The utility of lysine‐based aldehyde‐ruthenium red fixatives for the preservation and/or staining of the fibrous staphylococcal glycocalyx was improved by substitution of alternative forms of lysine for the free amino form. Paraformaldehyde‐glutaraldehyde fixatives containing alternative lysines, with or without ruthenium red, were compared at short 20‐minute prefixation times and at extended overnight fixation times. Although inclusion of paraformaldehyde made longer fixation times possible, the length of time for “safe” fixation varied per sample and could not be predicted. All alternative lysine forms permitted fixation of at least 24 hours without sample loss. The L‐lysine monohydrochloride or L‐lysine acetate forms permitted longer fixation times than the L‐lysine free amino form, and they had comparable or better preservation of the staphylococcal glycocalyx. Thus, the usefulness of aldehyde‐lysine‐based fixatives with minor changes has been enhanced. Microsc. Res. Tech. 41:291–297, 1998. © 1998 Wiley‐Liss, Inc.