120 BIOLOGY AND TECHNIQUE 



sufficient to saturate the fluid. The bottle is then shaken and allowed to 

 cool. Ten c.c. of this solution are added to 10 c.c. of freshly prepared ten 

 per cent tannic acid solution. To this add 5 c.c. carbol-fuchsin solution. 

 Mix and filter. 



To stain, filter the above mordant directly upon the fixed cover-slip 

 preparation. Heat gently for three minutes, but do not allow to boil. Wash 

 in water and stain with the following solution : 



Saturated alcoholic solution gentian >violet 1 c.c. 



Saturated solution ammonia alum 10 c.c. 



Filter the stain directly upon the preparation and heat for three or four 

 minutes. Wash in water, dry, and mount in balsam. 



Differential Stains. GRAM'S METHOD. IS By this method of 

 staining, which is extremely important in bacterial differentiation, 

 bacteria are divided into those which retain the initial stain and 

 those which are subsequently decolorized and take the counterstain. 

 The former are often spoken of as the Gram-positive, the latter as 

 Gram-negative bacteria. 



The reasons for the differential value of Gram's method are not 

 entirely clear, but must, of course, depend upon peculiarities of the 

 chemical constituents of the bacteria themselves. A considerable 

 amount of work has been done on the subject which has not been 

 entirely conclusive. A discussion of the subject may be found in 

 Wells' "Chemical Pathology," Second Edition, page 105. Wells 

 states that the results of Gram's method may be ascribed to the 

 formation of an iodin-pararosanilin-protein compound in the Gram- 

 positive bacteria. Only dyes of this group, namely, gentian violet, 

 methyl violet, etc., will form such combination. He quotes Burgers 16 

 as stating that trypsin will digest Gram-negative, but not Gram- 

 positive bacteria, and the gastric juice affects only a few of the 

 Gram-positive species. It has also been suggested that the fatty 

 substances in the bacterial bodies may have some relationship to 

 the Gram-stain, and that the bacterial cell wall is the part most 

 directly involved in the staining reaction. Benians 17 has found that 

 when Gram-positive bacteria are artificially disintegrated they lose 

 their characteristic reaction and become Gram-negative, and this 

 has been confirmed for tubercle bacilli in Wells' laboratory by 



15 Gram, Fortschr. d. Med., ii, 1884. 



18 Burgers and collaborators, Zeit. f. Hyg., 1911, 70'. 



17 Benians, Jour, of Pathol. and Bacter., 17, 1912, 199. 



