Improvement of lysine effect on aldehyde-ruthenium red preservation and staining of the staphylococcal glycocalyx

Abstract

The cationic reagent ruthenium red (RR) has been used to enhance ultrastructural preservation and staining of the bacterial glycocalyx. Lysine was needed in prefixation to preserve the polysaccharide glycocalyx material for the gram-positive, coagulase-negative staphylococci species. The inclusion of lysine in the prefixative of glutaraldehyde (GA)-RR enhanced observation of elaborate and extensive glycocalyx material. However, prefixation in 75 mM lysine in GA-RR is restricted to 20 minutes because gelling or solidification frequently occurs at longer intervals resulting in loss of sample. To improve the utility of this approach, the effect of the Karnovsky ratio of 2% paraformaldehyde to 2.5% GA was investigated.Cells of gram-positive, coagulase-negative staphylococci species, Staphylococcus epidermidis RP62, a polysaccharide producer, and Staphylococcus epidermidis M187-SN3, a polysaccharide negative mutant, were recovered from frozen storage and grown on blood agar plates. After 24 hours, cells were transferred to trypticase soy broth for 18 hours, and incubated at 35°C. Half of the cells were prefixed in 75 mM L-lysine, 0.075% RR, 2.5% GA in 0.1 M sodium cacodylate pH 7.2 for 20 minutes.