quickly and thus the dye on the slide was in solu- 

 tion for an insufficient period of time to give ade- 

 quate staining. For avian blood the first method 

 worked best, but more brilliant staining was ac- 

 complished when the mixture was held for at least 

 2 minutes before the smear was made. The same 

 reticle used for making thrombocyte counts can 

 be used for making reticulocyte counts. 



Graam (1934) used a method that is essen- 

 tially the same as given in the preceding para- 

 graph, but added 0.3 percent sodium citrate. 

 Six drops of this solution were mixed with about 

 two drops of fresh pigeon blood. The cells were 

 allowed to stain for 10 to 30 minutes before a 

 drop of the mixture was removed and spread 

 across a slide to dry. This long staining time 

 may have been the basis for the statement 

 (p. 202), "Practically every red cell of the 

 pigeon's blood contained some 'basophilic' sub- 

 stance in the protoplasm." She goes on to say, 

 "The younger cells contain the most of this so- 

 called 'basophilic' substance. One must learn 

 to be consistent and he must arbitrarily decide 

 which of these cells to call reticulocytes. I arbi- 

 trarily called those cells reticulocytes which had 

 a complete chain of basophilic substance around 

 the nucleus." 



Peabody and Neale (1933) used Loffler's 

 methylene blue in 0.85 percent solution of sodium 

 chloride. Air-dried smears were immersed in 

 this stain for about 3 minutes, washed briefly in 

 water and again air-dried. This stain was use- 

 ful for temporary mounts but the preparations 

 faded after a few hours. 



The basic method for staining reticulocytes 

 from which slight variations in procedure have 

 arisen, was given by Osgood and Wilhelm 

 (1931). They used, for mammalian blood, 

 equal parts of venous blood that had been mixed 

 with 2 mg. potassium oxalate per cc. and 1 per- 

 cent brilliant cresyl blue in 0.85 percent NaCl. 

 These substances were mixed in a test tube and 

 after one minute a drop was removed and a smear 

 made. This is the technic used by Magath and 

 Higgins (1934) to stain the i-eticulocytes of 

 mallard ducks. 



Ralpfl's modification of the benzidine tech- 

 nic for hemoglobin in cells 



The method given by Ralph (1941) is appli- 

 cable to air-dried blood smears. The slide is 



flooded with 1 -percent solution of benzidine in 

 absolute methyl alcohol and allowed to stand 1 

 minute. The solution is poured off the slide 

 and, without washing, it is replaced with a 25- 

 percent solution of superoxol in 70-percent ethyl 

 alcohol, which is allowed to stand IV2 minutes. 

 This is poured off and the slide rinsed for 15 

 seconds in distilled water, after which it is dried 

 by blotting and covered. Cells and other struc- 

 tures containing, hemoglobin stain dark or light 

 brown. The dried slide should be counter- 

 stained with Wright's in order to reveal the cells 

 and parts of cells that do not contain hemoglobin. 

 In this study it was found that fine, needle-shaped 

 crystals were sometimes deposited on the slide 

 but usually these did not interfere with study of 

 the cells. 



MISCELLANEOUS TECHNICS 



Method for hemoglobin determination 



This subject has been discussed by Denington 

 and Lucas (1955) and only a summarization is 

 presented here. 



Fill a test tube with 10 cc. of 0.4-percent solu- 

 tion of concentrated ammonium hydroxide in dis- 

 tilled water. Take up a 0.02-cc. sample of blood 

 with a hemoglobin pipette; immediately discharge 

 it into the ammonia solution; after rinsing the 

 pipette several times in the solution, seal the test 

 tube with a cork that has been washed, dried, and 

 infiltrated with paraffin. Invert the tube 2 or 3 

 times; after an hour add 0.36 cc. of concentrated 

 HCl to the tube and invert the tube 1 or 2 more 

 times. This amount of HCl, namely, ll/> times as 

 nuich as previously recommended, prevents for- 

 mation of cloudiness in the tube; the precipitate 

 that does fonn, aggregates into large masses, 

 which usually fall to the bottom of the tube or 

 float on top. Hemoglobin is not bound to this 

 precipitate. The density of color is determined 

 colorimetrically at a wavelength of 410mM- 

 These values are compared with those on a stand- 

 ard curve previously prepared ; the curve is based 

 on a series of 16 dilutions of hemin. Standard 

 cui-ves were made both from dried commercial 

 hemin and from fowl hemin prepared according 

 to the method given by Elvehjem (1931); the 

 results were the same. When the transmission 

 value, transposed into grams of hemin per cc. of 



231 



