Three hundred seventy-three milk samples were screened for Streptococcus agalactiae and Staphylococcus aureus. After sample storage at -20 C for 23 days, the frequency of Str agalactiae isolation increased 2.50 times. The frequency of S aureus isolation increased 1.48 times in the same interval. Increases in the proportion of these isolates were highly significant (P = 0.000006 and 0.0001, respectively). Results of the study indicate that optimal procedures for microbiological testing for these mastitis pathogens may include preculture freezing. The magnitude of the increase in the proportion of isolates indicates the existence of an important population of infected cattle shedding bacteria at concentrations not detected by use of standard microbiological techniques.