MACNEAL'S TETRACHROME 



188 



MAGDALA RED 



razor. Each segment is placed in cone, 

 hydrochloric acid, 75 cc, aq. dest. 25 cc. 

 1-7 days. Heat just below boiling 

 20-30 min. Tissue shrinks, turns dark 

 brown and softens. A sediment collects 

 in the dish. Part of acid is drawn off 

 with a pipette, boiled water is added 

 and the process is repeated until practi- 

 cally all of the acid is removed. The 

 water is boiled to prevent formation of 

 air bubbles along the tubules. It turns 

 the tubules a yellowish white color in 

 which condition they should be isolated 

 by careful teasing. When the tubules 

 cannot be easily lifted away from one 

 another, the maceration is insufficient. 

 When, on the other hand, they break 

 it is a sign of over maceration (Johnson, 

 F. P., Anat. Rec, 1934, 59, 187-199). 

 A similar method was used by Johnson 

 in 1916 to separate the lobules of the 

 pig's liver. 



Bone cells and lamellae. Treat a 

 thin bone section with cone, nitric 

 acid as long as 24 hrs. Mount on a 

 slide and squeeze out bone cells by pres- 

 sure on cover glass. The lamellae can 

 be pealed off easily from a piece of 

 decalcified bone which has been gently 

 boiled in water (Shipley, in McClung, 

 p. 348). 



Enamel rods. A piece of dental ena- 

 mel is dissociated with 5-10% hydro- 

 chloric acid for 24 hrs. When it has 

 become soft, remove a little with a 

 needle to a slide and tease out. Mount 

 in physiological salt solution under a 

 cover glass. Draw through a little 

 carmine stain with a blotter and wash 

 it out with 10% acetic acid. The 

 specimen can be ringed with paraffin 

 (Churchill, and Appleton, in McClung, 

 p. 372). 



Nerve cells. Pieces of gray matter 

 of ventral horn are soaked for 2 or 3 

 days in 0.02 formalin. The tissue 

 softens, the cells are dissected out and 

 stained with carmine or picro-carmine 

 (Addison, in McClung, p. 439). 



MacNeal's Tetrachrome is a blood stain 

 containing eosin, methylene azure A, 

 methylene blue and methylene violet. 

 It is employed like Wright's stain. 

 For details see MacNeal, W. J., J. A. 

 M. A., 1922, 78, 1122, and Conn, H. J., 

 Stain Technology, 1927, 2, 31. 



Macrophages. These are the free cells of 

 the reticulo-endothelial system. Al- 

 most any method of exposure to rela- 

 tively non-toxic, finely particulate 

 matter is sufficient to bring them out. 

 The simplest way is to inject mice with 

 trypan blue as described under Vital 

 Staining and to look for the macro- 

 phages in spreads of Loose Connective 

 Tissue. Another method, used by 



Maximow, is to give rabbits intra- 

 venous injections of saccharated iron 

 oxide or India ink and to examine blood 

 from right ventricle in smears (see 

 Cowdry's Histology, p. 69). Lines of 

 division between macrophages and 

 monocytes, if they exist, are difficult 

 to establish. Supravital staining with 

 Neutral Red and Janus Green is useful 

 to demonstrate neutral red granules 

 and mitochondria respectively. 



Madder Staining of bone. Madder is a red 

 dye, prepared from the plant Rubia 

 Tinctorum which has been used for 

 thousands of years. It is perhaps the 

 first dye to be used in camouflage in war. 

 With its help Alexander defeated the 

 Persians by staining the clothing of his 

 Greek soldiers red, each garment in a 

 different part so that the Persian leaders 

 at once concluded that all they had to 

 cope with was an already well damaged 

 army. (Leggett, W. F., Ancient and 

 Medieval Dyes. Brooklyn: Chemical 

 Publishing Co., Inc. 944, 95 pp.) 



Alizarin and purpurin, formed from 

 madder, are now made syntheticall3\ 

 Madder should be employed for the 

 vital staining of growing bone as de- 

 scribed by Macklin (C. C, Anat. Rec, 

 1917, 12, 403-405; J. Med. Res., 1917, 

 36, 493-507). Young rats are suggested 

 as material. Each should eat 1-5 gms. 

 of madder, thoroughly mixed with its 

 food, daily. The calcium deposited in 

 the growing bone while madder is thus 

 made available in the circulation is 

 colored red. Staining is noticeable 

 after 1 day but the feeding should be 

 continued for a week or more. 



The ventral ends of the ribs and the 

 epiphyseal lines of long bones are most 

 intensely colored. The bones selected 

 are fixed in 10% neutral formalin, 

 washed and cleaned in water, dehy- 

 drated thoroughly in alcohol, placed in 

 benzene for 24 hrs., cleared in oil of 

 wintergreen by the method of Spalteholz 

 and examined with binocular microscope 

 as whole objects. 



Chemistry of madder staining is dis- 

 cussed by Dr. Richter (Biochem. J., 

 1937, 31, 591-595). The substance giv- 

 ing the intense carmine red color is 

 apparently purpurin carboxylic acid. 

 Madder is one of the most classical of 

 stains. Its history extends back through 

 the centuries and has been well reviewed 

 by F. T. Lewis (Anat. Rec, 1942, 

 83, 229-253). See Line Test. 



Magdala Red (CI, 857) — naphthalene pink, 

 naphthalene red, naphthylamine pink, 

 Sudan red — According to Conn (p. 102) 

 this basic naphtho-safranin differs from 

 commercial magdala red which is an 

 acid dye belonging to an entirely dif- 



