GRAM'S STAIN 105 



Films may l>e stained with Loflier's blue by five minutes' exposure or 

 longer in the cold. They usually do not require decolorisation, as the 

 tissue elements are not overstained. 



2. Kiihnts Methylene-bluc. 



Methylene-blue .... l'5grm. 

 Absolute alcohol .... 10 c.c. 

 Carbolic acid solution (1-20) . . 100 ,, 



Stain and decolorise as with Loffler's blue, or decolorise with very weak 

 hydrochloric acid (a few drops in a bowl of water). 



3. Carbol'Thionin-blue.^lakc up a stock solution consisting of 1 

 gramme of thionin-blue dissolved in 100 c.c. carbolic acid solution (1-40). 

 For use, dilute one volume with three of water, and filter. Stain sections 

 for five minutes or upwards. Wash very thoroughly with water, other- 

 wise a deposit of crystals may occur in the subsequent stages. Decolorise 

 with very weak acetic acid. A few drops of the acid added to a bowl 

 of water are quite sufficient. Wash again thoroughly with water. 

 Dehydrate with absolute alcohol. Thionin-blue stains more deeply 

 than methylene-blue, and gives equally good differentiation. It is very 

 suitable for staining typhoid and glanders bacilli in sections. Cover- 

 glass preparations stained by this method do not usually require 

 decolorisation. As a contrast stain, 1 per cent, watery solution of eosin 

 may be used before staining with the thionin. 



4. Gentian-violet in Aniline Oil Water. Two solutions have here to 

 be made up. (a) Aniline oil water. Add about 5 c.c. aniline oil to 

 100 c.c. distilled water in a flask, and shake violently till as much as 

 possible of the oil has dissolved. Filter and keep in a covered bottle 

 to prevent access of light. (6) Make a saturated solution of gentian- 

 vi'.l.-t in alcohol. When the stain is to be used, 1 part of (b) is added 

 to 10 parts of (a), and the mixture filtered. The mixture should be made 

 not more than twenty- four hours before use. Stain sections for a few 

 minutes ; then decolorise with methylated spirit. Sometimes it is 

 advantageous to add to the methylated spirit a little hydrochloric acid 

 (2-3 minims to 100 c.c.). This staining solution is not so much used 

 by itself as in < J ram's method, which is presently to be described. 



5. Carbol-Gentian-Violet. 1 part of saturated alcoholic solution of 

 gentian-violet is mixed with 10 parts of 5 per cent, solution of carbolic 

 acid. It is used as No. 4. 



6. Carbol-Fuchsin (see p. 1C8). This is a very powerful stain, and, 

 when used in the undiluted condition, -1 minute's staining is usually 

 sufficient. It is better, however, to dilute with from five to ten times 

 its volume of water and stain for a few minutes. In this form it has a 

 very wide application. Methylated spirit with or without a few drops 

 of acetic acia is the most convenient decolorising agent. Then dehydrate 

 thoroughly, clear, and mount. 



Gram's Method and its Modifications. In the methods 

 alivady described, the tissues, and more especially the nuclei, 

 ivt;iiu some stain when decolorisation has reached the }>oint to 

 which it can safely go without the bacteria themselves bein^ 

 affected. In the method of Gram, now to be detailed, this does 

 not occur, for the stain can here be removed completely from 



