10/16/18 KJ/KC older freezer splits of 34H from -70C freezer taken to MSB to grow up in 1/2 2216 at -1C for future experimental stocks - started today (not rocking). P7E from growing stock up fresh from growing 2C stocks - same conditions.
10/1818 Lab organizing w/ Marcela
10/24/18 Checks on new SoLow benchtop freezer/ ship peristaltic pump for repair
10/25/18 Final trainings, Freezer troubleshooting, computer/printer troubleshooting, lab signage, UV lamp return and re-order.
11/1/18 Freeze glycerol splits of 34H (growing from KJ freezer split) at -1C; into 10% glycerol (pelleted in media -2216, for 5 min at 15K RCF); P7E this round was from 9/2018 grown culture at 2C as the -1C culture did not grow up. Will repeat to have a -1C culture of P7E, but have this one in 10% gly, pelleted as above - bringing to BH next week. RT. control was negative for growth (23°C).
12/5/18 Prepare new batch of P7E freezer splits with fresher cells. Same as above.
11/8/18 Group Meeting BH 512
11/14/18 Assist Marcela with Preliminary ; bring back 34H culture from deep freeze to grow up.
11/21/18 Grow up 34H in 45 ml 1/2 2216. Upright -1°C incubator, with rocker. Actual temp more like 0°C. Grow until 0.4 OD600. OD600 was 0.297 on 11/27 when added to the 300 ml fresh 1/2 2216 on 11/28. Growing with stir bar until Monday when I will take a final OD600 and an EFM count.
11/29/18 Assist Marcela with Expt details; she will start on Tuesday with Monday's 34H culture.
12/3/18 34H cells grown in more volume 1/2 2216 are ready with an OD600 of 0.404. Cell count by EFM = 1.04E+09 bact/ml! YAY!
1/7/2019 Counts calculated from end of Dec MES preps, counts on KJ preps, prep MES new samples if time allows.
A1 = 1.29E+08 b/ml (overloaded slide so underestimate but used entire vol so no reprep)
A2 = 3.42E+08 b/ml
A3 = 2.82E+08 b/ml
B1 = 3.21E+08 b/ml
B2 = 4.08E+08 b/ml
B3 = 2.89E+08 b/ml
D1 = 2.67E+08 b/ml
D2 = 2.65E+08 b/ml
D3 = 2.93E+08 b/ml
KJ sample counts:
AC1 = 1.36E+08 b/ml
AC2 = 9.11E+07 b/ml
AC3 = 1.41E+08 b/ml
B1 = 1.04E+08 b/ml
B2 = 9.29E+07 b/ml
B3 = 1.12E+08 b/ml
D1 = 9.08E+07 b/ml (clumping)
D2 = 4.65E+07 b/ml (clumping)
D3 = 5.77E+07 b/ml (clumping)
1/14/2019 Priority counts for MES
57 = 4.85E+08
56 = 3.51E+08
51 = 3.87E+08
48 = 4.42E+08
19 = 2.61E+07 (low field count - may reques reprep? ghosts in fields)
14 = 5.20E+07 (ghost cells in fields)
47 = 3.55E+08
55 = 3.59E+08
49 = 2.46E+08
46 = 2.02E+08
21 = 2.03E+08 (prep confusion; may want to repeat for a check) TOO HIGH - probably included ghosts in the count mistakenly though slide shows very bright cells - count on 2/26 seems clearer on ghost versus bright cells.
20 = 9.22E+07 (prep confusion; may want to repeat for a check) TOO HIGH - probably included ghosts in the count mistakenly though slide shows very bright cells - count on 2/26 seems clearer on ghost versus bright cells.
17 = 1.19E+08 (ghost cells in field)
18 = 8.85E+07
27 = 3.28E+08
26 = 2.03E+08
25 = 2.38E+08
2/19/2019 Continued counts - Many samples were clumpy even after addition of the trition X-100 which makes the StDev around the average high. Many samples also showed high numbers of "ghost" cells which don't stain brightly indicating a loss of DNA and likely nonviable cells in the sample. I do not count these, but a high number of ghosts in a sample make the preps hard to count accurately.
50 = 1.36E+08 (1/4 fd count; hi prep but no more vol)
21 = 9.37E+07 (reprep a lot lower than previous!! ??)
54 = 1.31E+08
53 = 1.58E+08
52 = 2.23E+08
60 = 1.93E+08
58 = 2.17E+08
59 = 2.33E+08
37 = 1.92E+08
34 = 1.68E+08
31 = 2.17E+08
22 = 1.02E+08
28 = 1.35E+08
40 = 1.98E+08
43 = 1.47E+08
1 = 7.29E+07
2/21/2019 Continued counts
35 = 2.38E+08
36 = 2.07E+08
38 = 9.70E+07
39 = 1.35E+08
41 = 1.83E+08
42 = 1.38E+08
44 = 1.75E+08
45 = 1.53E+08
32 = 1.06E+08
33 = 1.04E+08
13 = 1.67E+08
10 = 1.62E+08
2 = 2.78E+07
3 = 3.60E+07
23 = 1.53E+07
24 = 1.18E+08
30 = 8.92E+07
29 = 4.35E+07
19-2 = 1.80E+07 (Marcela fixed more cells on 2/20/19; many ghost cells) - matches previous count pretty well!
2/26/2019 Last of the counts for the -5/-10 experiment
7 = 8.33E+07
8 = 7.69E+07
9 = 6.07E+07
4 = 1.60E+07
6 = 1.40E+07
14 = 1.38E+08
15 = 1.24E+08
11 = 1.52E+08
12 = 1.43E+08
5 = 3.86E+06
20-2 = 2.59E+07 (Marcela fixed more cells on 2/20/19; many ghost cells)
21-2 = 7.22E+07 (Marcela fixed more cells on 2/20/19; many ghost cells but more brighter cells than in 20-2)
3/11/2019 Started freezer split of 34H in 1/2 2216 (35 ml) at -1C. Filtered and autoclaving large batch media; will cool at -1C and look for how I can stir it in BH unit (plug hole inadequate!). Started 34H on 3/12/2019 in 1200 ml 1/2 2216 stirring at -1C. Started a freezer split of P7E to get a head start on the next batch needed for April 1.
3/21/2019 Growth of culture started on 3/12 above. 3/19 OD600 3 pm reads 0.284, 3/21 OD600 1 pm reads 0.302 (1/2 2216 blank is 0.087 OD600). March 25 will do an OD600 at 10 am when fixing a count sample for Marcela.
3/26/2019 Final OD600 of 34H for Expt is 0.327 so still in exponential. DAPI count gives 1.00E+09 b/ml in the 1400 ml bottle. DAPI count on 1000 mL of the ASW-washed resuspension gives 1.01E+09 b/ml in ~500 ml volume.
4/3-8/2019 Counts on Expt 34H fixed samples by number:
Cell counts logged in spreadsheet (#1) LINK to MAR WORKSHEET #2 https://docs.google.com/spreadsheets/d/1fXa4h-4Q6psdL1GyDPXw-LGcGBnSei_lqsdJwVqJAR4/edit#gid=0 and to DEC WORKSHEET https://docs.google.com/spreadsheets/d/10YpceJZrS74TaND0mFITTdjXyDuL4u9xayQRvip01hc/edit?ts=5cc8a22b#gid=1102365721
Phase III: https://docs.google.com/spreadsheets/d/1v0D39sXWXmLJqQAIyH4KMUePJeF_ZTtsyyShYnedu54/edit?ts=5d5b3140#gid=950405272
C6 = 2.52E+08 b/ml
C3 = 2.85E+08
B6 = 2.62E+08
B3 = 3.22E+08
Z6 = 2.27E+08
Z3 = 1.61E+08
A6 = 2.54E+08
A3 = 2.48E+08
Z5 = 2.45E+08
A2 = 2.27E+08
A5 = 2.03E+08
Z2 = 1.99E+08
B2 = 2.62E+08
B5 = 2.67E+08
C2 = 3.53E+08
C2 = 2.87E+08
A1 = 2.48E+08
A4 = 2.37E+08
Z1 = 1.70E+08
Z4 = 2.28E+08
B1 = 3.21E+08
B4 = 2.75E+08
C1 = 2.31E+08
C4 = 2.66E+08
C9 = 3.05E+08
B9 = 3.01E+08
Z9 = 1.92E+08
A9 = 2.52E+08
Z7 = 2.31E+08
Z8 = 2.16E+08
A7 = 2.95E+08
A8 = 1.44E+08
B7 = 2.99E+08
B8 = 3.42E+08
C8 = 3.09E+08
C7 = 3.20E+08
May 6, 2019
On Thurs 5/2 started new P7E culture with a 25 ml inoculum of the 50cc grown tube from a freezer stock at -1C. Into 1200 ml of 1/2 2216 @ -1C. Todays count reveals all cells dividing and only a few examples of clumping cells (5-6 ct). Concentration about 3E+07 or early log growth.
May 9, 2019
P7E culture is now at 2E+08 and is starting to form larger clumps (10-20 ct). I don't feel there are enough clumpy areas to worry just yet, but Marcela was suspicious of the stirring and so we chatted and removed this culture from the stir plate @ –1°C. Marcela will take another look on Tues the 14th and will likely start the experiment at that time.
July 15, 2019
MES3 count at 1:10 dilution was 3.32E+07 bact/ml.
"I started a freezer split in a 50 cc tube on 6/25 to get it going. Marcela started the larger batch on July 1 so it's been going around 2 weeks. I don't know enough about MES3's growth at cold temperatures to give a guess about time to yield. You are forging on untrodden ground. -1C is pretty dang cold and the volume is pretty large so I'd imagine anywhere from 4 to 7 days to get up there more."
July 18, 2019
MES3 count at 1:100 dilution was 6.16E+07 bact/ml so still getting there! If this rate is accurate it should reach 1E+08 by next week!