MEMOIRS OF THK NATIONAL ACADEMY OF SGIEXOES. 429 



III the same table are tlie results obtained by coantiiig the colonies which (le\eh)|i in '• agar"' 

 l)Iates made from the same samples of water and, unless otherwise s|)ecilied, with the same (jnan- 

 tities of water as the gelatin jjlates, but unlike these kept at a temiieratnic of about 'Mi" ('. The 

 figures refer to the number of colonies in the plates as ascertained by actual count with the hand 

 lens, and it will be obser\ed that more colonies develop, from a given quantity of a sample of 

 water, at the room temperature than at a temperature of 31)° C. In the course of the work the 

 time of counting the plates has varied from the first to the ninth day, depending on the number 

 and character of the colonies and the condition of the plate. Counts made later than the sixth 

 day have been rather exceptional. In those cases where two or more counts were made of the 

 same plate on different days the maximum number was taken for the estimation of the number of 

 bacteria per cubic centimeter. 



Efforts have been made to stoi) the progress of licjuefaction in many cases, in order that later 

 counts could be niade, but these have been only partially successful. The best results seem to be 

 given by the use of a solution of mercuric chloride, niilligrani 1:20 or 1:100, applied to the 

 liquefying colony by means of a " swab." 



In the study and differentiation of the various organisms hereatter described the culture 

 media mainly employed have been nutrient gelatin (10 per cent), acid gelatin, sugar gelatin, agar- 

 agar, bouillon, tube potato cultures, Dunham's peptone solution, " rosolic acid" solution, litmus milk, 

 and to some extent sugar agar. The cultivations have been carried on in test tubes containing 

 these media. In the preparation of the plain gelatin, agar-agar, and bouillon, meat infusion or 

 Liebig's meat extract have been used. It would seem that the results are more satisfactory when 

 the medium is prepared with the former. This is probably less true of the bouillon tlian of the 

 other. The acid gelatin is prepared by adding to the medium, after neutralization in the usual 

 mauuer with caustic potash, pure concentrated hydrochloric acid in the proportion of about 1 : 1000 

 or 1:000 by volume. The sugar gelatin contains 2 per cent glucose. In the preparation of this 

 medium, as well as the preceding, Liebig's meat extract has been used. The Dunham's peptone 

 solution contains 1 per cent i)eptone and 0..5 per cent sodium chloride. The "rosolic acid" 

 solution consists of Dunham's solution colored with that indicator. The litmus milk is cow's milk, 

 colored with neutral litmus tincture. For the study of colonies, " Esmarch " cultures, rolled on ice, 

 and cultures in Petri's plates have been employed. The culture in "r/«7> stab'' mentioned in the 

 description of the different sjjecies consists in inoculating a sugar gelatin tube in the manner of 

 an ordinary "stab" and then filling the tube for some distance above the surface of the medium 

 with melted gelatin or agar, which is then made to solidify. The tubes used for this have been 

 rather narrow. The test for the production of indol has been made by adding a few drops of 

 pure concentrated sulphuric acid to a culture in Dunham's solution and then after a time about 

 1 cc. of a 0.01 per cent solution of sodium nitrite. The appearance of the red color after the addition 

 of the acid alone is considered as indicative of the coincident producti(m of nitrites. In the 

 staining of the flagella of the organisms the method of Loftier has been usually followed with slight 

 modifications. The cover glasses are easily freed from grease by heating in the Bunsen flame 

 It has not been found necessary to add alkali or acid to the mordant. Anilin water fuchsin has 

 been the staining fluid mostly used. The cover glass with the mordant should be only slightly 

 warmed over the flame and only for a few seconds. The chief cause of failure to obtain satisfactory 

 results seems to be the overheating of the preparation while being treated with the mordant. 

 Washing the preparation in water after the application of the mordant, and again after the 

 staining, which should be rather deep, seems to be all that is necessary to obtain clear prejjara- 

 tions, if there has not been overheating and consequent deposition of a tine i)recipitate. The 

 ferrous sulphate solution, as well as the mordant and the staining fluid, slumld be freshly juepared. 



Filtering the solution of ferrous sulphate and the solution of tannic acid before mixing in the 

 making of the mordant probably gives clearer preparations. 



RESULTS. 



The results of the analyses are given in tables showing the date, distribution district from 

 which the sample was collected, and the separate determinations, as well as the means and 

 averages. In addition to these, charts (Nos. I, II, III, and IV) have been prepared for the four 

 distribution districts to represent graphically the variations and approximate values of the 



