210 Silver Impregnation II (chap. 15) 



Hamlyii (1957) and Guillery et al. (1961) describe methods for paraf- 

 fin-embedded sections that are stained after mounting. 



Marchi Method B 



fixation: 10% formalin plus 1% of potassium chlorate: 24-48 hours, 

 no longer. (Sivank and Davenport, 1934) 



solution: 



Marchi fluid (Poirier et al., 1954): 



osmic acid, 0.5% aqueous II. ml. 



potassium chlorate 1% aqueous 16.0 ml. 



formalin 3.0 ml. 



acetic acid, 10% aqueous 3.0 ml. 



distilled water to make 100.0 ml. 



procedure: 



1. Cut tissue into thin slices — abotit 3 mm. for easier impregnation. 



2. Chromate, 2.5% potassium dichromate, aqtieous, in dark: 7-14 

 days, change twice. 



3. Transfer directly to Marchi fltiid, a vohime 15-20 times that of 

 tisstie: 1-2 weeks depending on size of tissue. Turn tissue over 

 every day to improve penetration. 



4. Wash in rtnining water: 24 hours. 



5. Dehydrate and embed. If using celloidin, keep embedding steps 

 to a minimum. If tising paraffin, avoid xylene and its solvent ac- 

 tion on osmic acid. Chloroform is safer; also keeps steps to mini- 

 mum. 



6. Deparaffinize slides with chloroform and mount in chloroform- 

 resin. 



results: 



degenerating myelin — black 

 background — brownish yellow 

 neutral fats — black 



comments: 



The principle behind the Marchi method is based on the fact that 

 myelin of medullated nerves oxidizes more easily than degenerated 

 myelin. Normal myelin is oxidized by c hromating and will not react 

 with osmic acid. Degenerate myelin contains oleic acid which does 

 not oxidize during chromating and therefore redtices the osmic acid 

 and stains black. 



