculate each 

 tube with 0.1 ml. basal medium which had been previously inoculated 

 with 2 drops of the washed bacterial suspension per 10 ml. Replace 

 cotton plugs and mix by tapping the tubes with the finger. 



7. Place rack of tubes in a vacuum desiccator containing damp 

 cotton swabs. Replace the air with carbon dioxide by alternately 

 reducing the pressure to about 150 mm. mercury and adding carbon 

 dioxide back to reach atmospheric pressure. Repeat four to five times 

 and leave pressure at about 700 mm. mercury. Place the desiccator 

 in an incubator for 3 days at 38°. 



8. After removing tubes from desiccator, add a minute droplet 

 of caprylic alcohol to each tube to prevent foaming, and blow off 

 the carbon dioxide by carefully bubbling air through the liquid for 

 1 min. by means of a capillary tube not over 0.5 mm. in diameter. 



