Journal of Probiotics & Health

Journal of Probiotics & Health
Open Access

ISSN: 2329-8901

+32 25889658

Abstract

Cell Wall Integrity and Protoplast Formation of the Probiotic Lactobacillus acidophilus through Fluorescent Staining and Fluorescence Microscopy

Ryan Page, David Burk, Kayanush Aryana

The identification of protoplast of bacterial cells has previously utilized phase contrast microscopy. This methoddetermines protoplast by their size and change in shape. A more verifiable method can be used utilizing fluorescentstains that target the specific cellular components. The goal of this study was to utilize fluorescence microscopytechniques to determine the presence or absence of bacterial cell walls in the probiotic Lactobacillus acidophilus, afterexposure to cell wall digestive enzymes. Bacterial cells were treated with different concentrations of lysozyme [0, 175,250, 425 μg/ml] and were incubated at 37°C for ten minutes. Following lysozyme treatment cells were fluorescentlystained with different concentrations (1x, 2x, 10x, and 100x) of two fluorescent dyes, Wheat-germ agglutinin (WGA)and Hoechst 33342. The WGA [CF®594 WGA, a red-fluorescent dye] was used to selectively bind to residues of thepeptidoglycan layer of the cell wall and Hoechst 33342, a blue fluorescent dye, was used for specifically binding tonucleic acids of double-stranded DNA of bacterial cells. The standard method for sample preparation forfluorescence microscopy was followed. Three fields were studied for each lysozyme and stain combination. A one-wayANOVA was performed to determine differences in lysozyme concentrations. A p-value < 0.05 was noted assignificantly different. Cell wall structural integrity began to deteriorate at 175 and 250 μg/ml of lysozyme and celllysis and striations of DNA increased at a concentration of 425 μg/ml. Lysozyme concentration of 175 μg/mlproduced an average of 41% protoplast or partial digestion of cell wall. An increase from 175 to 250 μg/mlconcentration of lysozyme resulted in a decreased average percentage of protoplast (4%). At a concentration of 425μg/ml, the average percentage of protoplast decreased to 1%, while also showing an increase in striations of DNA. At1x dye concentration, partial staining of the cell wall was observed. At 2x, complete staining of the cell wall wasrecorded. At 10x, complete staining of cell wall and nuclei was observed similar to dye concentrations at 2x with nosignificant saturation of dyes. Dye concentration at 100x produced an oversaturation of the dyes in the cell wall andnuclei causing them to mix and inhibit the efficacy of identifying bacterial cells and protoplasts. 2x was mostoptimum for complete staining of cell wall and nucleus. Background fluorescence noise was observed asconcentration of dye increased. In Lactobacillus acidophilus, a lysozyme concentration of 175 μg/ml was sufficient forcell wall digestion. Efficacy of dye concentration was best at 2x with the least amount of background noise

Published Date: 2020-05-18; Received Date: 2020-04-29

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