86 LABORATORY GUIDE IN BACTERIOLOGY 



2. Special study A. Inoculate fermentation-tubes in 

 the same manner as in the two preceding groups. Ob- 

 serve the absence of gas-formation but note growth in 

 both arms. Compare the results with those of the colon 

 and intermediate groups. 



3. Special study B. The staining of flagella. Read 

 carefully in the textbook the methods of staining flagella. 

 To demonstrate the presence of flagella on B. typhosus, 

 the following method will give good results (Loeffler's 

 method) : 



a) Prepare the mordant. (What is a mordant ? See 

 the textbook.) 



Tannic acid (25$ aqueous solution) 10 parts 



Ferrous sulphate (saturated aqueous solution) ... 5 parts 

 Fuchsin (saturated alcoholic solution) i part 



b) Prepare a number (six) of cover-slips by carefully 

 flaming them, and place them side by side on a piece of 

 filter-paper. (This paper must be burned after using). 



c) Place 4 or 5 loopfuls of water on a clean slide. 



d) Make a light suspension in this water of B. 

 typhosus from a 24-hour-pld agar-culture, taking par- 

 ticular care to stir the suspension as little as possible. 



e) Place a loopful of water on each of the cover-slips. 

 /) Carry over a loopful of the suspension on the slide 



to one of the cover-slips, from this to second, from the 

 second to the third, etc. 



g) Spread carefully and allow to dry in the air. 



h) Cover with the mordant. 



i) Heat over a small flame for ij minutes while 

 steam rises, or better heat for 5 minutes on a water-bath. 

 Replace the evaporated stain, to prevent its drying on 

 the cover-slip. 



