IV46-16 MANUAL OF METHODS FOR PURE CULTURE STUDY 



The following method, however, can be given as one of the most 

 satisfactory, especially for students who have not had previous 

 experience- with some other method: 



Use young and actively growing cultures (e.g. 18-22 hr. old) on 

 agar slants. Before proceding, check the culture for motility in 

 hanging drop. If motile, wash off the growth by gentle agitation with 

 2-3 ml. sterile distilled water. Transfer to a sterile test tube and 

 incubate at optimum temperature for 10 minutes (30 minutes for 

 those producinofslime). At this point, again check motility under 

 a microscope. Transfer a small drop from the top of the suspension 

 (where motile organisms are most numerous), by means of a capillary 

 pipette to one end of the slide prepared as above described. Tilt the 

 slide and allow the drop to run slowly to the other end. (Two or 

 three such streaks can be placed on a slide.) Place the slide in a 

 tilted position and allow it to dry in the air. 



Staining 'procedure. Good results can be obtained with any of the 

 following methods, especially after familiarity has been obtained with 

 it. Special recommendation must be given to the last of the four 

 procedures (modified Bailey method). Although seeming a little 

 more complicated, on first reading, it has been found to give the 

 most uniformly satisfactory results in inexperienced hands. 



casares-gil's flagella stain^ 



AS PUBLISHED BY PLIMMER AND PAINE (1921) 



Mordant: 



Tannic acid 10 g. 



AlCls-eHoO 18 g. 



ZnCls 10 g. 



Basic fuchsin* 1-5 g. 



Alcohol (60%) 40 ml. 



The solids are dissolved in the alcohol by trituration in a mortar, adding 10 ml. of the 

 alcohol first, and the rest slowly. This alcoholic solution may be kept several years. 

 For use, mix with an equal quantity of water (Thatcher, 1926) or dilute with four 

 parts of water (Casares-Gil), filter off precipitate and collect filtrate on the slide. 



Staining schedule: 



1. Prepare smears of young cultures, on scrupulously cleaned 



slides as above directed. 



2. Filter mordant onto slide as above directed (preferably using 



Thatcher's 1:1 dilution); allow to act for 60 sec. without heat- 

 ing. 



3. Wash in tap water. 



4. Flood slide with freshly filtered Ziehl's carbol fuchsin (p. 5), 



and allow to stand 5 min. without heating. 



5. Wash with tap water. 



6. Air-dry and examine. Sometimes considerable search may be 



needed before finding a satisfactorily stained part of the 

 smear. 

 Results: Fagella well stained (red) in the case of those bacteria (e.g. 



3See Galli-Valerio (1915). 



^The authors specify rosanilin hydrochloride. There are, however, other basic 

 fuchsins more universally available which ought to prove equally satisfactory. 



