Development of Hydrophilic Quantitative Reference Material of Escherichia coli for Application in Microbiology Laboratories
摘要
Reference materials (RM) play a critical role in addressing data accuracy situations and the usage of the same has been mandated by ISO 17025:2017 for accredited laboratories. The present study was conducted to identify the best possible combination of nutritive and protective agents to be incorporated in the production of viable and stable quantitative microbiological reference materials. Nutritive agents such as casein hydrolysate, soy protein hydrolysate, and fish protein hydrolysate were evaluated for long-term survival of Escherichia coli MTCC 1610 T. The effect of each of the hydrolysates was individually studied on E. coli at two different storage temperatures (5 ± 2 °C and − 18 ± 3 °C). Response surface methodology (RSM) was used for the optimization of the experimental run. Soy protein hydrolysate at 5 and 10% resulted in the long-term survival of E. coli MTCC 1610 T at 5 ± 2 °C with inoculum grown in broth. However, E. coli showed enhanced resistance and long-term survival at 5 ± 2 °C even in the presence of 5–15% fish protein hydrolysate when broth grown inoculum was replaced with agar grown inoculum. All three hydrolysates were found to be ineffective in the survival of E. coli at − 18 ± 3 °C, as they were incapable of imparting cryoprotection to E. coli. Hence for long-term survival, cryoprotectants were incorporated and assessed with RSM optimization. The final stage in the development of assigned value reference material involved encapsulation using carboxymethyl cellulose and gelatin. The produced reference material was then assessed for homogeneity, stability, and extended shelf life which was observed to be viable for 5 months and homogeneously stable for 4 months with average mean of 2.40 and 0.17 standard deviation.