EcoPlate™ Biolog Ecoplate Instructions
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Microbial Community Analysis EcoPlate™ A1 Water A2 -Methyl-DGlucoside A3 D-Galactonic Acid -Lactone A4 L-Arginine B1 Pyruvic Acid Methyl Ester B2 D-Xylose B3 DGalacturonic Acid C1 Tween 40 C2 i-Erythritol D1 Tween 80 A5 Water A6 -Methyl-DGlucoside A7 D-Galactonic Acid -Lactone A8 L-Arginine A9 Water A10 -Methyl-DGlucoside A11 D-Galactonic Acid -Lactone A12 L-Arginine B4 B5 L-Asparagine Pyruvic Acid Methyl Ester B6 D-Xylose B7 DGalacturonic Acid B8 B9 L-Asparagine Pyruvic Acid Methyl Ester B10 D-Xylose B11 DGalacturonic Acid B12 L-Asparagine C3 2-Hydroxy Benzoic Acid C4 C5 LTween 40 Phenylalanine C6 i-Erythritol C7 2-Hydroxy Benzoic Acid C8 C9 LTween 40 Phenylalanine C10 i-Erythritol C11 2-Hydroxy Benzoic Acid C12 LPhenylalanine D2 D-Mannitol D3 4-Hydroxy Benzoic Acid D4 L-Serine D5 Tween 80 D6 D-Mannitol D7 4-Hydroxy Benzoic Acid D8 L-Serine D9 Tween 80 D10 D-Mannitol D11 4-Hydroxy Benzoic Acid D12 L-Serine E1 Cyclodextrin E2 N-Acetyl-DGlucosamine E3 -Amino Butyric Acid E4 L-Threonine E5 Cyclodextrin E6 N-Acetyl-DGlucosamine E7 -Amino Butyric Acid E8 L-Threonine E9 Cyclodextrin E10 N-Acetyl-DGlucosamine E11 -Amino Butyric Acid E12 L-Threonine F1 Glycogen F2 F3 DItaconic Acid Glucosaminic Acid F4 F5 Glycyl-LGlycogen Glutamic Acid F6 F7 DItaconic Acid Glucosaminic Acid F8 F9 Glycyl-LGlycogen Glutamic Acid F10 F11 DItaconic Acid Glucosaminic Acid F12 Glycyl-LGlutamic Acid G1 D-Cellobiose G2 Glucose-1Phosphate G3 -Keto Butyric Acid G4 Phenylethylamine G5 D-Cellobiose G6 Glucose-1Phosphate G7 -Keto Butyric Acid G8 Phenylethylamine G9 D-Cellobiose G10 Glucose-1Phosphate G11 -Keto Butyric Acid G12 Phenylethylamine H1 -D-Lactose H2 D,L-Glycerol Phosphate H3 D-Malic Acid H4 Putrescine H5 -D-Lactose H6 D,L-Glycerol Phosphate H7 D-Malic Acid H8 Putrescine H9 -D-Lactose H10 D,L-Glycerol Phosphate H11 D-Malic Acid H12 Putrescine FIGURE 1. Carbon Sources in EcoPlate INTRODUCTION Microbial communities provide useful information about environmental change. Microorganisms are present in virtually all environments and are typically the first organisms to react to chemical and physical changes in the environment. Because they are near the bottom of the food chain, changes in microbial communities are often a precursor to changes in the health and viability of the environment as a whole. The Biolog EcoPlate™ (Figure 1) was created specifically for community analysis and microbial ecological studies. It was originally designed at the request of a group of microbial ecologists that had been using the Biolog GN MicroPlate, but wanted a panel that provided replicate sets of tests 1. Community analysis using Biolog MicroPlates was originally described in 1991 by J. Garland and A. Mills2. They and other researchers found that by inoculating Biolog GN MicroPlates with a mixed population of microorganisms and measuring the community metabolism over time, they could ascertain characteristics of that community. This approach, called community–level physiological profiling, or CLPP, has been demonstrated to be effective at distinguishing spatial and temporal changes in microbial communities. In applied ecological research EcoPlates are used as both an assay of the stability of a normal population and to detect and assess changes following the onset of an environmental variable. Studies have been done in diverse applications of microbial ecology and have demonstrated the fundamental utility of EcoPlates in detecting population changes in soil, water, wastewater, activated sludge, compost, and industrial waste. The utility of the information has been documented in hundreds of publications using Biolog technology to analyze microbial communities. A bibliography of publications is posted on the Biolog website at www.biolog.com/bibliography.php. ECOPLATE The EcoPlate contains 31 carbon sources that are useful for community analysis. These 31 carbon sources are repeated 3 times to give the scientist more replicates of the data. Communities of microorganisms will give a characteristic reaction pattern called a metabolic fingerprint. From a single EcoPlate, these fingerprint reaction patterns rapidly and easily characterize the community. Microbial Community Analysis The community reaction patterns are typically analyzed at defined time intervals over 2 to 5 days. The changes in the pattern are compared and analyzed using statistical analysis software. The most popular method of analysis of the data is via Principle Components Analysis (PCA) of average well color development (AWCD) data, but alternative methods may also offer advantages311. The changes observed in the fingerprint pattern provide useful data about the microbial population changes over time. [2] [3] [4] TYPICAL PROCEDURE3 STEP 1: Environmental samples are inoculated directly into EcoPlates either as aqueous samples or after suspension (soil, sludge, sediment, etc…). STEP 2: The EcoPlates are incubated and analyzed at defined time intervals. STEP 3: The community-level physiological profile is assessed for key characteristics: o Pattern development (similarity) o Rate of color change in each well (activity) o Richness of well response (diversity) Formation of purple color occurs when the microbes can utilize the carbon source and begin to respire. The respiration of the cells in the community reduces a tetrazolium dye that is included with the carbon source. The reaction patterns are most effectively analyzed using the MicroStation™ System or an OmniLog® Instrument configured for Phenotype MicroArray™ Analysis, which is especially useful when reading a large number of plates, or when kinetic analysis is required. However, any good microplate reader can be used to provide optical density (OD590) values. [5] [6] [7] [8] [9] Statistical analysis of the data is typically performed using standard software packages. Some researchers have found that PCA provides greater resolution than other methods of statistical analysis11. EcoPlates: Catalog No. 1506 (10/box) Wouterse, Sytske M. Drost, Anton M. Breure, Christian Mulder, Dorothy Stone, Rachel E. Creamer, Anne Winding and Jaap Bloem, Applied Soil Ecology, 2016, v. 97, p. 23-35. [10] REFERENCES [1] A new set of substrates proposed for community characterization in environmental samples. H. Insam, p. 260-261, In: Microbial Communities. Functional versus structural approaches, H. Insam and A. Rangger, editors, 1997, Springer. 21124 Cabot Blvd. Hayward, CA 94545 Classification and characterization of heterotrophic microbial communities on the basis of patterns of community level sole-carbon-source utilization. J.L. Garland, A.L. Mills, Applied and Environmental Microbiology, 1991, v.57, p. 2351-2359. Analysis and interpretation of community-level physiological profiles in microbial ecology. J.L. Garland, Federation of European Microbiological Societies, Microbiology Ecology, 1997, v. 24, p289300. Community analysis by Biolog: curve integration for statistical analysis of activated sludge microbial habitats, J.B. Guckert, G.J. Carr, T.D. Johnson, B.G. Hamm, D.H. Davidson, Y. Kumagai, Journal of Microbiological Methods, 1996, v. 27:2-3, p. 183187. Statistical analysis of the time-course of Biolog substrate utilization. C.A. Hackett, B.S. Griffiths, Journal of Microbiological Methods, 1997, v. 30, p. 63-69. Statistical comparisons of community catabolic profiles. E. Glimm, H. Heuer, B. Engelen, K. Smalla, H. Backhaus, Journal of Microbiological Methods, 1997, v. 30, p. 71-80. Application of multivariate analysis of variance and related techniques in soil studies with substrate utilization tests, W. Hitzl, M. Henrich, M. Kessel, and H. Insam, Journal of Microbiological Methods, 1997, v. 30, p. 81-89. Using the Gini coefficient with BIOLOG substrate utilization data to provide an alternative quantitative measure for comparing bacterial soil communities, B.D. Harch, R.L. Correll, W. Meech, C.A. Kirkby, and C.E. Pankhurst, Journal of Microbiological Methods, 1997, v. 30, p. 91-101. Monitoring soil bacteria with community-level physiological profiles using Biolog EcoPlates in the Netherlands and Europe, Michiel Rutgers, Marja [11] Community-level physiological profiling. K.P. Weber and R. L. Legge, p. 263-281, In: Bioremediation, Methods in Microbial Ecology v. 599, S.P. Cummings, editor, 2010, Springer. Defining soil quality in terms of microbial community structure. M. Firestone, T. Balser, D. Herman, Annual Reports of Research Projects, UC Berkeley, 1997 Telephone: 510-785-2564 Fax: 510-782-4639 www.biolog.com EcoPlate, MicroPlate, Phenotype MicroArray and MicroStation are trademarks; OmniLog is a registered trademark of Biolog, Inc., Hayward, CA Part# 00A 012, Rev. D, June 2018
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