A complex ofpolymyxin B, bovine serum albumin, and collodial gold was prepared and used for the ultrastructural localization of polymyxin B binding sites on thin sections of Epon-embedded Escherichia coli cells Gold partides were found on the outer membrane ofE coil, which is consistent with reported biochemical findings. We conduded that

Postembedding staining of intracellular carbohydrates on thin sections of Staphylococcus aureus was studied by the silver methenamine and the wheat germ agglutinin-gold techniques. Staining of silver grains was observed on both the cell wall and the cross wall. The staining was interpreted to be due to teichoic acid. Labeling by wheat germ agglutinin-gold particles was observed on both the cell wall and the cross wall, and the staining pattern resembled that of silver methenamine staining. Therefore, the labeling was considered to be due to N-acetylglucosamine of teichoic acid. The combination of two types of cytochemical techniques was useful to localize and characterize the carbohydrates of the bacterial cell.The silver proteinate and methenamine techniques have been used for the ultrastructural localization of carbohydrates in various tissues (3, 5) and bacteria (7,8,19,22) by postembedding staining. These techniques are based on oxidation of 1-2-ot-glycol bonds released by periodic acid (7,8).Recently, cytochemical techniques using lectins were employed for the localization of carbohydrates in mammalian tissues (6,12,18,20,21). The lectin-gold techniques particularly allows a high-resolution view of ultrastructural localization of carbohydrates on thin sections by postembedding staining (18, 21). This technique is based on the characteristic features of lectins, glycoproteins of nonimmune origin that bind specifically to certain sugar residues.The combination of these two techniques may be useful to localize and characterize the carbohydrates, since each technique relies on distinctively different mechanisms to detect carbohydrates. We examined the localization and characterization of the carbohydrates on thin sections of Staphylococcus aureus with silver methenamine and with colloidal gold coated with wheat germ agglutinin (WGA).Preparation of WGA-gold complex. Colloidal gold particles (15 nm in diameter) were prepared by the method of Frens (9). The WGA-gold complex was prepared by the method of Geoghegan and Ackerman (10). Briefly, the optimal amount of WGA (E. Y. Laboratories, San Mateo, Calif.) was determined to be 100 pg for stabilization of 10 ml of colloidal gold, as estimated by the salt flocculation test. After the WGAgold solution was mixed for 5 min, 1 ml of 1% (wt/vol) polyethylene glycol (molecular weight, 20,000) in 0.01 M phosphate-buffered saline (pH 7.4) was added. The mixture was centrifuged twice at 16,000 x g for 1 h and resuspended in 0.5 ml of phosphate-buffered saline containing 0.02% polyethylene glycol.Processing of bacteria. S. aureus 209P was grown in heart infusion broth at 37°C for 18 h. Cells were harvested by * Corresponding author. centrifugation and washed with 0.1 M phosphate buffer. The pellets were fixed with 1% glutaraldehyde in the buffer at 4°C for 2 h and embedded in 2% agar. After the cells were washed with the buffer at 4°C overnight, they were dehydrated by a graded series of ethanol and embedded in Spurr resin. Thin sections were cut on a Porter-Blum ultramicrotom...

We studied post- and pre-embedding staining of sugar-binding sites on thin sections of Staphylococcus aureus with an electron microscopic neoglycoprotein-gold technique. Although gold particles of cellobiosyl bovine serum albumin (BSA)-glycosylated BSA-, lactosyl BSA-, and melibiosyl BSA-gold did not label, heavy labeling of N-acetylglucosaminide-BSA-gold was observed in both the cell wall and the cytoplasm on Spurr-embedded thin sections of S. aureus. Inhibition of labeling with wheat germ agglutinin-biotin and N-acetylglucosaminidase indicated that the labeling was due to N-acetylglucosamine. These data suggested that molecules that bind specifically with N-acetylglucosamine occur in the cell wall and cytoplasm of S. aureus. Pre-embedding staining revealed that these molecules are abundant at the surface of the cell wall and that the abundance differs depending on the bacterial strain. An N-acetylglucosamine-specific lectin-like substance, glucosaminidase, and toxins are proposed as candidates for molecules responsible for the labeling, and the possible functional significance of the findings is discussed briefly.