ENTOMOLOGICAL TECHNIQUES 



117 



EOSIN -ORANGE G 



Forsch., 1925, 2, 506-535. See Small 

 Intestine. 



Entomological Techniques, see Mosquito, 

 Ticks, Insects, Arachnids, Parasites. 



Enzymes — Written by E. W. Dempsey, 

 Dept. of Anatomy, Washington Uni- 

 versity, St. Louis. February 26, 1951 — 

 Their name is legion. At present only 

 a few can be localized with any degree 

 of histological precision, but the number 

 of histochemical methods available is 

 increasing rapidly. There are no bet- 

 ter examples of felicitous association 

 between histological and biochemical 

 methods. Four principal kinds of tech- 

 niques are employed for localization: 



(1) By spectrographic identification in 

 the tissues — especially the metal-con- 

 taining Cytochromes and Peroxidase. 



(2) By close comparison of enzymic 

 activity with cellular composition of 

 tissues. See Glick for detailed meth- 

 ods, derived from the Linderstr0m- 

 Lang procedures — Amylase, Pepsin, 

 Peptidase, Esterase, Protease, Cho- 

 linesterase, Lipase, Urease, Carbonic 

 Anhydrase, etc. (3) By separation of 

 cellular components, such as nuclei, 

 granules and mitochondria, from homo- 

 genates of tissue. Such separation is 

 accomplished by differential flotation or 

 centrifugation, and is followed by esti- 

 mation of the enzymic activity of the 

 purified component fractions. Argi- 

 nase, Adenylpyrophosphatase, Phos- 

 phatase, etc. have been investigated by 

 such methods. (4) By the develop- 

 ment of characteristic insoluble prod- 

 ucts within tissues or cells — Aldolase, 

 Acid and Alkaline Phosphatase, Cholin- 

 esterase. Cytochrome Oxidase, Dehy- 

 drogenase, Dopa Oxidase, Esterase 

 Glucuronidase, Lipase, Peroxidase, 

 Phosphamidase, Sulfatase. 



Enzymes are coming into their own 

 as technical tools. Purified enzymes 

 may be used to destroy certain tissue 

 components. Ribonuclease selectively 

 destroys basophilic substances in cyto- 

 plasm and nucleus, Desoxyribonuclease 

 similarly attacks nuclear chromatin. 

 Hyaluronidase solubilizes certain meta- 

 chromatic ground substances, whereas 

 other mucoid materials react only with 

 more potent Mucinases. The connec- 

 tive tissue fibers have long been charac- 

 terized by their digestibility in Pepsin 

 and trypsin, and recently an Elas- 

 tase has been prepared. A purified 

 Collagenase has been reported. The 

 solubility of glycogen in solutions of 

 salivary Amylase is an old histochemical 

 procedure. The effects of Lysozyme 

 and other enzymes on the capsules of 

 pneumococci and upon the Gram stain 

 has been summarized by DuBos (The 



Bacterial Cell, Harvard Univ. Press, 

 1945). 



Eosin B or bluish (CI, 771)— eosin BN, BW, 

 or DHV, eosin scarlet, eosin scarlet B, 

 imperial red, nopalin G, saffrosin, 

 scarlet J, JJ, V — Dibrom derivative of 

 dinitro-fluorescein. Chemistry of 



(Holmes, W. C, Melin, C. G. and 

 Paterson, H. R., Stain Techn., 1932, 

 7, 121-127). 



There are several fluorescein dyes 

 and guidance may be needed in the 

 choice of the one best suited for a par- 

 ticular purpose. Conn, H. J. and 

 Holmes, W. C, Stain Tech.. 1926, 1, 

 87-95 ; 1928, 3, 94-104 have made a study 

 of color, acidity and chemical structure 

 and Conn (p. 145) gives further data. 

 Their color increases in depth in this 

 order: eosin Y, ethyl eosin, eosin B, 

 erythrosin B, phloxine and rose bengal. 

 This increase in color is proportional to 

 increase in number of hologen atoms. 

 Their acidity increases in a different 

 order: rose bengal, phloxine, erythrosin, 

 eosin Y and eosin B. (1) When the 

 eosin is to follow in alcoholic solution a 

 basic dye always in aqueous solution 

 (cf. hemato.xylin) the more acid and 

 lighter colors are recommended (eosin 

 Y, ethyl eosin and eosin B. (2) When 

 it is to precede in aq. solution a basic 

 dye (cf. methylene blue) also in aq. 

 solution, use phloxin or erythrosin (see 

 phloxine-methylene blue). 



Eosin lOB, see Phloxine B. 



Eosin BN, BW, or DHV, see Eosin B or 

 bluish. 



Eosin J, see Erythrosin, bluish. 



Eosin-Methyl Blue, see Mann's. 



Eosin-Methylene Blue has been employed 

 in many combinations for years. But 

 when the acid dye is applied first, 

 phloxine is preferred to eosin. See 

 therefore Phloxine Methylene Blue. 



Eosin-Orange G — Toluidine Blue for bone 

 marrow, spleen and connective tissue 

 (Dominici, M. C. rend. Soc. biol., 1902, 

 54, 221-223). Stain eosin-orange G 

 (eosin B. A. of Hollborn or eosin yellow- 

 ish of American manufacturers 0.5 gm. ; 

 aq. dest., 100 cc; orange G. 0.5 em.) 

 7 min. Rinse quickly in aq. dest. 

 Counterstain in 0.5% aq. toluidin blue 

 20-30 sec. Rinse again aci. dest. Dif- 

 ferentiate in 95% ale, dehydrate in 

 abs., clear in xylol and mount in balsam. 

 Instead of eosin, 0.5% aq. acid fuchsin 

 gives a little sharper contrast. In 

 place of toluidin blue 0.1% Azur A can 

 be employed to advantage. Phloxine- 

 orange G can be tried as a substitute for 

 eosin-orange G. (phloxine 0.12 gm., aq. 

 dest. 100 cc, orange G, 0.3 gm.). The 

 crucial point is the differentiation in 95% 

 ale. This should be quickly checked 



