Font Size

HOME > No.7, Nov 2016 > Research Highlights : Rapid and sensitive bacteria detection using microcolony method combining a fluorescent staining technique and a bioimaging system.

Rapid and sensitive bacteria detection using microcolony method combining a fluorescent staining technique and a bioimaging system

By Shigeki Nakauchi

We have developed a novel fluorescent staining method “metachronic observation”. This method consists of taking some metachronic fluorescent staining microcolony data, and comparing it to fluorescent signals in order to provide high definition microcolony signals. In addition, we have developed a novel method for detecting microcolonies which we have termed “membrane covering method”, . This method can be used for staining microcolonies above the agar surface, as well as for staining with fluorescence-labeled antibodies.

Microbiological contamination is a key issue for the pharmaceutical, food and beverage industries. However, traditional methods to detect physiologically active bacteria, such as the standard incubation method, take a long time to produce results. Thus, there is a significant demand for speedier results from microbiological detecting methods . To this end, we conducted research and development on a new method for rapid and defined bacteria detection. This research was supported by the Technological Development Project for Food Safety and Security which was a part of ‘The Knowledge Hub Aichi, Priority Research Project 2’.

Figure 1.
Fig.1 Appearance of Detecting Device “ColoColoMee” - Results of bacterial micro-colony detection by metachronic observation method. -

To begin with, we evaluated the standard rapid detection method for bacteria. Then, we focused on a membrane filtration method, which is one of the many rapid methods of bacterial detection. In this approach, bacterial cells were trapped on a membrane filter and cultured on nutrient medium. After a brief incubation, viable cells formed microcolonies and were stained with fluorescent staining solution. We identified two key deficiencies of this method as targets for improvement:

  1. Identification of falsepositive contamination.
  2. Standard colony incubation afterdetecting microcolonies.

As a result, we have developed a new fluorescent staining method called the “Metachronic observation method” and in addition the “Membrane covering method”. The “Metachronic observation method” consists of taking a number of fluorescent signals at intervals of a short period of around 1~3 min from fluorescent staining microcolonies. Comparison of the fluorescent signals provides high definition microcolony signals, removing noise from the contaminated debris and background. This technique has been used for developing a commercial fluorescent microcolony detection device named “ColoColoMee” produced by Tsuchiya Co., Ltd. Furthermore, we have also developed the “Membrane covering method” as a novel microcolony staining method, which can be used for staining microcolonies above the agar surface using a membrane immersed staining solution, allowing us to stain with fluorescence-labeled antibodies.

Figure 2.
Fig.2 Membrane covering method - Lactobacillus fructivorans on MRS agar plate surface and Staining of fluorescent labeled antibody

Shinichi KAIYA, postdoctoral researcher, said “This research was supported by Technological Development Project by Aichi Prefecture for the development of commercial products. In the beginning then, I worried about how I should approach the development. At that time, we first validated the system using well-known biological detecting methods. Also we targeted the microcolony method and tried to discover a way to resolve certain unsolved problems. Finally, we successfully developed two novel methods for microcolony detection: ‘metachronic observation method’ and ‘Membrane covering method’, and developed a commercial product ‘Microcolony detection system ColoColoMee’. I am very happy about that.”

This developed sytem “ColoColoMee” has now been improved by Tsuchiya Co., Ltd. for commercialization. “ColoColoMee” is the first microcolony detecting device using the “metachronic observation method” and further research into the “Membrane covering method” to develop a new function for the system.

Visual Perception and Cognition Laboratory, Toyohashi University of Technology

Tsuchiya Company Limited


  • 特願2015-058653 微生物検出装置、微生物検出プログラム及び微生物検出方法
  • 特願2015-151278 微生物コロニーの測定方法
  • 特願2016-179947 微生物コロニーの測定方法及び測定装置


Shinichi Kaiya, Shigeki Nakauchi (2015) . Development of fluorescent microcolony detecting system: The 2015 Annual Conference of the Japan Society for Bioscience, Biotechnology and Agrochemistry. Okayama, Japan, March 26-29, 2015

Shinichi Kaiya, Shigeki Nakauchi (2016) . Development of novel bacterial micro-colony staining method: The 2016 Annual Conference of the Japan Society for Bioscience, Biotechnology and Agrochemistry. Hokkaido, Japan, March 27-30, 2016

日本農芸化学会2015年度大会 光学式微生物微小コロニー検査装置の開発

日本農芸化学会2016年度大会 新規微生物微小コロニー染色法の開発



製薬、飲料、食品などの工場においては、微生物の汚染が非常に重要な問題として知られています。しかしながら、現在一般的な微生物の検査法(一般培養法)は、結果を得るまでに長い時間がかかり、そのため、迅速に結果を得られる微生物検査法は広い分野で求められています。それらの背景から、我々は、愛知県 食の安心安全技術開発プロジェクト重点研究プロジェクト2において、迅速かつ正確な微生物検査法に関わる研究開発を行ってきました。






Share this story

Researcher Profile

Shigeki Nakauchi
Name Shigeki Nakauchi
Affiliation Department of Computer Science and Engineering
Title Professor
Fields of Research Vision Science & Technology / Spectral Color Imaging