V49-6 MANUAL OF METHODS FOR PURE CULTURE STUDY 



Study of Cell Morphology 



The routine study of morphology should include examinations of 

 stained dried preparations and of unstained organisms in hanging 

 drop. Stained preparations to show the vegetative cells should be 

 made, preferably from agar slant cultures, from a few hours to two 

 days old, according to the rapidity of growth. The medium and 

 temperature used and the age of the culture should be recorded. The 

 examination of unstained organisms in hanging drop is a useful sup- 

 plementary procedure too often neglected. 



Motility. Hanging-drop preparations of young broth or agar cul- 

 tures should be examined for motility. Before drawing definite con- 

 clusions, cultures grown at several temperatures between 20° and 

 37°C. should be examined. It is important not to confuse Brownian 

 or molecular movement with true motility. The former consists of a 

 "to and fro" motion without change in position, except as influenced 

 by currents in the fluid. A phase microscope can prove useful in 

 studying motility. 



When interpreting results it is important to remember that whereas 

 definite motility in a hanging drop preparation is conclusive, weak 

 motility or none has little significance, and other means of confirma- 

 tion, such as those that follow, must be undertaken. In particular, 

 an increasing number of cases are found of organisms fully flagellated 

 as shown by staining methods and serology, but absolutely non- 

 motile by any other method — bacteria with so-called "paralyzed 

 flagella". 



Tittsler and Sandholzer (1936) have, in fact, proposed the use of 

 stabs in a semi-solid agar (meat extract 0.3%, peptone 0.5%, agar 

 0.5%). Motile organisms show a diffuse zone of growth spreading 

 from the line of inoculation; non-motile cultures do not. For this 

 test, incubation should be for 6 days at 30°C. unless positive results 

 are secured sooner. For Gram-negative non-spore-formers, 12-18 

 hour incubation gives more clear-cut results. This test is a good 

 check on the hanging drop method, but is slow and requires some 

 experience before one can be certain how to interpret results. 



For this reason Conn and Wolfe (1938) have recommended a 

 flagella stain even on cultures that do not appear motile upon exami- 

 nation in hanging drop. The modification of the Bailey flagella stain 

 given in Leaflet IV is simple and quick enough to be employed for 

 routine examinations; positive results cannot be misinterpreted, and 

 show the arrangement of flagella as well as the mere presence or 

 absence of motility. A few further refinements of the method, mak- 



