38 MORPHOLOGY 



the empirical discovery has been made that certain staining reactions are highly 

 characteristic of certain groups of bacteria. Two such staining reactions are 

 especially important in this respect. 



The Gram Stain. — Gram (1884) described a staining method which has been of tie 

 greatest service in differentiating bacterial groups, and which has been extensively studied, 

 and frequently modified, by subsequent workers. This reaction depends on the fact that, 

 when certain bacteria are stained with certain aniline dyes, such as gentian-violet, methyl- 

 violet and others, and are subsequently treated with a solution of iodine in potassium iodide, 

 a mordanting action occurs which prevents the subsequent decolorization of the bacteria on 

 treatment with alcohol. Other bacteria, after similar treatment, are readily decolorized. 

 This difference between the retention of the stain by Gram-positive bacteria and its loss 

 by Gram-negative forms is correlated with certain other characters. Thus (liruse 1910) 

 Gram-negative organisms are more susceptible to solution in alkaUes, or to digestion by 

 enzymes, than are Gram-positive organisms ; they are also more susceptible to lysis by 

 an immune serum in the presence of complement. According to Steam and Stearn ( 1930), 

 they are more resistant to the lethal action of oxidizing, and perhaps of reducing, agents. 

 Brudny (1908) and Eisenberg (1909) would ascribe the difference between Gram-positive- 

 ness and Gram-negativeness to a difference in the permeabihty of the cell-membrane ; 

 whUe Sander (1935), who has been able to render Gram-positive bacteria Gram-negative 

 by treatment with a variety of reagents and to demonstrate that this change is reversible, 

 regards the determining factor as the state of dispersion of the bacterial protoplasm. 

 Some observers, on the other hand, and notably Deussen (1918), incline towards a more 

 purely chemical theory, and Stearn and Stearn ( 1928) regard differences in intracellular pH 

 as the determining factor. 



Recently Henry and Stacey (1943), by extraction with bile salt, succeeded in removing 

 from Gram-positive organisms a substance they identify as magnesium ribonucleate, and 

 in thereby rendering the organisms Gram-negative. If the organisms are protected from 

 oxidation, the ribonucleate will recombine and the Gram-staining property will be restored. 

 Gram-negative organisms do not yield the salt and do not combine with it to become 

 Gram-positive. The Gram-positive material appears to be a high molecular complex 

 of a reduced basic protein and magnesium ribonucleate. It was noted too that removal 

 of the ribonucleate facilitated observation of stained nuclear bodies, a fact which supports 

 Knaysi's contention that the Gram-staining material is concentrated at the cytoplasmic 

 membrane. The difference between Gram-positive and Gram-negative organisms is 

 particularly evident in their susceptibility to various groups of antibacterial agents, both 

 inorganic and organic (see Chapter 6), and it is probable that investigation of these bio- 

 chemical differences will lead to a more precise elucidation of a staining reaction first 

 introduced on an entirely empirical basis. 



In employing Gram's method it must always be remembered that the differentiation 

 is not absolutely sharp and specific as regards a given bacterium at all stages of its growth, 

 or as regards all bacterial species. Those organisms which are completely Gram-negative 

 never retain the stain, but those organisms which are Gram-positive frequently fail to 

 retain the stain when preparations are made from old cultures. This is indeed easy to 

 understand, since such cultures consist largely of dead, dying or degenerate cells, whose 

 physical and chemical properties must be greatly altered. Certam bacterial species show 

 reactions to this method of staining which are of an mtermediate type, with the result 

 that they are extremely sensitive to small changes in techiuque, sometimes appearing to 

 be Gram-positive and at others Gram-negative. Due allowance must be made for aU these 

 points in determining the reaction of any given species. 



Acid -fastness. — The fact that certain bacteria, after being stained with warm solutions 

 of fuchsin, resist the decolorizing action of strong mineral acids was observed by Ehrhch 

 (1882) and confirmed by Ziejil (1882, 1883) who modified the technique of staining. This 

 reaction is highly characteristic of the tubercle bacillus, and of an allied group of organisms 



