P— OH P— O- P— O- 



III +H+ III -H+ III 



-NaH— NH3+< 1 NA |— NHo+ > | NA |— NHo 



C— OH C— OH C— OH 



POSITIVE CHARGE NUCLEIC ACID NEGATIVE CHARGE 



(Zwitterion) 



The isoelectric point of nucleic acids is considered to be pH 2 or less. 

 Because the dissociation of free acidic (i.e., COO~) groups of most 

 proteins is minimal at pH's less than 4.0, it is possible to selectively stain 

 nucleic acids with basic dyes such as Toluidine blue O and Azure B at 

 low pH levels (between pH 2.0 and 4.0) . 



After fixation, cell structures show a pronounced affinity for both 

 acidic and basic dyes. This is not unexpected when one recalls that the 

 primary action of a fixing agent is on the proteins of the cell and that, 

 in most instances, the proteins are precipitated and made insoluble by 

 the fixative. In addition to the general increase in stainability of cells 

 following fixation, there appears to be a preferential uptake of either 

 acidic or basic dye depending on the method of fixation employed. For 

 example, formaldehyde fixation enhances basic dye uptake whereas 

 mercuric chloride favors acid dye uptake. This selective effect of fixation 

 on dye uptake is mostly the result of combination of the fixing agent 

 with specific groups of proteins, thus making them unavailable for com- 

 bination with certain groups of the dye. For example, the increase in 

 acid dye uptake following fixation of tissues in salts of metallic ions 

 (HgClo) or dicromates (K2Cr04) is probably the result of combination 

 of the mercury or chromium ions with free acidic groups of proteins, 

 especially carboxyl and hydroxyl, and the phosphoric acid of nucleic 

 acids. In this way, more basic groups are made available for dissociation 

 and combination with acid dye. Acid fixatives such as acetic-alcohol 

 (Carnoy's) give excellent preservation of chromosomes by precipitating 

 the nucleoproteins and breaking the linkages between the nucleic acids 

 and proteins in these structures. As a result, the number of acidic groups 

 of chromosomal proteins and nucleic acids is increased to give strong 

 staining with basic dyes. 



Cytochemical Staining 

 Most of the dyes commonly used to reveal cell structure are nonspe- 

 cific in the sense that they do not stain selectively any one molecular 

 grouping or substance in the cell. The only exception is the Feulgen 

 reaction which is specific for DNA and is routinely employed as a 



218 / CHAPTER 11 



