108 Microbes and You 



more readily observable, and (2) Differential Stains in which 

 technics are used to divide bacteria into groups or to bring out 

 some specific structure of organisms. 



The idea of staining microbes was first introduced into micro- 

 biology by Carl von Weigert in 1871 when he first stained bacteria 

 with carmine and later aniline dves. Dozens of dyes have come 

 into common use since those early experiments of Weigert. A dye 

 is a colored organic compound which has the ability to combine with 

 certain substances and to impart color to them. It is possible to en- 

 hance staining ability by adding intensifiers. For example, if a 

 basic dye solution is made more alkaline, bacteria tend to stain 

 more intensely. The introduction of surface tension depressants 

 (wetting agents) may allow more intimate contact of dye with 

 protoplasm. By applying heat and by adding carbolic acid 

 (phenol) it is possible to intensify the dye-protoplasm union. 

 Staining is a chemical or a physical union between the dye and 

 components of the cell. If it is a chemical reaction, a new com- 

 pound is foiTned, and simple washing in water does not liberate 

 the bound dye. But if it is mere physical union, it is easier, as a 

 rule, to decolorize such organisms. Many staining reactions are 

 undoubtedly a combination of physical and chemical unions. 



NEGATIVE STAINS 



Negative stains, including India ink, nigrosine, and Congo red, 

 do not have an affinity for bacterial protoplasm. Negative staining 

 is also known as relief staining, since the background material 

 retains the dye while the bacteria stand out in relief as unstained 

 areas. To prevent bacterial growth in these dyes during storage— 

 a common occurrence and a troublesome one— the addition of 0.5% 

 formalin is advocated. 



India ink is a fine suspension of carbon particles in an aqueous 

 gelatinous medium, but because it lacks uniformity this dye is not 

 as popular as some of the others, particularly nigrosine which is 

 a colloidal suspension. When Congo red is the dye being em- 

 ployed, some persons recommend that the completed preparation 

 be dipped in acid-alcohol (1% hydrochloric acid in ethyl alcohol) 



