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='''Single E.coli cell micromanipulation test on 04-12-'11'''= ==Testing objectives== *Focus on the performance of fluorescently stained cells. **Question 1: Can we see the same number of samples under the BF GFP/DAPI filters? (If not, what do we missed, E.coli or unknown particles?) **Question 2: Do we observe flourescently stained particles in negative control (blank slide)? ==Cell resource== *Using old stored E.coli K-12 culture (4C, 1XPBS, March, 2010) **Estimated concentration = 10^9 CFU/mL ==E.coli stain (SYBR-safe / DAPI)== *Prepare dye working solution (10X) **Add 1uL SYBR-safe(10,000X) with 50uL 1X TE in a 1.7mL tube wrapped in foil => 200X **Add 10uL SYBR-safe(200X) with 190uL 1X TE in a 1.7mL Amber tube => 10X **Transfer 2uL E.coli culture into the 200uL dye working solution *Same method was prepared for DAPI staining. ==E.coli cell microscopy analysis== *Comparison types: 1. DAPI-stained cell(2uL, 10^7 CFU/mL) on regular slide with coverslip [[File:041211_dapi_stained_ecoli-bf1.png|400px]] [[File:041211_dapi_stained_ecoli-dapi100.png|400px]] 2. DAPI-stained cell(2uL, 10^7 CFU/mL) on micro-petri-dishes [[File:041211_dapi_stained_dish-bf1.png|400px]] [[File:041211_dapi_stained_dish-dapi200.png|400px]] 3. no cell on micro-petri-dishes [[File:041211_no_cells_dish-bf1.png|400px]] [[File:041211_no_cells_dish-dapi200.png|400px]] 4. no cell on regular slide [[File:041211_no_cells_slide-bf1.png|400px]] [[File:041211_no_cells_slide-gfp500.png|400px]] ==Discussions== *The number of particles (probably E.coli cells) are not consistent between BF and DAPI modes. **The missing particles have the similar size as non-missing particles. **It seems like the larger particles were stained better with DAPI and more observable under DAPI mode. *There are observable particles on no cell slide under both DAPI and SYBR modes. These particles showed similar size as the expected E.coli size (1~2um). ==Conclusion== *How do we tell if they are E.coli or non-Ecoli particles? **Based on the cell floating motion **Use high concentration of cells (density changed with our serial dilution) **Higher magnification of objective with phase-contrast setting. ==Next== *Try to improve the dye staining protocol **Using fixed cell **SSC hybridization buffer? **Higher dye concentration and longer staining time. **Use AntiFade reagent? *Optimize phase-contrast condition.
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