132 NITROGEN METABOLISM 



sodium salt of the nucleic acid can then be precipitated by 

 the addition of ethanol, or, alternatively, the free nucleic 

 acid by the addition of acidified ethanol or glacial acetic acid. 

 The staining procedure introduced by Christian Gram to 

 reveal the presence of bacteria in animal tissues was subse- 

 quently developed into an empirical technique for dividing 

 bacteria into two groups. After the fixed organisms have 

 been stained with a basic dye (crystal violet or methyl violet) 

 at pH 7-8, they are treated with a mordant, usually 1 2 in KI, 

 and then washed with ethanol or acetone. If the stain is 

 quickly removed, the organism is said to be Gram-negative; 

 whereas if the dye remains, it is regarded as being Gram- 

 positive. After the ethanol or acetone treatment, it is now 

 common practice to counterstain with a red dye, with the 

 result that in the final preparation Gram-positive organisms 

 are stained blue whilst Gram-negative organisms are red. 

 Though the mechanism of this staining reaction is still a 

 matter of dispute [see 3, 41], retention of the basic dye by 

 Gram-positive bacteria appears to be due to the presence of 

 a Mg'^'^-PNA complex in the peripheral layers of the cells. 

 The evidence for this belief rests on the observation that 

 after treatment of the dead cells with ribonuclease — an 

 enzyme depolymerizing PNA (p. 1 34) — or with a detergent 

 such as bile salts. Gram-positive organisms stain as though 

 they were Gram-negative. The action of the detergent is to 

 liberate PNA from the cells and this material has been iso- 

 lated by Henry and Stacey, who found that in the presence 

 of Mg"*"^ and a reducing substance, the isolated PNA, 

 or indeed yeast RNA, would restore Gram-positive stain- 

 ing properties to the appropriate Gram-negative cytoskele- 

 tons [21], but not to truly Gram-negative organisms. Though 

 the ratio of PNArDPNA was reported to be 8:1 in Gram- 

 positive as opposed to 1-3:1 in Gram-negative bacteria, these 

 figures are disputed by Mitchell and Moyle who claim that 

 the ratio is about 4:1 in all bacteria, irrespective of their 

 staining properties [41]. Moreover, the presence of a peri- 

 pheral layer containing PNA may not be a complete ex- 

 planation of the structure responsible for the Gram-staining 



