RING TEST FOR IN DOE. 
! 
(By S. B. Grubbs, Passed Assistant Surgeon, and Edward Francis, Assistant Sur(Jeon. 
United States Marine-Hospital Service.) 
3 
The method of applying the indol test, as ordinarily used in 
laboratories and described in our text-books, is certainly crude, 
and consequently the statement that an organism does or does not 
produce indol leaves us in most cases very uncertain as to just 
what is meant. 
As we know, the line between the indol producers and non- 
producers is not distinct, and several organisms “not producing 
indol” do give the reaction if grown long enough in suitable 
media. Thus, for example, we can get a faint but distinct reac- 
tion from cultures of Bacillus pestis and Bacillus typhosus, 
although these organisms under ordinary conditions are not con- 
sidered indol producers. 
So we must draw the line arbitrarily, and in this laboratory wo 
take cultures grown twenty-four hours at 37° C. in media contain- 
ing 1 per cent of peptone and use the ring or contact test, allowing 
it to remain in contact one hour. 
We have applied the ring test in determining the presence of 
indol on account of its simplicity, its clearness, and its delicacy. 
When the acid and solution of nitrites were mixed, wo found tlie 
color reaction was often unsatisfactory, tlie natural yellow tint 
masking the pink, or the color apjiearing on first contact witli tlie 
nitrite solution would disappear when the tube was sliaken. 
The following technique will always give a clear-cut reaction if 
indol is x^resent, or if not, there is no difficulty in deciding in tlie 
negative : 
To 7 c.c. of the twenty-four-hour culture add 8 to 10 drops of 
concentrated sulxffiuric acid and shake; then let 3 or 4 c.c. of a 
1-1000 sodium-nitrite solution run carefully down the side of the 
tube so as to form a sharp line of contact between the heavier 
culture and acid and the lighter nitrite solution, when the pink 
color will contrast sharply with the portions above and below. 
The adox^tion of this technique, with the time limits mentioned, 
will make the indol reaction very clear in its results and will 
increase its value as a means of recognizing species. 
( 3 ) 
