Validation of media hold time for Enterobacteria Enrichment Broth Mossel and Glucose oxidative fermentative media used in microbiological analysis.

Abstract

Media hold time validation is a crucial microbiological process that ensures the effectiveness of microbiological media in testing for a specific period, thereby ensuring accurate results and allowing for the confident release of products. The research aimed to determine the storage time for prepared culture media in specific conditions in the Microbiology laboratory, focusing on media required for routine activities. Bile-tolerant gram-negative bacteria, belonging to the Enterobacteriaceae, Pseudomonas, and Aeromonas families, grow in bile salts and utilize glucose. They are identified through selective enrichment, subculture onto solid media, and confirmed using biochemical and fermentation tests. Enterobacteria Enrichment Broth Mossel (MEEB), Violet Red Bile Glucose Agar (VRBGA), and Glucose Oxidative Fermentative media (GOF) are used for the tests. In the current study, three validation batches from MEEB and GOF medium were prepared and stored them at 5±3 °C. The stock cultures of test strains of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa) were diluted to prepare a dilution series. The selected dilutions for E. coli, S. aureus, and P. aeruginosa were 10-4, 10-4, and 10-3 respectively. The dilutions were predetermined and utilized for growth promotion and inhibitory tests. For GOF media, E. coli was used for the growth promotion while P. aeruginosa was used for the inhibitory test. For MEEB media, E. coli and P. aeruginosa were used for growth promotion while S. aureus was used for the inhibitory test. The color of the medium in three batches of GOF was changed from green to yellow due to E. coli anaerobic fermentation reactions, while inhibitory test tubes remained green in the sixth week. At the sixth-week, MEEB medium showed turbidity levels of 1 for E. coli, 1 for P. aeruginosa, and 0 for S. aureus, confirming all three batches of MEEB were approved. The study found that MEEB and GOF semi-solid media can be stored for 30 days at 5±3 °C in 200 mL glass reagent bottles and 10 mL test tubes with screw caps respectively.