Avaliação de crioprotetores na produção de material de referência para enumeração de coliformes em leite em pó a ser utilizado em ensaio de proficiência

The challenge in producing reference materials (RM) for microbiological assays is the natural instability of microorganisms. Freeze-drying is suitable for bacterial cultures cryopreservation when the number of cells has to be retained. Protective agents can be added before freezing to increase the material stability. This study aimed at evaluating the use of different cryoprotectants during the production of RM, by freeze-drying, to be used in proficiency testing for coliforms enumeration. Four batches were produced adding different cryoprotectants: 10 % skim milk solution (EC1), the same solution containing glycerol (EC2), sucrose (EC3), and trehalose (EC4). A strain of Escherichia coli was used for preparing the materials. Homogeneity was assessed according to the International Harmonized Protocol. Stability was analyzed during four months at ≤ -70 oC (long-term stability) and during five days at -20 oC, 4 oC, 25 oC and 35 oC (short-term stability), in accordance with the ISO guide 35. EC1 only was regarded as non-homogeneous. All of the lots remained stable at ≤ -70 oC during the four-month study. EC2 showed unsatisfactory results in the short-term study. EC3 and EC4 were homogeneous and stable at the studied temperatures. Sucrose and trehalose were regarded as suitable cryoprotectants to prepare these specific MR.