256 FERMENTS AND ANTIFERMENTS 



8. All shells should react negatively, i. e., they should not permit 

 the passage of unchanged albumin. If the ninhydrin test is used, the 

 tubes should be inspected one-half hour after boiling, and the contents 

 should be as clear as water or show but the faintest blue tint. If this is 

 not the case, shells should be discarded as being permeable to albumin. 

 Those that are satisfactory in this respect should be tested further as 

 follows : 



Testing the Shell for Permeability to Peptone. 1. The shells should 

 now be thoroughly cleansed, but not with a stiff brush, washed in 

 running water, and boiled for thirty seconds. 



2. Prepare a 1 per cent, solution of silk peptone (Hochst) in dis- 

 tilled water, and carefully pipet 2.5 c.c. into each shell, using every pre- 

 caution against contaminating the upper portion on the inside, and 

 especially of the outside, of the shell. 



3. Place the loaded shell in a sterile dialyzing cylinder containing 

 20 c.c. of sterile distilled water, and cover the contents of the shell and 

 water with toluol. Replace the cotton plug and incubate at 37 C. for 

 twenty-four hours. 



4. Remove 10 c.c. of the dialysate (avoid removing toluol) to a 

 clean, sterile, thin-walled test-tube, and add 0.2 c.c. of the 1 per cent, 

 ninhydrin solution. Insert a sterile boiling rod and boil for exactly one 

 minute. 



5. The boiling process is quite an important feature of this test. 

 Always boil in precisely the same manner. A high Bunsen flame should 

 be used, and about one minute after air-bubbles first appear on the sides 

 of the tube lively boiling commences. The flame should then be turned 

 down and the boiling continued for exactly one minute. 



6. Place the tube in a rack. With a fresh sterile pipet remove 10 

 c.c. of dialysate from the next cylinder and test in the same manner, 

 and repeat until the entire series have been finished. 



7. After half an hour inspect all the tubes; they should show a 

 deep blue color; if they do not do so they are impermeable or partly 

 permeable to peptone and should be discarded. There is usually a 

 difference in the degree of color reaction among a number of shells, as 

 their permeability varies. 



8. Those shells that have withstood both tests are now thoroughly 

 washed in running water, boiled for from thirty seconds to one minute, 

 placed in a jar of sterile distilled water containing a few drops of chloro- 

 form, and covered with toluol. From this time on they should not be 

 handled with the fingers, but only with forceps that have been sterilized 



