500 BACTERIOLOGY. 



prepared (6 and d) are then placed in distilled water in a wide test-tube 

 on foot (Fig. 71 c). The sacs are now sterilized by exposure to steam 

 for 15 minutes, on each of three successive days; of, they may be 

 sterilized in an autoclave at 115° for 15 minutes. In this condition 

 the sacs can be preserved for several weeks. 



The filling of the sac with water or bouillon, and the withdrawal 

 of that present, is done by means of a bulb pipette (Fig. 61 e). This 

 should be bent at right angles, just below the bulb. The tip of the 

 pipette is cut off and the end is flamed. Special care must be taken 

 to heat the cut end of the pipette till the sharp edges are rounded 

 off. 



The sac is now ready for the final steps — inoculation, sealing and 

 placing in the abdominal cavity. The cotton is removed and the 

 mouth of the test-tube is then flamed. The narrowed end of the ster- 

 ile pipette, prepared as just mentioned, is inserted through the con- 

 stricted portion of the test-tube into the sac. The water is now care- 

 fully drawn up into the bulb. The pipette is then withdrawn and the 

 cotton plug replaced. The water is blown out of the pipette which is 

 then flUed with the bouillon that has been inoculated with the organ- 

 ism to be tested. The pipette is again inserted into the sac and 

 this is now filled up to the threaded portion, with the inoculated 

 bouillon. 



Nothing now remains but to seal the sac. For this purpose, it is 

 taken out of the wide test-tube and the constricted portion is wiped 

 dry with sterile filter-paper. The sac is then wrapped in a piece of 

 sterile paper which should not project above the silk thread. One 

 can thus grasp the glass end of the sac without causing contamiha- 

 tion. A very narrow flame is then directed against the constricted 

 tube till it is sealed. The sac is held in the sterile paper, avoiding 

 unnecessary pressure, until the glass cools. The, glass end is then 

 coated several times with collodium, and, when this has dried, the sac 

 is picked up by a pair of sterile forceps and placed in a sterile dish 

 (Fig. 43, p. 265).. It is now ready to be inserted into an animal. 



The guinea-pig or rabbit is usually used for sac experiments, 

 although other animals may be employed. The animal is placed on a 

 holder (Fig. 45, p. 268), and the hair over the abdomen is removed 

 with a pair of curved scissors. The surface is then soaped and 

 shaved, after which it is washed with alcohol and mercuric chloride. 

 A piece of filter-paper soaked in' mercuric chldride is then spread 

 over the prepared surface. The animal is now anesthetized with 

 ether. An incision, about 3-5 cm. long', is made through the skin of 

 the upper part of the abdomen. A similar incision is made on one 



