Effect of preculture freezing and incubation on bacteriological isolation from subclinical mastitis samples

In this study the sensitivity of three methods of isolation of udder pathogens from milk samples from subclinical mastitis cases was compared. For analysis 1827 quarter milk samples were selected. Milk was cultured using a standard culture technique (0.01 ml of fresh milk streaked on a sheep blood agar plate and on Edward's medium). In addition, an inoculum of 0.01 ml of the original milk sample was incubated for 24h at 37 degrees C in broth, followed by culture using the standard culture technique. In the third method, the whole milk sample was frozen for 24h, and then incubated for 24h at 37 degrees C, followed by culture using standard culture technique. The isolation percentage of Staphylococcus aureus was 4.7% for standard culture technique, 14.2% for incubation in broth, and 21.5% for the combination of freezing plus incubation. Isolation percentage of Streptococcus dysgalactiae and Streptococcus agalactiae was highest using the standard culture technique, while isolation rate of Streptococcus uberis was not different among the three methods used. With increasing somatic cell count, the likelihood of S. aureus, S. dysgalactiae and S. uberis isolation increased. Based on the relative sensitivity, defined as the isolation rate using a single technique compared to the isolation rate of the three techniques together, a combination of standard culture technique and freezing plus incubation was most attractive for achieving a high isolation rate of S. agalactiae and S. dysgalactiae. Relative sensitivity of S. uberis isolation was highest using the standard culture technique and incubation in broth, while S. aureus was most often isolated using a combination of incubation in broth and freezing plus incubation. A combination of the three methods increased the isolation rate for S. dysgalactiae, S. uberis and S. aureus. The standard culture technique, together with the combination of freezing plus incubation, can be recommended for isolating major udder pathogens. If S. aureus is the pathogen of main interest, using incubation in broth together with the combination of freezing plus incubation performed best.