TREPONEMA PALLIDUM 



357 



TREPONEMA PALLIDUM 



mortar slowly adding 100 cc. hexylre- 

 sorcinol. Filter and store filtrate in 

 stock bottle. Stain smears 30 min. 

 Wash in water, dry and examine. Stain 

 on slide must not be heated. Trep- 

 onenias, light purple. 



2. Wright's stain for smears (Mallory, 

 p. 289). To make stain add 1 cc. 

 Wright's stain and 1 cc. 1% aq. potas- 

 sium carbonate to 10 cc. aq. dest. in 

 test tube and heat to boiling. Spread 

 material thinly on cover glass (not slide) 

 and hold level with forceps. Cover 

 with hot stain 3-4 min. After fluid has 

 turned violet, and a yellow metallic 

 scum has formed over it, pour off and 

 repeat process twice with hot stain. 

 Wash in water, dry and mount in balsam. 

 Treponemas, intensely violet. 



3. Giemsa's stain for smears (Giemsa, 

 G., Deut. med. Wochn., 1909, 35, 1751- 

 1752) after Mallory (p. 290). Fix 

 smears for 15 min. in absolute alcohol 

 or pass them through flame thrice. 

 Pour on freshly diluted stain (1 cc. aq. 

 dest. + 1 drop stock Giemsa). Steam 

 gently and leave 15 sec. Decant and 

 add immediately fresh diluted stain, 

 warm and let cool 15 sec. Repeat 4 

 times leaving 1 min. last time. Rinse 

 quickly in running water. Blot. 

 Mount in balsam. Treponemas, dark 

 red. 



4. Fontana-Tribondeau silver method 

 for serum (Fontana, A., Dermat. Zeits., 

 1925-26, 46, 291-293) after Mallory 

 (p. 291). To make silver solution add 

 ammonia water (diluted 1:20) drop by 

 drop to 50-100 cc. 1% aq. silver nitrate 

 until a coffee colored clouding takes 

 place. Air dry thin smears of serum. 

 Pour on few drops Ruge's sol. (aq. dest., 

 100 CO.; glacial acetic, 1 cc; formalin, 

 2 cc.) and change several times during 

 1 min. Rinse in running water. Mor- 

 dant witha little aq. dest., 100 cc; tan- 

 nic acid, 5 gm. ; liquid carbolic acid, 1 cc. 

 for 20 sec. warming to steaming. Rinse 

 in aq. dest. Treat with silver solution 

 30 sec. heating slightly. Wash in tap 

 water. Dry in air. Mount in balsam. 

 Treponemas, brown to deep black. 



5. Burri's India Ink method for lesion 

 fluid (Mallory, p. 291). Make 1:4 

 suspension of India ink in aq. dest. 

 Sterilize in autoclave, 15 min. Mix 

 this in equal parts with fluid from lesion 

 on slide with platinum loop. Spread 

 thinly. Dry and examine. Trep- 

 onema (and bacteria if present), white 

 in brown to black background. 



6. Quick method for demonstration 

 in fresh autopsy tissues. This is 

 Krajian's modification of Dieterle's 

 method (Am. J. Syphilis, 1933, 17, 127) 

 as amplified in Stain Techn., 1935, 10, 



68. Fix tissue 5 mm. thick 10 min. in 

 10% formalin, 70°C. Cut frozen sec- 

 tions 5-7 microns. Place in 2% aq. 

 sodium cobalti nitrite 5 min. Wash 2 

 changes aq. dest. Mordant for 15 min. 

 at 70°C. in uranium nitrate 1 gm. ; 85% 

 formic acid, 3 cc; glycerin, 5 cc; 

 acetone, 10 cc; 95% alcohol, 10 cc. 

 Wash quickly in aq. dest. Develop 

 5 min. in 10 cc. of following mixture -f- 

 1 drop albumin-glycerin before use 

 (hydroquinone, 0.62 gm. ; sodium sulfite, 

 0.12 gm.; acetone, 5 cc. ; 40% neutral 

 formaldehyde, 5 cc; pyridine, 5cc.; 

 sat. gum mastic in 95% alcohol, 5 cc, 

 aq. dest., 30 cc). Wash few sec. aq. 

 dest. Then warm silver solution 15-25 

 sec. and wash in aq. dest. Keep all 

 solutions in cool place. (Original gives 

 treatment with 0.75% aq. silver nitrate 

 at 70°C. for 1 hr. upon the development 

 in hydroquinone mixture.) 



7. Levaditi's block silver method 

 (Mallory, p. 293). Fix tissue pieces 

 (1 mm. thick) in 10% formalin, 24 hrs. 

 Rinse in aq. dest. 95% alcohol, 24 hrs. 

 Transfer to aq. dest. and leave until 

 tissue sinks to bottom. Fresh 1.5-3% 

 aq. silver nitrate at 37°C. in dark 3-5 

 days changing 3 times. (The stronger 

 silver is advised for tissues excised 

 during life.) Wash in aq. dest. Re- 

 duce 24-72 hrs. in dark at room tempera- 

 ture in:aq. dest., 100 cc. ; formalin, 

 5 cc; pyrogallic acid, 2-4 gms. Wash 

 in aq. dest. Dehydrate in 80, 95 and 

 absolute alcohol. Clear in oil of cedar 

 wood, imbed in paraffin, mount 5fx sec- 

 tions on slides, remove paraffin and 

 mount in balsam. Treponemas, black. 



8. Heitzman's modification of the 

 Warthin-Starry and Nieto's methods as 

 given by Mallory (p. 293). Cut frozen 

 sections 15/n or less of 10% formalin fixed 

 tissue. Place directly in pyridine, 

 10 min. Wash in aq. dest., 3 changes. 

 1% aq. uranium nitrate at 37°C., 15 

 min. Wash quickly in aq. dest., 2 

 changes. 0.25% aq. silver nitrate at 

 56°C., 15-30 min. Develop until dark 

 brown in following mixture made im- 

 mediately beforeliand by pipetting into 

 a beaker: (1) 15 cc. 5% aq. gelatin at 

 56°C.; (2) 3 cc. 2% an. silver nitrate; 

 (3) 0.5 cc. 1% aq. hydroquinone. Re- 

 move and thoroughly wash in warm aq. 

 dest. Dehydrate on slide adding by 

 pipette increasing alcohols to absolute. 

 Clear in benzol and mount in balsam. 

 A heavy black ppt. indicates too long 

 development. Treponenmas, black. 

 See Warthin-Starry method. 



9. For routine paraffin sections, 

 Steiner, G., J. Lab. & Clin. Med., 1939 

 25, 204-210. Fix in 10% formalin and 

 make sections 9-10 microns. Remove 



