Post on 13-Jan-2016
description
2006 Chesapeake Bay Monitoring Program Split Sample Data Analysis
Phytoplankton
Morgan State University Estuarine Research Center
Ann Marie HartsigRichard Lacouture
Stella Sellner
IntroductionIntroduction• In 2005, a standard method for phytoplankton identification and enumeration was adopted, yet, analysis of 2006 phytoplankton split-samples show major discrepancies.
• Differences are observed for major groups at the high magnification counts as well as for a variety of groups at the primary (mid) magnification.
• Commonly identified species such as Cylindrotheca closterium, Skeletonema costatum, and Heterocapsa rotundatum show significant differences in densities.
• For 2006, MSU and ODU are examining exactly the same subvolume or % of total sample. ODU determined that 1/64 of their concentrated sample equals 7.81mls of sample settled for MSU.
• Possible reasons for this discrepancy might include calculations, sample preparation techniques and resulting cellular optical properties.
The Canary in the Coal MineThe Canary in the Coal Mine
Cylindrotheca closterium
MSU Calculation:
Number of individuals counted Constant 1000 = Number of Number of Fields Counted Number of mls settled Cells/Liter
ODU Calculation:
Number of individuals counted Constant 1 1 = Number of Number of Fields Counted 1 concentrate volume Cells/Liter
X X
X X X
Constant= Number of Fields in Chamber at magnification used(Note: Percent of sample analyzed equal between MSU and ODU for 2006)
Ex: 120cells * 1214.9 (312.5mag) X 1000= 1866683.7cells/liter 10 7.81ml
Calculation of Normalized DensitiesCalculation of Normalized Densities
48hsiphon
until~40ml
72hsiphon~250ml
Fill up to 4/4 mark w/formalin waterNow 1/16dilution
Pour ¼ into chamberNow 1/64 dilutionSettle in chamber16-24h
Pipette 1-10mlssettle 2-24h
Sample Preparation MethodsSample Preparation Methods
MSU
ODU
~40mls in vialsettle 16-24h
Pour ¼ of orig. into next vial
Potential Errors Related to Sample Preparation MethodsPotential Errors Related to Sample Preparation Methods
MSU:1) Calibration of pipettes2) Non-settling cells
ODU:1) Loss of cells during siphoning phases2) Error associated with transfer of subsample during dilution process3) Effects of dilution process on optical properties of cells (bleaching)4) Non-settling cells
Photo of Cells Found in 50mls of SupernatantPhoto of Cells Found in 50mls of Supernatant
300X
Cylindrotheca
Cryptomonas sp.
Skeletonema
(Sample settled >72 hours before siphoning)
Mean Densities of Dominant Taxa in Supernatants
Cylindrotheca
closterium
Skeletonema
costatum
Cryptomonas spp.Heterocapsa
rotundatum
Chaetoceros spp.
Total Density
0
200000
400000
600000
800000
1000000
1200000
Cells
/Lite
rLoss of Cells during Siphoning ProcedureLoss of Cells during Siphoning Procedure
MSU prep 300X
ODU prep300X
ODU prep300X
Alteration of Cellular Appearance by Alteration of Cellular Appearance by ‘Bleaching’ Effect of Dilution Water at 300X‘Bleaching’ Effect of Dilution Water at 300X
Heterocapsa rotundatum
Cryptomonas spp.Pyramimonas sp.and Heterocapsa rotundatum
Loss of stainHard to see pigment
MSU prep – 500X
ODU prep – 500X
Alteration of Cellular Alteration of Cellular Appearance by ‘Bleaching’ Appearance by ‘Bleaching’ Effect of Dilution Water at 500XEffect of Dilution Water at 500X
Alteration of Cellular Appearance by ‘Bleaching’ Effect of Dilution WaterAlteration of Cellular Appearance by ‘Bleaching’ Effect of Dilution Water500X500X
Mean Cylindrotheca Densities
0
2000000
4000000
6000000
8000000
10000000
MSU ODU MD3 MD4
Cells
/L
(ODU method) (ODU method)
Canary in the Coal Mine DifferencesCanary in the Coal Mine Differences
Total Densities of Samples by Comparison
MSU
MSU
ODU ODU MSU (ODU method)
MSU (ODU method)
0
5000000
10000000
15000000
20000000
25000000
Sample 3 Sample 4
Ce
lls/L
Results SummaryResults Summary
There are several sources of error related to the preparation of samples for There are several sources of error related to the preparation of samples for each laboratory.each laboratory.
The pipettes used by MSU to draw off the subsample for taxonomic The pipettes used by MSU to draw off the subsample for taxonomic enumeration are macropipettes (1-5mls or 5-10mls) and could introduce an enumeration are macropipettes (1-5mls or 5-10mls) and could introduce an error but not likely a bias.error but not likely a bias.
The siphoning of the sample during the concentration phase of ODU sample The siphoning of the sample during the concentration phase of ODU sample preparation may be a source of cell loss and therefore a negative bias (loss preparation may be a source of cell loss and therefore a negative bias (loss was ~ 10% for total cell densities and 3-18% for five of the dominant taxa).was ~ 10% for total cell densities and 3-18% for five of the dominant taxa).
The transfer of subsamples during the dilution phase of the ODU sample The transfer of subsamples during the dilution phase of the ODU sample preparation may be a source of error but not likely a bias.preparation may be a source of error but not likely a bias.
The ‘bleaching’ effect induced by the transfer of subsamples during the The ‘bleaching’ effect induced by the transfer of subsamples during the ODU sample preparation may be a source of cell loss and therefore a ODU sample preparation may be a source of cell loss and therefore a negative bias.negative bias.
There may be cells that do not settle out of the subsample completely and There may be cells that do not settle out of the subsample completely and may be a source of cell loss and therefore a negative bias.may be a source of cell loss and therefore a negative bias.
CONCLUSIONSCONCLUSIONS Several potential sources of error related to sample preparation have been Several potential sources of error related to sample preparation have been
identified for each laboratory.identified for each laboratory. Cell loss caused by the siphoning phase of the ODU sample preparation only Cell loss caused by the siphoning phase of the ODU sample preparation only
accounts for a small portion of the discrepancy between the 2 labs’ total cell accounts for a small portion of the discrepancy between the 2 labs’ total cell densities but showed higher proportional losses for several dominant taxa.densities but showed higher proportional losses for several dominant taxa.
The transfer of subsample during the dilution phase of the ODU sample The transfer of subsample during the dilution phase of the ODU sample preparation technique may lead to a certain amount of error but this was not preparation technique may lead to a certain amount of error but this was not quantifiable in the current split sample analysis.quantifiable in the current split sample analysis.
The source of intra-laboratory variability remains a mystery.The source of intra-laboratory variability remains a mystery. Several recommendations are proposed:Several recommendations are proposed:
1) Identify other potential sources of error and a means of quantifying the 1) Identify other potential sources of error and a means of quantifying the potential error associated with these issues.potential error associated with these issues.
2) Adopt a common sample preparation technique so that the entire protocol 2) Adopt a common sample preparation technique so that the entire protocol for enumerating phytoplankton is ‘identical’ between the two laboratories.for enumerating phytoplankton is ‘identical’ between the two laboratories.
3) Bring in outside experts in the field to assist with this standardization of 3) Bring in outside experts in the field to assist with this standardization of methodologies. methodologies.