74 PRINCIPLES OF BACTERIOLOGY 



bolic acid the saturated alcoholic solution of fuchsin until a metallic 

 lustre appears on the surface of the fluid. The carbolic acid, like the 

 alkali, favors the penetration of the stain. 



The last two methods, combined with heating, are used to stain spores 

 and certain resistant bacteria intensely, so that they retain their color 

 when exposed to decolorizing agents. 



Carbolic methylene blue, first used by Kiihne, consists of 1.5 gm. 

 of methylene blue, 10 gm. of absolute alcohol, and 100 c.c. of a 5 

 per cent, solution of carbolic acid. Carbolic thionin consists of 10 

 parts of a saturated alcoholic solution of thionin and 100 parts of a 

 1 per cent, solution of carbolic acid. 



GRAM'S STAIN. Another differential method of staining which is 

 employed is that known as Gram's method. In this method the objects 

 to be stained are floated on or covered with the aniline or carbolic gen- 

 tian-violet solution. After remaining in this for a few minutes they 

 are rinsed in water and then immersed in an iodine solution (LugoPs), 

 composed of iodine, 1 gm.; potassium iodide, 2 gm.; distilled water, 

 300 c.c. In this they remain for from one to three minutes and are 

 again rinsed in water. They are then placed in strong alcohol until 

 most of the dye has been washed out. If the cover-glass as a whole 

 still shows a violet color, it is again treated with the iodine solution, 

 followed by alcohol, and this is continued until no trace of violet color 

 is visible to the naked eye. They may then be washed in water and 

 examined, or a contrasting color of eosin, fuchsin, carmine, or Bis- 

 marck brown may be given them by inserting them in weak solutions 

 of these dyes for a few minutes. This method is useful in demonstrat- 

 ing the capsule which is seen to surround some bacteria particularly 

 the pneumococcus and also in differentiating between varieties of 

 bacteria, for some do and others do not retain their stain when put in 

 the iodine solution for a suitable time. 



Staining of Capsules. Many methods of demonstrating the cap- 

 sule have deen devised. Two only will be given here. The glacial 

 acetic acid method, as described by Welch, is as follows: 1. Cover the 

 preparation with glacial acetic acid for a few seconds. 2. Drain 

 off and replace with aniline gentian-violet solution; this is to be repeat- 

 edly added until all the acid is replaced. 3. Wash in 1 to 2 per cent, 

 solution of sodium chloride and mount in the same. 



Hiss' COPPER SULPHATE METHOD. The organisms are grown, if 

 possible, on ascitic fluid or serum media. If not, spread the organisms 

 on the cover-glass by mixing with a drop of serihn, or, better, a drop 

 of one of the diluted serum media. Dry in the air and fix by heat. 



The capsules are stained by the following method: A 5 per cent, or 

 10 per cent, aqueous solution of gentian violet or fuchsin (5 c.c. saturated 

 alcoholic solution gentian violet to 95 c.c. distilled water) is used. This 

 is placed on the dried and fixed cover-glass preparation and gently 

 heated for a few seconds until steam arises. The dye is washed off 

 with a 20 per cent, solution of copper sulphate (crystals). The prep- 

 aration is then placed between filter-paper and thoroughly dried. 



