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.