VISIBLE AND U.V. ABSORPTION HISTOSPECTROSCOPY 125 



2. Place the needle in a cold muffle furnace and turn on the heat. 

 \Mien the temperature reaches 500° turn off the heat and remove the 

 needle when the furnace is cool. As an alternative, place the needle in 

 the furnace at 500° and remove it after 20-30 min. at this temper- 

 ature. 



3. Hydrolyze the pyro- and metaphosphate to orthophosphate 

 by pipetting 1-2 fA. 1 X hydrochloric acid onto the needle tip and 

 heating for 1 hr. at 100° in the hydrolysis chamber (page 123) . Allow 

 to dry at room temperature. 



4. For the Deniges method, add 0.05 ml. of the stannous chloride 

 soln. to 10 ml. of the sodium molybdate soln. This mixture nmst be 

 used within 3 min. after its preparation. Pipette a 0.6 to 1.0 fxl. drop 

 of the reagent mixture on a prepared slide near one of the inked 

 hnes (page 123) as a blank. Then pipette a suitable vol. (0.6-1.0 ^,1.) 

 of the reagent mixture onto the needle tip bearing the ashed sample. 

 Use the end of the pipette to mix the drop on the needle tip for 10-15 

 sec. in order to dissolve the sample and obtain a homogeneous soln. 

 During the next 15 sec, transfer the drop from the needle to the 

 slide, placing it beside the other iliked line a few mm. from the blank 

 drop. Surround the drops with paraffin oil and place cover glass as 

 described on page 122. The entire process from the addition of the 

 reagent to placing the cover glass can be performed in 35-45 sec. 

 Standardize the manipulations to maintain a constant evaporation 

 effect. Carry out the photometry after 5 min. from the beginning of 

 the color development, and finish within 45 min. 



5. For the Fiske and Subbarow method, mix 24 ml. of the molyb- 

 date soln. with 1 ml. of the aminonaphtholsulfonic acid soln. This 

 mixture may be used for at least 1 hr. after its preparation, and it 

 is applied in the same manner as the Deniges reagent. Let the drop 

 stand for 30 min. before starting the photometry, and finish the 

 measurement within 2 hr. from the beginning of the color develop- 

 ment. 



3. Stowell Technique 



The apparatus consists of a lamp, a microscope, a photocell, and 

 amplification and recording equipment. A 50 c.p. automobile head- 

 lamp operated by a .storage battery is used as the source of light. 



