150 MANUAL OF MICROBIOLOGICAL METHODS 



organisms in hanging drop is a useful supplementary procedure too often 

 neglected. 



Studies of morphology using methods such as those described by Knaysi 

 (1951) and Bisset (1950) reveal a more natural morphology than do 

 classical procedures. The majority of morphological descriptions of bac- 

 terial species are based upon studies of air-dried, heat-fixed, stained 

 smears. If morphological descriptions obtained from improved cy to- 

 logical procedures (see page 30) are published, these should be accom- 

 panied by similar descriptions based upon classical methodology. 



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

 should be examined for motility. Before drawing definite conclusions, 

 cultures grown at several temperatures between 20 and 37°C should be 

 examined. It is important not to confuse Brownian or molecular move- 

 ment 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 motil- 

 ity or none has little significance and other means of confirmation, 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 nonmotile by any other 

 method — bacteria with so-called '^ paralyzed flagella." 



Tittsler and Sandholzer (1936) have proposed the use of stabs in a semi- 

 solid agar (meat extract 0.3 per cent, peptone 0.5 per cent, agar 0.5 per 

 cent). Motile organisms show a diffuse zone of growth spreading from 

 the line of inoculation; nonmotile cultures do not. For this test, incuba- 

 tion should be for 6 days at 30° C Unless positive results are secured sooner. 

 For gram-negative nonsporeformers, 12- to 18-hr 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. The medium can now be obtained in dehydrated form 

 under the name Motility Test Medium. 



Conn and Wolfe (1938), moreover, have recommended a flagella stain 

 even on cultures that do not appear motile upon examination in hanging 

 drop. The modification of the Bailey flagella stain given in Chap. II is 

 simple and quick enough to be employed for routine examinations; posi- 

 tive results cannot be misinterpreted and show the arrangement of flagella 

 as well as the mere presence or absence of motility. A few further refine- 

 ments of the method, making it more adaptable to routine use on bacteria 

 of various types, published by Fisher and Conn (1942), are also given in 

 Chap. II. The Leifson (1951) procedure is also well suited for routine 

 use. 



