102 BIOLOGY AND TECHNIQUE 



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.'— 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. 



Preparations are made on cover-slips or slides in the usual way. 



The preparation is then covered with an anilin gentian-violet solu- 

 tion which is best made up freshly before use. 



The staining fluid is made up, according to Gram's original direc- 

 tions,^ as follows: 



Five c.c. of anilin oil are shaken up thoroughly with 125 c.c. of dis- 

 tilled water. This solution is then filtered through a moist filter paper. 



To 108 c.c. of this anihn water, add 12 c.c. of a saturated alcohoUc 

 solution of gentian-violet. The stain acts best when twelve to twenty- 

 four hours old, but may be used at once. It lasts, if well stoppered, for 

 three to five days. A more convenient and simple method of making up 

 . the stain is as follows: 



To 10 c.c. of distilled water in a test tube add anilin oil until on 

 shaking the emulsion is opaque; roughly, one to ten. Filter this through 

 a wet paper until the filtrate is clear. To this add saturated alcoholic 

 solution of gentian-violet until the mixture is no longer transparent, 

 and a metallic film on the surface indicates saturation. One part of 

 alcoholic saturated geiitian-violet to nine parts of the anilin water 

 will give this result. This mixture may be used immediately and lasts 

 two to five days if kept in a stoppered bottle. 



Cover the preparation with this; leave on for 5 minutes. Pour off 

 excess stain and cover with Gram's iodin solution for 2 to 3 minutes. 



lodin 1 gm. 



Potassium iodid 2 gm. 



Distilled water 300 c.c. 



1 Gram, Fortschr. d. Med., ii, 1884. ^ Qrg,^^ Jqc. cit. 



