THE TWO GREAT GROUPS OF CONNECTIVE-TISSUE CELLS. 19 



Protocol: Rat 325, injected intraperitoneally with a 0.1 per cent aqueous solution of trypan 

 blue, February 13, 16, 19, 22, 25, and 28, 1 c. c. each day. 



March 1: A faint blue shows at the base of the ears. Skin over the body is stained light blue. 

 Subcutaneous tissues arc light blue, deeper blue at the injection-point. Films from the abdo- 

 men show, under the low-power, sharp, bright-blue dye deposits in cells which are apparently 

 macrophages. 



Under the oil the macrophages are seen to be filled with bright-blue crystalline deposits. 

 Vacuoles are present in these cells also and are stained often a light gray-blue, though some are 

 without color. Macrophages from the thigh are similar to those from the abdomen. Neutral red 

 stains the vacuoles and tinges parts of the blue crystals. Preparations so stained with neutral red 

 (1:5,000) show a segregation-apparatus which is not greatly, if at all, increased over the normal 

 in size or number of vacuoles (fig. 38). Janus green (1 : 10,000) stains numerous filiform mitochon- 

 dria in the macrophages. 



Under the oil, fibroblasts in films from the abdomen show a few dye deposits. These are minute 

 granules, or vacuoles, and at times crystal-like deposits are present. Janus green stains normal 

 mitochondria. 



There are a number of these most perfectly segregated and crystallizable dyes, 

 such as trypan blue, among which we may mention in particular two bright-blue 

 dyestuffs from amido napthol disulphonic acids isomeric with the H acid, namely, 

 the 1.8.3.5 and 1.8.2.4 acids. The 1.8.4.6 isomer (the so-called K acid) produces 

 a dye whose traits in this respect are less marked. The experiments which we have 

 just detailed are interesting in another direction, for they demonstrate that minute 

 quantities of these quickly and perfectly segregated dyes are "fixed" by macro- 

 phages at such a rate that none of them remains in the tissue-juices to affect the 

 fibroblasts, always slower in their reaction. It is thus possible, by means of an 

 extremely low dosage with such dyes, to produce deposits sharply confined to the one 

 cell type, just as it is invariably possible to satisfy oneself that one has affected the 

 same cells alone, or to an overwhelmingly greater extent, by very concentrated 

 acute dosage and the examination of tissue before the fibroblasts can react. Our 

 attention was first directed to the phenomenon of minute crystal production in the 

 macrophages by feeding experiments. Contrary to the assertion of Goldmann and 

 others, it is not difficult to obtain a light general vital stain by the introduction of 

 these dyes with food. So effectual, however, is the alimentary epithelial barrier 

 that relatively enormous amounts of dye must be present to gain entrance by this 

 route. We have achieved it by the employment of rather concentrated solutions of 

 diffusible dyes in milk, or by the admixture of the actual dye powder with flour 

 made into small biscuits. A microscopic examination of the intestinal tract in such 

 animals shows an extensive cell necrosis of the epithelial investiture which is con- 

 tinually repaired through the abundant mitoses found in the necks of Lieberkuhn's 

 crypts. It is our conviction that at no time is the efficacy of the epithelial barrier 

 seriously impaired, but that minute amounts of the dye diffuse into the mucosal 

 capillaries at these many injured points. (See figs. 39, 40, and 41.) 



Protocol: Rat 4, fed Niagara blue BB (the benzidine homologue of trypan blue) from 

 March 3 to March 26 (total of 5 to 6 grams of dye) in milk and egg mixture. 



March 26: Animal is stained a light purple; abdominal skin is light blue. Low-power view 

 shows tissue juices and fibers are free of any diffuse stain. Deep-blue discrete deposits are present. 



Under the oil the deep-blue deposits are seen to be predominantly crystalline and are confined 

 to the macrophages. The crystalline needles, or sheaves, are rather large structures (fig. 39). 



