The continued prevalence of nosocomially acquired infections and concomitant clinical and epidemiological interest in typing isolates have resulted in many large clinical laboratories using molecular epidemiological methods to investigate the relatedness of clinically isolated microorganisms. In the Microbiology Service, we are currently evaluating different approaches to molecular strain typing, including plasmid analysis, restriction endonuclease analysis of genomic DNA, pulsed-field gel electrophoresis (PFGE) of genomic DNA, and typing methods based on the polymerase chain reaction. The goal of this project is to develop a coordinated battery of typing methods that can be used to provide information on a wide variety of clinically significant microorganisms, permitting us to respond promptly in potential outbreak or nosocomial spread situations. Our technical development of these methods includes simplifying procedures so that they can be done rapidly and thereby be of greater epidemiological use. The use of molecular typing methods greatly expands the species of organisms for which we will be able to offer typing. Additional uses of typing availability will be to look at the relatedness of species common to particular patient populations. Thus far, the use of plasmid analysis and PFGE looks reliable and sufficiently discriminatory for staphylococci. PFGE also will be suitable for a variety of Gram-negative rods, such as Serratia. PFGE is somewhat slow and labor intensive, and we have found that, for some organisms, getting an adequate number of bands for sufficient discrimination is difficult. Our recent work with random amplified polymorphic DNA assays may prove to be of greater use for these organisms, such as roseomonas and burkholderia species. We have already used our molecular typing procedures to investigate several situations, and as a result have identified a cluster of bacteremias related to a single strain of Serratia aureus and have ruled out the possibility of nosocomial spread in another cluster of patients.