Introduction to Quantitative Microbiological Testing
📌 The training focuses on demonstrating the quantitative microbiological testing for the enumeration of *Listeria monocytogenes* in fresh fruit using ready-to-use culture media kits.
🔬 The objective is to enhance the knowledge and skills of pharmaceutical and food quality control inspectors in performing this quantitative microbiological testing.
📜 The principle involves cultivating and counting specific bacterial colonies after inoculating the sample into ready-to-use media via pipetting 1 mL of homogenized suspension.

Characteristics of *Listeria monocytogenes*
🦠 *Listeria monocytogenes* is a Gram-positive bacterium capable of causing serious infections, especially in infants, the elderly, and immunocompromised individuals.
🔬 Its size ranges from 0.4 to 0.5 $\mu$m in width and 0.5 to 2 $\mu$m in length, possessing flagella that aid in mobility.
🌬️ This bacterium is a facultative anaerobe, meaning it can survive and grow in the absence of oxygen.

Sample Preparation and Inoculation
🧼 Sample preparation begins by cleaning the packaging with alcohol, weighing 10 grams, and homogenizing it with 90 mL of Peptone Water Buffer (PWB) to achieve a $10^{-1}$ dilution.
🧪 Serial dilutions (e.g., up to $10^{-2}$ or as needed) must be performed and clearly labeled with the sample code and dilution level.
💧 Inoculate 1 mL of the homogenized sample (and subsequent dilutions) into the specific ready-to-use media kit designated for *Listeria monocytogenes* enumeration, performing tests in duplicate.

Incubation and Preliminary Result Interpretation
🌡️ Incubate the inoculated media at $37 \pm 1^\circ$C for 24 to 48 $\pm 2$ hours, following the kit manufacturer's instructions.
🔴 Preliminary colonies of *L. monocytogenes* are typically observed as red colonies, potentially surrounded by a blue halo, or they may appear orange/reddish-brown or purple/reddish, also possibly with a blue halo.

Confirmatory Testing Procedures
📝 Suspected specific colonies must undergo confirmation testing, following ISO 11290/2 or conventional biochemical tests.
🔪 Subculture confirmed colonies onto TSA agar and incubate at $37 \pm 1^\circ$C for 24 $\pm 2$ hours before proceeding to biochemical confirmation.
🧪 Key conventional biochemical tests include: positive $\beta$-hemolysis on blood agar, positive L-rhamnose fermentation (media turns yellow), and negative D-xylose fermentation (media color remains unchanged).

Key Points & Insights
➡️ Use the newest analysis method, specifically the National Drug and Food Testing Development Center's guidelines (BIA No. 34 of 2024), for current testing standards.
➡️ Sterilize all glassware and ensure measuring equipment is properly calibrated before sample preparation.
➡️ Confirmed *Listeria monocytogenes* characteristics include positive $\beta$-hemolysis and positive L-rhamnose fermentation in biochemical testing.
➡️ Final conclusions on test results must align with applicable regulations.

📸 Video summarized with SummaryTube.com on Dec 01, 2025, 02:34 UTC