Effect of sample preparation on the discrimination of bacterial isolates cultured in liquid nutrient media using laser induced breakdown spectroscopy

Authors: Gamble, Gary; Park, Bosoon; Yoon, Seung-Chul; Lawrence, Kurt

Submitted to: Applied Spectroscopy
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/21/2015
Publication Date: 1/27/2016
Citation: Gamble, G.R., Park, B., Yoon, S.C., Lawrence, K.C. 2016. Effect of sample preparation on the discrimination of bacterial isolates cultured in liquid nutrient media using laser induced breakdown spectroscopy. Applied Spectroscopy. 70(3):494-504.

Interpretive Summary: Laser Induced Breakdown Spectroscopy (LIBS) is an atomic emission analytical technique useful for discriminating materials based upon their respective mineral contents. In this study, the technique is used as the basis for discrimination between 2 genera of gram-negative bacteria and 2 genera of gram-positive bacteria representing pathogenic threats commonly found in poultry processing rinse waters. Because LIBS relies on the relative proportions of minerals present in a sample, this study aims to determine the effects of trace mineral content and pH found in the water source used to culture the bacteria upon their respective mineral compositions. All four genera were cultured using tryptic soy agar (TSA) as the nutrient medium, and were grown under identical environmental conditions. The only variable introduced was the source water used to make the TSA growth medium and isolate the cultured bacteria. Three cultures of each bacterium were produced using deionized water, distilled water, and tap water. After three days of culture growth, the bacteria were centrifuged and washed three times in the same water source. Bacteria were then freeze dried, mixed with microcrystalline cellulose, and a pellet was made for LIBS analysis. Resultant LIBS spectral data were treated by computational methods to extract relationships and variability among the 4 bacteria types. Results indicate not only that the four genera can be discriminated from each other in each water type, but that each genus can be discriminated by water type. It is concluded that in order for LIBS to be a reliable and repeatable method for discrimination of bacteria grown in liquid nutrient media, care must be taken to insure that the water source used in purification of the culture be precisely controlled regarding pH, ionic strength, and proportionate amounts of mineral cations.

Technical Abstract: Laser induced breakdown spectroscopy (LIBS) is used as the basis for discrimination between 2 genera of gram-negative bacteria and 2 genera of gram-positive bacteria representing pathogenic threats commonly found in poultry processing rinse waters. Because LIBS-based discrimination relies primarily upon the relative proportions of inorganic cell components including sodium, potassium, magnesium and calcium, this study aims to determine the effects of trace mineral content and pH found in the water source used to culture the bacteria upon the reliability of the resulting discriminant analysis. All four genera were cultured using tryptic soy agar (TSA) as the nutrient medium, and were grown under identical environmental conditions. The only variable introduced was the source water used to make the TSA growth medium and isolate the cultured bacteria. Three cultures of each bacterium were produced using deionized water, distilled water, and tap water. After three days of culture growth, the bacteria were centrifuged and washed three times in the same water source. Bacteria were then freeze dried, mixed with microcrystalline cellulose, and a pellet was made for LIBS analysis. Principal component analysis (PCA) was used to extract related variations in LIBS spectral data among the four bacteria genera and three water types used to culture and wash the bacteria. Results indicate not only that the four genera can be discriminated from each other in each water type, but that each genus can be discriminated by water type. It is concluded that in order for LIBS to be a reliable and repeatable method for discrimination of bacteria grown in liquid nutrient media, care must be taken to insure that the water source used in purification of the culture be precisely controlled regarding pH, ionic strength, and proportionate amounts of mineral cations.