PURE CULTURE METHODS 117 
Smith (1905) regards the iodine starch test as most satisfactory for 
microbiologists. He gives the procedure.as follows: ‘‘ Twenty-five c.c. 
of distilled water are added to 4 gm. (more or less) of pure potato 
starch and the fluid boiled. One cubic centimeter of this starch water 
and 1 c.c. of freshly prepared potassium iodide water (1:250) are now 
put into the culture fluid, to which is then added a few drops of strong 
sulphuric acid water (2:1). If any appreciable quantity of nitrite is 
present the culture immediately becomes blue black from the libera- 
tion of free iodine which acts upon the starch. Old potassium iodide 
should never be used ~vithout first testing carefully as it usually con- 
tains some free iodine.”” Blank determination should be made. 
Silicate Jelly. Make the required observations and record on the 
Chart. 
Fermentation Reactions. The various compounds upon which it is 
desired to study the action of microorganisms, are added to plain broth. 
Great care should be used in the sterilization of these media since 
Mudge (1917) and Hasseltine (1917) have shown that polysaccharides 
are easily hydrolyzed to monosaccharides. The latter of these investi- 
gators found that serious errors were introduced in the study of B. 
proteus, for instance, on common carbohydrates. For investigations 
requiring accurate data other methods of sterilization than moist heat 
should be used. The broth may be sterilized by filtration after the 
carbohydrates have been added, or the carbohydrate solutions may be 
sterilized by filtration and added to the broth by means of a sterile 
pipette just before the tubes are inoculated. In determinating the 
reaction of the fermentation tubes, it has been the custom to titrate 
5 ¢.c. of the medium with N/20 NaOH or HCI with phenolphthalein 
as the indicator. The work of Clark and Lubs has shown that this 
method does not allow accurate results. They have advised the deter- 
mination of H-ion concentration, but such a procedure is not adapted 
to routine work on account of the time consumed. In place of this, 
these workers have prepared a series of indicators with a sufficient 
range to meet the requirements of bacteriological media. These 
should be used where it is possible. They are given in the appendix. 
Pathogenicity to Animals. The suspected organism should be fed 
to different laboratory animals and also injected into different tissues. 
The animal should be kept under observation for a short period before 
the experiment begins. After injection, it should be constantly watched 
and weighed. 
The Committee recommends the use of these indicators and makes 
the following statement: 
