Example 7 



Demonstration of Gram Positive Bacteria in Smear Preparation 



by the Method of Gram 



For the benefit of those who are not acquainted with bacteriology, it may 

 be said by way of introduction that it has been customary since the time of 

 Gram to utihze the reactions of bacteria to iodine mordanting as a basis of 

 diagnostic classification. All bacteria, without reference to their nature, may 

 be stained by the method given in Example 6, but there are some bacteria 

 from which this stain can be removed by the action of an iodine-potassium- 

 iodide solution reinforced with alcohol. The bacteria from which the stain is 

 not removed are known as Gram positive; those from which the stain is re- 

 moved are known as Gram negative. The solutions required are the violet 

 used in Example 6 and Gram's iodine solution, the formula for which is given 

 on page 12. Iodine is very soluble in strong solutions of potassium iodide but 

 is only slightly soluble in weak solutions. If the total quantities of iodine and 

 potassium iodide are placed in the total quantity of water, a period of as long 

 as a week may elapse before a solution is complete; if the dry iodine and the 

 dry potassium iodide are mixed together and a few drops of water added, the 

 whole will go almost instantly into solution. 



Presuming that one is working with a pure culture of bacteria, a smear is 

 prepared as described in Example 6, taking the same precautions as to dilu- 

 tion mentioned there if the material is obtained from a bacterial colony. The 

 smear is dried, flamed, and a drop of crystal violet poured on it from a drop 

 bottle exactly as in the previous example. In this instance, however, it is not 

 desirable to extract too much of the stain with water. After the stain has been 

 acting for two minutes or so, it is not removed, as in Example 6, with a jet 

 from a wash bottle, but the entire slide is rinsed rapidly in a fairly large vol- 

 ume of water and a drop or two of the iodine solution poured over it. If many 

 slides are being stained, it is probably simpler to drop each slide into a coplin 

 jar containing the iodine solution rather than to pour iodine on it. After the 

 iodine has been permitted to act for a period of 1 minute, the whole slide is 

 given a quick rinse to remove the excess iodine and then placed into absolute 

 alcohol until no more color comes away; unless the film is very thick, this will 

 appear to decolorize it completely. It is passed from alcohol to water, which 

 instantly stops differentiation, and then dried. Varying types of bacteria require 

 varying periods of differentiation, but it is best for the beginner to use absolute 

 alcohol until no more color comes away rather than to endeavor to control 

 the differentiation under the microscope. 



102 



