Comparing effects of freezing at -196 ºC and -20 ºC on the viability of mastitis pathogens

The aim of this study was to compare the effects of cryopreservation at approximately -196 ºC in liquid nitrogen (N) and freezing at approximately -20 ºC in a freezer, on the viability and survival of eight different mastitogenic bacteria inoculated in milk. Bacteria were frozen at approximately -20 ºC in a freezer and cryopreserved at approximately -196 ºC in liquid nitrogen. An effective preservation method was needed for follow-up samples from cows identified in the South African National Milk Recording Scheme (NMRS) with somatic cell counts above 250 000 cells/mL milk. The organisation responsible for sample collection of the NMRS milk samples also provides producers with liquid nitrogen for their semen flasks at the collection sites. This existing mode of storage and transport could therefore be utilised. Ten samples of each organism were thawed and cultured bi-weekly until week 18 for both temperature treatments. An additional sampling was performed at week 30 for samples frozen at approximately -20 ºC. Freezing and cryopreservation did not impair subsequent isolation of Streptococcus dysgalactiae, Streptococcus uberis, Enterococcus faecalis, Staphylococcus aureus (STH) (phage type lytic group III) or Sta. aureus (STA) (phage typed, other than lytic group III). Survival was indicated by the isolation of bacteria from samples, and viability by the strength of growth of the bacteria isolated. The survival of Streptococcus agalactiae decreased after week 12 and Escherichia coli after week 16 of freezing, but both organisms survived under cryogenic preservation until week 18. Coagulase-negative staphylococci survived until week 18 for both freezing and cryogenic preservation. Both storage methods could thus contribute to the improvement of a pro-active approach towards udder health management in South African dairy herds.