Developments and applications with signature lipid and exopolysaccharide (EPS) methodologies covering a thirty year period in the DC White laboratories at Florida State University and the University of Tennessee at Knoxville are illustrated. These powerful techniques were used to gain new insight into microbial communities, not obtainable by classical approaches. Selected case examples are highlighted and include: use of a specific dimethyl disulphide (DMDS) derivitization procedure with monounsaturated fatty acids (MUFA) to precisely determine double bond position and geometry; application of the DMDS procedure in taxonomic and environmental studies including the degradation of pollutant halogenated hydrocarbons in groundwater and subsurface aquifers; exploiting the ubiquitous nature of uronic acids in microbial EPS to quantify these exopolymers in complex environmental samples; development of rapid and non-destructive approaches including FT-IR to follow biofilm formation in a unique manner not possible with other approaches. The foundations laid in the DC White laboratories have seen a wide suite of applications in modern microbial ecology and associated fields. The training of young scientists by DC White will also ensure that his unique approach and quest for new and or novel methodologies for use in environmental microbiology will continue.