Do not filter. 
Sterilize at 120° (autoclave) for 10 minutes. 
Reaction of Media. 
Gelatin, Agar, Broth and Milk were adjusted to +1 reaction. 
Sugars and glycerin broth were neutralized. 
Peptone water and Nitrate broth were left unadjusted (.4 per cent. acid 
to phenol phthalein). 
Sterilization of Media. 
All media except milk and gelatin were sterilized in the autoclave for 15 
minutes under 15 pounds pressure. 
Gelatin and milk were sterilized by the discontinuous method in the Arnold 
Steam Sterilizer. 
SEPARATION OF BLACKENING ORGANISMS. 
The first phase of the problem was the search for blackening organisms. 
Three methods were employed: 
Method 1. 
Streak cultures were made upon plates of lead carbonate gelatin. The 
result was unsatisfactory. Complete liquefaction followed before definite con- 
clusions could be drawn with regard to blackening. Cultures i.j.r.s.t.u. produced 
slight darkening of the lead carbonate after two days, then rapidly liquefied the 
plates. 
Method 2. 
Cultures were transferred from 24 hour broth cultures to the strong peptone 
solution, recommended by Pake, as a test for the production of Hydrogen 
sulphide. 
Filteen organisms, viz.; 1, 3,211, 12; 1o,\17;528;.29, dil, brew inni smc turned 
the precipitate black and were thus differentiated as H2S formers. Some others 
darkened the precipitate to brown only. All cultures were kept under observa- 
tion for more than four weeks. 
The fifteen organisms were transferred from broth cultures to other media, 
astudy beingmade of preparations in Dextrose broth, Gelatin stick, Litmus milk 
and potato. : 
Method 3. 
Myers’ called attention to the fact that some organisms produce hydrogen 
sulphide on one brand of peptone and not on another. This suggested the 
advisability of testing all organisms in clam meat itself. Tubes were prepared 
according to Dr. Harrison’s suggestion, using clam meat and sea water in the 
proportions of one to two. They were prepared in triplicate—about seven 
hundred in all—arranged in the following series: 
A. Clam meat+sea water+iron. 
B. Clam meat+sea water+tin. 
C. Clam meat+sea water+iron-+tin. 
Chemically pure iron wire was used in small pieces (1/8 in. to a tube) and 
chemically pure flaked tin. Sterilization was done in the autoclave 15 minutes 
at 15 pounds pressure. 
