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Methods Confocal Microscopy Staining Filtration Collection.

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Presentation on theme: "Methods Confocal Microscopy Staining Filtration Collection."— Presentation transcript:

1

2 Methods Confocal Microscopy Staining Filtration Collection

3 1 sample collection from the Ballona Wetlands during the ebb flow 1 sample collection during the flood flow Determination of salinity, turbidity, oxygen level, water temperature

4 Filtration Dilutions Using 0.02 um and 0.4 um filtered water Water contamination most common contamination 1 ml of each dilution through 0.02 um filter

5 Staining SYBR Green I Binds to double stranded DNA and RNA “Virus particles were distinctly shaped ‘pinpricks’ and fluoresced bright green, and bacterial cells could easily be distinguished from viruses because of their relative size and brightness” [Noble, R.T. & Fuhrman, J.A “Use of SYBR Green I for rapid epifluorescence counts…” Anti-fade/ glycerol mix

6 Confocal Microscopy EbbFlood 6/21/12 EBB 3 10 -1 (g)6/21/12 FLD 2 10 -1 (i)

7 Protist 6/21/12 EBB 2 10 -2 (b)

8 Data: Average Bacteria per 10 images EBB 1 (-1)EBB 2 (-1)EBB 3 (-1)AVERAGEFLD 1 (-1)FLD 2 (-1)FLD 3 (-1)AVERAGE 21-May25.71826.623.43N/A 30-May24.815.54.614.9723.1834.621.90 4-Jun24.810.610.315.23481.14.37 19-Jun10.512.88.210.507.95.84.15.93 21-Jun8.312.88.29.777.87.39.48.17 14.78 10.09 EBB 1 (-2)EBB 2 (-2)EBB 3 (-2)AVERAGEFLD 1 (-2)FLD 2 (-2)FLD 3 (-2)AVERAGE 21-May22.715.58.115.43N/A 30-May22.42.914.213.1733.830.31626.70 4-Jun22.46.92.310.534.37.12.34.57 19-Jun0.50.31.20.670.61.1 0.93 21-Jun1.11.92.91.971.81.61.11.50 8.35 8.43

9 Data: Average Bacteria per mL EBB 1 (-1)EBB 2 (-1)EBB 3 (-1)AVERAGEFLD 1 (-1)FLD 2 (-1)FLD 3 (-1)AVERAGE 21-May3.50E+092.45E+093.62E+093.19E+09N/A 30-May3.37E+092.11E+096.26E+082.04E+093.14E+093.54E+094.71E+093.80E+09 4-Jun3.37E+091.44E+091.40E+092.07E+095.44E+081.09E+091.50E+085.95E+08 19-Jun1.43E+091.10E+091.20E+091.24E+091.07E+097.89E+085.58E+088.06E+08 21-Jun1.13E+091.74E+091.12E+091.33E+091.06E+099.93E+081.28E+091.11E+09 1.97E+09 1.58E+09 EBB 1 (-2)EBB 2 (-2)EBB 3 (-2)AVERAGEFLD 1 (-2)FLD 2 (-2)FLD 3 (-2)AVERAGE 21-May3.09E+102.11E+101.10E+102.10E+10N/A 30-May3.05E+103.94E+091.93E+101.79E+104.60E+104.12E+102.18E+103.63E+10 4-Jun3.05E+109.38E+093.12E+091.43E+105.85E+099.66E+093.13E+096.21E+09 19-Jun6.80E+084.08E+081.63E+099.06E+088.16E+081.50E+09 1.27E+09 21-Jun1.50E+092.58E+093.94E+092.67E+092.45E+092.18E+091.50E+092.04E+09 1.14E+10 1.15E+10

10 Data: Average Bacteriophage per 10 images EBB 1 (-1)EBB 2 (-1)EBB 3 (-1)AVERAGEFLD 1 (-1)FLD 2 (-1)FLD 3 (-1)AVERAGE 21-May323.2290.1168.4260.57N/A 30-May245.4214.165.4174.97203.3232.1293.5242.97 4-Jun245.4127.3215195.9083.457.810.550.57 19-Jun232.1216.9204.1217.702535833.2114.73 21-Jun164.8232.7217204.8349.233.9136.873.30 210.79 120.39 EBB 1 (-2)EBB 2 (-2)EBB 3 (-2)AVERAGEFLD 1 (-2)FLD 2 (-2)FLD 3 (-2)AVERAGE 21-May11.114.721.815.87N/A 30-May13.341.832.829.3046.921.833.434.03 4-Jun22.423.12.315.9311.7154.713.159.83 19-Jun6.54.53.34.776.68.822.612.67 21-Jun6.82.53.34.202.81.35.23.10 14.01 27.41

11 Data: Average Bacteriophage per mL EBB 1 (-1)EBB 2 (-1)EBB 3 (-1)AVERAGEFLD 1 (-1)FLD 2 (-1)FLD 3 (-1)AVERAGE 21-May4.40E+103.95E+102.29E+103.55E+10N/A 30-May3.43E+102.91E+108.89E+092.41E+102.76E+103.16E+103.99E+103.30E+10 4-Jun3.34E+101.73E+102.92E+102.66E+101.13E+107.86E+091.43E+096.86E+09 19-Jun3.16E+102.95E+102.78E+102.96E+103.44E+107.89E+094.52E+091.56E+10 21-Jun2.24E+103.16E+102.95E+102.78E+106.69E+094.61E+091.86E+109.97E+09 2.87E+10 1.64E+10 EBB 1 (-2)EBB 2 (-2)EBB 3 (-2)AVERAGEFLD 1 (-2)FLD 2 (-2)FLD 3 (-2)AVERAGE 21-May1.51E+102.00E+102.96E+102.16E+10N/A 30-May1.81E+105.68E+104.46E+103.98E+106.38E+102.96E+104.54E+104.63E+10 4-Jun1.81E+103.12E+103.60E+102.84E+101.59E+102.10E+111.78E+108.12E+10 19-Jun8.84E+096.12E+094.49E+096.48E+098.98E+091.20E+103.07E+101.72E+10 21-Jun9.25E+093.20E+094.49E+095.65E+093.81E+091.77E+097.07E+094.22E+09 2.04E+10 3.72E+10

12 Data: Average Protists per 10 images EBB 1 (-1)EBB 2 (-1)EBB 3 (-1)AVERAGEFLD 1 (-1)FLD 2 (-1)FLD 3 (-1)AVERAGE 21-May0.1000.030000.00 30-May0.2000.070000.00 4-Jun0.2000.0700.100.03 19-Jun0.20.100.100000.00 21-Jun0.1 00.070000.00 0.07 0.01 EBB 1 (-2)EBB 2 (-2)EBB 3 (-2)AVERAGEFLD 1 (-2)FLD 2 (-2)FLD 3 (-2)AVERAGE 21-May0000.00000 30-May0000.00000 4-Jun00.400.13000.10.03 19-Jun0000.00000 21-Jun0.1000.030000.00 0.03 0.01

13 Data: Average Protists per mL EBB 1 (-1)EBB 2 (-1)EBB 3 (-1)AVERAGEFLD 1 (-1)FLD 2 (-1)FLD 3 (-1)AVERAGE 21-May1.36E+07004.53E+060000 30-May2.72E+07009.07E+060000 4-Jun2.72E+07009.07E+0601.36E+0704.53E+06 19-Jun2.72E+071.36E+070 0000 21-Jun1.36E+07 09.07E+060000 9.07E+05 EBB 1 (-2)EBB 2 (-2)EBB 3 (-2)AVERAGEFLD 1 (-2)FLD 2 (-2)FLD 3 (-2)AVERAGE 21-May00000000 30-May00000000 4-Jun05.44E+0801.81E+08002.72E+089.07E+07 19-Jun00000000 21-Jun1.36E+08004.53E+070000 2.27E+07

14 Data: Turbidity 5/30/2012 EBB [NTU]5/30/2012 FLD [NTU]6/4/12 EBB [NTU]6/4/12 FLD [NTU] Turbidity 1:5.971.581.521.04 Turbidity 2:2.521.432.061.01 Turbidity 3:2.411.61.361.03

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