AH Mcap2021 Physiology Processing

AHuffmyer Mcap 2021 physiology processing

MCAP2021 Sample processing master list

Physiology master list

Contents:

Cell counts

Sample location: Putnam Lab -80C AH Rack; “Physiology Host/Sym/Holo Separations”.

Google drive folder for cell counts
Google sheet for data input
Protocol

We’re trying the symbiont pellet first to resuspend in small volumes of 1X PBS.
From the ‘Symbiont’ tube (LETTER-Symb).

Protocol:

  1. Thaw symbiont pellet.
  2. Add 400 uL cold 1X PBS.
  3. Homogenize with small bit on half speed for 10-15 seconds.
  4. Count.
TubeID Date_counted Tech Squares_counted Count1 Count2 Count3 Count4 Count5 Count6 Notes CV
A-Symb 20220425 ES 2 166 149 188 167 195 185   9.86
B-Symb 20220426 ES 4 147 152 167 188 172 187   10.16
C-Symb 20220425 ES 3 201 170 168 193 197 181   7.62
D-Symb 20220426 ES 3 118 120 105 120 97 114   8.36
E-Symb 20220426 ES 3 140 129 125 145 130 122   6.74
F-Symb 20220426 ES 9 109 135 143 124 125 144 dropped count: 93 10.27
G-Symb 20220426 KD 6 166 169 142 147 144 143 dropped count: 112 8.09
H-Symb 20220426 ES 9 27 15 16 28 23 12   33.40
I-Symb 20220420 ES 3 170 151 156 168 157 151   5.21
J-Symb 20220425 ES 3 156 162 177 154 179 176 dropped count: 126 6.76
K-Symb 20220426 KD 3 174 149 139 132 146 177   12.13
L-Symb 20220425 ES 3 176 148 166 153 174 191   9.44
M-Symb 20220425 ES 4 198 204 191 191 191 198   2.76
N-Symb 20220425 ES 2 125 110 111 121 138 121   8.48
O-Symb 20220425 ES 3 218 182 184 187 197 182   7.34
P-Symb 20220426 ES 4 123 117 143 146 128 140   8.90
Q-Symb 20220420 ES 1 127 126 143 133 110 137   8.81
R-Symb 20220425 ES 3 190 176 198 159 203 179   8.78
S-Symb 20220420 ES 4 149 159 142 167 144 171   7.84
T-Symb 20220425 ES 4 173 195 146 157 157 164   10.31
U-Symb 20220426 ES 2 211 204 206 194 223 237   7.20
V-Symb 20220426 ES 2 230 230 238 251 228 205   6.54
W-Symb 20220425 ES 3 172 190 193 188 181 180   4.22
X-Symb 20220426 KD 9 121 151 128 108 116 111   12.80

Chlorophyll concentration

Sample location: Putnam Lab -80C AH Rack; “Physiology Host/Sym/Holo Separations”.

Google drive folder for chlorophyll
Protocol

From 200 uL leftover from cell count ‘Sym’ tube.

  1. Thaw homogenate
  2. Take 200 uL leftover from cell count ‘Sym’ tube and place in new 1.5 mL microcentrifuge tube.
  3. Spin down at 13,000 rpm for 3 minutes.
  4. Remove supernatant and throw away (since this is 1X PBS not host supernatant).
  5. Add 1 mL 100% acetone to each tube.
  6. Vortex for 15 seconds.
  7. Place in 4C flammable fridge for 24 hours.

Placed in fridge at 2:56 pm 20220427. Read out 20220428 at 3 pm.

  1. Vortex 15 seconds
  2. Centrifuge at 13,000 rpm for 3 minutes.
  3. Take 200 uL of the supernatant and place in well in 96 well quartz plate.
  4. Repeat Step 10 to create a duplicate (so you have 2 wells per sample). Use silicone pad to cover up wells with sample already in them.
  5. 200 uL 100% acetone as blank.
  6. Read on microplate reader (protocol installed on computer).

Platemap:

All files on google drive.

Larval Size Photographs

Putnam Lab 4C Fridge, Plastic box in plastic bag; “Mcap 2021 Fixed Larvae”

Sample list for photographs
Photos google drive
Protocol
Larval size datasheet

Notes:

  • We were having trouble with the brightness and focus on 20220419. These images may not be as sharp but should still be able to get length and width out of them.
Timepoint Response Tube ID Date Collected Time Treatment Tank Larval volume Sample method Storage Photograph ID’s Date Photographed Initials Photo Uploaded?
Baseline Size P1 20210614 1840 Ambient Pool 100uL 4% PFA 4C P1-1, P1-2, P1-3, P1-4, P1-5 20220419 ES Yes
Baseline Size P2 20210614 1840 Ambient Pool 100uL 4% PFA 4C P2-1, P2-2, P2-3 20220419 ES Yes
Baseline Size P3 20210614 1840 Ambient Pool 100uL 4% PFA 4C P3-1, P3-2, P3-3 20220419 ES Yes
Baseline Size P4 20210614 1840 Ambient Pool 100uL 4% PFA 4C P4-1, P4-2, P4-3 20220419 ES Yes
Sampling Size P5 20210618 1545 Ambient A1 100uL 4% PFA 4C P5-1, P5-2, P5-3, P5-4 20220426 KD Yes
Sampling Size P6 20210618 1545 Ambient A2 100uL 4% PFA 4C P6-1, P6-1, P6-3, P6-4, P6-5 20220426 KD Yes
Sampling Size P7 20210618 1545 Ambient A3 100uL 4% PFA 4C P7-1, P7-2, P7-3, P7-4, P7-5, P7-6 20220426 KD Yes
Sampling Size P8 20210618 1545 Ambient A4 100uL 4% PFA 4C P8-1, P8-2, P8-3, P8-4, P8-5 20220426 KD Yes
Sampling Size P9 20210618 1545 Ambient A5 100uL 4% PFA 4C P9-1, P9-2, P9-3, P9-4, P9-5 20220426 KD Yes
Sampling Size P10 20210618 1545 Ambient A6 100uL 4% PFA 4C P10-1, P10-2, P10-3, P-10-4 20220426 KD Yes
Sampling Size P11 20210618 1545 High H1 100uL 4% PFA 4C P11-1, P11-2, P11-3, P11-4, P11-5 20220426 KD Yes
Sampling Size P12 20210618 1545 High H2 100uL 4% PFA 4C P12-1, P12-2, P12-3, P12-4 20220426 KD Yes
Sampling Size P13 20210618 1545 High H3 100uL 4% PFA 4C P13-1, P13-2, P13-3, P13-4, P13-5 20220426 KD Yes
Sampling Size P14 20210618 1545 High H4 100uL 4% PFA 4C P14-1, P14-2, P14-3, P14-4 20220426 KD Yes
Sampling Size P15 20210618 1545 High H5 100uL 4% PFA 4C P15-1, P15-2, P15-3, P15-4 20220426 KD Yes
Sampling Size P16 20210618 1545 High H6 100uL 4% PFA 4C P16-1, P16-2, P16-3, P16-4, P16-5 20220426 KD Yes
Written on April 13, 2022