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Criteria for diagnosing intramammary infections (IMI) have been debated for many years. Factors that may be considered in making a diagnosis include the organism of interest being found on culture, the number of colonies isolated, whether or not the organism was recovered in pure or mixed culture, and whether or not concurrent evidence of inflammation existed (often measured by somatic cell count). However, research using these criteria has been hampered by the lack of a "gold standard" test (i.e., a perfect test against which the criteria can be evaluated) and the need for very large data sets of culture results to have sufficient numbers of quarters with infections with a variety of organisms. This manuscript used 2 large data sets of culture results to evaluate several definitions (sets of criteria) for classifying a quarter as having, or not having an IMI by comparing the results from a single culture to a gold standard diagnosis based on a set of 3 milk samples. The first consisted of 38,376 milk samples from which 25,886 triplicate sets of milk samples taken 1 wk apart were extracted. The second consisted of 784 quarters that were classified as infected or not based on a set of 3 milk samples collected at 2-d intervals. From these quarters, a total of 3,136 additional samples were evaluated. A total of 12 definitions (named A to L) based on combinations of the number of colonies isolated, whether or not the organism was recovered in pure or mixed culture, and the somatic cell count were evaluated for each organism (or group of organisms) with sufficient data. The sensitivity (ability of a definition to detect IMI) and the specificity (Sp; ability of a definition to correctly classify noninfected quarters) were both computed. For all species, except Staphylococcus aureus, the sensitivity of all definitions was <90% (and in many cases<50%). Consequently, if identifying as many existing infections as possible is important, then the criteria for considering a quarter positive should be a single colony (from a 0.01-mL milk sample) isolated (definition A). With the exception of "any organism" and coagulase-negative staphylococci, all Sp estimates were over 94% in the daily data and over 97% in the weekly data, suggesting that for most species, definition A may be acceptable. For coagulase-negative staphylococci, definitions B (2 colonies from a 0.01-mL milk sample) raised the Sp to 92 and 95% in the daily and weekly data, respectively. For "any organism," using definition B raised the Sp to 88 and 93% in the 2 data sets, respectively. The final choice of definition will depend on the objectives of study or control program for which the sample was collected.