(a) |— N— H + HCHO — > — NH-CH.OH 



H 



AMINO FORMALDEHYDE METHYLOL 



GROUP 



(b) I— NH-CHoOH + H— N— I — > |— NH— CHo— HN— j + H.O 



H 



METHYLENE 

 BRIDGE 



The primary fixation effect of chromium salts (e.g., potassium dichro- 

 mate) is considered to result from formation of chromium linkages be- 

 tween proteins; however, some oxidation of protein may take place 

 during the fixation process. Chromium solutions appear to fix polysac- 

 charides by combining with aliphatic hydroxyl groups and whh lipids 

 by oxidation of unsaturated double bonds. Mercuric chloride appears to 

 fix protein structures by combination of the divalent mercuric ion with 

 sulfhydryl, carboxyl, and amino groups of proteins to produce inter- 

 molecular mercury linkages. 



Alcohol and acetic acid are components of many of the so-called 

 "chromosome" fixatives. Carnoy's fluid (acetic-alcohol) contains only 

 these two substances combined in the proportions of 3:1 and is one of 

 the most reliable and widely used fixatives for the preservation of chro- 

 mosomes. The mechanism of fixation of protein by acetic acid and al- 

 cohol apparently is somewhat different from that of formaldehyde, chro- 

 mates, and metallic ions. One of the primary effects of acetic acid on 

 tissues is the loss of bound water associated with the ionized groups of 

 proteins. This disappearance of bound water probably brings reactive 

 groups into closer proximity with each other, with the result that new 

 cross linkages are formed. Alcohol produces a similar effect, but appears 

 also to function in rendering proteins insoluble following their precipita- 

 tion. In dealing with highly hydrated structures such as chromosomes, 

 the use of fixatives that contain substances which not only dehydrate or 

 precipitate nucleoproteins but also render them insoluble, is desirable. 

 For the fixation of cytoplasmic structures, quite the opposite effect is 

 required for cytological study. Fixatives such as formaldehyde, chro- 

 mium, and osmium tetroxide solutions used in one combination or an- 

 other generally give poor preservation of chromosome structure but are 

 useful for the preservation of cytoplasmic structures such as the mito- 

 chondria. In addition to forming cross linkages between proteins, formal- 

 dehyde and chromates also fix lipids when used under appropriate con- 

 ditions. Treatment of tissues with chromates brings about oxidation of 



SURVEY OF CYTOLOGICAL TECHNIQUES / 213 



