BACTERIOLOGICAL TECHNIC. 



73 



with a solution of iodine and washed in alcohol, give up the stain. Such 

 microbes are known as Gram-negative, whereas those which do not give up 

 the stain are said to be Gram-positive. 



The method of using this stain is somewhat complicated, requires care, 

 and, with a beginner, often yields disappointing results. Keeping in mind 

 the following will minimize the disappointments: 



a. Long-continued (one year or more) subcultures frequently lose the 

 Gram-stain behavior. 



b. Old cultures, that is, those which have been growing in the same 

 medium for several days or more, as a rule do not stain characteristically. 

 With such cultures the results are often neither negative nor positive, just 

 enough to be confusing and perplexing. 



c. The solutions used must be fresh. The gentian-aniline solution, as 

 well as the iodine solution, deteriorates quite rapidly. 



d. Do not overstain, and do not decolorize too long. Stop decolorizing 

 as soon as no more violet color comes away. 



In the Gram method two solutions are used, namely: 



1. Aniline gentian-violet, and 



2. Gram's iodine solution. 



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 minute' 

 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 dropping 

 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 



