st-amand-algal-methods by ajizai

VIEWS: 12 PAGES: 44

									Algal Laboratory Methodology
           Where are we going
                 And

        how do we not screw it up   ?
 Algal Laboratory Methodology


Sampling and
Analysis should
  be based on
 project Goals,
NOT goals based
 Sampling and
   Analysis
     Algal Laboratory Methodology

   For Example:

RBP is a stream
 sampling and
 analysis protocol,
 NOT a plankton
 protocol
   Algal Laboratory Methodology
Preservation – NO perfect preservative
  – Formaldehyde - hazardous fumes, distorts many
    cells, bleaches color, added at 2-5% as formalin
    (40% formaldehyde)
  – Lugol’s Iodine - stains starch, may cause loss of
    flagella, loses preservative power over time-must
    re-add every 6 months, added at 1-2%, can cause
    30-40% shrinkage from live biovolume
  – Glutaraldehyde - hazardous fumes, retains color
    and fluorescence, limited cell distortion, added at
    only 0.25-1%
  Algal Laboratory Methodology

Concentration Methods
  – Concentrate to get a higher
    density for counting
  – Don’t have to count in the
    same chamber you
    concentrate in, so you can
    build your own settling
    tower or buy an utermohl
  Algal Laboratory Methodology


 More Concentration
      Methods
•Centrifugation (not
recommended for counting,
disrupts flagellates, hard to
make it quantitative)

•Filtration & Reverse
Filtration-not often used
       Algal Laboratory Methodology

   Temporary Mounting Methods*
       – Less investment of materials/equipment
       – Can be faster if sample already concentrated
       – No mounting distortion
       – Settle 1 cm/hr, can take days to settle a sample
       – Buoyant blue-green algae/nannoplankton don’t settle
         well
       – Limited to certain microscope choices because
         generally a thick preparation

*I use a permanent mounting method, see me if you are interested
  Algal Laboratory Methodology

Which Counting Chamber?

Density of sample
Particulates/color
Size distribution of algae
Type of microscope
Cost
    Algal Laboratory Methodology

Temporary Chambers
• Sedgwick-Rafter Cells (grid
  or non-grid)
• Nanoplankton Counting
  Chambers
• Palmer-Maloney Cells




                                Remember: Concentrated Sample!
   Algal Laboratory Methodology
                 Temporary Chambers
              Utermohl Chambers-just a note!
Buoyant blue-greens present a
problem and don’t settle randomly or
don’t settle at all.
Percussive techniques help them
settle, you literally hit your chamber
with a rubber mallet (VERY
CAREFULLY).
   Algal Laboratory Methodology
               Primary Optical Enhancements


• Brightfield
   – No enhancement, produces little detail, real color
• Phase
   – Uses annuli to scatter light at different angles through sample, all
     color distorted
• Differential Interference Contrast (Nomarski DIC)
   – Uses crystal prisms as beam splitters, eliminates halo effect get
     with phase contrast, looks 3D, good color
• Epifluorescence
   – Uses specified light wavelength to excite pigment or dye which
     then emits light back at a different wavelength
Algal Methodology Bright Field
Algal Methodology   Phase
DIC   Algal Methodology
 Algal Laboratory Methodology

            Epifluorescence
• Blue excitation causes chlorophyll a
  to fluoresce back red.
• Green excitation causes
  phycoerythrin to fluoresce back
  bright orange-red and phycocyanin
  to fluoresce back orange yellow.
Algal Laboratory Methodology
Epifluorescence vs other optics
    Algal Laboratory Methodology

Epifluorescence
vs other optics




                  Required for
                  picoplankton
Algal Laboratory Methodology
 Epifluorescence vs other optics
Algal Laboratory Methodology
  Epifluorescence vs other optics
                     phycocyanin
                         vs.
                    phycoerythrin
  Algal Laboratory Methodology
                      Quantification
• Counting Unit
   – Natural units
   – Total cells
   – Areal standard units
• Precision vs. Accuracy
• Counting Method
   –   Fields
   –   Passes
   –   Strips
   –   Whole sample
  Algal Laboratory Methodology


    Quantification
• Biomass Estimates
  – Biovolume
  – Biomass (assume a
    specific gravity of 1)
  – Carbon estimates
    (based on biovolume)
                             Whipple Grid
  Algal Laboratory Methodology

               Biovolume Estimates
• Allow you to estimate the importance of
  individual taxa based on size AND abundance
• Calculate the volume of living protoplasm based
  on common geometric figures
   – Cube, sphere, cone, cylinder, sickle
• Exclusive of sheaths, spines, setae
• Everyone measures differently!
Algal Laboratory Methodology
    Shape Estimates
Algal Laboratory Methodology
   Shape Estimates
Algal Laboratory Methodology
    Shape Estimates
Algal Laboratory Methodology
   Shape Estimates
     Algal Laboratory Methodology
         Shape Estimates

Different growth forms,
      same taxon!!!
    Algal Laboratory Methodology
              Quantifying Algae
             Evaluation of Quantity
• Depends upon units of measure
• Depends on counting threshold, 100 NU-80%, 400
  NU 90%
• Can have data in cells, areal units, natural units, or
  biovolume/biomass per unit volume
• May also have pigment (usually chlorophyll) data
• Filter run times may be a useful measure in water
  supply
• Some knowledge of algal types (quality) is essential
   Algal Laboratory Methodology
             Quantifying Algae
  Limitations on Quantitative Relationships
• Variable cell size affects interpretation
  • Everyone measures differently!
• Chlorophyll per unit of algae varies with
  division, species, cell health, and phase of
  growth
• Ash-free dry weight to chlorophyll a ratio
  varies from 50:1 (healthy greens) to 300:1
  (many blue-greens) – average is about 100:1
                                       Algal Laboratory Methodology
                                                             Quantifying Algae
                                   Bar graph for showing assemblages over time
                                                       Concentration - Station 1
                                   5e+5
Concentration (Natural Units/mL)




                                   4e+5
                                                                                                                                               Blue-greens
                                   5e+3                                                                                                        Diatoms
                                                                                                                                               Euglenoids
                                   4e+3                                                                                                        Haptophytes
                                                                                                                                               Dinoflagellates
                                   3e+3                                                                                                        Chrysophytes
                                                                                                                                               Cryptophytes
                                   2e+3                                                                                                        Golden-Browns
                                                                                                                                               Greens
                                   1e+3

                                   0
                                          05/29/2002 06/11/2002 07/11/2002 07/25/2002 08/08/2002 08/22/2002 09/03/2002 09/17/2002 10/03/2002


                                                                           Sample Date
Algal Laboratory Methodology
                               Quantifying Algae
             Percent Algal Abundance by Division: Eucha, EUC16-1
             100
              90
   Percent
              80
              70
              60
              50
              40
              30
              20
              10
               0       98 98 98 98 98 98 98 98 98 98 98 98 98 98 98 98 98 98 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 00 00 00
                    /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /20 /20 /20
                 /24 /24 /14 /30 /13 /28 /10 /23 /14 /28 /12 /25 /09 /23 /14 /27 /12 /09 /12 /17 /10 /13 /28 /13 /25 /09 /28 /13 /27 /10 /24 /15 /30 /13 /03 /08 /18 /08 /09
               02 03 04 04 05 05 06 06 07 07 08 08 09 09 10 10 11 12 01 02 03 04 04 05 05 06 06 07 07 08 08 09 09 10 11 12 01 02 03




             Percent Algal Biovolume by Division: Eucha, EUC16-1
             100
              90
   Percent




              80
              70
              60
              50
              40
              30
              20
              10
               0       98 98 98 98 98 98 98 98 98 98 98 98 98 98 98 98 98 98 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 00 00 00
                    /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /19 /20 /20 /20
                 /24 /24 /14 /30 /13 /28 /10 /23 /14 /28 /12 /25 /09 /23 /14 /27 /12 /09 /12 /17 /10 /13 /28 /13 /25 /09 /28 /13 /27 /10 /24 /15 /30 /13 /03 /08 /18 /08 /09
               02 03 04 04 05 05 06 06 07 07 08 08 09 09 10 10 11 12 01 02 03 04 04 05 05 06 06 07 07 08 08 09 09 10 11 12 01 02 03


                                                                                          DATE
  Algal Laboratory Methodology
                                         Quantifying Algae
Area graph for showing assemblages over time –
   NOT continuous data, can’t express as solid
                     fill….
                            2000000
                                                                  Station 3
   Biovolume (cubic um/L)




                            1500000



                            1000000

                                                                                    CYBIOVOL
                             500000                                                 CHLBIOVOL
                                                                                    BABIOVOL
                                                                                    CRBIOVOL
                                                                                    CHBIOVOL
                                  0                                                 PYBIOVOL
                                         02 02 02 02 02 02 02 02 02
                                      /20 1/20 5/20 8/20 2/20 3/20 7/20 3/20 4/20
                                   /29 /1     /2   /0   /2   /0   /1   /0   /0
                                 05 06 07 08 08 09 09 10 12
                                                  SAMPLEDATE
         Algal Laboratory Methodology
                    QA/QC
•   Sample Collection Methods
     – True Replicates
     – Pseudo-replicates
•   Sub-sampling
     – Multiple chambers/mounts (at least 3)
•   Counting Methods
     – Threshold
          • Time, Cells encountered, NU
             encountered (100, 400, 600, 800, error
             on slide) or combination
     – Fields/strips/whole slides
     – Magnification
•   Measurement Methods
     – 10-15 of each taxa
     – Entire assemblage
     – NU, Cells or both – NEVER mix and match
•   Identification preferences/skill
     – Fatigue
Algal Laboratory Methodology QA/QC
                                                            Total Concentration


                                              1.4e+6


                                              1.2e+6
           Concentration (Natural Units/mL)




                                                                                          Sample 1
                                              1.0e+6                                      Sample 2
                                                                                          Sample 3
                                                                                          Sample 4
                                              8.0e+5                                      Sample 5


                                              6.0e+5


                                              4.0e+5


                                              2.0e+5


                                              0.0
                                                       AS      A          B       C


                                                       1      1Laboratory 2           3
Look for consistency among taxonomists:
Stations in same relative order
Same list of dominants
Total numbers within 20-30%, 10% is VERY difficult to meet when using different mounting
and counting methods
                                             Top 10 Dominant Concentration


                                    1.4e+6


                                    1.2e+6
                                                                                                                            Algal Laboratory
 Concentration (Natural Units/mL)




                                                                             Sample 1
                                    1.0e+6


                                    8.0e+5
                                                                             Sample 2
                                                                             Sample 3
                                                                             Sample 4
                                                                             Sample 5
                                                                                                                              Methodology
                                    6.0e+5


                                    4.0e+5


                                    2.0e+5


                                    0.0
                                             AS            B            C                                                             QA/QC
                                             1             2
                                                       Laboratory
                                                                        3
• Difference between counters                                                                                                     Top 10 Dominant Biovolume

  can be substantial, even with                                                                                      9e+7


  acceptable technique                                                                                               8e+7




                                                                                  Biovolume (cubic micrometers/mL)
                                                                                                                     7e+7                                     Sample 1
                                                                                                                                                              Sample 2
                                                                                                                     6e+7                                     Sample 3
                                                                                                                                                              Sample 4


• Difference between units of                                                                                        5e+7                                     Sample 5


                                                                                                                     4e+7

  measurement can also be                                                                                            3e+7


  substantial, just by nature of                                                                                     2e+7

                                                                                                                     1e+7
  measurement, but also by                                                                                           0

  variation among counters                                                                                                   1               2            3
                                                                                                                                 AS           B           C

                                                                                                                                          Laboratory
Algal Laboratory                                                                                         1e+6
                                                                                                                 Cylindrospermopsis raciborskii (Straight), Concentration




  Methodology                                                                                                         Sample 1




                                                                      Concentration (Natural Units/mL)
                                                                                                         8e+5




        QA/QC                                                                                            6e+5




                                                                                                         4e+5




                                                                                                         2e+5




                                                                                                         0


                                                                                                                               1              2           3
                                                                                                                               AS             B              C

                                                                                                                                        Laboratory


                                                                                                                Cylindrospermopsis raciborskii (Straight), Biovolume


                                                                1.4e+7


                                                                1.2e+7
Mixing Natural Units and     Biovolume (cubic micrometers/mL)
                                                                                                                   Sample 1
                                                                1.0e+7
Cells during counting and
                                                                8.0e+6

data reporting confuses                                         6.0e+6

analysis…..is this a true                                       4.0e+6

difference or a mis-report                                      2.0e+6


of cells as NU?                                                 0.0


                                                                                                                          1              2              3
                                                                                                                          AS              B              C

                                                                                                                                     Laboratory
Algal Laboratory Methodology
                                                              QA/QC
                                            Aphanocapsa (incerta), Microcystis aeruginosa - Biovolume


                                     8e+7
  Biovolume (cubic micrometers/mL)




                                     6e+7




                                     4e+7




                                     2e+7




                                     0
                                                         AS             B            C
                                                         1             2
                                                                    Laboratory       3
                                     Identification Issues- Toxic Microcystis or
                                                    Aphanocapsa?
Algal Laboratory Methodology QA/QC

 • Taxonomic consistency trumps
   true taxonomic accuracy if
   ecological interpretation or risk
   assessment does not change!
     • As long as well documented and
       still meet project goals
     • If you must change, side by side
       QA/QC recommended for at least a
       season if you can (year is better)
     Algal Laboratory Methodology
         Quantifying Algal Quality


•   Dominance
•   Richness
•   Diversity
•   Quality Indices
    Algal Laboratory Methodology
        Quantifying Algal Quality


Programs using entire assemblage
• Primer E
• PC Ord


                                    Primer E6
      Algal Laboratory Methodology
            Quantifying Algal Quality
             Richness as an indicator of quality
•   Richness usually relates more to counting effort than
    to ecological indications. 10 - 25 normal without great
    effort, high sample concentration, and/or lots of taxa
    at low abundance.
•   Richness depends on level of taxonomic separation;
    more taxa (higher richness) with more detailed
    identification.
•   With consistent effort, higher richness should be an
    indication of higher diversity.
•   Presence/absence data at the genus/species level can
    be used to determine the similarity of samples
      Algal Laboratory Methodology
            Quantifying Algal Quality
        Diversity as an indicator of quality – Part I
•   Measures the distribution of numbers over the number
    of taxa present; highest when numbers evenly
    distributed among taxa.
•   Higher diversity usually taken to indicate more
    desirable ecological conditions, but concept is
    controversial.
•   Low diversity indicates high dominance by one or a
    few taxa; conditions beyond dominance threshold(s).
•   Proper diversity calculation varies with type of sample
    and approach to identification/enumeration.
     Algal Laboratory Methodology
          Quantifying Algal Quality
     Diversity as an indicator of quality – Part II
• Expression of diversity as a function of maximum
  diversity possible for a given sample tends to
  minimize statistical artifacts, creates scale of 0-1,
  more easily comprehended.
• Rare species may be neglected in diversity indices, but
  can become important later in time.
• Expression of detailed sample analysis as a single
  number oversimplifies community features and
  may not be dependable by itself.
     Algal Laboratory Methodology
 Microscopy Educational Sites and Trouble Shooting Information

• http://www.mic-d.com/gallery/index.html
• http://www.olympusamerica.com/seg_section/seg_faq.asp
• http://www.microscopy-uk.org.uk/full_menu.html

								
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