AN IMPROVED SILVER METHENAMINE TECHNIQUE FOR THE DETECTION OF PERIODIC ACID-REACTIVE COMPLEX CARBOHYDRATES WITH THE ELECTRON MICROSCOPE

Itt 194li, ( onssoni (_lmei. ,J, (‘tin. Path. 10: 177. 1946) ititrodutced a histochsenical test bmused onu the itse of silver noethenamitse tufter chromic acid oixidtit ion for I he demonsst rat lomi tif conplcx cturbohydrates ins the bight nsicroscope. Later, ti-ic silver nethenamimic techmuiqimc wtts mudapted to the dcctromu microscope, butt the oxidmutiots doimsc with periodic muck! rmuther thmutu with chsronic acid (L)ettmer amid Schwarz, Z. 1I’iss. llikr. 61: 423. 1954; Jonues, Amer. J. Path. 33: 313. 1957; Chuurg et al., . 1. Biophys. Biochem. (lytol. 4: 841. 1958; Marl noizzi, Proceedings of the European Conference on Electron Microscopy, edited! by Ilouuwitik and Spit, Vol. 2. 1)etiedcrlmumudsc Venemuiging voor Elect rotimicrosctipie, 1)ebft ,1960, 626; . J. Biophys. Biochem. (‘ytol. 9: 121. 1961; .J. Roy. Micr. Soc. 81: 141. 1963; Movat, Amer. J. Clin. Path. 35: 528. 1961; Sutzuuki ansd Sekiyama, J. Electronmicr. (Tokyo) 10: 36. 1961; Thiery, Proceedings of the Enropean Conference on Electron . llicroscopy, edited by Ilouwitsk and Spit, Vol. 6, I)cmuedcrlattdse Veremiigitug voor Elect romu microscopic, Delft, 1964, l 209; ilobbmatstu, J. 111cr. 4: 701. 1965; Marx anid Molbert, J. Micr. 4: 799. 1965; Van Heytuingen, J. Histochem. Cytochem. 13: 286. 1965; Foblemsiuss amid 1)oerr-Schott, C. II. Acad. Sci. Paris 262: 912. 1966). however, itt 1954, Lillic (.1. Histochem. (‘ytochein. 2: 127. 1954) cast douubt oti tue specificity of the peni(idic acid-silver methenamine techmtiquse; itsdeed, his opitsiotu as well as the difficunlties encointstcrcd in the uuse of the techstsiquse discredited it among histochemists. Thums Revel states (J. Histochem. Cytochem. 14: 740. 1966): “Efforts to develop more specific staimis, based on periodate oxydatioms of diols followed by redumction of silver, have nstit yet beemt especially sumccessfutl. ” its our owts experience difficulties were imideed emicoututered, yet it was poissible to make effective umse tif the periodic acid-silver methetsansimic techmsiquue toi demot-istrate the carbohydrates of the cell coat (Ramboutrg amid Leblonsd, J. Cell Biol. 32: 27. 1967). More recemitly, the stainsimug proicedure has beemu futrther insproved and conssistently reprodutcibbe resmbts were tibtained with a minimum of precipitates. Ins view of the importansce of technical details, a meticutlotus descniptiots of our improved procedutre is msow given. Soltttiotss: Methenamine (hexamethylenetetra in me): freshly nade 3% sobtutioni in-i listilbed water. Silver nitrate: freshly made 5% solution imu (histilled wmutcr. Sodium borate: 2 3 soltutitits itt distilled wmiter, which may be kept for 1 week at rooni tcmpcrtuutunc. Periodic acid: 1% solumtioms its distilled wtuter which may be kept itudefimsitely at rtiom temisl)ertut Smre. Sodium thiosulfate: solut lots its dist illeth wmuten which mmuy he kept itsdcfinsitely at room temperatitre. Glassware: Three ttew 100-mb Pyrex cyhitudcns are umsed toi preptunc the nsethetiamitsc, silver niltnatc amid sodiuum borate solumtions, respectively. The san-ic cylimuder shouuld abwtuys be utsed for the sanse soluttiotu. They tune washed with somup after itse atsd rimised with tap amsd distilled water. A 10-mb Pyrex cylimsdcn which-i serves to add silver tsitratc tumid sodiuun borate to methctsamimse is cleamsed its the same mantsen. A 100-mI Pyrex cylimider amid tutu Enlemunseyer flask which will consttuin the silver methemutumimse reagent amid its filtrmute, respectively, nuuust be clemutied its the stmlfochnomic mixtumre atid! nimise(l thoiroumghly with tap tumid distilled water. The Petni dishes used for stmuimsinug tune of Pyrex hintum-id gltuss (no. 8-747 im Fisher’s catmuloguc), 60mm in-i dianseten x 15 n-im its height, which arc muot tuflccted chenically. They are kept its the suulfochnomic mixture mum-id thoroughly rinssed wit is tap arid distilled wtuter just before umse. They muust be discarded as sooits as silver precipitations against the walls shoiws a tcndemscy to imscnease, uusuiably after 10-15 days. The dimettsioimss of these Petni dishes tune critical sinsce the surface-volumme-temperatutne rtutio of the staining solution must be kept constant to avoid precipitation. Silver methenamine reagent: The preparation of the reagetut tund the staining proeedumre tune done in-i a dark room. Five milliliters of the silver nitrate solutions are measutned in-i a 10-ml cylinder and added to 45 cc methetamitse soluttion. A white precipitate appears which may be dissolved by shaking. The final soltutioin is obtained by adding 5 ml sodium