A Rapid Microcolony Analysis for the Count of Viable Bacteria in Solid Commercial Products
It takes one to several days to determine the number of viable microbial cells by plate count analysis. Microcolony analysis is based on plate count analysis, and is used to detect the early stage of colony formation by staining with a fluorescent dye and observation under a fluorescence microscope....
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Published in | Chōnai saikingaku zasshi Vol. 28; no. 4; pp. 165 - 172 |
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Main Authors | , , , , |
Format | Journal Article |
Language | Japanese English |
Published |
Tokyo
The Intestinal Microbiology Society
01.01.2014
Japan Science and Technology Agency |
Subjects | |
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Abstract | It takes one to several days to determine the number of viable microbial cells by plate count analysis. Microcolony analysis is based on plate count analysis, and is used to detect the early stage of colony formation by staining with a fluorescent dye and observation under a fluorescence microscope. Using this method, it is possible to measure the number of bacteria with growth ability in a short time. As this analysis collects bacteria by filtration, it is used to measure the number of viable bacteria in liquid samples rather than solid samples containing insoluble materials. We usually use the plate count analysis to count the number of viable bacteria in solid commercial products such as pharmaceuticals and quasi drugs. However, a more rapid, accurate and simple technique is required. In this study, we investigated the feasibility of microcolony analysis for the quantification of viable bacteria in solid products, and obtained the following results. 1. SYTO®9 is an optical fluorescent dye which can be used to prevent the staining of nonspecific insoluble excipients in products. 2. When the samples were centrifuged for 5 min at 150×g, the insoluble excipients were mostly removed. Therefore, it became possible to prevent the collapse of the shape of microcolonies. 3. The number of viable bacteria was accurately estimated when the microcolonies were counted in over 10 microscopic fields with 5×objective. Under these measurement conditions, we demonstrated that microcolony analysis can determine more rapidly and accurately than plate count analysis the number of viable bacteria in a Bifidobacterium solid product as well as several commercial products. In conclusion, we have demonstrated that modified microcolony analysis can be used as an alternative analysis to plate count analysis. |
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AbstractList | It takes one to several days to determine the number of viable microbial cells by plate count analysis. Microcolony analysis is based on plate count analysis, and is used to detect the early stage of colony formation by staining with a fluorescent dye and observation under a fluorescence microscope. Using this method, it is possible to measure the number of bacteria with growth ability in a short time. As this analysis collects bacteria by filtration, it is used to measure the number of viable bacteria in liquid samples rather than solid samples containing insoluble materials. We usually use the plate count analysis to count the number of viable bacteria in solid commercial products such as pharmaceuticals and quasi drugs. However, a more rapid, accurate and simple technique is required. In this study, we investigated the feasibility of microcolony analysis for the quantification of viable bacteria in solid products, and obtained the following results. 1. SYTO registered 9 is an optical fluorescent dye which can be used to prevent the staining of nonspecific insoluble excipients in products. 2. When the samples were centrifuged for 5 min at 150g, the insoluble excipients were mostly removed. Therefore, it became possible to prevent the collapse of the shape of microcolonies. 3. The number of viable bacteria was accurately estimated when the microcolonies were counted in over 10 microscopic fields with 5objective. Under these measurement conditions, we demonstrated that microcolony analysis can determine more rapidly and accurately than plate count analysis the number of viable bacteria in a Bifidobacterium solid product as well as several commercial products. In conclusion, we have demonstrated that modified microcolony analysis can be used as an alternative analysis to plate count analysis. It takes one to several days to determine the number of viable microbial cells by plate count analysis. Microcolony analysis is based on plate count analysis, and is used to detect the early stage of colony formation by staining with a fluorescent dye and observation under a fluorescence microscope. Using this method, it is possible to measure the number of bacteria with growth ability in a short time. As this analysis collects bacteria by filtration, it is used to measure the number of viable bacteria in liquid samples rather than solid samples containing insoluble materials. We usually use the plate count analysis to count the number of viable bacteria in solid commercial products such as pharmaceuticals and quasi drugs. However, a more rapid, accurate and simple technique is required. In this study, we investigated the feasibility of microcolony analysis for the quantification of viable bacteria in solid products, and obtained the following results. 1. SYTO®9 is an optical fluorescent dye which can be used to prevent the staining of nonspecific insoluble excipients in products. 2. When the samples were centrifuged for 5 min at 150×g, the insoluble excipients were mostly removed. Therefore, it became possible to prevent the collapse of the shape of microcolonies. 3. The number of viable bacteria was accurately estimated when the microcolonies were counted in over 10 microscopic fields with 5×objective. Under these measurement conditions, we demonstrated that microcolony analysis can determine more rapidly and accurately than plate count analysis the number of viable bacteria in a Bifidobacterium solid product as well as several commercial products. In conclusion, we have demonstrated that modified microcolony analysis can be used as an alternative analysis to plate count analysis. |
Author | SHIMAKAWA, Masaki OZAKI, Toru MAEDA, Ayako OHNO, Hiroshi YAMAMURA, Hideki |
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References | 6) 見坂武彦,馬場貴志,山口進康,那須正夫.マイクロコロニー法による食材中の増殖能をもつ細菌数の迅速測定.日本食品微生物学会雑誌.2008; 25: 148-152. 3) 馬場貴志,那須正夫.医薬品製造用水の微生物管理.クリーンテクノロジー3月号.東京:日本工業出版;2006. p.6-9. 4) Kawai M, Yamaguchi N, Nasu M. Rapid enumeration of physiologically active bacteria in purified water used in the pharmaceutical manufacturing process. J Appl Microbiol. 1999; 86: 496-504. 7) Nakajima K, Nonaka K, Yamamoto K, Yamaguchi N, Tani K, Nasu M. Rapid monitoring of microbial contamination on herbal medicines by fluorescent staining method. Lett Appl Microbiol. 2005; 40: 128-132. 2) 楢村友隆,佐藤和弘,松浦浩美,十倉秀臣,井ノ口亜紀,古閑尚栄,井出孝夫.蛍光染色法を用いたRO水製造工程中に存在する細菌の迅速評価.透析会誌.2007; 40: 1051-1056. 5) Asano S, Iijima K, Suzuki K, Motoyama Y, Ogata T, Kitagawa Y. Rapid detection and identification of beer-spoilage lactic acid bacteria by microcolony method. J Biosci Bioeng. 2009; 108: 124-129. 1) 山口進康,那須正夫.微生物の迅速高精度検出法の現状とその可能性.日本PDA学術誌.2005; 7: 94-105. 8) Rodrigues UM, Kroll RG. Rapid selective enumeration of bacteria in foods using a microcolony epifluorescence microscopy technique. J Appl Bacteriol. 1988; 64: 65-78. 9) Yamaguchi N, Kitaguchi A, Nasu M. Selective enumeration of viable Enterobacteriaceae and Pseudomonas spp. in milk within 7 h by multicolor fluorescence in situ hybridization following microcolony formation. J Biosci Bioeng. 2012; 113: 746-750. |
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SubjectTerms | Bacteria Bifidobacterium Microcolony analysis Plate count analysis Rapid detection Solid commercial products Viable bacteria number |
Title | A Rapid Microcolony Analysis for the Count of Viable Bacteria in Solid Commercial Products |
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