Microbial Community Analysis

EcoPlate™
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12
Water -Methyl-D- D-Galactonic L-Arginine Water -Methyl-D- D-Galactonic L-Arginine Water -Methyl-D- D-Galactonic L-Arginine
Glucoside Acid Glucoside Acid Glucoside Acid
-Lactone -Lactone -Lactone

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12

Pyruvic Acid D-Xylose D- L-Asparagine Pyruvic Acid D-Xylose D- L-Asparagine Pyruvic Acid D-Xylose D- L-Asparagine
Methyl Ester Galacturonic Methyl Ester Galacturonic Methyl Ester Galacturonic
Acid Acid Acid

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12

Tween 40 i-Erythritol 2-Hydroxy L- Tween 40 i-Erythritol 2-Hydroxy L- Tween 40 i-Erythritol 2-Hydroxy L-
Benzoic Acid Phenylalanine Benzoic Acid Phenylalanine Benzoic Acid Phenylalanine

D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12

Tween 80 D-Mannitol 4-Hydroxy L-Serine Tween 80 D-Mannitol 4-Hydroxy L-Serine Tween 80 D-Mannitol 4-Hydroxy L-Serine
Benzoic Acid Benzoic Acid Benzoic Acid

E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12

- N-Acetyl-D- -Amino L-Threonine - N-Acetyl-D- -Amino L-Threonine - N-Acetyl-D- -Amino L-Threonine
Cyclodextrin Glucosamine Butyric Acid Cyclodextrin Glucosamine Butyric Acid Cyclodextrin Glucosamine Butyric Acid

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12

Glycogen D- Itaconic Acid Glycyl-L- Glycogen D- Itaconic Acid Glycyl-L- Glycogen D- Itaconic Acid Glycyl-L-
Glucosaminic Glutamic Acid Glucosaminic Glutamic Acid Glucosaminic Glutamic Acid
Acid Acid Acid

G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12

D-Cellobiose Glucose-1- -Keto Phenylethyl- D-Cellobiose Glucose-1- -Keto Phenylethyl- D-Cellobiose Glucose-1- -Keto Phenylethyl-
Phosphate Butyric Acid amine Phosphate Butyric Acid amine Phosphate Butyric Acid amine

H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12

-D-Lactose D,L-- D-Malic Acid Putrescine -D-Lactose D,L-- D-Malic Acid Putrescine -D-Lactose D,L-- D-Malic Acid Putrescine
Glycerol Glycerol Glycerol
Phosphate Phosphate Phosphate

FIGURE 1. Carbon Sources in EcoPlate

INTRODUCTION temporal changes in microbial communities. In applied

ecological research EcoPlates are used as both an assay of the
Microbial communities provide useful information about stability of a normal population and to detect and assess
environmental change. Microorganisms are present in changes following the onset of an environmental variable.
virtually all environments and are typically the first organisms
to react to chemical and physical changes in the environment. Studies have been done in diverse applications of microbial
Because they are near the bottom of the food chain, changes in ecology and have demonstrated the fundamental utility of
microbial communities are often a precursor to changes in the EcoPlates in detecting population changes in soil, water,
health and viability of the environment as a whole. wastewater, activated sludge, compost, and industrial waste.
The utility of the information has been documented in
The Biolog EcoPlate™ (Figure 1) was created specifically for hundreds of publications using Biolog technology to analyze
community analysis and microbial ecological studies. It was microbial communities. A bibliography of publications is
originally designed at the request of a group of microbial posted on the Biolog website at
ecologists that had been using the Biolog GN MicroPlate, www.biolog.com/bibliography.php.
but wanted a panel that provided replicate sets of tests 1.

Community analysis using Biolog MicroPlates was originally ECOPLATE

described in 1991 by J. Garland and A. Mills2. They and other The EcoPlate contains 31 carbon sources that are useful for
researchers found that by inoculating Biolog GN MicroPlates community analysis. These 31 carbon sources are repeated 3
with a mixed population of microorganisms and measuring the times to give the scientist more replicates of the data.
community metabolism over time, they could ascertain Communities of microorganisms will give a characteristic
characteristics of that community. This approach, called reaction pattern called a metabolic fingerprint. From a single
community–level physiological profiling, or CLPP, has been EcoPlate, these fingerprint reaction patterns rapidly and easily
demonstrated to be effective at distinguishing spatial and characterize the community.
 Microbial Community Analysis

The community reaction patterns are typically analyzed at [2] Classification and characterization of heterotrophic
defined time intervals over 2 to 5 days. The changes in the microbial communities on the basis of patterns of
pattern are compared and analyzed using statistical analysis community level sole-carbon-source utilization. J.L.
software. The most popular method of analysis of the data is Garland, A.L. Mills, Applied and Environmental
via Principle Components Analysis (PCA) of average well Microbiology, 1991, v.57, p. 2351-2359.
color development (AWCD) data, but alternative methods [3] Analysis and interpretation of community-level
may also offer advantages311. The changes observed in the physiological profiles in microbial ecology. J.L.
fingerprint pattern provide useful data about the microbial Garland, Federation of European Microbiological
population changes over time. Societies, Microbiology Ecology, 1997, v. 24, p289-
300.
[4] Community analysis by Biolog: curve integration for
TYPICAL PROCEDURE3 statistical analysis of activated sludge microbial
habitats, J.B. Guckert, G.J. Carr, T.D. Johnson, B.G.
STEP 1: Environmental samples are inoculated directly into Hamm, D.H. Davidson, Y. Kumagai, Journal of
EcoPlates either as aqueous samples or after suspension (soil, Microbiological Methods, 1996, v. 27:2-3, p. 183-
sludge, sediment, etc…). 187.
STEP 2: The EcoPlates are incubated and analyzed at defined [5] Statistical analysis of the time-course of Biolog
time intervals. substrate utilization. C.A. Hackett, B.S. Griffiths,
Journal of Microbiological Methods, 1997, v. 30, p.
STEP 3: The community-level physiological profile is 63-69.
assessed for key characteristics: [6] Statistical comparisons of community catabolic
profiles. E. Glimm, H. Heuer, B. Engelen, K. Smalla,
o Pattern development (similarity)
H. Backhaus, Journal of Microbiological Methods,
o Rate of color change in each well (activity) 1997, v. 30, p. 71-80.
[7] Application of multivariate analysis of variance and
o Richness of well response (diversity)
related techniques in soil studies with substrate
Formation of purple color occurs when the microbes can utilization tests, W. Hitzl, M. Henrich, M. Kessel,
utilize the carbon source and begin to respire. The respiration and H. Insam, Journal of Microbiological Methods,
of the cells in the community reduces a tetrazolium dye that is 1997, v. 30, p. 81-89.
included with the carbon source. [8] Using the Gini coefficient with BIOLOG substrate
utilization data to provide an alternative quantitative
The reaction patterns are most effectively analyzed using the
measure for comparing bacterial soil communities,
MicroStation™ System or an OmniLog® Instrument configured
B.D. Harch, R.L. Correll, W. Meech, C.A. Kirkby,
for Phenotype MicroArray™ Analysis, which is especially
and C.E. Pankhurst, Journal of Microbiological
useful when reading a large number of plates, or when kinetic
Methods, 1997, v. 30, p. 91-101.
analysis is required. However, any good microplate reader can
[9] Monitoring soil bacteria with community-level
be used to provide optical density (OD590) values.
physiological profiles using Biolog EcoPlates in the
Statistical analysis of the data is typically performed using Netherlands and Europe, Michiel Rutgers, Marja
standard software packages. Some researchers have found Wouterse, Sytske M. Drost, Anton M. Breure,
that PCA provides greater resolution than other methods of Christian Mulder, Dorothy Stone, Rachel E.
statistical analysis11. Creamer, Anne Winding and Jaap Bloem, Applied
Soil Ecology, 2016, v. 97, p. 23-35.
EcoPlates: Catalog No. 1506 (10/box) [10] Community-level physiological profiling. K.P.
Weber and R. L. Legge, p. 263-281, In:
REFERENCES Bioremediation, Methods in Microbial Ecology v.
599, S.P. Cummings, editor, 2010, Springer.
[1] A new set of substrates proposed for community [11] Defining soil quality in terms of microbial
characterization in environmental samples. H. Insam, community structure. M. Firestone, T. Balser, D.
p. 260-261, In: Microbial Communities. Functional Herman, Annual Reports of Research Projects, UC
versus structural approaches, H. Insam and A. Berkeley, 1997
Rangger, editors, 1997, Springer.

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Part# 00A 012, Rev. D, June 2018