Practical Improvement in the Detection and Enumeration of Microbial Colonies on Membrane Filters by Using a Fully Automated Microbial Detection System Based on Time-lapse Shadow Image Analysis

Introduction: Microbial colonies on/in agar plates can be detected at their initial stage and counted precisely with MicroBio μ3D^TM AutoScanner (Microbio Corporation), a fully automated microbial detection system based on time-lapse shadow image analysis, which identifies only growing spots with three-dimensionally convex shadow gradient as microbial colonies. Although membrane filter (MF) method is preferably used in beverage industry to inspect a trace of contaminant in a large volume of liquid sample, the interference of light penetration by MF often causes critical disturbance.

Purpose: The main purpose of this study is to reduce the time for detecting slow-growing bacteria with the system by using a semi-transparent MF.

Methods: Cell suspension of a wild strain of Mycobacterium sp. was prepared with PBS and approximately 100 cells were filtrated with either a white MF made of cellulose-mixed ester (ADVANTEC, pore size 0.45μm) or a semi-transparent MF made of polycarbonates (Millipore, pore size 0.4 μm). These MFs were placed on 1/10 Trypticase Soy Agar (TSA) plates and incubated at 28°C. Microbial colonies on the MFs were counted by the system every 30 minutes or operator’s daily observation and we compared the time for detecting 90% of the final counts observed at 120 h.

Results: When a white MF was used, it took more than 96 h in operator’s observation and 108 h with the system, while we succeeded in reducing the time to 70 h by replacing it with a semi-transparent MF which became almost transparent when it got wet. It should also be emphasized that manual counts often involved misjudgment of foreign particles as small colonies especially at the early time of incubation, while no miscounting has occurred with the system.

Significance: We expect that non-human operations of microbiological testing with the system will achieve both rapid results and relief of operator’s tension.