68 CYTOLOGICAL TECHNIQUE 



tissue, and thus stop its progress. Among the common primary 

 fixatives, however, only formaldehyde and osmium tetroxide fix 

 gelatine. 



Gelatine is a chemically modified form of collagen, an insoluble 

 protein distinguished by its high content of glycine, proline, and 

 hydroxyproline. 



I 

 N-CH3 



\ 

 CHo 



/ 

 HC— CH2 



1 



1 

 Proline as part of a protein chain 



{The middle ^CH, group is replaced by 



^CH.OH in hydroxyproline.) 



The collagen used in the preparation of gelatine is derived from 

 the organic matter in the bones of cattle and from the white con- 

 nective tissue fibres in the skin of cattle and pigs. Bones are first 

 decalcified with hydrochloric acid. The skin or bone is 'limed' for 

 several weeks in an aqueous solution of calcium hydroxide; the 

 soluble proteins are thus dissolved away. After having been 

 washed with water, the material that has resisted solution is kept 

 in clean water at about 60^ C for several hours. In this it dissolves. 

 It is filtered, cooled to produce a gel, and dried. ^°- The dry 

 material is sold in the form of sheets or powder. The latter is the 

 more convenient form for use in the laboratory. 



The transformation that is undergone by collagen during the 

 long treatment with limewater is reflected in the change of the 

 iso-electric point from about pH 7-8 to pH 4-7.^^ This results from 

 the conversion of amide-groups in the asparagine and glutamic 

 acid of the protein chain to the carboxyl groups of aspartic acid 

 and glutamic acid respectively. It is this change that makes gela- 

 tine so much more readily colourable by basic dyes than collagen 

 is. Various bonds that tie the protein chains together, such 



