Advanced Phenotypic Analysis


Phenotype MicroArray Services


What is a Phenotype MicroArray™ (PM) analysis?

PM experiments test cellular phenotypes. Included in the tests are assays of basic cellular nutritional pathways for C, N, P, and S metabolism, osmotic and pH and sensitivity, and sensitivity to chemical agents. The most common application is to assess the phenotypic effects of mutations. A change in genotype of a cell should lead to one or more changes in phenotype. PMs allow testing of knock-out or knock-in mutants to help discern the biological changes that occur consequent to genetic changes. Another common application is phenotypic characterization of a collection of related strains. For example, it is possible determine the phenotypic relatedness of a collection of isolates of a given species. PM analysis has been successfully implemented for a wide variety of model microbial cells including Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Pseudomonas putida, Sinorhizobium meliloti, Saccharomyces cerevisiae, Bacillus subtilis, Bacillus cereus, Shewanella oneidensis, Proteus mirabilis, and many other species.

How is a Phenotype MicroArray™ experiment executed?

Bacteria are grown on Biolog BUG plus blood agar plates. Single colonies are swabbed and suspended in inoculating fluid. Cell suspensions are pipetted at 100 ul per well into half area/96 well Biolog PM panels. Panels are incubated in the OmniLog® incubator reader and data is collected, organized and stored over a timecourse.

What is an inoculating fluid (IF)?

Complete inoculating fluid typically contains cells, redox dye, buffer, salts, detergent, gelling agent, and other components. Inoculating fluids are prepared specifically for the category of PM plates and species or cell type being studied. For example, inoculating fluid for PM1 and PM2 cannot contain any carbon source, since we are testing carbon source utilization in those PM panels.

What is a Phenotype MicroArray™ panel?

A PM panel is a Biolog half area 96 well plate. Each well contains a different formulation in order to test substrate utilization or sensitivity in various categories of tests including carbon, nitrogen, sulfur, phosphorus, nutrient stimulation, osmotic sensitivity, and chemical sensitivities. Each category contains a base formulation plus either a metabolic substrate or chemical. PM1-8 are metabolic panels testing substrate utilization or stimulation. PM9-40 test a variety of stress conditions or chemical sensitivities.

What is the exact media formulation?

Biolog PM media formulation is proprietary. However, it is similar, but not identical, to published formulations. Bochner et al., 2001 published defined minimal media used in EN, EPS, and EA microplates, which are predecessors of PM plates. The following is an excerpt from p. 1252: "The complete minimal medium used in the EN, EPS, and EA microplates contained 100 mM NaCl, 30 mM triethanolamine HCI (pH 7.1), 25 mM sodium pyruvate, 5.0 mM NH4Cl, 2.0 mM NaH2PO4, 0.25 mM Na2SO4, 0.05 mM MgCl2, 1.0 mM KCl, 1.0 µM ferric chloride, and 0.01% tetrazolium violet. Nitrogen-free, phosphorus-free, and sulfur-free versions of this medium were used in the EN and EPS microplates keeping the same concentrations for nitrogen, phosphorus, and sulfur sources." Zhou et al., 2003 reported methods for PM analysis of two-component mutants of E. coli using PM1-20. The following is an excerpt from p. 4957: "PM tests were performed essentially as described elsewhere (5), except that IF-0 inoculating fluid was used for PM1-8 and IF-10 was used for PM9 to PM20. PM1 and PM2 are similar to ES. PM3 to PM8 are similar to EN, EPS, and EA, which used a defined medium containing 100 mM NaCl, 30 mM triethanolamine HCI (pH 7.1), 5.0 mM NH4Cl, 2.0 mM NaH2PO4, 0.25 mM Na2SO4, 0.05 mM MgCl2, 1.0 mM KCl, and 0.01% tetrazolium violet. PM3, PM4, and PM6 to PM8 contain various N, P, or S sources which are omitted from the defined medium. PM9 to PM20 are similar to ES1, ES2, and ES3, which used a rich medium containing 2.0 g of tryptone, 1.0 g of yeast extract, and 1.0 g of NaCl per liter." The methods also indicate a preparation that specifies initial cell density and inclusion of 20 mM disodium succinate and 0.2 mM ferric citrate.

How can I independently verify PM phenotypes?

Media formulations similar to those used by the Bochner and Zhou publications can be used to grow cells in microplate format. Carbon sources can be supplemented in the 5-50 mM range. Nitrogen sources can be supplemented in the 2-20 mM range. Phosphorus sources can be supplemented in the 1 mM range and sulfur sources in the 100 uM range. Some species will require additional nutrient supplements. You must omit the corresponding substrate source from the base medium for the category of substrate you are testing. Contact Biolog to learn the midpoint of the range for chemical tests. Most chemicals can be purchased from Sigma.

What is PM data and how is it analyzed?

The OmniLog® instrument is an incubator reader. Using video-based detection and data collection software, the OmniLog® can read PM panels every 15 minutes, and organize, and store the data into computer files. Data can then be read and analyzed with OmniLog® PM software. Data is stored as read number (time) versus OmniLog® signal, which can be graphed as a kinetic response curve. In order to compare kinetic responses, it is convenient to reduce the curve data into a "parameter". Area under the curve, for example, is a "parameter" of the kinetic response. "Parameters" can then be compared by difference, ratio, cluster analysis, etc.



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