VITAL STAINING 341 



found that by staining with methylene blue it was possible to 

 demonstrate the Golgi a])paratus as a colourless reticulum against 

 tlie blue stained eyto])lasin. 



766. The Staining of Cytoplasmic Granules and Vacuoles. 

 Neutral red is one of the best dyes for this purpose. Aquatic 

 animals, e.g. tadpoles, can be stained by keeping them in weak 

 solutions — 1 in 200,000, or less. With stronger concentrations 

 staining is more rapid, but segregation of the dye may occur. 



The various methods of staining with Janus green (see § 761) 

 are also applicable to neutral red. Neutral red is frequently used 

 togetiier with Janus green. Mitochondria are then stained with 

 the latter, and vacuoles and granules with the former. A mixture 

 of neutral red and methylen blue has been found to give similar 

 results. A temporary fixation of the staining is possible with the 

 iodine vapour method of Lewis. 



Supravital. Use dilutions of 1 in 20,000 to 1 in 50,000, or less. 

 For staining blood, bone marrow and isolated cells, prepare 

 slides as described in § 761, employing neutral red in a 1 per cent, 

 solution in absolute alcohol. For double staining, with Janus 

 green, mix 1 per cent, alcoholic solutions of the dyes before 

 applying to the slides. 



Intra-vitam. For staining connective tissue cells, inject 

 solutions of 1 in 50,000, or less, in saline, subcutaneously. Kill 

 animals and remove tissues for examination after ten to twenty 

 minutes. 



In order to stain pancreas and liver cells of small laboratory 

 animals stronger solutions are usually injected intraperitoneally. 

 Small injections at intervals of thirty minutes, or 1 c.c. of a 0-5 

 per cent, solution have been employed. 



In Vitro. Solutions of 1 in 50,000 to 1 in 100,000 in saline 

 usually give the best results. For the coverslip method of staining 

 blood and Protozoa, see under " Blood," § 872. This is Simpson's 

 method. 



767. Preservation of Neutral Red Staining. The preservation of 

 neutral red staining presents considerable difficulties. Most of 

 the methods which have been described have a limited range of 

 application. Ludford {Proc. R. S. B., cvii, 1930, p. 101) has 

 employed Gardner's method for cytological work {Proc. Soc. 

 Expt. Biol. Med., xxiv, 1926, p. 646), making up the fixative as 

 follows : — 



43 c.c. of Zenker's fluid (without acetic acid). 

 7 c.c. of formol to which 2 drops of normal NaOH have been 

 added. 



Gardener's Technique. (I) Fix for twenty-four hours. (2) Cut 

 up tissues into small blocks, not more than 2 mm. in thickness, and 

 transfer to fresh Zenker's liuid (without acetic acid). (3) Conmience 

 dehydration any time within the next two days by washing for fifteen 

 minutes, then blot the small blocks of tissue. (4) Transfer to 80 per 



