STAINING REAGENTS 57 



method above described, but as soon as the films have become 

 saponified, instead of at once proceeding to stain with the desired 

 dye, the film is thoroughly cleared by several alternate washings 

 with the 5 per cent, solution of acetic acid, and distilled water. 

 The subsequent procedure is as in (a). In our hands this simple 

 plan has yielded excellent results. The organisms are both clearly 

 defined and well stained. 



Staining Reagents 



There is no need for a multiplicity of staining reagents in 

 ordinary work upon milk or its bacteria.^ We have found in 

 practice that, except when engaged in special work, the following 

 concentrated solutions are quite sufficient as stock : — 



Red . . . Carbol-fuchsin. 



gi /Methylene-blue, in saturated alcoholic solution. 



I „ „ Loffler's alkaline blue. 



r Gentian violet, in saturated alcoholic solution. 

 *Jol^t • • • j 11 •! Nicolle's carbol-gentian violet (for 



'^ Gram's method). 



The above, when made up, will keep for a lengthened period, 

 and from them the watery solutions employed in staining ordinary 

 bacteria can be made up in a few moments when required for use. 

 It is advisable, however, as they will probably be exposed to the 

 full light of the working bench, that they should be stored in drop 

 bottles of a deep amber colour. Convenient forms of these bottles 

 are illustrated on Plate 8. The stains are made up as follows : — 



CarboU-fitchsiH (Ziehl-Neeken). 

 Fuchsin ....... i part. 



2 Carbolic acid (cryst) . . . . .5 parts. 



Absolute alcohol , . . . . . 10 „ 



Distilled water . . . . . . 90 „ 



Triturate the fuchsin and the alcohol in a small glass mortar ; 

 add the carbolic acid and well mix. Add by degrees to the above 

 two-thirds of the distilled water and pour into a small conical flask 

 or other convenient receptacle. Wash out the mortar with the 

 remainder of the water, and add it to the bulk. Shake well and 



^ Care should be taken, however, that whatever stains are used are fresh. 

 We have found that the best stains are those which bear the name of Griiber of 

 Leipsig, or Marck of Darmstadt. 



- Solution of carbolic acid (i in 20) may be employed in the proportion of 

 90 parts, and the carbolic crystals and distilled water omitted. The authors 

 however, prefer the formula as above. 



