108 BACTERIOLOGICAL METHODS 



highly motile Bacillus typhosus will tend to remain near the middle 

 and upper parts of the tube. 



3. Cultural Separation on Basis of Motility. By means of a 

 sterile pipette take up the upper half or third of the contents 

 of the centrifugalized tube (2) and place in the special loop tube 

 with phenol-broth and incubate at 37 C. for 24 hr., or longer if 

 necessary. 



4. Plate Cultures. Take up several platinum loopfuls from 

 the loop tube (the opening opposite the inoculated end) and 

 plant in lactose-litmus-agar (at 37 C.) and note the character 

 of the colonies which form. Compare with the colon bacillus 

 colonies. Examine colonies microscopically. 



5. Other Cultural Tests. Test for absence or presence of gas 

 formation. Enrichment in liver broth may be tried, etc. 



6. Agglutination Tests. Two methods may be used. The 

 microscopical and the macroscopical. The usual routine mi- 

 croscopical method is carried out as follows: By means of a 

 clean sterile pipette place o.i cc. of the typhoid serum and 0.9 

 cc. of physiological salt solution (salt is necessary to bring about 

 agglutination) in a clean sterile Syracuse watch crystal and 

 mix thoroughly by means of a clean sterile glass rod. This 

 gives a serum dilution of i-io. Place one platinum loopful of a 

 24-hr, bouillon culture of the typhoid bacillus on a clean cover 

 glass and add one loopful of the mixture from the Syracuse watch 

 glass. This gives a dilution of 1-20. Two loopfuls of the 

 culture and one of the serum mixture gives a dilution of 1-40. 

 Three loopfuls of culture and one of serum mixture gives a dilu- 

 tion of 1-80. Make the dilutions one at a time and place the 

 cover glass holding them (inverted) on a vaselined hollow or 

 concave slide and examine at once under the high power, con- 

 tinuing the observation for 30 min. if necessary. The first 

 change noticeable will be a gradual loss of motility, followed 

 by a clumping of the now non-motile germs. This constitutes 

 a positive agglutination reaction. Clumping with the lower 



