Brent said wiki is public so won't post ms here...but where???
Nov 7, 2011

Aliquoted ciliates from larval abs into 16 wells of 24 well plate (250 uL of ciliates per well with ambient CO2 SW (1 um filtered). Added 2 mL ambient or ~750ppm CO2 SW to each well.

Added 2X4 mm plug of 10% Tryptone agar per well as food. parafilmed the wells, added lid.
Incubate at RT.
After about 5 mins some ciliates were around tryptone plug.
Will count in morning to see if difference and if tryptone agar can sustain the ciliates.

Note may add antibiotics if too much bacterial growth.

Nov 8, 2011

Ciliates still alive

Nov 10, 2001

Ciliates still growing as are bacteria.
Made 0.2 um Filtered seawater (FSW)
Added 100IU Pen and 100 ug Strep per ml to some (expired in 2008...yikes) FSW-PS
Added same to another plus 0.5x fungizone with PS: FSW- FPS
Added ~500uL original culture to each of 24 well of 24 well plate
Added ~1.5 mL FSW or FSW-PS or FSW-FPS to 8 wells each
Added 10% tryptone plug to each well and parafilmed plate--> RT

Reduced each well of 11 - 7 plate to 1 ml and then added the 3 buffers to 1/3 wells on that plate--> parafilm and RT inc.

Froze ~10 mL aliquots of the 3 FSWs at -20 in 236


Nov 14, 2011

Ciliates grew best (much higher density) in FSW than either FSW with PS or FPS.
Used ciliates in FSW to inoculate new plates to test impact of CO2 level on ciliate growth...took 1 mL from several ambient ciliate wells and pooled. Removed 30 uL aliquots (n=3) and added to 96 well plate, immobilized with 10 uL 10% bleach and counted: 45, 36,38=119/3=40/30uL=1.33/uLx 50uL=~67/well.

Nitzchia: Took 30 uL from Nitzchia aliquot used to inoculate wells, and then also did 1:10 dilution. Counted both wells:
107 Nitzchia in 10^-1 dilution on hemocytometer: 1.07x10^7 ciliates per mL. counted 5 squares at full concentration = 76 cellsx5x10^4=3.6x10^6/mL with a mean of 7.2x10^6/mL x 0.05 mL/well =3.6x10^5/well with 50 uL added and 3.6x10^4 per well with 5 uL added.

Pinto abalone larvae: Collected pinto ab larvae spawned on Nov 8, 2011, hatched Nov 9 (=Day1 larvae), so today = day 6 post fertilization (Day 6 larvae). Captured larvae from 3 families onto 64 uM submerged screen and brought to UW in plastic bottles (n=2). At UW, rescreened (30um screen), pooled and placed in 50 mL conical tube ambient seawater. Mean number of larvae in 50 uL = 35.

CO2 Experiment:
0.22 um FSW bubbled with ambient air, 750 ppm or 2000 ppm CO2 from ~11am to ~230pm. Then added SW to each of 6 square boxes (~8x8inches and about 1.5 inches deep) with 2 per CO2 level (ambient -~400-, 750 and 2000 ppm) and placed in a sweater box with lid. To one to each SE box, air, 750 or 2000ppm CO2 was bubbled. Box without gas is to check pH change over night and those receiving gas bubbled are to make new SW for water changes.

Used 3 waters to inoculate one 24 well plate (1mL per well - double check this), 12 well plates, 4 mL per well and 6 well plates (5mL per well) x 2 plates each CO2 level. Other plates: 1 plate per CO2 level (e.g. 12 or 24 well) at 3pm.

Added 50 uL abalone larvae to each well in all 6 and 12 well plates (~35 per well....note: inverted falcon tube 2x and then removed two successive aliquots and then re- inverted). then placed plated in Rm 6 with proper CO2 gas blowing into sweater box at 3pm.

Took spec pH of the 3 waters at 4pm: Ambient was pH 7.9, 750 ppm was 7.5 and 2000ppm was 7.4. Salinity is 32 ppt and temperature is 16.8C.

At 5 pm Brent added 5 uL of 2 mM GABA per well in one 6 well plate and 6 wells of 12 well plate at each CO2 level to obtain a final concentration of 2 uM GABA per well.

Nov 15, 2011
845am 2000 not bubbling…likely leaking into room but some into box. Changed tubing and got gas flowing to SW. Brent bought fermentation locks to add to each box.
10:45 am Lisa took spec pH of all 6 SW boxes:
11/15/2011
10:45am
400
0
7.87


400
1
7.86


750
0
7.6


750
1
7.67


2000
0
7.39


2000
1
7.37
3pm Sammi took spec pH all boxes.
Carolyn counted until nearly 715pm..ugh. See excel file:


Ciliates not much growth so Brent fed at 745 pm 1 ~2x3 mm plug of 10% tryptone agar.

Nov 16, 2011
Carolyn counted from ~1230-6pm (oh joy) and data is in file above
but summary here:
% ALIVE 6
%ALIVE 6G
%ALIVE 12
% ALIVE 12G
%SET 6
% SET 6G
%SET 12
%SET 12G
CO2
Time
0.80
0.88
0.90
0.91
0.07
0.21
0.07
0.17
400
24
0.83
0.85
0.84
0.81
0.14
0.16
0.07
0.13
750
24
0.83
0.83
0.82
0.81
0.03
0.10
0.04
0.12
2000
24
0.57
0.45
0.60
0.41
0.08
0.09
0.05
0.06
400
48
0.41
0.31
0.51
0.22
0.02
0.02
0.04
0.00
750
48
0.57
0.18
0.41
0.08
0.03
0.01
0.02
0.01
2000
48
Crud too many factors....time for Brent's magic analyses :-)

Ciliates don't like CO2 (see graph below)...time for Sam to ID via PCR...samples to Sam??
Axes are Y=ciliates per well and X=treatment.
Ciliates_like_low_CO2.jpg
Ciliates at 3 CO2 levels

Nov 17, 2011
Different, larger ciliates growing in some of 750 wells and 1-2 2000 wells with pinto abs....kept going and will count tomorrow and also culture these.

December 2011

Grew ciliates more: Now have:

1) small ambient ones from above study
2) small 2000 ppm
3) large will anterior and posterior cilia and no spot
4) large as in 3 but with spot
5) vermiform slow growing protist

Redid CO2 study with ambient, 750 and 2000 ppm for 72 hrs. Included ciliates 1-4 as well as liquid culture of 4 sea grass laby cultures.

Robyn passed cultures pm 12 22 11 and Carolyn passed on Dec 29 2011. At RT in 240 on Carolyn's bench.

January 4, 2012
Robyn entering and analyzing ciliate data. Sammi to take pics and analyze laby data.