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Tryptic soy broth (TSB)-grown cells of Staphylococcus aureus isolated from acute and chronic bovine mastitis bound mainly 125I-fibronectin (125I-Fn), whereas strains of nine species of coagulase-negative staphylococci showed a predominant interaction with 125I-collagen (125I-Cn) type I. A particle agglutination assay (PAA) was used to examine the interaction of coagulase-negative staphylococci with 1251-Fn and 125I-Cn immobilized on latex. All 368 coagulase-negative staphylococci demonstrated high 125I-Cn and moderate to low 125I-Fn interactions in the PAA. Cn-PAA reactivity was high among strains of Staphylococcus xylosus (84.2%), Staphylococcus simulans (77.8%), Staphylococcus epidermidis (76.7%), and Staphylococcus hyicus (74.3%), whereas all six Staphylococcus capitis strains clumped Cn-PAA reagent. Incubating TSB-grown cells in 10% skim milk for 1 h decreased the 125I-Fn- and 125I-Cn-binding affinity in most of the S. aureus and coagulase-negative staphylococci, while growth in 10% skim milk for 18 h resulted in more than 90% decrease or complete loss of interaction with these proteins. Decreased 1251-Fn binding in the presence of milk was correlated with protease production but not with 125I-Cn binding.