236 MANUAL OF MICROBIOLOGICAL METHODS 



if available, for the collection of specimens from larger animals, such as 

 rabbits, dogs, and monkeys. 



The aseptic collection of blood for purposes of culture is a relatively 

 simple procedure, but certain essential precautions must be observed. 

 Particularly important is adequate sterilization of the injection site, and 

 for this purpose it is advisable to use one of the stronger germicides, as 

 tincture of iodine. A suitable location for collection of blood for culture 

 is directly from the heart, and this may be done in a manner analogous to 

 that described for intracardiac inoculation. Animals may be anesthe- 

 tized with ether to ensure their remaining quiet during the procedure. 

 Anticoagulants, as heparin or citrate, are toxic to certain organisms, and 

 wherever possible blood should be collected without preservative and 

 immediately inoculated into culture media. For this reason it is often 

 desirable to bring the culture materials directly to the site of bleeding. 



For routine cultures, liquid media, such as a tryptophane broth, should 

 be previously sterilized in 60- to 80-ml volumes in 100-ml glass serum 

 bottles which are stoppered with a rubber vaccine cap. The use of such a 

 container makes it relatively simple to inject the blood directly through 

 the closure into the broth medium upon removing the hypodermic needle 

 from the animal. An important consideration is the ratio of the volume 

 of blood to that of the culture fluid. In general, one should try to main- 

 tain a ratio of 1 part of blood to 15 or 20 parts of medium. It is well to 

 prepare multiple cultures so that anaerobic, aerobic, or other special pro- 

 cedures can be performed. It is sometimes also desirable to place 0.1- 

 0.5 ml of the blood specimen on the surface of a blood agar or similar 

 plate and observe for surface growth. It is not good procedure to delay 

 blood cultures until after death, since a massive bacteremia can occur 

 shortly before or immediately after the death of an animal. This may 

 even develop with some saprophytic strains, such as PseudomonaSj thus 

 giving a false result. 



The collection of exudates, especially peritoneal, is frequently desired 

 to obtain material for microscopic examination. The recovery of peri- 

 toneal fluid is readily performed in the living animal by aspirating a small 

 amount of fluid with a syringe and hypodermic needle while the animal 

 is held with its ventral abdominal wall parallel with the working surface. 

 When the needle has been inserted at an angle tangential to the surface, 

 the point of the needle is then depressed with the bevel upward and the 

 peritoneal fluid will run into the pocket thus formed and can be with- 

 drawn. If the material is viscous or small in quantity, it may be neces- 

 sary first to inject 3-5 ml of sterile saline solution to flush out the peri- 

 toneal cavity. 



Animal-autopsy procedures constitute one of the backbones of bac- 

 teriological technique, and it is essential that basic techniques be properly 



