STAINING METHODS 25 



Use new slides if possible, preferably of Pyrex glass or similar heat- 

 resistant properties. (This is because under the drastic method of clean- 

 ing to remove grease, old slides have a greater tendency to break.) Clean 

 first in a dichromate cleaning fluid, wash in water, and rinse in 95 per 

 cent alcohol; then wipe with a clean piece of cheesecloth. (Wiping is not 

 always necessary but is advisable unless fresh alcohol is used after every 

 few slides.) Pass each shde back and forth through a flame for some 

 time, ordinarily until the appearance of an orange color in the flame ; some 

 experience is necessary before the proper amount of heating can be 

 accurately judged. 



Unless heat-resistant slides are used, cool sHdes gradually in order to 

 minimize breakage. An ordinarily satisfactory method of doing this is to 

 place the flamed slides on a metal plate (flamed side up) standing on a 

 vessel of boiling water and then to remove the flame under the water so as 

 to allow gradual cooling. (Too rapid cooling may result in breakage, 

 sometimes as long as 2 weeks after the heating.) 



Methods of handling cultures. Of various methods proposed, it is not 

 possible to recommend any one as uniformly the best. As any laboratory 

 worker becomes familiar with one particular method, he soon finds he can 

 get better results with that than with any other. 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 proceeding, check the culture for motility in hanging 

 drop. If motile, wash off the growth by gentle agitation with 2-3 ml of 

 sterile distilled water. Transfer to a sterile test tube, and incubate at 

 optimum temperature for 10 min (30 min for those producing slime). 

 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 pipet to one end of the sHde 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 shde.) 

 Place the shde 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 famiharity has been obtained with it. Special recom- 

 mendation 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. 



