lOO PHARMACEUTICAL BACTERIOLOGY 



d. Donotoverstain, and do not decolorize too long. Stop decolorizing 

 as soon as no more violet color conies away. 



In the Gram method two solutions are used, namely: 



1 . Aniline gentian- violet, and 



2. Gram's iodine solution. 



Iodine, i gm. 



Potassium Iodide, 2 gm. 



Distilled Water, 300 cc. 



The method, briefly outlined, is as follows : 



a. Spread the bacteria evenly and thinly over the cover-glass (the 

 usual smear preparation). Stain with the aniline gentian- violet for from 

 two to five minutes. Warming will hasten and intensify the staining. 

 Wash in water to remove excess of stain. 



b. Drop on the iodine solution and allow it to act for about one min- 

 ute or until the preparation assumes a coffee-brown color. It may be 

 desirable to apply the iodine a second time. 



c. Wash off the excess of iodine in water and then decolorize by drop- 

 ping on 95 per cent, alcohol. Tip the slide and allow alcohol to run over 

 the preparation; continue until the violet color ceases to stream away. 



d. Finally rinse in water and examine in water. If desired, dry and 

 mount permanently in Canada balsam or some other suitable mounting 

 medium. 



e. A contrast stain, such as eosin, fuchsin, safranin, or Bismarck 

 brown, may be used, following (c). 



Keeping in mind the difficulties already referred to in using the Gram 

 method, and the additional possible source of error due to the fact that 

 one and the same microbe will stain but feebly at one time and very 

 intensely at another time, we now name the principal organisms which 

 are Gram-positive or Gram-negative. 



Bacteria and other Organisms Stained by the Gram Method 



Staphylococcus pyogenes aureus. 



Staphylococcus pyogenes albus. 



Streptococcus pyogenes. 



Micrococcus tetragenus. 



Micrococcus lanceolatus. 



Bacillus diphtheriae. 



BacUlus tuberculosis. 



Bacillus of anthrax. 



Bacillus of tetanus. 



Bacillus of leprosy. 



Bacillus aerogenes capsulatus. 



Oidium albicans. 



Actinomyces (of Actinomycosis). 



