Journal of Applied Microscopy. 



113 



case it is extremely difficult to secure the 

 proper decoloration. Rapid staining in a 

 strong dye is preferable to slow, pro- 

 longed staining in a weak dye. 



The section is placed in the stain for 

 from ten to fifteen minutes. It is then 

 washed in water to remove the excess of 

 dye. The formation of unsightly deposits 

 on subsequent contact with iodine is thus 

 avoided. The section is then placed in 

 the solution of iodine for Ave minutes. 

 It is now transferred to absolute alcohol, 

 in which it is gently moved about till 

 most of the stain is removed. The sec- 

 tion should not be wholly decolored, but 

 should still show a distinct violet color. 



It is now placed in very dilute eosin 

 for about twenty or thirtj' seconds. 

 Overstaining with eosin will impart to 

 the tissue a deep red color, which will 

 thus make an unfavorable contrast for 

 the violet colored organism. It is prefer- 

 able to stain with eosin so that the tissue 

 has a light pink color. 



The section is transferred from eosin 

 to absolute alcohol for one or two 

 minutes. When thoroughly dehydrated 

 it should be placed in oil of cloves and 

 should be allowed to remain in this oil 

 till all the violet color has been taken out 

 of the section. The sections may re- 

 main in the oil for some hours, or even 

 over night. The oil of cloves will decolor 

 the section perfectly and will not affect 

 the colored bacteria. The section is then 

 passed through xylol, transferred to a 

 slide and mounted in Canada balsam. The 

 deep violet organism should stand out 

 in bold relief against a light pink back- 

 ground. 



The following summary will show the 

 several steps in the method: 



Anilin-water gentian violet, 10 to 15 

 minutes. 



Water. 



Iodine solution, 5 minutes. 



Water. 



Absolute alcohol, 5 to 10 minutes. 



Water. 



Very dilute eosin, 1-4 to 1-2 minute. 



Water. 



Absolute alcohol, 1-2 minute. 



Oil of cloves, till declored. 



Xylol. 



Canada balsam. 



LEPROST AND TUBERCLE BACILLI. 



These two organisms can be demon- 

 strated in tissues by Gram's method. 

 They can, moreover, be stained by a 

 special method similar to that already 

 described in connection with the detec- 

 tion of the tubercle bacillus. This method 

 as applied to sections is as follows: 



The section is floated on cold, fresh, 

 carbolic fuchsin over night, or for about 

 thirty minutes on the stain, which has 



been warmed to about 40 degrees C. It 

 should be remembered that the more 

 deeply the section is stained, the more 

 difficult it will be to properly decolor it. 



The section is then transferred to 

 water in order to remove the excess of 

 the dye. It is then placed in sixty per 

 cent, alcohol for one to two minutes, and 

 then into Ebner's solution for about one- 

 half minute, after which it is returned to 

 the sixty per cent, alcohol for another 

 minute or two, or until it is almost wholly 

 decolored. A light pink color of the sec- 

 tion will be displaced on subsequent 

 staining with inethylene blue. 



The almost decolored section is placed 

 in Loeffler's methylene blue for one half 

 minute, after which it is washed in water. 

 It is then placed in absolute alcohol for 

 about twenty seconds in order to dehy- 

 drate. A longer exposure to alcohol will 

 remove the blue stain. The section is 

 then transferred to xylol for some 

 minutes, after which it can be mounted 

 in Canada balsam. A properly stained 

 section will show the deep red bacilli on 

 a light blue background. 



Ebner's solution is ordinarily used for 

 decalcifying purposes. The acid and 

 alcohol present make it very useful for 

 decolorizing sections, and it is to be pre- 

 ferred to the common method of treat- 

 ment with nitric or sulphuric acids. It is 

 prepared according to the formula: 



Sodium Chloride 0.5 



Hydrochloric acid 0.5 



Distilled water 30 



Alcohol 100 



The leprosy and tubercle bacilli will 

 give beautiful stains by this method when 

 fresh tissues are employed. The preser- 

 vation of the tissue in alcohol seems to 

 destroy the capacity of these organisms 

 to stain by this method. This is probably 

 due to the removal or alteration of the 

 fat which is present in these bacilli. The 

 protoplasm of the organisms is, however, 

 unchanged and consequently they can be 

 stained by Gram's method when the 

 other process fails. It is advisable to 

 preserve leprous and tubercular tissue 

 in paraffin rather than in alcohol. 



The special method just described can 

 be summarized as follows: 



Carbolic fuchsin, warm, 15 to 30 min- 

 utes. 



Water. 



Sixty per cent, alcohol, 1-2 minute. 



Ebner's solution, 1-2 minute. 



Sixty per cent, alcohol, 1-2 minute. 



Loeffler's methylene blue, 1-2 minute. 



Water. 



Absolute alcohol, 20 seconds. 



Xylol. 



Canada balsam. 



University of Michigan. 



( To he continued.) 



