METHODS FOR MICROSCOPIC STUDY OF BACTERIA 229 
5. Prepare double strength agar; 24 gm. agar in 1000 cc. tap water. 
(). Mix equal volumes of filtrate from Step 4 and double strength 
agar (Step 5). 
7. Tube and sterilize in autoclave. 
This medium is particularly suited for cultures of the more fastidious 
organisms; the pnemnococcus, gonococcus, meningococcus and B. 
influenziie. 
Lbffler's Blood Serum.— Add 1 part by volume of 1 per cent nutrient 
glucose broth^ to 3 parts of clear, hemoglobin-free beef or sheep serum, 
and distribute in test-tubes. The tubes are placed in a Koch's serum 
inspissator or in specially designed racks in an autocla^'e in an inclined 
position to produce a slanted surface, and slowly heated to 80° C. 
This temperature is maintained until the medium is firmly coagulated. 
The temperature is then raised to 95° or 100° C, and maintained for 
one hour on each of three successive days, or to 115° in the autoclave, 
and maintained for one hour. The medium is opaque and white and 
the surface is smooth and should be free from a metallic luster when 
viewed by reflected light. The luster indicates an accumulation of 
salts, which are inimical to the growth of many bacteria. 
Coagulated Serum. — Clear blood serum from the dog, sheep, cow 
or other animal, preferably sterilized by filtration through Berkefeld 
filters, and with or without the addition of glycerin, is placed in test- 
tubes and slantefl and coagulated in a serum inspissator at a tempera- 
ture of 75° to 80° C. An exposure of one hour to this temperature 
on each of six successive days is necessary to insure sterility. The 
mediiun should be translucent, free from bubbles and firm. 
Hiss Serum-water Media.— Hiss- has recommended a serum-water 
medium for the cultivation of pneumococci and similar organisms. 
It is prepared in the following manner: 
Sheep or beef serum,^ clear and free from hemoglobin, is added to 
water in the proportion of 1 volume of serum to 3 of water. Ten per 
cent aqueous solutions of various sugars are prepared and sterilized, 
and a sufficient amount of the desired sugar to make a final concentra- 
tion of 1 per cent is added to the sterile serum solution. Sufficient 
sterile 5 per cent litmus solution is added for an indicator. Fermenta- 
tion of the carbohydrate is shown by the development of an acid reac- 
tion, and frequently by a well-definerl coagulation of the medium as 
well. 
Endo Medium^ for the Isolation of Typhoid, Paratyphoid and Dysentery 
Bacilli.— I. Preparation uf Agar. — (a) Prepare plain, sugar-free nutri- 
ent agar as described on page 227, using 15 gm. of agar per liter. 
1 If the liquefaction of blood serum by bacteria is to be tested, sugar-free broth must 
be used in place of glucose broth. 
•■! Jour. Exper. Med., 1905, 7, 22.3. 
' It is advisable to sterilize the serum by passage through an unglazed porcelain filter. 
^ Plating media for the isolation of bacteria from the feces or intestinal contents 
are fundamentally composed of nutrient lactose agar and an indicator which will react 
by a definite color change to fermentation acid. 
