METHODS FOR MICROSCOPIC STUDY OF BACTERIA 233 
Hiss'' Semi-solid Medium.— 
FORMULA. 
Water 1000 cc. 
Agar S gm. 
Peptone 10 " 
Meat extract -^ " 
NaCl 5 " 
Gelatin-^ 40 " 
When all the ingredients are dissolved, adjust the reaction to + 0.5 
(phenolphthalein), filter, and add sufficient litmus solution to impart 
a clear blue and sterilize in the autoclave. Add sufficient sterile 
aqueous solution (20 per cent) of glucose, lactose, saccharose or 
mannitol, or other carbohydrate to the medium to obtain at least a 
concentration of 0.5 of 1 per cent. 
Semi-solid media are inoculated by the stab method. A change 
in reaction is indicated by the litmus; gas-forming organisms generate 
bubbles of gas in the depth of the medium. 
Russell Glucose-lactose Medium.— To 1 liter of nutrient agar, slightly 
alkaline to litmus, add sufficient sterile 5 per cent litmus solution to 
impart a distinct clear blue color. Add 1 per cent of C. P. lactose 
and 0.1 per cent glucose, and distribute in test-tubes. The Andrade 
indicator is far more sensitive than ordinary litmus, and it is preferable 
for this medium. 
Sterilize in the Arnold sterilizer for three successive days, and allow 
to harden in a slanted position. 
Saccharose-mannitol Medium.^— The glucose-lactose medium, and 
the saccharose-mannitol medium together are of importance for the 
cultural diagnosis of many intestinal bacteria, particularly the alca- 
ligenes, typhoid, paratyphoid, dysentery and coli groups. To 1 
liter of nutrient agar, neutral to the Andrade indicator, add 1 per 
cent saccharose and 0.1 per cent mannitol. Distribute in test-tubes, 
add 1 per cent Andrade indicator, sterilize and allow to harden in the 
slanted position. 
Media for the Cultivation of Aciduric Bacteria. — .Icir/ Broth.— Add 
sufficient glacial acetic acid to a liter of 2 per cent glucose broth 
to make the reaction equal to 50 cc. of normal acid. A precipitate 
forms, which will settle out, leaving a clear supernatant fluid that may 
be removed to sterile test-tubes with a sterile 10 cc. pipette. 
Oleate .l.gar.— The addition of 0.2 per cent sodium oleate to glu- 
cose agar makes a favorable medium for the cultivation of aciduric 
l)acteria. 
Oleate Agar for the Influenza Bacillus.^— Prepare as follows: 1. Two 
per cent meat infusion agar, reaction + 0.3 to 0.5 to phenolphthalein 
(pii = 7.3 — 7.5). 
1 Jour. Exp. Med., 1897, 2, 677. 
2 Add after the other ingredients are in solution. 
^ Kendall and Ryan: Jour. Infec. Dis., 1919, 24, 400. 
* Avery: Jour. Am. Med. Assn., 1918, 71, 2050. 
