METHODS FOR MICROSCOPIC STUDY OF BACTERIA 2.31 
the medium, ])ut sharper reactions are obtained when it is added sepa- 
rately as a sterile solution. The medium should be adjusted to the 
exact neutral point of this indicator. One larj);e drop of ^ HCl should 
produce in the medium with the Andrade indicator a distinct red color, 
which is discharged with one drop of ^ XaOH. 
Lactose Litmus Agar.~I. Prepare 1 per cent lactose nutrient agar 
by adding 10 gm. of C. P. lactose (free from glucose) to 1 liter of 
plain nutrient agar. Adjust the reaction to a point slightly alkaline 
to litmus. Tube and sterilize in the Arnold sterilizer. 
11. Prepare an aqueous solution of litmus— either a 5 per cent 
solution of purified litmus (Merck) or a 1 per cent solution of azo- 
litmin (Kahlbaum) and sterilize. 
To Use Lactose Litmus Agar.— Add about 1 cc. of sterile litmus solu- 
tion to a sterile Petri dish and pour over it the melted lactose agar, 
previously inoculated with the desired material. For water and milk, 
add 1 cc. of water or milk (diluted to the proper degree) to the Petri 
dish before adding the lactose agar. Mix intimately by rotating gently, 
allow to harden and incubate. 
Those bacteria which ferment lactose with the production of acid 
appear as red colonies. Xon-lactose-fermenting organisms appear as 
blue colonies. 
Blood-agar.— Blood is drawn with aseptic precautions from the 
carotid or femoral artery of a dog or rabbit into a sterile flask con- 
taining beads. The blood is defibrinated by prolonged agitation and 
added to plain (not glucose) nutrient agar previously melted and 
cooled to 45° C, in the proportion of 2 cc. of blood to 10 cc. of agar. 
Small amounts of blood may be withdrawn directly from the heart 
of an animal without difficulty, provided a small hypodermic needle 
is used. The blood may be injected directly into the melted agar 
without defibrination. 
Occasionally human blood is added to agar; if a series of agar slants 
are prepared it is possible to convert them into blood-agar with a small 
amount of blood, as follows: 
Withdraw 10 cc. of blood, using aseptic precautions, from the 
median basilic vein, in a large syringe. Inject the blood at once into 
four times the volume of plain nutrient agar melted and cooled to 
45° C. Mix at once and run 2 cc. of this agar-blood mixture over the 
slanted surface of previously prepared agar slants, and allow to harden 
in the inclined position in such a manner that a uniform layer of the 
blood-agar mixture is obtained. Incubate to prove sterility. 
Ascitic and Hydrocele Fluid Media,. — Ascitic and Hydrocele Agar.^ — 
Collect ascitic or hydrocele fluid in a sterile bottle, using aseptic 
precautions. Allow to stand in the ice-box until clear, and heat to 
1 Ascitic and hydrocele fluids may be sterilized by passage through an unglazed 
porcelain filter. 
