Example 6 



Staining a Bacterial Film with Crystal Violet by 

 the Technique of Lillie 



This technique is so simple that it would be scarcely worth the trouble to 

 describe it were it not necessary for the benefit of those who have never pre- 

 viously handled bacterial material and who may wish to attempt this for the 

 first time. 



The only tools and reagents necessary are a clean glass slide, a wire loop of the 

 type used normally in bacteriology, a drop bottle containing the crystal violet 

 stain, and a wash bottle containing distilled water. 



The freshly flamed wire loop is touched as lightly as possible either to the 

 surface of the medium in a test tube, or to the surface of the colony in an agar 

 petri-dish culture. Then the loop is touched lightly to the center of the clean 

 slide to transfer the bacteria. The only mistakes likely to be made by the be- 

 ginner are in securing too great a quantity of material or in making too large 

 an area on the slide. It must be remembered that the specimen is to be exam- 

 ined under an oil-immersion lens, so that the smallest possible smear derived 

 just by touching the slide with the moist platinum loop will have an ample 

 area for the purpose required. 



If the microorganisms have been taken from a liquid culture which has not 

 yet reached a very thick stage of growth, the spot on the slide now may be 

 permitted to dry in air; but if the bacteria have been taken from a colony on 

 the surface of a dish, it is necessary to dilute the drop on the slide. For this 

 purpose, a small quantity of water in the same platinum loop is touched to 

 the same spot, rubbed backward or forward once or twice, and then the slide 

 permitted to dry. 



As soon as the slide has dried, which should only be a moment or two if a suf 

 ficiently small quantity of suspension of the specimen has been used, it is taken 

 and "heat fixed" in the flame of a bunsen burner or a spirit lamp. This is done by 

 passing the slide quite rapidly twice through the flame. The actual temperature 

 should not exceed about 80° C, and it is customary to hold the slide smear down- 

 ward as it passes through the flame. Care must be taken that the slide is dried be- 

 fore it is thus quickly flamed or, of course, the bacteria will burst and be worthless. 



On the flamed and dried smear is placed a drop of the selected stain, leaving 

 this in place for about 30 seconds. The time is not critical, and any time be- 

 tween a half and one minute is perfectly satisfactory. It will be noticed that 

 the stain frequently evaporates slightly, leaving a greenish film on the surface, 

 and therefore, it is much better to wash it off" with a jet from a wash bottle 



100 



