192 



Journal of Applied Microscopy. 



growth is transferred to the water. The 

 latter should be rendered just barely 

 cloudy by the bacteria thus introduced. 

 By means of a very small loop some of 

 this suspension is taken up, transferred 

 to a clean cover-glass, and spread out as 

 evenly as possible. 



The specimen is allowed to dry in the 

 air or by gently waving it over a flame. 

 The next step is to fix the material. This 

 should not be done in the ordinary way 

 inasmuch as there is danger of overheat- 

 ing, which would destroy the delicate 

 whips. The cover-glass should be held 

 between the thumb and forefinger, speci- 

 men side up, and quickly passed through 

 a flame once or twice. There is thus no 

 risk of over-heating. 



The staining process requires the use cf 

 two solutions. The first one employed 

 serves as a mordant, and, as used by 

 Fischer, is prepared as follows: Two 

 grams of dry tannin are dissolved in 

 twenty cubic centimeters of water, and 

 to this liquid four cubic centimeters of 

 a ferrous sulphate solution (1:2), and one 

 cubic centimeter of a concentrated alco- 

 holic solution of fuchsin are added. The 

 mixture is thoroughly stirred and the 

 resultant precipitate is removed by filtra- 

 tion. The filtered mordant will keep for 

 some time, and is said, indeed, to 

 improve with age. 



The stain proper is a hot saturated 

 aqueous solution of fuchsin (1:50). An 

 anilin-water fuchsin may be used to 

 advantage. 



This is prepared by adding two to three 

 grams of fuchsin to 100 cubic centimeters 

 of anilin water and heating till solution 

 results. 



The fixed specimen is held in a pair of 

 forceps and the surface moistened with a 

 drop of water. It is then covered with 

 the mordant and gently heated over a 

 low Bunsen flame so that vapors are 

 slowly given off. At no time should the 

 liquid boil. After heating thus for one 

 to two minutes the cover-glass is washed 

 thoroughly under the tap. If the speci- 

 men has not been overheated, every trace 

 of the mordant will wash off and leave a 

 perfectly clear, colorless cover-glass. If 

 a ring of deposit forms on the edge of 

 the glass, and even this can be prevented 

 by careful heating, it must be removed 

 by scraping with the blade of the 

 forceps. 



The clean, mordanted cover-glass, 

 moistened if necessary with a droplet of 

 water, is then covered with the fuchsin 

 stain, and slowly and gently heated over 

 a flame, for one to two minutes. Actual 

 boiling of the liquid should be avoided. 

 The specimen is then washed thoroughly 

 and examined. 



On examination with a one-eighth inch, 

 or, better, with a one-twelfth inch homo- 



geneous oil immersion objective, the 

 bacilli will be seen to be provided with a 

 number of very flne, wavy lines, the 

 flagella. If much granular matter has 

 been deposited on the cover-glass, it is as 

 a rule due to overheating while mordant- 

 ing, or while staining. Not a little 

 patience and intelligent manipulation is 

 necessary in order to obtain stained 

 flagella on a clear, colorless background. 



The method of staining flagella can be 

 summarized as follows: 



Dilution, 



Cover-glass preparation, 



Dry in air. 



Fix in flame by touching once. 



Mordant, hot (one minute). 



Water, 



Concentrated aqueous or anilin-water 

 fuchsin, hot (one minute). 



Water, and examine. 



Dry in air, 



Canada balsam. 

 University of Michigan. 



{To be continued.) 



Low Temperatures for Physiologi- 

 cal Experimentation. 



The use of cold as a factor is rapidly 

 extending to several phases of physiologi- 

 cal experimentation. Ordinarily, lower 

 temperatures are obtained by freezing 

 mixtures. Salt and ice will give, if prop- 

 erly manipulated, a temperature of -ISC, 

 and calcium chloride with snow will give 

 -42C. In northern latitudes, however, 

 advantage may often be taken of outside 

 cold, but this has its disadvantages since 

 it is not always agreeable or even con- 

 venient to carry on experiments in 

 winter weather. 



In work of this character, in the labo- 

 ratory for plant physiology of the Uni- 

 versity of Minnesota, the following 

 methods are used: 



In the use of freezing mixtures, such 

 as that of ice and salt, the mixture is 

 placed in one vessel several feet above 

 the floor and the fluid allowed to flow 

 through the jacket of an ordinary chemi- 

 cal condenser to a lower vessel, from 

 which it is returned to the flrst by means 

 of a hand pump or by pouring. The 

 material to be frozen is placed in the con- 

 densing tube, either free or in small 

 phials, and through the upper, stopper a 

 thermometer is inserted. 



It is possible to reduce the tempera- 

 ture of ten cubic centimeters of material 

 from 10 degrees C to -18 degrees C 

 in a few minutes by this method, but it 

 is necessary that the ice be very finely 



