Use of augmented cultural techniques in the diagnosis of the bacterial cause of clinical bovine mastitis.

Preculture incubation, preculture freezing, and increased plate inoculation volumes were tested in an attempt to increase the recovery rate of pathogens in milk from cases of clinical bovine mastitis. Culture of milk from 291 cases of clinical bovine mastitis was performed using standard milk culture techniques (.01 ml of fresh milk streaked on trypticase soy agar plates with 5% sheep blood and .1% esculin). The sensitivity of this method was compared with that of cultures performed using augmented techniques: 4 and 18 h of preculture incubation; preculture freezing of samples overnight at -20 degrees C; and increasing the plate inoculation volume to .05 and .1 ml for fresh, incubated, and frozen samples. Preculture incubation and larger plate inoculation volumes significantly increased the recovery rate of bacterial pathogens over the standard culture method. The greatest improvement in sensitivity without a concomitant increase in contamination was achieved when samples were incubated for 4 h and plates were inoculated with .1 ml of the sample. Recovery was enhanced significantly by this method for several organisms, including environmental streptococci and coliform bacteria. Freezing milk before culture yielded a significantly higher positive culture rate overall, but freezing did not affect the positive culture rate of any individual bacterial species.