Avaliação de crioprotetores na produção de material de referência para enumeração de coliformes em leite em pó a ser utilizado em ensaio de proficiência / Evaluation of cryoprotectants in the production of reference material for coliforms counting in milk powder to be used for proficiency testing

O desafio na produção de materiais de referência (MR) destinados a ensaios microbiológicos é a instabilidade natural dos micro-organismos. A liofilização é indicada para criopreservação de culturas bacterianas quando o número de células deve ser resguardado. Agentes protetores podem ser adicionados antes do congelamento para aumentar a estabilidade do material. Neste trabalho foram avaliados os crioprotetores na produção de MR liofilizados a serem utilizados em ensaio de proficiência para contagem de coliformes. Foram produzidos quatro lotes utilizando-se diferentes crioprotetores: solução de leite desnatado a 10 % (EC1), a mesma solução contendo glicerol (EC2), sacarose (EC3) e trealose (EC4). Umacepa de Escherichia coli foi empregada no preparo dos materiais. A homogeneidade foi avaliada conforme o Protocolo Internacional Harmonizado. A estabilidade foi estudada durante quatro meses à ≤ -70 ºC (longa duração) e às temperaturas de -20 ºC, 4 ºC, 25 ºC e 35 ºC durante cinco dias (curta duração), segundo aISO/GUIDE 35. Apenas EC1 foi considerado não homogêneo. Os lotes permaneceram estáveis à ≤ -70 ºC durante quatro meses. EC2 apresentou resultados insatisfatórios na estabilidade de curta duração. EC3 eEC4 foram homogêneos e estáveis nas temperaturas estudadas. A sacarose e a trealose foram consideradas crioprotetores adequados para o preparo do MR em questão.(AU)

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.(AU)

Isolation of Staphylococcus from minas frescal type cheese and detection of enterotoxin genes

This work aimed at isolating Staphylococcus spp. from minas frescal type cheese, and for this purpose a Multiplex PCR (M-PCR) was standardized for detecting the classical Staphylococcus aureus enterotoxingenes (sea, seb, sec, sed and see), using the femA gene as a positive control for S. aureus strains. Bacterial detection directly from the minas frescal type cheese and from artificially contaminated food was tested.One hundred eleven colonies (104 coagulase-positive and seven coagulase-negative) were selected for performing M-PCR assay. Thirty-four colonies (30.62%) were positive for at least one of the fiveente rotoxin genes analyzed; enterotoxins sea and seb were the most frequently detected. The study on Staphylococci coagulase-positive samples revealed that 40% of the samples showed bacterial counts above the limit established by Brazilian legislation. (AU)