20090720
Counting Tetraselmis: experimented with different methods of slowing/killing tetraselmis for counting. 300 uL of ethanol per 1 mL of culture kills cells without lysing.

Testing within/between sample variation of ATP assay
sample variation ATP 7.20.09.xls



20090717

Rachel separated brine shrimp in a multiwell plate into 1's, 3's, & 5's, 5 samples each. 1 mL of DMSO added to each tube and crushed with a pestle. Entire mL is added to 5 mL of Tris-Acetate. 10uL of this mixture is transferred to 100 uL of reagent and read immediately.
Some samples were not included in the analysis because we lost a few of the brine shrimp during the transfer process between counting and separating and extracting. Using a multiwell plate for separation is not really neccessary. It is easier to separate them in droplets of water on a petri dish and transfer directly to an epitube and then suck the water out very carefully with a syringe. In some cases the BS got sucked up into the syringe.

zooplankton extractions(update)7.17.09.xls

20090716

Brine shrimp hatched after 24 hours. Removed & placed in a glass jar at room temperature. Will hold for 24 hours before extracting ATP. (Following the protocol the ballast water testing facilities use. (Note* brine shrimp must be removed after 24 hours because their capsules contain hyrogen sulfide which will kill the hatched shrimp if left in the same water.)


20090715

froze 1 ml aliquots of 7.14.09 extraction to test after being frozen for 24 hours. RLU values were higher, but within the variablity that we are seeing in the assay. ATP does not seem to diminish after being frozen for 24 hours.

Freezing ATP extract
retested extract from
B1 151
B2 95
B3 113

1-1 941,828
1-2 590,666
1-3 650,872
1-4 507,628
1-5 279,814


20090714
Zooplankton Extraction
Obtained some Tigriopus californicus collected in Anacortes by Adelade on ~ 7/10/09. (With some Tetraselmis in the media)
Put at 4°C for ~ 10 min. to slow them down before separating them.

Proposed Protocol for Separation & Assay:
  1. Suck some out with plastic transfer pipet onto nylon mesh screen, rinse with sterile filtered seawater(This didn't really work, so we decided to transfer them to an epitube, suck out the water, rinse with sterile seawater and then suck that out and add the DMSO)
  2. Backwash into a petri dish and pick out sets of 1, 3, & 5 into 24 well plates. (Numbers were checked under the dissecting scope but it was difficult to be sure with the 3's and 5's)
  3. Use a syringe to suck out all the water and then transfer to epitube for extraction. (Some organisms could have been lost during the transfer process)
  4. Add 1 mL of DMSO and crush with pestle for 5 min.
  5. Transfer entire 1 mL to 5 mL of Tris-Acetate.
  6. Transfer 10 uL to 100 uL of reagent.
  7. Read on Luminometer.

Blank readings were all over the place, waiting 10 minutes for machine to warm up and redid them. The numbers were on the high side but reproducible at that point. Avg. blank RLU was 227. So the lower numbers on the standard curve are negative values, still possible to determine moles of ATP from the zooplankton.

zooplankton extractions(update)7.14.09.xls

Phytoplankton Cultures
Rachel transferred tetraselmis culture. Culture was last transferred on 6/23/09. Checked under scope & there are still many motile cells but there are some dead. 3 weeks too long between transfers. 10% inoculum, 2 mL into 18 mL fresh media. Will count cells tomorrow and in 24 hour increments to determine log phase growth. May need to obtain fresh culture.

Brine Shrimp
Restarted brine shrimp in ~ 5L of seawater, checked salinity it is 29 ppt. Using heater set to 86°F or 30°C & 3 air stones for constant aeration & a microscope light as a light source. Will check in 24 hr. Turn off air and let unhatched cysts sink and you will see brine shrimp hovering just above the bottom.

20090710
Phytoplankton ATP determination #2(again)
TW1336 (3 wk old culture)

1. Rachel made serial dilutions of culture in the hood. Same as setup on 20090619. Culture is less dense so we counted the 1:10 dilution using the field of view method.
  1. 1:10 #cells=214 /fields of view = 9.
  2. 1:10 #cells=217 /fields of view = 10.
  3. 1:10 #cells=210 /fields of view = 13.
  4. 1:10 #cells=200 /fields of view = 11.
  5. 1:10 #cells=208 /fields of view = 13.


phytoplankton extractions7.10.09.xls

Results:
This time the RLU's measured did not differ much between dilutions in most of the samples and the blank was higher than most of the 1:10000 & 1:100000 dilutions, so this experiment will have to be redone.
Conclusions:
Adelade suggested cells may be in a resting phase altering their ATP levels. It is possible the counts were off and/or the cultures are too old. Maybe many of the cells are dead. We also discovered that we have been maintaining the TW in f/2 w/out Silicate when they in fact require Si. We purchased the correct media and will repeat this experiment.

20090623 Phytoplankton Cultures
Transferred 3 phytoplankton cultures.
TW1052 : 10% inoculum in f/2 (w/out Si), 3 fresh flasks at 16°C
TW1336 : 10% inoculum in f/2 (w/out Si), 3 fresh flasks at 16°C
Tetraselmis908 : 10% inoculum in L1, 3 fresh flasks at 24°C

12 hr light:12 hr dark
save old cultures until growth is confirmed in new ones.


20090619 Phytoplankton ATP determination #2

W1336 (2 wk old culture)
Will count all 5 1:1000 dilutions.

Protocol for ATP extractions for Phytos
1. Serial dilutions of culture in 8 mL sterile tubes. (.5 mL into 5 mL media)
1:10, 1:100, 1:1000, 1:10,000, 1:100,000
2. Count 1 mL of the 1:1000 dilution using the field of view method (count for all 5 replicates)

  1. 1:1000 - 200 cells in 43 fields.
  2. 1:1000 - 201 cells in 43 fields.
  3. 1:1000 - 201 cells in 38 fields.
  4. 1:1000 - 211 cells in 50 fields.
  5. 1:1000 - 204 cells in 42 fields
3. Spin down 1 mL of last 3 dilutions in an epitube at 3000 rpm for 30 minutes.
4. Add 1 mL of DMSO & crush with pestle.
5. Transfer to 5 mL of Tris-Acetate.
6. add 10 uL to 100 uL reagent and read immediately

(Counting took too long did not have time to complete extraction, need to start early am)

20090615 ATP in Zooplankton
Need to determine which zooplankton species to use for extraction, where to obtain them & how to grow or maintain them for multiple experiments.
According to the Screening of Surrogate Species for Ballast Water Treatment Report the best potential zooplankton surrogate species are:
1) Artermia salina (marine)
2)Tigriopus californicus (marine)
3)Ostracod species (freshwater)

- Can order Artemia & growth/hatching chamber from brineshrimp.direct.com.
- Tigriopus can be collected in Deception Pass.
- Ostracod can be bought live from Carolina Biologic Supply.
- Need to determine maintenance media and container and conditions for all 3 species.

20090604
ATP results for TW1052
DMSO standard

Moles ATP
 RLU
 Adj RLU



1.00E-18
 140
 22



1.00E-17
 154
 36



1.00E-16
 656
 538



1.00E-15
 3764
 3646



1.00E-14
 31632
 31514



1.00E-13
 330301
 330183



1.00E-12
 5384206
 5384088



Blank
 134



Blank
 81



Blank
 141



Average
 118.66667



St. Dev.
 32.807519






DMSO STD
Moles ATP




1.00E-18
 22



1.00E-17
 36



1.00E-16
 538



1.00E-15
 3646



1.00E-14
 31514



1.00E-13
 330183



1.00E-12
 5384088

Phytoplankton Extractions














Dilution
 RLU
 Adjusted RLU
 Slope
 Y-intercept
 Moles per 10uL extract
 Moles per mL
 Moles per 6mL extract
 #Cells
0.001
 902
 792
 5.000E+18
 0
 1.584E-16
 1.584E-14
 9.504E-14
 174
0.0001
 178
 68
 5.000E+18
 0
 1.360E-17
 1.360E-15
 8.160E-15
 30
0.00001
 110
 0
 5.000E+18
 0
 0.000E+00
 0.000E+00
 0.000E+00
 2
0.001
 984
 874
 5.000E+18
 0
 1.748E-16
 1.748E-14
 1.049E-13
 174
0.0001
 222
 112
 5.000E+18
 0
 2.240E-17
 2.240E-15
 1.344E-14
 30
0.00001
 108
 -2
 5.000E+18
 0
 -4.000E-19
 -4.000E-17
 -2.400E-16
 2
0.001
 1,349
 1239
 5.000E+18
 0
 2.478E-16
 2.478E-14
 1.487E-13
 174
0.0001
 183
 73
 5.000E+18
 0
 1.460E-17
 1.460E-15
 8.760E-15
 30
0.00001
 113
 3
 5.000E+18
 0
 6.000E-19
 6.000E-17
 3.600E-16
 2
0.001
 1022
 912
 5.000E+18
 0
 1.824E-16
 1.824E-14
 1.094E-13
 174
0.0001
 305
 195
 5.000E+18
 0
 3.900E-17
 3.900E-15
 2.340E-14
 30
0.00001
 197
 87
 5.000E+18
 0
 1.740E-17
 1.740E-15
 1.044E-14
 2
0.001
 1200
 1090
 5.000E+18
 0
 2.180E-16
 2.180E-14
 1.308E-13
 174
0.0001
 281
 171
 5.000E+18
 0
 3.420E-17
 3.420E-15
 2.052E-14
 30
0.00001
 154
 44
 5.000E+18
 0
 8.800E-18
 8.800E-16
 5.280E-15
 2
Blank1
 124






Blank2
 90






Blank3
 118






avg blank
 110.6667







phytoplankton extractions6.2.09.xls

notes: set y-intercept to 0 & used line from standard to determine moles of ATP for each RLU value.

Conclusions for 1st attempt:

Even at the largest dilution, (based on counting ~200 cells) in the 1:1000 dilution, so far the fewest number of cells ~2, RLU can be measured and is above the background level and moles of ATP can be determined.

Would be more accurate if all 5 replicates were counted in the counting chamber prior to extraction, but this is difficult with the time constraint to complete in one day. In the next extraction with attempt to count all 5, of the 1:1000 dilution, using the field of view method and counting at least 200 cells.

20090605

Phytoplankton Cultures
Tetraselmis #908 in L1 - (5/15/09) culture, transferred to 3 new flasks. 1 mL into 10 mL of media @ 24°C.

TW 1336 in f/2 -(5/15/09) culture, transferred to 3 new flasks. 1 mL into 10 mL of media @ 16°C.

TW1052 in f/2 -(5/15/09) culture, transferred to 3 new flasks. 1 mL into 10 mL of media @ 16°C.

*save old cultures until can confirm growth in new culture.

20090602
ATP measurements for phytoplankton

Thalassiosira weissflogii strain 1052:
Diluted 2 week old culture .5 mL/5mL fresh media (serial dilutions)
Planned to count all 3 dilutions for 5 replicates w/ Sedgwick rafter. Counting the entire slide.
replicate 1: 1:1000 = 174 cells (x1000 = 1.74 x 10>5)
1:10000 = 30 cells (x10000 = 3.0 x 10>5)
1:100000= 2 cells (x100000 = 2.0 x 10>5)

took too long to count, so will just use the numbers from the first replicate

RLU readings:
blank:
B1: 134
B2: 81
B3: 141
standard:
10<-7: 5,384,206
10<-8: 330,301
10<-9: 31,632
10<-10:3,764
10<-11:656
10<-12:154
10<-13:140

1) 10<-3: 902
10<-4:178
10<-5:110
2) 10<-3: 984
10<-4:222
10<-5:108
3) 10<-3: 1,349
10<-4:183
10<-5:113
4) 10<-3: 1,022
10<-4:305
10<-5:197
5) 10<-3: 1,200
10<-4:281
10<-5:154

B1:124
B2:90
B3:118

20090601

Plan for Phytoplankton ATP measurements

10 mL Culture (serially diluted)

↓

1:10, 1:100, 1:1000, 1:10000, 1:100000 (based on previous counts the 1:100000 should have ~ 1 cell)

↓

Count last 3 dilutions for 5 replicates (dilutions done in 8 mL sterile tubes, then transfer 1 mL to sedgwick rafter)

↓

Extract and assay last 3 dilutions for 5 replicates. (1)transfer 1 mL to epitube and spin down, then remove supernatant, add 1 mL DMSO & immediately crush with sterile pestle.(2) transfer entire mL to 5 mL of Tris-acetate. (3) add 10 uL of this to 100 uL of reagent and read on luminometer.

*will freeze left over atp extract for later retesting, to determine stability of the atp once extracted.

Determining the lowest speed to centrifuge that will result in a pellet

Used the 1:10 dilution to make sure I can visualize a pellet.
5 min @ 2000 rpm (no pellet)
10 min @ 2000 rpm (no pellet)
5 min @ 3000 rpm (no pellet)
10 min @ 2000 rpm (no pellet)
20 min @ 3000 rpm (no pellet)
30 min @ 3000 rpm (Pellet..yeah!)

20090528

Measuring typical atp in Phytoplanton

20 mL culture
↓ (dilute 1 mL aliquot)
count, determine cells per mL
↓(serial dilutions)
1000, 100, 1 cell (confirm dilutions by counting)
↓
Spin down (slowest possible speed)
↓
Extract ATP
↓
Assay


Counted TW culture again to compare methods:

Hemacytometer: TW1052 : 13 cells/21 cells = 17 x 10<4 = 1.7 x 10<5 cells/mL

Sedgwick-rafter: 1:10 dilution: (field of view method) : 279 in 20 fields = 279 x 1000/1 x 1 x 20 = 1.395 x 10<4
1.395 x 10<4 x 10 = 1.395 x 10<5 cells/mL

1:1000 dilution: 108 cells in entire chamber. 108 x 1000 = 1.08 x 10<5 cells/mL











20090526

Phytoplankton Cultures

Examined cultures with inverted scope. Media is turbid and appear to be growing well. Need to determine best method for counting cells & dilution to extinction. Will test Sedgwick Rafter cell and Hemacytometer with TW culture (no need to fix cells since they are non-motile).

In biosafety cabinet:
1. transfer aliquot of ~ 5 mL from one tissue culture flask to be used for counting experiments.
2. transfer some sterile media to 50 mL centrifuge tube for dilutions.
Upstairs by inverted scope:
1. test a 1:10 dilution first.
2. mix aliquot well by inversion, then transfer 500 uL to 5 mL sterile media in 15 mL centrifuge tube.
3. using wide bore pipet tips, add 1 mL to sedgewick rafter chamber.
4. Let settle ~ 15 -30 min.
5. count cells on the bottom of the chamber.
1st attempt there is 10+ cells in a 1mm square, too many to count the entire chamber.
Can dilute further, count selected random fields of view, or try different counting chamber.

-First tested the hemacytometer.
1. transferred 10 uL of diluted culture to both sides of hemacytometer.
2. Only a few cells visible.
3. Tried again with undiluted culture.
4. 14 cells in chamber one = 1.4 x 10<5 cells/mL ; 5 cells in chamber 2 = 5.0 x 10<4.

Will use sedgewick rafter chamber and after determining that the cells are evenly distributed will make field counts. Number of phytoplankton per mL will be calculated as follows:

no. per mL = C x 1000/A x D x F where: C = # of organisms counted, A = area of field (1 mm), D = depth of field (1 mm), F = # of fields counted.

(and multiplied by dilution factor).


20090515

Phytoplankton Cultures
Examined with inverted scope and transferred.

Transferred TW Culture #1: 1mL into 10 mL in T25 flask, f/2 media, 2 flasks at 14 C, 12:12 light:dark

Transferred TW Culture #2: 1mL into 10 mL in T25 flask, f/2 media, 2 flasks at 14 C, 12:12 light:dark

Tetraselmis CCMP908 : (transferred stock culture from 14 C) 1 mL into 10 mL in T25 flask, L1 media, 1 flask at 14 C, 2 flasks at 23 C. 12:12 light :dark.