Escherichia coli O157:H7 is a major cause of foodborne illness and methods for rapid and sensitive detection of this deadly pathogen are needed to protect consumers. The use of immunomagnetic separation (IMS) for capturing and detecting foodborne pathogens has gained popularity, partially due to the introduction of automated and high throughput IMS instrumentation. Three methods for automated IMS that test different sample volumes, Kingfisher mL, Pathatrix Auto, and Pathatrix Ultra, were compared using microbiological detection of E. coli O157:H7 from buffered peptone water (BPW), in the presence of background microbial flora derived from spinach leaves, and from culture enrichments from artificially contaminated spinach leaves. The average efficiencies of capture of E. coli O157:H7 using the three methods were 32.1%, 3.7%, and 1.3%, respectively, in BPW; 43.4%, 8.8%, 2.9%, respectively, in the presence of spinach microbial flora; and 63.0%, 7.0%, and 6.3%, respectively, from artificially contaminated spinach. Despite the large differences in IMS capture efficiencies between the KingFisher and two Pathatrix methods, all three methods allowed the detection of E. coli O157:H7 from spinach that was artificially contaminated with the pathogen at relatively high (25 cfu/30 g sample) and low (1 cfu/30 g sample) levels after 4-6h of culture enrichment. The differences in capture efficiency were compensated for by the differences in sample volume used by the KingFisher mL (1 mL), Pathatrix Auto (50 mL) and Pathatrix Ultra (250 mL) instruments. Thus, despite the reduced capture efficiencies observed for the Pathatrix methods, the large increase in sample volume results in a greater number of captured cells for downstream detection resulting in improved detection sensitivity.