EXAMPLE 8 



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 utilize 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 7, 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 removed are known as Gram negative. The solu- 

 tions required are the violet used in Example 7 and Gram's iodine solution, 

 the formula for which is given in Chapter 7. Iodine is 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 7, taking the same precautions as 

 to dilution 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 in- 

 stance, however, it is not desirable to extract too much of the stain with 

 water. After the stain has been acting for 2 min or so, it is not removed, 

 as in Example 7, with a jet from a wash bottle, but the entire slide is rinsed 

 rapidly in a fairly large volume 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 

 than to pour iodine on it. After the iodine has been permitted to act for a 

 period of 1 min, the whole slide is given a quick rinse to remove the 



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