1892.] MICROSCOPICAL JOURNAL. 7 



it to act from two to three minutes. A convenient method of heating 

 the mordant on the cover-ghiss is to pass it several times through the 

 lower portion of the flame, which iieats it sufficiently and prevents 

 spattering. A safer method is to heat the preparation for the required 

 time in a cover-glass containing the mordant. 



The heating of the cover-class to Hx the film is also an important 

 condition in the success of the operation. Loeffler's method has the 

 objection that it is difficult to heat the preparations uniformly. I have 

 employed a hot-air chamber for this purpose, and after a series of ex- 

 periments varying in temperature from 90 to iSo° C. and in duration 

 from one-fourth to five minutes, have found that I could obtain the 

 best results by heating the preparations for one minute at a temperature 

 of about 135^ C. Little if any difference was noticed in preparations 

 heated at a temperature varying from 120-140° C. 



(3) The Reaction of the Mordant. — Experiments have shown 

 that the statement made by LoefHer, that an alkali producing organism 

 required an acid mordant and an acid producing organism an akaline 

 mordant need not be taken in a very strict sense. This is illustrated 

 with the hog cholera bacillus. As it is an alkali producing germ, it 

 would be necessary, according to Loeffler's statement, to add a certain 

 quantity of the acid solution to the mordant in order to stain its flagella. 

 Dr. Theobald Smith stained the flagella on this bacillus by the use of 

 the neuti'al or standard mordant.* Further investigation has shown that 

 its flagella can be stained by the use of the mordant containing a varia- 

 ble quantity of either the acid or sodium solution, good results being 

 obtained when as much as 3 cc. of either solution was added to the 16 cc. 

 of the mordant. It is better to add the sodium solution just befoi'e the 

 mordant is to be used. I have also found that the flagella on the ty- 

 phoid bacillus, an acid producing germ, can be stained by the use of 

 either the acid or alkaline mordant. This deviation from Loeffler's re- 

 sults with the typhoid bacillus may possibly be due to the age of the 

 germ, as the one I used had been preserved, by means of subcultures, 

 for several years. The same I'ange in the reaction of the mordant was 

 found to be applicable to a few other bacteria. 



(4) The Stainitio- Fluid. — 1 have found that carbol fuchsin (10 cc. 

 of a saturated alcoholic solution of fuchsin, 100 cc. of a 5 percent, solu- 

 tion of carbolic acid) gives equally as good, if not better, results than 

 the aniline water fuchsin recommended by Loeffler. It also has the ad- 

 vantage that it can be kept for a much longer time, and, consequently, 

 is ready for use at any moment. It has a less tendency to form a pre- 

 cipitate with any trace of the mordant that might be left on the cover. 

 I have also stained the ffagella very nicely with Loeffler's alkaline 

 methyline blue. 



(5) The Age of the Culture to be Used. — A careful examination of 

 the growth from cultures of certain bacteria shows that the flagella can 

 be stained on these germs from cultures varying in age from 20 hours to 

 several weeks. For simply demonstrating their presence any aged 

 culture, within the limits mentioned, can be used. In old cultures the 

 flagella are more broken, and a larger number of detached appendages 



* Dr. Smith pointed out that the reaction of the culture liquid with sume bacteria may be cither ai.id 

 or alkaline, according as glucose or other sugar-i are present or absent. These undergo fermentation 

 with the formation of acids. In liquids free fj om sugars the reaction becomes speedily alkaline. 

 Fetruschky's classification of bacteria as acid and alkali producing is thus shown to depend largely on 

 the composition of the culture medium. 



