CULTURAL CHARACTERISTICS 541 



iodine-alcohol, and pure alcohol, and dried. The steps of the iron 

 hematoxylin method are the following : 



1. Stain smears for three to five minutes in a 10 per cent, watery 

 solution of ferric chloride. 



2. Drain and blot the cover-glass, then pour over it a few drops of 

 a freshly prepared 1 per cent, aqueous solution of hematoxylin. If 

 all the hematoxylin is precipitated by the excess of ferric chloride, 

 pour off the solution and add a fresh supply. In three to five minutes 

 the sections will be colored a dark bluish black. 



3. Wash in water. 



4. Decolorize and differentiate in a J per cent, aqueous solution 

 of ferric chloride. The cover-glass must be kept constantly moving 

 in the solution. The differentiation will be complete in a few seconds 

 to several minutes. 



5. Wash in water, dry, and examine. 



If the differentiation is not sufficient the preparation must again 

 be washed in the \ per cent, solution of ferric chloride. Mallory 

 states that the principal point in this method is first to stain very deeply 

 and then to differentiate properly. The nuclei of amebse stain sharply 

 with this method. 



Cultural Characteristics. Attempts to cultivate amebse had been 

 made for a number of years, but not much progress was made until 

 Musgrave and Clegg devised a method which is now generally used, 

 either according to the original recommendation or with some slight 

 modification. The principle of this method consists in preparing a 

 culture soil, comparatively poor for bacteria, which will enable them 

 to thrive only moderately, but sufficiently to serve as food for the 

 amebse, which require proteids for their metabolism and cannot 

 utilize simple compounds like plants. Musgrave and Clegg's medium 

 consists of: 



Agar . . ' . ' . . . 20 gr. 



Sodium chloride . . -. . . 3 to . 5 gr. 



Extract of beef . . . . .. . . , . . . . ... . , 0.3to0.5gr. 



Water. . ..... . . . . , ' . . . . . . . . 1000 c.c. 



This is prepared in the same manner as ordinary agar and made 1 per 

 cent, alkaline to phenolphthalein. The medium is sterilized in tubes 

 and from these Petri dishes are filled in the ordinary manner, but, 

 of course, without inoculating anything into the sterile, melted agar 

 before it is poured out into the lower pla'te of the Petri dish. The agar 

 is there allowed to solidify. If amebse are to be cultivated from water 

 or from water mixed with vegetable material, 100 to 500 c.c. of the 

 fluid are placed in a flask and 0.5 to 1 c.c. of ordinary nutrient bouillon 

 is added for each 100 c.c. of amebse-containing fluid. The flasks 

 are then set aside and kept at room temperature for from twenty-four 

 to seventy-two hours, when a few loopful of fluid may be removed 

 from the surface, spread on a slide and examined fresh for the presence 



