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

The identification of protoplast of bacterial cells has previously utilized phase contrast microscopy. This method determines protoplast by their size and change in shape. A more verifiable method can be used utilizing fluorescent stains that target the specific cellular components. The goal of this study was to utilize fluorescence microscopy techniques to determine the presence or absence of bacterial cell walls in the probiotic Lactobacillus acidophilus, after exposure 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 fluorescently stained 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 the peptidoglycan layer of the cell wall and Hoechst 33342, a blue fluorescent dye, was used for specifically binding to nucleic acids of double-stranded DNA of bacterial cells. The standard method for sample preparation for fluorescence microscopy was followed. Three fields were studied for each lysozyme and stain combination. A one-way ANOVA was performed to determine differences in lysozyme concentrations. A p-value < 0.05 was noted as significantly different. Cell wall structural integrity began to deteriorate at 175 and 250 μg/ml of lysozyme and cell lysis and striations of DNA increased at a concentration of 425 μg/ml. Lysozyme concentration of 175 μg/ml produced an average of 41% protoplast or partial digestion of cell wall. An increase from 175 to 250 μg/ml concentration of lysozyme resulted in a decreased average percentage of protoplast (4%). At a concentration of 425 μg/ml, the average percentage of protoplast decreased to 1%, while also showing an increase in striations of DNA. At 1x dye concentration, partial staining of the cell wall was observed. At 2x, complete staining of the cell wall was recorded. At 10x, complete staining of cell wall and nuclei was observed similar to dye concentrations at 2x with no significant saturation of dyes. Dye concentration at 100x produced an oversaturation of the dyes in the cell wall and nuclei causing them to mix and inhibit the efficacy of identifying bacterial cells and protoplasts. 2x was most optimum for complete staining of cell wall and nucleus. Background fluorescence noise was observed as concentration of dye increased. In Lactobacillus acidophilus, a lysozyme concentration of 175 μg/ml was sufficient for cell wall digestion. Efficacy of dye concentration was best at 2x with the least amount of background noise.