238 CtfVEtTE COLORIMETRY 



75 ml. distilled water to 350 ml. 10% sodium hydroxide (within 

 1% of 2.5 A^) to make the final Nessler reagent. 



PROCEDURE 



1. Place a microtome section of tissue in 7 jxl. water in the bottom 

 of a digestion tube (Fig. 39, page 167). This tube has a total capac- 

 ity of about 2.5 ml. Add 50 fA. of the Levy digestion mixture and 

 drive off most of the water at 130° (about 1.5 hr. required). Digest 

 until water- white (Levy uses a small mobile gas flame), 



2. After the tube has cooled, hold almost horizontally and pipette 

 700 fA. distilled water directly into the digest, delivering the water 

 with as much force as possible. If complete solution is not attained 

 at once, it will occur on standing. 



3. Add the Nessler solution in a standardized manner (Levy, 

 1936) : First, "A piece of glass tubing is drawn out to a long capil- 

 lary and clamped vertically above a platform which may be raised 

 and lowered by a rack and pinion. It is connected to a source of 

 air supply at 20 cm. water pressure. The capillary is of such diam- 

 eter that a rapid stream of air bubbles passes through when im- 

 mersed in water," Then, the reaction tube is placed in a holder and 

 set on the platform under the capillary tube with air flowing through 

 it. Fill a 300 fA. constriction pipette with Nessler reagent and place 

 one end of a rubber tube connected to the pipette in the mouth, and 

 hold the pipette in the right hand. "The pinion of the platform is 

 now turned with the left hand to raise the tube about the bubbler so 

 that a stream of air bubbles passes from bottom to top of the solu- 

 tion. Just as soon as the bubbler reaches the bottom the Nessler 

 reagent is blown in. The platform is then lowered. The entire process 

 takes five seconds." Tilt the tube back and forth to collect any 

 solution in the bulb, 



4. Make the colorimetric measurement from 10-90 min. after 

 Nesslerizing. Prepare a blank by following the preceding steps, but 

 omitting the sample. (Levy used a Pulfrich photometer with filter 

 S43. The microcuvettes which hold 0.2 ml. were employed.) 



Russell Plienol-Hypoclilorite Method for Ammonia 



SPECIAL REAGENTS 



Alkaline Phenol Reagent. Add some water to 25 g. crystalline 

 phenol and pour in with stirring 54 ml, 5.0 A^ sodium hydroxide. 



