Io8 BACTERIOLOGICAL METHODS 



highly motile Bacillus typhosus will tend to remain near the middle 

 and upper parts of the tube. 



3. Culttiral 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 Cvilttiral 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 t3^hoid 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 vaseUned 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 



