T H E U S E O F PERIODATE I N MICROBIOLOGICAL STAINING DERROL PENNINGTON Department o f Microbiology, University Washington School o f Medicine, Seattle 6 , Washington Received f o r publication November 1 , 1948 T h e publications o f Hotchkiss (1948) a n d McManus (1948) relating t o theuse o f periodic acid i n detecting polysaccharide structures i n fixed tissue prepara- tions prompt u s t o record t h e results o f a similar technique applied t o micro- organisms. T h e staining procedure developed independently i n this laboratory i s essentially t h e same a s that o f Hotchkiss with t h e exception that aqueous solutions a r e used a n d a reducing rinse h a s beenfound unnecessary. T h e staining o f polysaccharide structures depends upon t h e specific oxidizing action o f sodium periodate toward such chemical configurations a s a,#-glycols, a-hydroxy ketones, a n d related structures (Jackson, 1946). I n these configura- tions a t least on e o f t h e oxidation products will b e an aldehyde that c a n b e d e - tected b y treating t h e oxidized material with sulfite-decolorized basic fuchsin (Feulgen's reagent). Those portions of t h e cell that contain material oxidizable b y periodate t o yield an aldehyde will thus appear stained r e d while t h e remain- d e r o f t h e cell will remain colorless. I t c a n b e presumed from o u r knowledge o f t h e specificity o f t h e reactions involved that only polysaccharide material such a s cellulose, dextrans, levans, starch, glycogen, pectins, a n d hyaluronic acid will b e stained i n this manner. An excellent discussion o f t h e specificity o f t h e r e - action i s given b y Hotchkiss (1948). Bacteria t o b e examined a r e fixedeither b y heat, i n Bouin's solution, o r other fixative. They a r e then immersed i n a 1 p e r cent aqueous solution o f sodium metaperiodate f o r 5 t o 1 5 minutes a t room temperature. After thorough wash- i n g with water t h e preparations a r e stained f o r 1 5 minutes i n sulfite-decolorized basic fuchsin solution (Coleman, 1938). T h e slides a r e then washed f r 1 0 minutes i n sulfur dioxide water ( 5 m l o f 1 0 p e r cent K2520r a n d 5 m l 0 . 1 N H C I i n 1 0 0 m l water), finally i n water, a n d dried. RESULTS AND DISCUSSION I n applying this technique t o a variety o f organisms a number o f apparently significant structures have been observed. None o f t h e structures described below a r e stainable with iodine, n o r a r e they stained with sulfite-decolorized basic fuchsin prior t o periodate oxidation. T h e following observations illus- trate t h e results obtained with this procedure. Further work will b e necessary t o identify a nd determine t h e significance f many o f t he structures observed. Unless otherwise stated, preparations were made o n 24-hour cultures o f t h e organisms grown n nutrient agar a t t h e optimum temperature f o r each organism. Saccharomyces cereiswe. (Yeast extract glucose agar.) T h e surface o f t h e 163
