DIPHTHERIA BACILLI 



81 



DOPA, OXIDASE REACTION 



Then B, 30 sec. Wash in water, dry 

 and mount. Bacilli brown with dark 

 blue dots at either end. Better results 

 can be secured by adding 1 part of 

 crystal violet (Hocchst) 1 gm.; 95% 

 ale, 10 cc; aq. dest., 300 cc. to 2 parts 

 of A before using. Chrysoidin 1 gm. 

 in hot aq. dest. 300 cc. is more satis- 

 factory counterstain than Bismark 

 brown. Most American brands of crys- 

 tal violet are satisfactory. 



2. Pander's stain (Stitt, p. 863). 

 Toluidin blue (Grubler) 0.02 gm.; aq. 

 dest., 100 cc; glacial acetic acid, 1 cc; 

 abs. ale, 2 cc. Add small amount to 

 fixed film on cover glass. Invert and 

 mount on slide. Diphtheria bacilli 

 recognizable by metachromatic granules 

 intensely stained, diphtheroids by their 

 strong color in contrast with ordinary 

 cocci and bacilli the bodies of which 

 are only faintly blue. 



3. Laybourn's modification of Albert's 

 stain (Stitt, p. 863). A = toluidin 

 blue, 0.15 gm. ; malachite green, 0.2 gm. ; 

 glacial acetic acid, 1 cc. ; 95% ale, 2 

 cc, aq. dest., 100 cc. B = iodine 

 crystals, 2 gm. ; potassium iodide, 3 

 gm.; aq. dest., 300 cc. Let both stand 

 24 hrs. and use filtrate. Apply A to 

 heat fixed smears 3-5 min. Wash in 

 water. Apply B for 1 min. Wash, 

 blot and dry. Granules of diphtheria 

 bacilli, black; bars, dark green; inter- 

 mediate parts, light green and all three 

 in sharp contrast. 



Diplosome, a double centrosome. 



Direct Fast Orange (CI, 326)— Erie Fast 

 Orange (NAC), Erie Fast Scarlet YA 

 (NAC) — a direct disazo dye of light 

 fastness 3 (Emig, p. 38). 



Direct Fast Scarlet 4 BS (CI, 327)— Pont- 

 amine Fast Scarlet 4 BS of DuPont — , a 

 disa.zo direct dye of light fastness 3, can 

 be employed instead of carmine as a 

 general stain. Details of use in study 

 of plant and animal tissues are de- 

 scribed (Emig, p. 38). 



Direct Garnet R, see Erie Garnet B. 



Direct Green B (CI, 593)— Diazine Green 

 B — a direct disazo dye of light fastness 

 3 to 4. Recommended as counterstain 

 for Crocein Scarlet 7 B of invertebrates 

 or paraffin sections, time 5 min. (Emig, 

 p. 43). 



Direct Green G (CI, 594)— Alkali Green D— 

 a direct disazo dye of light fastness 3 to 

 4. Formula for blue green algae and 

 whole mounts is given (Emig, p. 43). 



Direct Red 4B, see Benzopurpurin 4B. 



Direct Red, C, R, or Y, see Congo Red. 



Direct Sky Blue, see Niagara Blue 4B. 



Direct Steel Blue BB, see Dianil Blue 2R. 



Direct Violet B, see Azo Blue. 



Direct Violet C, see Erie Garnet B. 



Dis-Azo Dyes. Azo blue, benzopurpurin 

 4B, Biebrich scarlet, Bismark brown 



Y and R, brilliant purpurin R, congo 

 red, dianil blue 2R, Erie garnet B, 

 Niagara blue 4B, orseillin, trypan blue, 

 trypan red, sudan III, sudan IV, 

 vital new red, vital red, etc 



Dissociation, see Maceration. 



Distrene 80 is a polysterene which forms a 

 water clear solution in xylol. It is 

 recommended by Kirkpatrick and Len- 

 drum (J. and A. C, J. Path, and Bact., 

 1939, 49, 592-594) as a mounting medium 

 giving good preservation of color in 

 microscopic slides. See also Ivirk- 

 patrick, J. and Lendrum, A. C, J. Path. 

 & Bact., 1941, 53, 441. 



Dominici's Stain, see Eosin-Orange G and 

 Toluidin blue. 



Donaldson's lodine-Eosin Method, see lo- 

 dine-Eosin. 



Dopa, Oxidase Reaction for Melanoblasts 

 (Laidlaw, G. F., Anat. Rec, 1932, 53, 

 399-407). Dopa is short for 3.4-c/ihy- 

 droxyphenylalanin, a substance which 

 when applied in a certain way picks out 

 the melanoblasts by blackening them. 

 Use frozen sections of fresh material or 

 of tissues fixed 2 to 3 hours but not 

 longer in 5% formalin. Rinse 4 or 5 

 seconds in aq. dest. and immerse in 

 buffered dopa. (To make dopa stock 

 solution dissolve 0.3 gm. dopa powder — 

 manufactured by Hoffmann-La Roche, 

 Nutley, New Jersey — in 300 cc cold 

 aq. dest. Keep in refrigerator and dis- 

 card when solution becomes dark red. 

 To make buffers dissolve 11.87 gms. di- 

 sodium hydrogen phosphate (NaaHP04 

 -f 2H2O) — or what would be better 

 9.47 gm. anhydrous Na2HP04— in 1000 

 cc. aq. dest. and 9.08 gms. anhydrous 

 potassium dihydrogen phosphate 

 (KH2PO4) in an equal amount aq. dest. 

 Immediately before use buffer to pH 7.4 

 by adding 2 cc. potassium phosphate 

 solution, and 6 cc. sodium phosphate 

 solution to 25 cc. dopa solution). The 

 reaction is slow for 3-4 hours at room 

 temperature. If solution becomes 

 sepia brown it is likely to overstain. 

 Observe under microscope. Wash in 

 aq. dest., dehydrate and counterstain 

 if desired with alcoholic crystal violet, 

 clear and mount in balsam. Melan- 

 oblasts should be black. 



This much used method has been 

 criticized by H. Sharlit et al. (Arch. 

 Dermat. and Syph., 1942, 45, 103-111) 

 chiefly on the ground that the incuba- 

 tion for 3 hrs. at room temperature may 

 itself increase the amount of melanin 

 present which happened in their ex- 

 perience at 37°C. See also remarks 

 by Blaschko and Jacobson (Bourne, 

 p. 198) on specificity of the reaction. 

 It is given by pheuoloxidases but thus 

 far they have not been found in mam- 

 malian tissues. 



