Paraformaldehyde effect on ruthenium red and lysine preservation and staining of the staphylococcal glycocalyx

Abstract

The utility of lysine in glutaraldehyde-ruthenium red fixatives for the preservation and/or staining of the fibrous staphylococci glycocalyx was improved by inclusion of paraformaldehyde. Short, 20 min prefixation times for paraformaldehyde-glutaraldehyde fixatives containing lysine, with or without ruthenium red, were compared to an extended overnight fixation. Samples were often lost in fixatives that did not contain paraformaldehyde at extended fixation times hampering the effective use of these fixatives for clinical or environmental applications. Inclusion of paraformaldehyde in the fixation with lysine permitted longer fixation times as well as stabilized the staphylococcal glycocalyx. Thus, the technical usefulness of fixatives employing lysine was significantly improved.

Microridges are apical projections formed of branched F-actin networks that organize the glycan layer

“…S 1 B). To observe the organization of the glycan layer, we fixed samples in the presence of alcian blue and lysine, which has been shown to preserve the glycan layer better (Fassel et al, 1997(Fassel et al, , 1992Fischer et al, 2012). SEM of heads of animals fixed in such a way showed that the peridermal surface was covered by a thick glycan layer, which completely obscures the underlying microridges on the head epidermis (Figure 1 B).…”

Improved ultrastructural preservation of yeast cells for scanning electron microscopy

“…Additionally, surface structures seem to be better conserved, because the surfaces of the yeast cells were never smooth, as after conventional fixation procedures. However, preservation of glycocalyx-like structures requires special fixation techniques (Fassel et al, 1993). So far, glycocalyx-like structures have been described only for Candida albicans (Kusamichi et al.…”

“…Method D. Glycocalyx-like surface layers were preserved according to Fassel et al (1993) by adding ruthenium red and L-lysine to the glutaraldehyde fixative and to the 0.1 M cacodylate buffer. The further procedure was performed as described for method B.…”

“…Samples prepared and dehydrated as described above were transferred from 100% dehydrated ethanol through a graded series of isoamyl acetate/ethanol ( 2 5 , 50, 75, three times 100% 10min each). Then the samples were critical-point dried in a Polaron CPD7501 fixation by glutaraldehyde and osmium tetroxide: b, additional sequential fixation with 1% tannic acid and 0.5% uranyl acetate: c, conservation of surface structures after fixation according to Fassel et al (1993) followed by a fixation with 1% tannic acid and 0.5% uranyl acetate. Scale bars = 1 pm.…”

“…However, these surface layers were thinner than the surface structures which we found in Rhodosporidium toruloides, Rhodotorula spec. and Crgptocorrus albidus after using the glycocalyx preserving technique of Fassel et al (1993) (cf. Fig.…”