DS 23.20 



DYE STAINS OF SPECIAL APPLICATION 



469 



those who are not acquainted with bac- 

 teriolog}^ it may be added that it has been 

 customary, since the time of Gram, to 

 utiHze the reactions of bacteria to iodine 

 mordanting as a basis of diagnostic classi- 

 fication. All bacteria, without reference 

 to their nature, may be stained by the 

 method given in the last example, but 

 there are some bacteria from which the 

 stain can be removed by the action of 

 an iodine-potassium iodide solution rein- 

 forced 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 solutions required are crys- 

 tal violet (Gram himself used the mixture 

 of dyes known as gentian violet) which 

 is usually prepared as a phenol solution 

 by the method given in Chapter 20 (DS 

 12.15). There is often a sharp argument 

 between technicians as to whether or not 

 this method of preparation is essential, but 

 it is in customary usage and should be 

 followed b}' the beginner. Gram's iodine 

 solution, the formula for which is given as 

 ADS 12.2 Gram 1884 (Chapter 22) is 

 difficult to prepare unless the technique 

 is exactly followed. Iodine is very soluble 

 in strong solutions of potassium iodide, 

 but is only slightly soluble in weak solu- 

 tions. If the total quantities of iodine and 

 potassium iodide shown are placed in the 

 total quantity of water, a period as long 

 as a week may elapse before a solution is 

 complete; but if the dry iodine and the 

 drj' potassium iodide are mixed together, 

 and a few drops of water are added, a solu- 

 tion will be produced almost instantly. 



A smear is prepared as described in the 

 last example. The same precautions as to 

 dilution there mentioned must be taken 

 if the material is obtained from a bacterial 

 colony. The smear is then dried, flamed, 

 and a drop of crystal violet poured onto 

 it from a drop bottle, exactly as in the 

 previous example. In this instance, how- 

 ever, it is not desirable to extract too much 

 of the stain with water, hence, after the 

 stain has been acting for two minutes or 

 so, the entire sUde is rinsed rapidly in 

 water, and a drop or two of the iodine 

 solution poured over it. If manj' slides are 

 being stained, it is probably simpler to 



drop the shde into a coplin jar containing 

 the iodine solution than to pour iodine on 

 it. After the iodine has acted for one min- 

 ute the 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 completely to decolorize 

 it. It is then passed from alcohol to water, 

 which instantly stops differentiation, and 

 then dried. Varying types of bacteria re- 

 quire varj'ing periods of differentiation, 

 but it is better for the beginner to use 

 absolute alcohol until no more color comes 

 away, than to endeavor to control the 

 differentiation under the microscope. 



Though such a preparation is a Gram's 

 preparation by the original technique, it is 

 customary nowadays to pro\'ide a counter- 

 stain of a contrasting color to bring clearly 

 into evidence any Gram-negative organ- 

 isms which may be mixed with Gram- 

 positive. A 1% solution of safranin is 

 widely employed, though Kopelloff and 

 Beerman 1922 (DS 23.212 below) recom- 

 mend a 0.1% solution of magenta for the 

 same purpose. In either case, the second 

 red contrasting stain is allowed to act for 

 five to ten seconds and is then washed off 

 with water. 



Demonstration of tubercle bacilli 



in sputum by the technique 



of Neelsen 1883 



When Neelsen pubUshed his original 

 technique for the demonstration of tuber- 

 cle bacilli (DS 23.213 Neelsen 1883), the 

 standard magenta solution used for the 

 staining of bacteria was that proposed in 

 the previous year by Ziehl. Because of this 

 Neelsen's technique was referred to as a 

 modification of Ziehl, and to this day the 

 hyphenated term Ziehl-Xeelsen is applied 

 to almost any method for the demonstra- 

 tion of tubercle bacilh in sputum, irrespec- 

 tive of the author of the technique. 



It is proposed here to describe the 

 original technique of Neelsen, leaving it 

 to the technician to determine which of 

 the other methods given in this section is 

 more readily applicable to his problem. It 

 may be said in favor of the technique of 

 Neelsen that it gives a better differentia- 



