190 



Journal of Applied Microscopy. 



Laboratory Methods in Bacteriol- 

 ogy. 



Dr. F. G. Now. 

 III.— Gram's Method. 



With the exception of the tubercle 

 bacillus, the methods of staining hereto- 

 fore described are simple in character. 

 That is to say, the germ and background 

 are stained alike. In the case of the 

 tubercle bacillus, special double-staining 

 was resorted to, because this reaction is 

 so characteristic as to enable immediate 

 recognition of the organism. Only two 

 or three other organisms are known 

 which will double stain when treated by 

 the same method. 



There is, however, a method of double 

 staining which is applicable to a large 

 number of bacteria. This process, known 

 as Gram's method, is based upon the fact 

 that the protoplasm of certain bacteria 

 forms a difficultly soluble compound, 

 when stained with anilin-water gentian 

 violet and subsequently treated with 

 iodine. On treatment with alcohol the 

 dye is washed out of everything on the 

 specimen except out of the germs. The 

 deeply stained violet bacteria lie now on 

 a colorless background which, on stain- 

 ing with a contrast color, such as eosin, 

 becomes stained a light pink. The 

 method is as follows: 



A solution of anilin-water gentian 

 violet is first prepared. Anilin oil is 

 placed in a test tube to a depth of about 

 half an inch. The tube is then filled with 

 water, closed with the thumb, and thor- 

 oughly shaken in order to obtain a satu- 

 rated aqueous solution of anilin. The 

 liquid is then passed through a small 

 filter, and collected in another test tube. 

 The filtrate should be perfectly clear, not 

 cloudy. To the anilin water thus 

 obtained a saturated alcoholic solution of 

 gentian violet is added till the fluid is 

 deeply colored, rendered opaque. This 

 result is obtained when about one-half 

 cubic centimeter of the gentian violet 

 solution is added to ten cubic centimeters 

 of the anilin water. 



Some of the anilin water gentian violet 

 thus prepared is poured out into a watch 

 glass. The cover-glass preparation is 

 prepared in the usual way, dried in the 

 air and then fixed by passing through a 

 flame. The fixed cover-glass is placed 

 between the thumb and forefinger, with 

 the specimen side down, and then care- 

 fully dropped upon the surface of the 

 stain. It is allowed to float on the dye 

 for three to five minutes. 



The cover-glass is then picked up with 

 the forceps, thoroughly washed with 

 water, and immersed in a solution of 

 iodine in potassium iodide. This is made 

 by dissolving two grams of potassium 



iodide, and one gram of iodine in 300 

 cubic centimeters of distilled water. The 

 specimen is allowed to remain in the 

 iodine solution for three to five minutes. 

 It is then removed, washed with water, 

 and placed in 95 per cent, or in absolute 

 alcohol. If the specimen has not been 

 overstained, decoloration will take place 

 rapidly. This may be assisted by gently 

 tilting the dish, or by moving the 

 specimen. 



From time to time the cover-glass 

 should be washed with water, placed on 

 a slide, and examined with a one-sixth 

 inch objective to ascertain the progress 

 in decoloration. If the material has been 

 spread out in a thin, even layer the de- 

 coloration will be rapid and thorough. On 

 the other hand, if thick masses are pres- 

 .ent it will not be possible to obtain com- 

 plete decoloration, without decoloring at 

 the same time many of the bacteria. 

 When, therefore, the greater part of the 

 background has been decolored the treat- 

 ment with alcohol should be discontinued. 



The cover-glass is then washed with 

 water, and stained with dilute eosin for 

 one-fourth to one-half minute. Care 

 must be taken not to overstain the pre- 

 paration with eosin, since this would 

 diminish the sharp contrast desired. 

 After staining with eosin the specimen is 

 thoroughly washed with water, and 

 examined under the microscope. It 

 should show the deeply stained violet 

 bacteria on a light pink background. 



Gram's method is applicable to many 

 non-pathogenic and pathogenic bacilli 

 and micrococci. A number of important 

 disease bacteria are not stained by this 

 method. Among these may be mentioned 

 the gonococcus, the germs of typhoid 

 fever, Asiatic cholera, influenza, black 

 plague, glanders, and chicken cholera. 



The method is applicable for staining 

 the micrococci present in pus, the germs 

 of erysipelas, diphtheria, tuberculosis, 

 leprosy, actinomycosis, anthrax, etc. It 

 is especially valuable when endeavoring 

 to detect these organisms in material rich 

 in organic matter, such as blood and pus. 

 Most excellent results are obtained when 

 the method is applied to sections of 

 tissue. 



The beginner can familiarize himself 

 with the method by applying it to the 

 staining of tubercle bacilli in sputum. 

 It can also be tried for the detection of 

 the diplococcus of pneumonia in the 

 "rusty" sputum of that disease. 



The method described may be summar- 

 ized as follows: 



Anilin-water gentian violet (three to 

 five minutes). 



Water, 



Iodine in potassium iodide (three to 

 five minutes), 



