Wipavadee Sangadkit, Anat Deepatana and Aluck Thipayarat
Department of Food Engineering, King Mongkut''s University of Technology Thonburi, Thailand
Unlike clinical samples, food product and environmental samples were often characterized by low initial cell count and large volume of samples that call for different strategies to handle, especially in low-resource settings and less advanced food industrial laboratory. This paper was to compare three popular industrial routines (i.e., MPN method, PetrifilmTM by 3M, and standard pour plate technique) against a modified surface spread technique using 96-well microtiter plate format. Chromocult Coliform Agar (CCA) was utilized as the chromogenic agar medium enabling the formation of purple colonies from Escherichia coli and pink colonies from coliforms and possible E. coli O157:H7 - cells. The obvious drawback of the proposed miniaturized protocol was the limitation of using small inoculums volume (approximately 10-20 μl) generating the minimum detection limit at 102 CFU/ml. However, the common practice of using a glass pipette in most industrial laboratories slightly produced better minimum detection limit, approximately 101 CFU/ml. The cell enumeration results obtained from each technique showed good agreements. The miniaturized rapid protocol was able to effectively manage large number of samples with the use of multichannel autopipettes and high-throughput design of 96-well microtiter plates and able to conclude the colony count within 12-16 hr. The analytical efficiency of miniaturized protocol surpassed those of the three conventional routines. The colony counts from the ready-to-eat product samples showed similar and comparable results to the pour plate technique displaying good agreement to the universally-accepted standard techniques. The feedbacks by QA staffs from a local Thai food factory revealed good overall method acceptance (e.g., usability, protocol design and method efficiency). The proposed miniaturized technique gave highly consistent results of colony count numbers and good colony separation. This cell enumeration consistency supported that the miniaturized rapid protocol can replace the complex standard protocols as an in-house protocol for less restricted samples, like environmental swabs, to enhance its sampling frequency due to the ease and cost effectiveness of the technique.
Keywords: Practical miniaturized technique, rapid colony enumeration, Escherichia coli, coliforms, environmental sample.