Title

Development of Gram Stain Alternatives for Use with Haloalkaliphilic Bacteria

Presenter Information

Shao Lu

Document Type

Oral Presentation

Location

SURC Ballroom A

Start Date

17-5-2012

End Date

17-5-2012

Abstract

The ability to microscopically differentiate bacteria is an important first step in the identification of bacterial species. Introduced in 1884, the Gram Stain is commonly used to differentiate between 2 groups of bacteria, termed either gram-negative or gram-positive based on their cell wall architecture. It is useful for bacteria inhabiting “normal” environments, but often unreliable for characterizing bacteria isolated from sites exhibiting extremes of pH, temperature, salinity, etc. The goals of this study were to develop alternative differential staining methods suitable for bacteria found in “extreme” environments then evaluate their effectiveness using bacteria isolated from Soap Lake, a haloalkaline lake located in Grant Co.,WA. Wheat germ agglutinin (WGA) is a compound that selectively binds to cell wall molecules (N-acetylglucosamine and N-acetylneuraminic acid) present on the surface of gram-positive bacteria. WGA was linked to a second molecule, either HRP (visualized with light microscopy), or FITC (visualized with fluorescence microscopy). Common species of gram positive and gram negative neutrophilic bacteria were used to verify the specificity of the stains. Thirty Soap Lake bacterial strains with previously determined cell wall structures were used to test the efficacy of WGA-HRP and WGA-FITC for haloalkaliphilic bacteria grown at pH 10. While both stains were effective at differentiating between the 2 cell wall types, the WGA-FITC method was better in terms of accuracy and visual quality, and suffered less background interference compared to the WGA-HRP-metal conjugates. Future work will utilize additional extremophiles (acidophiles, thermophiles, etc) to determine its utility in other extreme environments.

Poster Number

17

Faculty Mentor(s)

Holly Pinkart

Additional Mentoring Department

Biological Sciences

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May 17th, 8:30 AM May 17th, 11:00 AM

Development of Gram Stain Alternatives for Use with Haloalkaliphilic Bacteria

SURC Ballroom A

The ability to microscopically differentiate bacteria is an important first step in the identification of bacterial species. Introduced in 1884, the Gram Stain is commonly used to differentiate between 2 groups of bacteria, termed either gram-negative or gram-positive based on their cell wall architecture. It is useful for bacteria inhabiting “normal” environments, but often unreliable for characterizing bacteria isolated from sites exhibiting extremes of pH, temperature, salinity, etc. The goals of this study were to develop alternative differential staining methods suitable for bacteria found in “extreme” environments then evaluate their effectiveness using bacteria isolated from Soap Lake, a haloalkaline lake located in Grant Co.,WA. Wheat germ agglutinin (WGA) is a compound that selectively binds to cell wall molecules (N-acetylglucosamine and N-acetylneuraminic acid) present on the surface of gram-positive bacteria. WGA was linked to a second molecule, either HRP (visualized with light microscopy), or FITC (visualized with fluorescence microscopy). Common species of gram positive and gram negative neutrophilic bacteria were used to verify the specificity of the stains. Thirty Soap Lake bacterial strains with previously determined cell wall structures were used to test the efficacy of WGA-HRP and WGA-FITC for haloalkaliphilic bacteria grown at pH 10. While both stains were effective at differentiating between the 2 cell wall types, the WGA-FITC method was better in terms of accuracy and visual quality, and suffered less background interference compared to the WGA-HRP-metal conjugates. Future work will utilize additional extremophiles (acidophiles, thermophiles, etc) to determine its utility in other extreme environments.