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.
