468 



METHODS AND FORMULAS 



DS 23.20 



logical techniques into a separate branch 

 of the science. This is very unfortunate 

 and has given rise to many misunder- 

 standings in the literature. Bacterial 

 stains can be broadly divided, as they are 

 below, into those which are applied 

 customarily to smears, and those which 

 are applied to the differential staining of 

 bacteria in cells. The former class is very 

 much the larger of the two, for the staining 

 of bacteria in cells is rarely used for diag- 

 nostic purposes, being generally confined 

 to demonstrations in bacteriology and 

 pathology classes. 



23.20 TYPICAL EXAMPLES 



Staining a bacterial film with crystal 



violet by the technique of Hucker 



(1929) 



The 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 previously 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 crystal violet stain (DS 

 23.211 Hucker 1929), and a wash bottle 

 containing distilled water. 



Take a culture from which it is desired 

 to stain the organisms, and touch the 

 freshly flamed wire loop as lightly as pos- 

 sible either to the surface of the medium 

 in a test tube or to the surface of the col- 

 ony in an agar petri-dish culture. The loop 

 is then touched lightly to the center of the 

 clean slide to transfer the bacteria. The 

 only mistakes likely to be made by the 

 beginner is securing too great a quantity 

 of material, or making too large an area. 

 It must be remembered that the specimen 

 is to be examined 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 micro-organisms have been taken 

 from a test tube containing a liquid cul- 

 ture which has not yet reached a very 

 thick stage of growth, the spot may now 



be allowed to dry in air; but if the bacteria 

 were taken from a colony on the surface 

 of agar, it is necessary to dilute them 

 with water, taken in the same platinum 

 loop, and touched to the same spot. The 

 spot is then spread with the loop, and the 

 slide is dried. 



As soon as the shde has dried, which 

 should take only a moment or two if a 

 sufficiently small quantity of fluid has 

 been used, it is taken and "heat-fixed" in 

 the flame of a bunsen burner or a spirit 

 lamp. This is done by taking the slide and 

 passing it twice quite rapidly through the 

 flame of the bunsen. The actual tempera- 

 ture should not exceed about 80°C., and 

 it is customary to hold the slide smear 

 downward as it passes through the flame. 

 Care must be taken that the slide is quite 

 dried before being thus quickly flamed, or 

 the bacteria will burst and be worthless. 



Now take the flamed and dried smear 

 and place on it a drop of the selected stain 

 (in this case crystal violet), leaving this in 

 place for about 30 seconds. The time is 

 not critical; any time between a half and 

 one minute is satisfactory. It will be 

 noticed that the stain frequently evapo- 

 rates slightly, leaving a greenish film on the 

 surface. It is, therefore, better to wash it 

 off with a jet from a wash bottle than to 

 endeavor to rinse it off. Tliis jet should be 

 directed from the fine orifice of the wash 

 bottle, at an angle of about 30°, to the 

 slide and should be intended to hit the 

 edge of the drop. This will instantly hft 

 off and float away the surface film, and 

 will also wash excess stain out of the 

 preparation. The preparation is now dried, 

 touched with immersion oil, and examined 

 under the oil-immersion lens. 



Demonstration of Gram-positive 



bacteria in smear preparations 



by the method of Gram 1884 



The name of Gram is so firmly fixed to 

 these iodine-differential techniques, that 

 it seems ■well to describe the original 

 method without modification, leaving it 

 to the technician to determine for himself 

 which of the numerous modifications, pro- 

 posed in section DS 23.212 below, best fits 

 his particular problem. For the benefit of 



