326 BACTERIA PATHOGENIC TO MAN 



showed a total absence of influenza bacilli. The pneumococcus was 

 present in almost pure culture. 



Examination of Sputum for Influenza Bacilli. 1. Sputum coughed 

 from the deeper air passages and not from throat scraping should be 

 used. 



2. The sputum should be received in a sterile bottle, which should 

 then be placed immediately in cracked ice. 



3. Blood-agar plates should be made by dropping a drop of fresh rab- 

 bit's blood, obtained aseptically, on the centre of a hardened agar plate. 



4. One of the more solid masses of the sputum should be taken from 

 the bottle with sterile forceps and placed on a plain agar plate. A 

 small portion of this mass should be separated with a sterile platinum 

 needle and drawn through the blood on the blood-agar plate from the 

 centre out in different directions. The larger part of what is left of 

 this small portion is then placed in a similar manner over a second 

 blood agar, and from this to a third, sterilizing the needle between the 

 transfers. The plates should be placed in the thermostat for twenty- 

 four hours. 



5. After the plates are planted two smears should be made, one 

 stained by Gram and the other by weak carbol-fuchsin. 



6. After twenty-four hours the plates are examined under low power. 

 The influenza colonies use up the hemoglobin, and in parts of the 

 blood-agar plate where the blood is of right thickness such colonies 

 show as almost clear areas surrounded by the red blood. With a 

 higher power (No. 6 or 7 objective), if such areas seem to be made up 

 of fine indefinite granulations, they are practically sure to be influenza 

 colonies. Most influenza colonies are more highly refractive than 

 other light colonies, and they show this characteristic best when they 

 grow on the edge of a blood mass. Many influenza colonies also show 

 heapings in the centre. Influenza colonies growing away from the 

 blood cells are less characteristic in appearance and less easily differ- 

 entiated from other bacteria. 



7. Fishings from the influenza-like colonies should be planted on 

 blood-agar tubes, and if, after twenty-four hours in the thermostat, the 

 resulting growth should consist of influenza-like organisms, plantings 

 should be made on plain agar. The first generation on plain agar 

 may show slight growth because of the blood carried over from the 

 original tube, but the second generation should show no growth if the 

 organism is the influenza bacillus. 



8. The agglutination characteristics of the cultures should be tested 

 in the serum from a rabbit injected with a single typical culture, and in 

 the serum from one injected with a number of cultures. The aggluti- 

 nation tests should be carried out in order to gain information. The 

 cultures tested in the Research Laboratory have shown considerable 

 variation. 



For Testing the Agglutination of Influenza Bacilli in the Hanging Drop. 

 Grow the cultures on slanted agar tubes to which, after cooling to 

 40 C., i c.c. of defibrinated blood has been added. When twenty to 



