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Light scattering to Biomass calibration experiment

1. List of potential cultures (21 strains)

Axenic strains

0.5 - 1.5 micron diameter

  • Prochlorococcus MED4 (HLI) - smallest
  • Prochlorococcus AS9601 (HLII)
  • Prochlorococcus 1314 (HLII)
  • Prochlorococcus Natl2A (LLI)
  • Prochlorococcus 9303 (LLIV)
  • Prochlorococcus 9313 (LLIV) - largest
  • Synechococcus WH8102
  • Synechococcus 7803
  • Synechococcus 8012
  • Synechococcus 8109

2-5 micron diameter

  • Crocosphaera watsonii 8501

Non Axenic:

>3 micron diameter

  • TAPS CCMP1335 - smallest
  • Emiliania hux CCMP1742
  • TAPS CCMP3367
  • Phaeodactylum tricornutum CCMP632
  • Licmophora paradoxa
  • Micromonas pusilla
  • Minutocellus sp. CCMP3330
  • Navicula transitans
  • Thalassiosira weissflogii CCMP3365 - largest

2. Experimental setup

Culture will be grown under continuous light (100 µmol photon m-2 s-1). Cultures will be harvested in exponential phase.

  • Triplicate samples of 50 ml will be filtered on pre-combusted GFF filter for CHN analysis. 150 ml
  • Triplicate 1 ml will be used for counting (Influx). 3 ml
  • Triplicate 10X diluted 100 ml will be used for FSC measurement on SeaFlow (3 instruments). 90 ml.
  • Triplicate 10X diluted 100 ml will be used for FSC measurement on LISST (1 instrument). Will only analyze cells > 2 micron in diameter. 30 ml
  • Triplicate 50 ml will be used for Volume measurement (Coulter Counter). All but Prochlorococcus cultures. 150 ml
  • 100 ml will be aliquoted into 2 ml cryovials, fixed with Glutaraldehyde + Pluronic F68 and kept at -80 degC. These aliquots will be sent to other instruments for FSC calibration. 100ml

Total volume needed < 500 mL

To be more manageable, the calibration experiment will be divided in two parts:

  • Cells >3 µm (11 strains) FEBRUARY 21st-24th - NON AXENIC
  • Cells 0.5-1.5 µm (10 strains) MARCH 14th - 16th - AXENIC

3. Preparation - LARGE CELLS

>3 micron diameter

  • TAPS CCMP1335 - smallest
  • Emiliania hux CCMP1742
  • TAPS CCMP3367
  • Phaeodactylum tricornutum CCMP632
  • Licmophora paradoxa
  • Micromonas pusilla
  • Minutocellus sp. CCMP3330
  • Navicula transitans
  • Thalassiosira weissflogii CCMP3365 - largest

February 8

2.5 mL from stock diluted 10X in 25 mL of F/2 medium for non-axenic cultures - Duplicate cultures

February 10

1 ml collected from duplicate A for counting (Influx) using 2 µm beads:

  • TAPS CCMP3367 = 1,242,440 cells mL-1
  • TAPS CCMP1335 = 651,800 cells mL-1
  • Navicula transitans = 616,690 cells mL-1
  • Thalassiosira weissflogii CCMP3365 = 305,660 cells mL-1
  • Licmophora paradoxa = 87 cells mL-1
  • Minutocellus sp. CCMP3330 = 40,350 cells mL-1
  • Phaeodactylum tricornutum CCMP632 = 634,200 cells mL-1
  • Emiliania hux CCMP1742 = 300,340 cells mL-1
  • Micromonas = 2,787,500 cells mL-1

Dilution of duplicate A with F/2 medium as follow (duplicate B as backup):

  • Dilution 100 for CCMP3367, Micromonas
  • Dilution 50 for CCMP1335, Navicula, CCMP3365, CCMP632, CCMP1742
  • Dilution 10 for CCMP3330

February 15

1 ml collected from Diluted cultures for counting (Influx) using 1 µm beads:

  • TAPS CCMP3367 = 2,355,180 cells mL-1
  • TAPS CCMP1335 = 1,439,900 cells mL-1
  • Navicula transitans = 1,597,640 cells mL-1
  • Thalassiosira weissflogii CCMP3365 = 282,450 cells mL-1
  • Licmophora paradoxa = 1,410 cells mL-1
  • Minutocellus sp. CCMP3330 = 863,380 cells mL-1
  • Phaeodactylum tricornutum CCMP632 = 2,290,940 cells mL-1
  • Emiliania hux CCMP1742 = 23,820 cells mL-1
  • Micromonas = ** 181,400** cells mL-1

February 16

Dilution with F/2 medium, final volume 100 mL

  • TAPS CCMP3367 = D100 (2 ml in 200 ml)
  • TAPS CCMP1335 = D100 (2 ml in 200 ml)
  • Navicula transitans = D100 (2 ml in 200 ml)
  • Thalassiosira weissflogii CCMP3365 = D20 (10 ml in 200 ml)
  • Licmophora paradoxa = merge all cultures together (50 ml) and add 150 ml F/2
  • Minutocellus sp. CCMP3330 = D100 (2 ml in 200 ml)
  • Phaeodactylum tricornutum CCMP632 = D100 (2 ml in 200 ml)
  • Emiliania hux CCMP1742 = D8 (50 ml in 200 ml)
  • Micromonas = D20 (10 ml in 200 ml)

February 17

1 ml collected from 200-ml cultures for counting (Influx) using 2 µm beads:

  • TAPS CCMP3367 = 35,130 cells mL-1
  • TAPS CCMP1335 = 38,680 cells mL-1
  • Navicula transitans = 33,250 cells mL-1
  • Thalassiosira weissflogii CCMP3365 = 75,330 cells mL-1
  • Licmophora paradoxa = 145 cells mL-1
  • Minutocellus sp. CCMP3330 = 3,680 cells mL-1
  • Phaeodactylum tricornutum CCMP632 = 72,720 cells mL-1
  • Emiliania hux CCMP1742 = 1,453 cells mL-1
  • Micromonas = 8,390 cells mL-1

D2 for TW CCMP3365 (remove 100 ml of culture and replaced by F/2 medium)

February 21

1 ml collected from 200-ml cultures for counting (Influx) using 2 µm beads:

  • TAPS CCMP3367 = 1,264,600 cells mL-1
  • TAPS CCMP1335 = 1,033,920 cells mL-1
  • Navicula transitans = 1,372,740 cells mL-1
  • Thalassiosira weissflogii CCMP3365 = 335,260 cells mL-1
  • Licmophora paradoxa = 700 cells mL-1
  • Minutocellus sp. CCMP3330 = 339,120 cells mL-1
  • Phaeodactylum tricornutum CCMP632 = 2,524,320 cells mL-1
  • Emiliania hux CCMP1742 = 1,64 cells mL-1
  • Micromonas = 15,260 cells mL-1

Dilutions (F/2 medium)

  • D3 for TAPS-1335, TAPS-3367, Navicula, TW3365, Minutocellus (Add 400 ml F/2)
  • D5 PT-632 (remove 100 ml, add 400 ml F/2)
  • No dilution for Ehux, Micromonas, Licmophora

February 22

  • D3 of Navicula and Minutocellus (collected 200 ml of surface bottle and added 400 ml F/2)

February 23

1 ml collected from 600-ml cultures for counting (Influx) using 2 µm beads:

  • TAPS CCMP3367 = 828,060 cells mL-1
  • TAPS CCMP1335 = 651,16 cells mL-1
  • Navicula transitans - D3= 277,280 cells mL-1
  • Navicula transitans = 651,140 cells mL-1
  • Thalassiosira weissflogii CCMP3365 = 164,180 cells mL-1
  • Licmophora paradoxa = 660 cells mL-1
  • Minutocellus sp. CCMP3330 = 27,320 cells mL-1
  • Minutocellus sp. CCMP3330 - D3 = 28,520 cells mL-1
  • Phaeodactylum tricornutum CCMP632 = 651,140 cells mL-1
  • Emiliania hux CCMP1742 = 1,840 cells mL-1
  • Micromonas = 21,980 cells mL-1

Dilutions (filtered seawater)

  • D50 for TAPS3367, TAPS1335, PT632
  • D10 for Navicula - D3 and TW3365
POC HARVEST 1

  1. Counting 100-300 µL with Influx using 1 µm beads

  2. filtration on pre-combusted GFF, triplicate:

  • Phaeodactylum tricornutum CCMP632 = 50 ml
  • TAPS CCMP3367 = 50 ml
  • Blank = 75 ml
  • TAPS CCMP1335 = 100 ml (a fourth filter for 1335 is either 100ml or 75ml, not sure, but written on the box)
  • Navicula transitans = 75 ml
  • Thalassiosira weissflogii CCMP3365 = 50 ml
  1. Counting for 6 minutes (2 files) with SeaFlow #740 and #751 using 1 µm beads

February 24

POC HARVEST 2

  1. Counting 100 µL with Influx using 1 µm beads

  2. filtration on pre-combusted 0.7 µm GFF, triplicate:

  • Micromonas = 50 ml
  • Blank = 75 ml
  • Phaeodactylum tricornutum CCMP632 = 50 ml
  • Emiliania hux CCMP1742 = 26 ml (only 1 filter for Ehux)
  • Licmophora paradoxa = 50 ml
  1. Counting for 6 minutes (2 files) with SeaFlow #740 and #751 using 1 µm beads

4. Preparation - SMALL CELLS

0.5 - 1.5 micron diameter

  • Prochlorococcus MED4 (HLI) - smallest
  • Prochlorococcus AS9601 (HLII)
  • Prochlorococcus 1314 (HLII)
  • Prochlorococcus Natl2A (LLI) - largest
  • Synechococcus WH8102
  • Synechococcus 7803

April 25

1 mL from stock grown in Pro99 medium for axenic cultures under continuous light D100 before counting

  • MED4 = 676 x 106 cells mL-1
  • AS9601 = 199 x 106 cells mL-1 -- 2 populations (contamination?)
  • 1314 = 291 x 106 cells mL-1
  • Natl2A = 32 x 106 cells mL-1
  • WH8102 = 38 x 106 cells mL-1 -- unhealthy
  • 7803 = 48 x 106 cells mL-1

D5 for all stocks with Pro99

April 27

1 mL from stock, D100 before counting

  • MED4 = 302 x 106 cells mL-1
  • AS9601 = 181 x 106 cells mL-1 -- 2 populations (contamination?)
  • 1314 = 115 x 106 cells mL-1
  • Natl2A = 32 x 106 cells mL-1
  • WH8102 = 24 x 106 cells mL-1 -- unhealthy
  • 7803 = 35 x 106 cells mL-1

May 1st

D5 of all cultures

May 2nd

1 mL from stock, D100 before counting

  • MED4 = 140 x 106 cells mL-1 -- Low FSC pop
  • AS9601 = 227 x 106 cells mL-1 =
  • 1314 = 34 x 106 cells mL-1
  • Natl2A = 7 x 106 cells mL-1 -- small pop
  • WH8102 = 24 x 106 cells mL-1 -- Low CHL pop
  • 7803 = 15 x 106 cells mL-1 -- Ok, but low CHL pop

Washing 6 - 500 ml glass Erlen with 10% HCL

May 3

1 mL from stock, D100 before counting

  • MED4 = 263 x 106 cells mL-1
  • AS9601 = 556 x 106 cells mL-1
  • 1314 = 45 x 106 cells mL-1
  • Natl2A = 11 x 106 cells mL-1
  • WH8102 = 46 x 106 cells mL-1 -- Ok, but low CHL pop
  • 7803 = 41 x 106 cells mL-1 -- Ok, but low CHL pop

May 4

1 mL from stock, D100 before counting

  • MED4 = 377 x 106 cells mL-1
  • AS9601 = 469 x 106 cells mL-1
  • 1314 = 79 x 106 cells mL-1
  • Natl2A = 15 x 106 cells mL-1
  • WH8102 = 70 x 106 cells mL-1
  • 7803 = 48 x 106 cells mL-1

Take 30 ml stock into 120 ml of Pro99 medium in flask (flask was washed with 10%HCl, rinsed with MQ and autoclaved)

May 5

POC HARVEST 3

  1. Influx count: 985 µL media , 5 µL beads + 10 µL cultures Counting 100 µL with Influx using 1 µm beads

  2. filtration on pre-combusted 0.3 µm GF-75 (Advantec), triplicate:

  • 30 ml for cultures
  • 30 ml for Blank (Pro99)
  1. Counting for 6 minutes (2 files) with SeaFlow #740 and #751 using 1 µm beads

NB: 100-300 dilution before counting cells on SeaFlow; Trigger on FSC (Red noisy on #751)

NB2: Pressure sensor not working on #751