Improved methods for staining spirochaeta pallida in tissue. 1

The two methods outlined here present the first satisfactory ones devised for the staining of spirochetes in single sections of tissue mounted upon cover-glasses. We consider them of great value for the following reasons: They are more certain than the Levaditi method of staining in bulk; the time required is shortened to hours, instead of the days required by that method; they have a much greater applicability to practical diagnostic work in that they can be used for single sections, thus permitting a closer control of histological findings.

1. Warthin And Starry's Cover-Glass Method.

I. Fix tissues in 4 per cent. formol.

2. Wash thoroughly in distilled water.

3. Imbed in paraffin (alcohol, xylol, paraffin).

4. Cut; mount sections on cover-glasses with albumin fixative.

5. Remove paraffin from section (xylol, alcohol, water).

6. Place cover-glass in a saturated solution of ferric alum, or a 4 per cent. solution of ferrous ammonium sulphate, in incubator for I to 2 hours.

7. Wash in distilled water.

8. Rinse cover-glass with section in a 2 per cent. silver nitrate solution. Cover section with another perfectly clean cover-glass which has also been rinsed in the silver solution, so that the coverglasses are held together by capillary attraction. Then place them carefully on the bottom of a wide-mouthed dark bottle covered with black paper, and cover them with the silver nitrate solution. Cork tightly, and put into incubator for 3 to 24 hours.

9. After impregnation pour off the silver nitrate solution and rinse in distilled water without removing cover-glasses from bottle, by pouring the water into the bottle, shaking gently, and then pouring off.

10. Pour reducing fluid (pyrogallic acid, 4 grams; 40 per cent. formol, 5 cc.; distilled water, 100 cc.) into the bottle.