NEUTRAL RED 



227 NEUTRAL RED AND JANUS GREEN 



(2) Potassium bichromate 2.5 ems.; 

 mercuric chloride, 5 gms.; aq. dest., 

 100 cc. (3) Zenker's fluid less acetic 

 90 cc, neutral formalin 10 cc. or (4) 

 2% osmic acid 2 cc; 2.5% potassium 

 bichromate 8 cc. ; glacial acetic acid 1 

 drop. In the case of the last the paraf- 

 fin sections are treated with 1% aq. 

 potassium permanganate 1 min.; 5% 

 aq. oxalic acid 1 min. and are washed 

 thoroughly in water before staining. 

 Stain 4/i sections 24 hrs. Blot with 

 several layers filter paper. Dehydrate 

 in acetone. Place in toluol. Dif- 

 ferentiate in 1 part abs. ale. and 3 parts 

 oil of cloves. Wash in toluol and mount 

 in balsam. Zymogen granules, purple; 

 cytoplasm and nucleus, yellow; chromo- 

 phile material, lavender. 

 Neutral Red (CI, 825)— toluylene red— This 

 weakly basic amino-azin dye is used for 

 many purposes . It is a chloride. Some 

 advocate the iodide as more easily 

 purified but neutral red sold by any 

 reliable manufacturer is satisfactory. 

 Vital neutral red is recommended by 

 Conn. The principal uses of neutral 

 red are to stain: 



1. Islets of Langerhans of the pancreas 

 (Bensley, R. R., Am. J. Anat., 1911, 

 12, 297-388). Ad.d 2 cc. of a previously 

 prepared 1% aq. neutral red to 300 cc. 

 physiological salt solution (0.85% NaCl) 

 thus making a concentration of neutral 

 red of 1:15,000. Place this, and as 

 much more as may be required in a 

 bottle from the bottom of which a glass 

 tube leads off, or in an ordinary bottle 

 with a bent glass tube to serve as a 

 siphon. The tube is connected with a 

 glass cannula by about 5 feet of rubber 

 tubing. A freshly killed guinea pig is 

 bled from the throat. Insert the can- 

 nula in the thoracic aorta and inject 

 the solution by raising the bottle to 

 a height of 4 or 5 feet. Expose the 

 pancreas. Cut the inferior vena cava 

 near the heart so that the blood, followed 

 by the solution, can easily escape. The 

 pancreas will take on a deep rose red 

 color. Remove pieces, mount in phys- 

 iological salt solution under cover glasses 

 and examine at low magnification. The 

 optimum depth of staining must be 

 determined experimentally. The islets 

 of Langerhans appear as deep yellow 

 red irregular masses of difi'erent sizes 

 in a pale red background. After a time 

 the aye is bleached from the background 

 and the islets become more sharply 

 stained. 



A wonderfully fine color contrast can 

 be secured when methylene blue is 

 added to the neutral red solution in a 

 concentration of 1:10,000 and both are 

 injected in the same way. The islets 



are stained yellow red and the ducts 

 blue. But it is desirable first to obtain 

 satisfactory results with the methylene 

 blue alone. 



2. Parietal cells in the stomach, 

 (Harvey, B. C. H. and Bensley, R. R., 

 Biol. Bull., 1912, 23, 225-249). These 

 are beautifully stained by injection 

 with neutral red as described above. 



3. Granules in blood cells. Touch a 

 drop of fresh blood to a little 1:15,000 

 neutral red on a slide and cover imme- 

 diately without attempting to mix. 

 When the size of the drop of blood and 

 the amount of stain are properly 

 estimated the cover glass will press out 

 the fluid into a thin film suitable for 

 examination. The specific granules of 

 leucocytes are stained red. In the 

 monocytes red stained granules appear 

 and sometimes increase in size. When 

 the staining is fairly intense, or after 

 a sufficient interval the nuclei of the 

 leucocytes become colored and also a 

 basophilic material in young reticulated 

 red blood cells. Simultaneous colora- 

 tion with Neutral Red and Janus Green 

 is frequently carried out by hema- 

 tologists. 



Fluorescent X is a special type of 

 reduced neutral red (Lewis, M. R., 

 1935, 17, 96-105). See Nerve Fiber 

 Degeneration and Nissl Bodies. 

 Neutral Red and Janus Green. These are 

 often employed together as a supravital 

 stain for blood cells. A recent com- 

 prehensive statement of the technique 

 is given by Cunningham and Tompkins 

 (Downey, pp. 555-579). They add 3 

 drops cone janus green in absolute 

 alcohol to 1 cc dilute neutral red, which, 

 latter, is 20-30 drops cone neutral red 

 in absolute alcohol. This mixture is 

 spread evenly on slides and evaporated. 

 They caution that for exudates, tissue 

 scrapings, leucemic blood, bone marrow 

 and lymph nodes it is necessary to use 

 stronger solutions. Neutral red CC. 

 (Commission Certified) is satisfactory 

 in place of the neutral red-iodide advised 

 by Sabin. Fresh blood is mounted on 

 the dye deposit, and is ringed with 

 vaseline to prevent evaporation. This 

 technique has had a profound influence 

 on cytology. Obviously it must be 

 cautiously used and observations dis- 

 continued as soon as evidences are seen 

 of experimental modifications in the 

 cells. It affords valuable information 

 on the mitochondria and neutral red 

 granules not stainable together by other 

 methods, but it will not supplant the 

 staining of blood smears by the methods 

 of Gierasa, Wright and others. See 

 critical evaluation by Hall (Downey, 

 pp. 643-698). See application in studiy 



