analisis air ( teknik lingkungan) ist akprind yogyakarta

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ANALISIS AIR DANAU Oleh Sri Sunarsih Jurusan Teknik Lingkungan, Fak Sains Terapan, IST AKPRIND Yogyakarta

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  • ANALISIS AIR DANAUOleh

    Sri SunarsihJurusan Teknik Lingkungan, Fak Sains Terapan,

    IST AKPRIND Yogyakarta

  • Alkalinitas dan kesadahan Alkalinitas: ukuran kemampuan air untuk menetralkan

    asam kuat (menerima dan dinetralkan oleh proton) Dinyatakan dalam mg CaCO3 per liter mikroekivalen Alkalinitas air alamiah berkisar 20 - 200 mg/L

    Dalam air alamiah yang dominan bikarbonat dan karbonat Kesadahan : Ukuran total konsentrasi ion Ca dan Mg

    Dinyatakan dalam mg CaCO3 per liter Sebenarnya ion Ca dan Mg diperlukan untuk pertumbuhan

    normal dan daya tahan tumbuhan dan binatang. Kesadahan dapat mempengaruhi toleransi ikan terhadap

    logam toksik

    Alkalinitas: ukuran kemampuan air untuk menetralkanasam kuat (menerima dan dinetralkan oleh proton) Dinyatakan dalam mg CaCO3 per liter mikroekivalen Alkalinitas air alamiah berkisar 20 - 200 mg/L

    Dalam air alamiah yang dominan bikarbonat dan karbonat Kesadahan : Ukuran total konsentrasi ion Ca dan Mg

    Dinyatakan dalam mg CaCO3 per liter Sebenarnya ion Ca dan Mg diperlukan untuk pertumbuhan

    normal dan daya tahan tumbuhan dan binatang. Kesadahan dapat mempengaruhi toleransi ikan terhadap

    logam toksik

  • Analisis Alkalinitas pH meter Buret* Termometer Pengaduk Magnetik

    dan stirer Top loading balance

    pH meter Buret* Termometer Pengaduk Magnetik

    dan stirer Top loading balance

  • Analisis Alkalinitas Reagen

    0.04 N H2SO4 Analisis total alkalinitas dengan titrasi sampai sampel

    mencapai pH tertentu (disebut titik akhir titrasi) Pada pH tersebut, semua senyawa basa dalam sampel

    sudah habis Jumlah asam yang digunakan sesuai dengan

    alkalinitas total sampel Hasilnya dinyatakan dalam milligram per liter kalsium

    karbonat (mg/L CaCO3) Juga dapat dinyatakan dalam milliekuivalen dengan

    dibagi 50

    Reagen 0.04 N H2SO4

    Analisis total alkalinitas dengan titrasi sampai sampelmencapai pH tertentu (disebut titik akhir titrasi)

    Pada pH tersebut, semua senyawa basa dalam sampelsudah habis

    Jumlah asam yang digunakan sesuai denganalkalinitas total sampel

    Hasilnya dinyatakan dalam milligram per liter kalsiumkarbonat (mg/L CaCO3)

    Juga dapat dinyatakan dalam milliekuivalen dengandibagi 50

  • Analisis Alkalinitas

    samplemLNCBLCaCOmgalkalinitytotal 50000)2(/, 3

    samplemLNCBLeqalkalinitytotal 999100)2(/,

    or

    samplemLNCBLeqalkalinitytotal 999100)2(/,

    Where:B = mL titrant first recorded pH (i.e., to pH = 4.5)C = total mL titrant to reach pH 0.3 unit lower, andN = normality of acid (titrant)

  • Analisis kesadahan Idealnya kesadahan ditentukan dengan perhitungan secara

    terpisah antara kalsium dan magnesium.

    Satuan kesadahan adalah mg CaCO3/L][118.4][497.2 MgCa

    Dengan Ca dan Mg dalam mg/L

    ][118.4][497.2 MgCa

  • Analisis alkalinitas dan kesadahanAda kit tes titrasi yangtersedia untukalkalinitas dan kesadahan

    Ada kit tes titrasi yangtersedia untukalkalinitas dan kesadahan

    www.hach.com

    www.lamotte.com

  • ANALISIS NUTRIEN Kolorimetri & spektrofotometri nitrogen and phosphorus using

    spectrophotometry Specific techniques for students to review

    in or out of class included: developing calibration curves QA/QC : standards, spikes, etc

    Kolorimetri & spektrofotometri nitrogen and phosphorus using

    spectrophotometry Specific techniques for students to review

    in or out of class included: developing calibration curves QA/QC : standards, spikes, etc

  • Kolorimetri & spektrofotometri

    Prinsip:1.Makin tinggi kosentrasi

    warna = absorbansimakin tinggi, add a dye that binds

    specifically to nutrientof interest

    measure the increasein color as anestimate of analyteconcentration

    2. Menyiapkan larutankalibrasi standar(konsentrasi sampelharus berada dlmkisaran kalibrasi

    3. Membandingknabsorbansi sampeldengan absoebansistandard danmengestimasikonsentrasi sampel

    Prinsip:1.Makin tinggi kosentrasi

    warna = absorbansimakin tinggi, add a dye that binds

    specifically to nutrientof interest

    measure the increasein color as anestimate of analyteconcentration

    2. Menyiapkan larutankalibrasi standar(konsentrasi sampelharus berada dlmkisaran kalibrasi

    3. Membandingknabsorbansi sampeldengan absoebansistandard danmengestimasikonsentrasi sampel

  • Kolorimetri & spektrofotometri4. Menambahkan

    reagen untukmembentuk warna

    5. Membandingkan Menggunakan peta

    warna Mengguakan

    kolorimeter Menentuan

    absorbansinya denganspektrofotometer

    4. Menambahkanreagen untukmembentuk warna

    5. Membandingkan Menggunakan peta

    warna Mengguakan

    kolorimeter Menentuan

    absorbansinya denganspektrofotometer

    rendah.. ke . tinggiKonsentrasi fosfat

  • Komparator warna dankolorimetri

    Test Kits Ada banyak merek

    Images from www.hach.com

    Tabung warna Cakram warna Kolorimeter saku

  • Instrumen pengukur warna

    Hach DR2400 portablespectrophotometer

    Bausch & Lombspectrophotometer 20

    Hach DR2400 portablespectrophotometer

    Bausch & Lombspectrophotometer 20

  • Standard Kalibrasi

    Standard dibuat dari lrutn stndr yang lebih pekat, diencerkandengan presisi tinggi

    Ortho-P:Use dried KH2PO4, K2HPO4,NaH2PO4 or Na2HPO4

    NH4-N dan NO3-N:Menggunakan NH4NO3 keringsebagai standar (masing-masing50%)

  • Water chemistry 101Procedure: See specific analyses

    Reagents are added to eachsample and standardidentically

    Mix after each step

    Incubate at room temp or inwater bath for 20 min to ~ 2hrs, depending on the analyte

    Procedure: See specific analyses

    Reagents are added to eachsample and standardidentically

    Mix after each step

    Incubate at room temp or inwater bath for 20 min to ~ 2hrs, depending on the analyte

  • Kurva kalibrasi Standar

    NH4-N standards

    Garis lurus:A = a + b*c

  • Estimasi konsentrasi

    maka, jika sampel memilikiabsorbansi 0.290

    N

    Konsentrasinya kira-kira~ 0.33 ppm N

  • #2

    Standard curves troubleshooting

    Errors in preparing the 0.25 and 0.50ppm standards perhaps ?

    Example #1 Live with it or re-run the batch#1

    The line becomes non-linear after ABS ~ 1.0 (~

    1000 ugN/L)

    Example #2 Fit a straight line from 0-1000 and a 2nd line from 1200-2000ugN/L

    Use non-linear quadratic instead ofa line for 0-2000 ugN/L

    Re-read in smaller cuvette or diluteand re-run

  • Some data from northern Minnesotalakes

    0.000

    0.100

    0.200

    0.300

    0.400

    0.500

    0.600

    0 50 100 150 200 250

    ortho-P (ug/L)

    Abs

    orba

    nce

    @ 8

    80 n

    m Calibration curve

    = std

    ABS = (-0.0010) + (0.00254)* PR2 = 0.9997 n=12

    Sample #1 = 11.2 ugP/LSample #1 - Replicate = 12.6 ugP/LSample #1 + 50 Spike = 59.4 ugP/L

    0.000

    0.100

    0.200

    0.300

    0.400

    0.500

    0.600

    0 50 100 150 200 250

    ortho-P (ug/L)

    Abs

    orba

    nce

    @ 8

    80 n

    m

    Sample #1 = 11.2 ugP/LSample #1 - Replicate = 12.6 ugP/LSample #1 + 50 Spike = 59.4 ugP/L

    % RPD = 100* (1.4)/ 11.9 = 12%% R = 100* (59.4-11.9)/50 = 95%

    Conclusion:The data are valid

  • ANALISIS TSS DAN KEKERUHAN

  • Analisis total padatan tersuspensi1. Air yang volumenya tertentu

    disaring dengan kertas saringyang sudah dicuci, dikeringkan(pada 103-105oC), dan ditimbang(~ + 0.5 mg)

    2. dibilas, dikeringkan dalam oven,ditimbang dengan neraca analitisuntuk mengukur berat TSS mg/L(ppm)

    3. Kertas saring disimpan untukanalisis lain misal padatantersuspensi volatil (VSS) yangmemprediksi senyawa organik

    1. Air yang volumenya tertentudisaring dengan kertas saringyang sudah dicuci, dikeringkan(pada 103-105oC), dan ditimbang(~ + 0.5 mg)

    2. dibilas, dikeringkan dalam oven,ditimbang dengan neraca analitisuntuk mengukur berat TSS mg/L(ppm)

    3. Kertas saring disimpan untukanalisis lain misal padatantersuspensi volatil (VSS) yangmemprediksi senyawa organik

  • Analisis total padatan tersuspensiPenyaring jenis apa yang

    digunakan?

  • Analisis total padatan tersuspensi Jenis penyaring:

    Filter membranmenahan partikulat danorganisme sub-mikron

    Filter gelas mikrofiber100% terbuat dari gelasborosilikat.

    Polikarbonat memilikiukuran pori yang tepattetapi alirannya lambat

    Jenis penyaring: Filter membran

    menahan partikulat danorganisme sub-mikron

    Filter gelas mikrofiber100% terbuat dari gelasborosilikat.

    Polikarbonat memilikiukuran pori yang tepattetapi alirannya lambat

    www.whatman.com

  • Total padatan tersuspensi Ada beberapa set peralatan

    Corong yang terikat klem, disekrup, atau dgn magnetic Peralatan plstik yg bermanfaat unt di lapangan

    multiple towers

  • Peralatan yg diperlukan

    oven pengering

    Neraca Analitik

    Total padtn tersuspensi

    oven pengering

    Penyaring dan petri dish

  • Menghitung TSS:

    Total padatan tersuspensi

    TSS (mg/L) = ([A-B]*1000)/Cdengan

    A = berat akhir kertas saring (mg)B = berat awal kertas saring (mg)C = Volume air yang disaring (Liter)

    TSS (mg/L) = ([A-B]*1000)/Cdengan

    A = berat akhir kertas saring (mg)B = berat awal kertas saring (mg)C = Volume air yang disaring (Liter)

  • Hubungan kekeruhan denganTSS

    Aturan umum :1 mg TSS/L ~ 1.0 - 1.5 NTU kekeruhan

    BUT hamburan oleh kekeruhan bergantung pada ukuranpartikel sehingga perkiraan tersebut hanya merupakanpendekatan kasar

  • Turbidity - meter Kebanyakan menggunakan nefelometrik

    optik dan daibaca dlm satuan NTU(nephelometric turbidity units)

    Pengukuran kekeruhan dilakukan dengan: Turbidimeter (untuk sampel diskrit) Sensor kekeruhan yg dapat dibenamkan

    (USGS menganggap sbg metoda qualitative)

    Instrumen Laboratorium : Turbidimeter

    Kebanyakan menggunakan nefelometrikoptik dan daibaca dlm satuan NTU(nephelometric turbidity units)

    Pengukuran kekeruhan dilakukan dengan: Turbidimeter (untuk sampel diskrit) Sensor kekeruhan yg dapat dibenamkan

    (USGS menganggap sbg metoda qualitative)

    Instrumen Laboratorium : Turbidimeter

  • KekeruhanTurbidimeter

    Nefelometrik optik Kekeruhan nefelometrik

    ditaksir menggunakan efekhamburan sinar oleh partikeltersuspensi

    detektor terletak pada sudut 90oterhadap sumber sinar

    http://www.bradwoods.org/eagles/turbidity.htm

    TurbidimeterNefelometrik optik

    Kekeruhan nefelometrikditaksir menggunakan efekhamburan sinar oleh partikeltersuspensi

    detektor terletak pada sudut 90oterhadap sumber sinar

  • Satuan Kekeruhan Nephelometric Turbidity

    Units (NTU) Standardnya adalah

    formazin atau material lainyg tersertifikasi

    Satuan JTU berasal dariteknologi yang lebih awalmenggunakan nyala lilin ygdilihat melalui tabung air

    1 NTU = 1 JTU (JacksonTurbidity Unit)

    Nephelometric TurbidityUnits (NTU)

    Standardnya adalahformazin atau material lainyg tersertifikasi

    Satuan JTU berasal dariteknologi yang lebih awalmenggunakan nyala lilin ygdilihat melalui tabung air

    1 NTU = 1 JTU (JacksonTurbidity Unit)

  • Kekeruhan standard formazin

    Contoh 1 set standard formazin

  • Kekeruhan

    Here is a range of NTUs using clay

  • Bench and portable instruments and kits vs.Turbidi meters dan probes

    Submersible Turbidimeters

    YSI wiping turbidityYSI 6820 withunwiped turbidity

    Hydrolab

    Submersible Turbidimeters

  • Turbidity - methods

    Comparability of different methods: With the proliferation of automated in situ turbidity

    sensors there is concern about the comparabilityof measurements taken using very differentoptical geometries, light sources and lightsensors.

    The US Geological Survey and US EnvironmentalProtection Agency are currently (August 2002)developing testing procedures for a fieldcomparison of a number of instruments producedby different manufacturers.

    Comparability of different methods: With the proliferation of automated in situ turbidity

    sensors there is concern about the comparabilityof measurements taken using very differentoptical geometries, light sources and lightsensors.

    The US Geological Survey and US EnvironmentalProtection Agency are currently (August 2002)developing testing procedures for a fieldcomparison of a number of instruments producedby different manufacturers.

  • Turbidity - calibration Turbidity free water = zero (0

    NTU) standard USGS recommends filtering

    either sample water ordeionized water through a 0.2um or smaller filter to removeparticles

    WOW uses deionized waterthat is degassed by sparging(bubbling) with helium, tominimize air bubbles that maygive false turbidity readings

    Turbidity free water = zero (0NTU) standard USGS recommends filtering

    either sample water ordeionized water through a 0.2um or smaller filter to removeparticles

    WOW uses deionized waterthat is degassed by sparging(bubbling) with helium, tominimize air bubbles that maygive false turbidity readings

  • Turbidity - standards Standards range depends on anticipated sample

    values Lakes - typically 0-20 NTU Streams and wetlands - 0-20, 0-50 or 0-100 NTU 2 non-zero standards typically adequate

    (response is linear) Types of standards

    Formazin particles (either from a recipe orpurchase a certified, concentrated stock solution -usually 4000 NTU)

    Other commercially available materials, e.g.,polystyrene

    Standards range depends on anticipated samplevalues Lakes - typically 0-20 NTU Streams and wetlands - 0-20, 0-50 or 0-100 NTU 2 non-zero standards typically adequate

    (response is linear) Types of standards

    Formazin particles (either from a recipe orpurchase a certified, concentrated stock solution -usually 4000 NTU)

    Other commercially available materials, e.g.,polystyrene

  • Table of standards Prepare daily2 to 20 NTUHach Company

    Prepare dailyAll dilutionsStandard Methods(APHA 1995)

    Prepare monthly20 to 40 NTU

    Suggested holding timesConcentrationsSourceTurbidity standards

    Prepare weeklyAll dilutionsEPA Region 5

    Prepare dailyAll dilutionsStandard Methods(APHA 1995)

  • ANALISIS BOD

  • BOD

    BOD mengukur jumlah oksigen yangdiperlukn oleh mikroorganisme untukmenguraikan senyawa organik, termasukuntuk mengoksidasi senyawa anorganik

    Uji BOD mengukur jumlah oksigen ygdiperlukan selama waktu tertentu(biasanya 5 days at 20o C)

    BOD mengukur jumlah oksigen yangdiperlukn oleh mikroorganisme untukmenguraikan senyawa organik, termasukuntuk mengoksidasi senyawa anorganik

    Uji BOD mengukur jumlah oksigen ygdiperlukan selama waktu tertentu(biasanya 5 days at 20o C)

  • BOD 5 DO is measured initially and again after a

    5-day incubation at 20o C BOD is computed from the difference

    between initial and final DO The rate of oxygen consumption is

    affected by a number of variables: temperature pH the presence of certain kinds of

    microorganisms the type of organic and inorganic material in

    the water

    DO is measured initially and again after a5-day incubation at 20o C BOD is computed from the difference

    between initial and final DO The rate of oxygen consumption is

    affected by a number of variables: temperature pH the presence of certain kinds of

    microorganisms the type of organic and inorganic material in

    the water

  • BOD - analysis Equipment needed:

    Incubation bottles Air incubator or water bath

    thermostatically controlled at20 +/- 1o C

    DO meter and probe

    Equipment needed: Incubation bottles Air incubator or water bath

    thermostatically controlled at20 +/- 1o C

    DO meter and probe

  • BOD Reagents:

    Dilution water provides nutrients necessaryfor microorganism growth

    Seed a population of microorganismscapable of oxidizing the organic matter in thesample

    Commercially available or freeze-driedculture

    A conditioned bacteria source (effluentfrom a biological treatment source such asa wastewater treatment plant).

    Glucose-glutamic acid standard

    Reagents: Dilution water provides nutrients necessary

    for microorganism growth Seed a population of microorganisms

    capable of oxidizing the organic matter in thesample

    Commercially available or freeze-driedculture

    A conditioned bacteria source (effluentfrom a biological treatment source such asa wastewater treatment plant).

    Glucose-glutamic acid standard

  • BOD QA/QC Assure quality with:

    Seed control determine the BOD of the seedingsource

    Dilution water blank used to check for quality ofunseeded dilution water and incubation bottlecleanliness

    Steps to Include: Read and record temperature of incubator Prepare replicate bottles for dilution water blanks and

    seed controls Include at least one set of replicate samples per

    analysis

    Assure quality with: Seed control determine the BOD of the seeding

    source Dilution water blank used to check for quality of

    unseeded dilution water and incubation bottlecleanliness

    Steps to Include: Read and record temperature of incubator Prepare replicate bottles for dilution water blanks and

    seed controls Include at least one set of replicate samples per

    analysis

  • BOD - procedure

    Blanks Prepare dilution water, bring to 20o C and

    aerate Add sufficient seeding material to produce a

    DO uptake of 0.05 to 0.1 mg/L in 5 d (dilutionwater)

    Samples Add sample to bottle and dilute. Dilutions should result in a residual DO of at

    least 1 mg/L and DO uptake of at least 2 mg/Lafter 5 day incubation

    Blanks Prepare dilution water, bring to 20o C and

    aerate Add sufficient seeding material to produce a

    DO uptake of 0.05 to 0.1 mg/L in 5 d (dilutionwater)

    Samples Add sample to bottle and dilute. Dilutions should result in a residual DO of at

    least 1 mg/L and DO uptake of at least 2 mg/Lafter 5 day incubation

  • BOD procedure Steps in procedure:

    Fill bottles with enough dilution water so thestopper displaces all of the air, leaving NO airbubbles

    Read initial DO Incubate for 5 days at 20o C Read final DO Calculate BOD5 correcting for the exact duration

    Steps in procedure: Fill bottles with enough dilution water so the

    stopper displaces all of the air, leaving NO airbubbles

    Read initial DO Incubate for 5 days at 20o C Read final DO Calculate BOD5 correcting for the exact duration

  • Phytoplankton/Algae countingmethods

  • BOD

    Calculations When dilution water is not seeded:

    When dilution water is seeded:

    PDDLmgBOD day 215 )/(

    Calculations When dilution water is not seeded:

    When dilution water is seeded:

    PDDLmgBOD day 215 )/(

    PfBBDDLmgBOD day )()()/( 21215

  • Algae chlorophyllinstrumentation

    Spectrophotometer: Visible with 1-2 nm

    bandwidth Matched cuvettes, 1-5

    cm

    Fluorometer: Requires excitation and

    emission filtersspecifically forchlorophyll measurement

    Spectrophotometer: Visible with 1-2 nm

    bandwidth Matched cuvettes, 1-5

    cm

    Fluorometer: Requires excitation and

    emission filtersspecifically forchlorophyll measurement

  • Algae chlorophyll filtration

    Apparatus - extraction Prewashed 47 mm glass fiber filters (GF/C,

    GF/F, AE, or equivalent) Gelman polycarbonate filtration tower or

    equivalent Vacuum pump (5 to 7.5 psi) Centrifuge (clinical) DIW/acetone (90%) washed 15 mL Corex

    centrifuge tubes with caps

    Apparatus - extraction Prewashed 47 mm glass fiber filters (GF/C,

    GF/F, AE, or equivalent) Gelman polycarbonate filtration tower or

    equivalent Vacuum pump (5 to 7.5 psi) Centrifuge (clinical) DIW/acetone (90%) washed 15 mL Corex

    centrifuge tubes with caps

  • Algae chlorophyll filtration(cont.)

    Filter a known volume ofwater through a GF/C filter

    Volume filtered dependsupon algal density

    Add a few drops of saturatedMgCO3 solution near the end

    When all the water has beenpulled through, fold the filterinto quarters and wrap in foil

    Filter a known volume ofwater through a GF/C filter

    Volume filtered dependsupon algal density

    Add a few drops of saturatedMgCO3 solution near the end

    When all the water has beenpulled through, fold the filterinto quarters and wrap in foil

  • Algae chlorophyll storage Wrap the folded filter in a

    square of foil, label, thenfreeze

    Record the volume filtered,date, site, depth, replicate# all with permanentmarker

    Store the filter in thefreezer at < 20o C

    EPA holding time for afrozen chlorophyll filter is 2weeks

    Wrap the folded filter in asquare of foil, label, thenfreeze

    Record the volume filtered,date, site, depth, replicate# all with permanentmarker

    Store the filter in thefreezer at < 20o C

    EPA holding time for afrozen chlorophyll filter is 2weeks

  • Algae chlorophyll extraction &analysis

    Chlorophyll extraction: Tear filter into several pieces Place in a test tube Add 10 mLs of 90% acetone Extract overnight at 4oC

    Chlorophyll analysis: After 18-24 hr extraction,

    centrifuge to settle filter debris Read absorbance or

    fluorescence of the supernatant

    Chlorophyll extraction: Tear filter into several pieces Place in a test tube Add 10 mLs of 90% acetone Extract overnight at 4oC

    Chlorophyll analysis: After 18-24 hr extraction,

    centrifuge to settle filter debris Read absorbance or

    fluorescence of the supernatant

  • Algae chlorophyllmeasurement

    Measure absorbance of a 90% acetone solutionblank at 750 nm and at 664 nm to correct forprimary pigment absorbance

    Record sample absorbance at 750 nm and 664nm

    Estimate phaeophytin by acidifying the sample.Record the absorbance at 665 nm and again at750 nm

    Run working standard solutions of purifiedchlorophyll-a (Sigma Chemical Co. Anacystisnidulans by the procedure used for the blank)

    Measure absorbance of a 90% acetone solutionblank at 750 nm and at 664 nm to correct forprimary pigment absorbance

    Record sample absorbance at 750 nm and 664nm

    Estimate phaeophytin by acidifying the sample.Record the absorbance at 665 nm and again at750 nm

    Run working standard solutions of purifiedchlorophyll-a (Sigma Chemical Co. Anacystisnidulans by the procedure used for the blank)

  • Algae chlorophyll andphaeophytin

    What is phaeophytin? Degradation product of

    chlorophyll Absorbance wavelength

    (665 nm) is very close tothat of chlorophyll (664nm)

    acid

    What is phaeophytin? Degradation product of

    chlorophyll Absorbance wavelength

    (665 nm) is very close tothat of chlorophyll (664nm)

    H

  • Algae spectrophotometrycalculations

    LVVEELgalchlorophyl

    sample

    extab

    ][7.26)/( 665664

    LVVEELgnphaeophyti

    sample

    extab

    ]7.1[7.26)/( 664665

    Where:b = before acidificationa = after acidificationE664b - [{Abs664b(sample)Abs664b(blank)}-{Abs750b(sample)Abs750b(blank)}]E665a - [{A665a(sample)-Abs665a(blank)}-{Abs750a(sample)-Abs750a(blank)}]Vext = Volume of 90% Acetone used in the extraction (mL)Vsample = Volume of water filtered (L)L = Cuvette path length (cm)

    LVVEELgnphaeophyti

    sample

    extab

    ]7.1[7.26)/( 664665

  • Algae chlorophyll QA

    Quality assurance There are no commercial QA check standards Lab replicates are usually not done Essentially, the analysis is a one-shot deal,

    you dont get a second chance, so be careful Field replicates should be done every 10

    samples Cut filters in half and save one half if nervous

    Quality assurance There are no commercial QA check standards Lab replicates are usually not done Essentially, the analysis is a one-shot deal,

    you dont get a second chance, so be careful Field replicates should be done every 10

    samples Cut filters in half and save one half if nervous

  • Algae- counting methods Wet mounts Filter Counting chambers Utermohl

    requires an invertedmicroscope (light fromabove)

    Sedgewick rafterchamber

    Hemocytometer

    Wet mounts Filter Counting chambers Utermohl

    requires an invertedmicroscope (light fromabove)

    Sedgewick rafterchamber

    Hemocytometer

  • Microscopes capable of magnifications of 100X to 1000X

    Algae counting methods

    Inverted microscopeCompound microscope Less expensiveinverted microscope

  • Algae- taxonomy Use an algal taxonomic key that shows

    species from your geographical area

    Phytoplankton are continually beingdescribed and re-classified so its essentialfor a good taxonomist to keep current (noteasy by any means)

    Its a good idea to take photographs ofslides for cataloging

    Use an algal taxonomic key that showsspecies from your geographical area

    Phytoplankton are continually beingdescribed and re-classified so its essentialfor a good taxonomist to keep current (noteasy by any means)

    Its a good idea to take photographs ofslides for cataloging

  • Algae determining biomass Algal biomass (standing crop):

    A quantitative estimate of the total mass of livingorganisms within a given area or volume

    Biovolume estimates: Identification to genus and species level Calculate cell volume by approximation to

    nearest geometrical shape Count cells over a known area of the slide so

    cells per unit volume can be determined Chlorophyll

    Algal biomass (standing crop): A quantitative estimate of the total mass of living

    organisms within a given area or volume Biovolume estimates:

    Identification to genus and species level Calculate cell volume by approximation to

    nearest geometrical shape Count cells over a known area of the slide so

    cells per unit volume can be determined Chlorophyll

  • Algae determining biovolume

    Taxonomic keys often include questionsabout size

    Determining size is basically like using aruler. The standard ruler for a microscope is called

    an "ocular micrometer," which is fitted into theeyepiece of your microscope

    Taxonomic keys often include questionsabout size

    Determining size is basically like using aruler. The standard ruler for a microscope is called

    an "ocular micrometer," which is fitted into theeyepiece of your microscope

  • Algae determining biovolume

    Some formulas to estimate biovolume fromcell dimensions (Wetzel & Likens 2000)

    B

    A

    Rod4/2AB

    BA

    6/3ASphere

    A

    Ellipsoid6/2AB

    A

  • Algae chlorophylldetermination

  • Algae chlorophylldetermination

    Measuring chlorophyll-a concentrationremains the most common method forestimating algal biomass

    Chlorophyll-a concentration has also beenshown generally, when comparing lakes,to relate to primary productivity (Wetzel1983)

    Can be used to assess the physiologicalhealth of algae by examining itsdegradation product, phaeophytin

    Measuring chlorophyll-a concentrationremains the most common method forestimating algal biomass

    Chlorophyll-a concentration has also beenshown generally, when comparing lakes,to relate to primary productivity (Wetzel1983)

    Can be used to assess the physiologicalhealth of algae by examining itsdegradation product, phaeophytin

  • Algae chlorophyll basics Algal biomass is most commonly

    estimated by chlorophyll-a. Units are ug/L or mg/L (ppb and ppm) Detection limit depends upon method used

  • Algae chlorophyllmethodology

    Spectrophotometry and fluorometry,utilizing 90% acetone extraction, remainthe most commonly used methods

    Spectrophotometry is most widely usedbut fluorometry is more sensitive and maybe used when low levels of chlorophyll areanticipated or when handling largevolumes of water is logistically difficult

    Spectrophotometry and fluorometry,utilizing 90% acetone extraction, remainthe most commonly used methods

    Spectrophotometry is most widely usedbut fluorometry is more sensitive and maybe used when low levels of chlorophyll areanticipated or when handling largevolumes of water is logistically difficult

  • Microscopes capable of magnifications of 100X to 1000X

    Algae counting methods

    Inverted microscopeCompound microscope Less expensiveinverted microscope

  • Bacteria 2 indicator methodsTwo basic methods:1. membrane filtration 2. multiple-tube

    fermentation

    http://www.intelligence.gov/2-community_examples.shtml

    http://picturethis.pnl.gov/picturet.nsf/f/uv?open&SMAA-3V9T37

  • Bacteria membrane filtertechnique

    The fecal coliform MF procedure uses anenriched lactose medium and incubationtemperature of 44.5 0.2o C for selectivity.

    Results in 93% accuracy (APHA 1995) indifferentiating between coliforms found in thefeces of warm-blooded animals and thosefrom other environmental sources.

    Fecal Coliform is reported as colony formingunits per 100 mL (CFU/100 mL).

    The fecal coliform MF procedure uses anenriched lactose medium and incubationtemperature of 44.5 0.2o C for selectivity.

    Results in 93% accuracy (APHA 1995) indifferentiating between coliforms found in thefeces of warm-blooded animals and thosefrom other environmental sources.

    Fecal Coliform is reported as colony formingunits per 100 mL (CFU/100 mL).

  • Bacteria membrane filter equipment Materials needed for MF

    method: Air incubator or water

    bath Non-corrugated forceps Heat sterilizer (Bacti-

    Cinerator) Filter flask and tower

    (Autoclavable) Vacuum pump or water

    aspirator

    Materials needed for MFmethod: Air incubator or water

    bath Non-corrugated forceps Heat sterilizer (Bacti-

    Cinerator) Filter flask and tower

    (Autoclavable) Vacuum pump or water

    aspirator

  • Bacteria membrane filter equipment MF materials

    (continued): Sterile 50 mm petri

    plates (with tight-fittinglids)

    Sterile 0.45 um griddedmembrane filters

    Sterile absorbent pads Autoclave (121o C at

    15-17 psi)

    MF materials(continued):

    Sterile 50 mm petriplates (with tight-fittinglids)

    Sterile 0.45 um griddedmembrane filters

    Sterile absorbent pads Autoclave (121o C at

    15-17 psi)

    http://www.nbtc.cornell.edu/biofacility/autoclave.html

  • Bacteria membrane filterprocedureProcedure:

    Saturate the absorbent pad with M-FC brothSelect a sample volume that will yield 20-60

    colonies/filterFilter sample and dilution water through padPlace pad into petri dishInvert plates and place in incubator for 24 hrs

    Procedure:Saturate the absorbent pad with M-FC brothSelect a sample volume that will yield 20-60

    colonies/filterFilter sample and dilution water through padPlace pad into petri dishInvert plates and place in incubator for 24 hrs

  • Bacteria membrane filtercounting

    Fecal coliformcolonies bacteria arevarious shades ofblue.

    Non-fecal coloniesare gray to creamcolored. normally, few of these

    are present.

    Fecal coliformcolonies bacteria arevarious shades ofblue.

    Non-fecal coloniesare gray to creamcolored. normally, few of these

    are present.

  • image showing method of countinghttp://water.usgs.gov/owq/FieldManual/Chapter7.1/images/Fig7.1-3.gif

    Bacteria MF counting (cont.)

  • MTF image process

    Bacteria multiple tubefermentation

    http://water.usgs.gov/owq/FieldManual/Chapter7.1/images/Fig7.1-3.gif

  • Bacteria cleaning and sterilizingAll equipment Wash equipment thoroughly with dilute nonphosphate, laboratory-grade detergent.

    Rinse 3 X with hot tap waterRinse again 3-5 X with deionized or glass-distilled water.

    Glass,polypropylene,or Teflonbottles

    If sample will contain residual chlorine or other halogens, add Na2S2O3.If sample will contain > 10 ug/L trace elements, add EDTA.Autoclave at 121 C for 15 min or bake glass jars at 170 C for 2 hrs.

    Stainless-steelfield units

    Flame sterilize with methanol (Millipore Hydrosol units only), or autoclave, orbake at 170 C for 2 hrs

    Portablesubmersiblepumps andpump tubing

    Autoclavable equipment (preferred): autoclave at 121 C for 15 min.Non-autoclavable equipment:Submerge sampling system in a 200 mg/L laundry bleah solution and circulatesolution through pump and tubing for 30 min; follow with thorough rinsing, insideand out, with sample water pumped from the well. **SEE NOTES

  • Bacteria USGS summaryTest (media type) Ideal count range

    (colonies per filter)Typical colony color, size, and morphology

    Total coliform bacteria(m-Endo)

    20-80 Colonies are round, raised and smooth; 1 to 4 mm di; andred with golden-green metallic sheen.

    Escherichia coliAfter primary culture astotal coliform colonieson m-Endo (NA-MUG)

    None given but muchfewer in number thantotal coliformson the same filter

    Colonies are cultured on m-Endo media as total coliformcolonies. After incubation on NA-MUG, colonies have blueflorescent margins with a dark center. Count under a longwave ultra violet lamp in a completely dark room.

    Fecal coliform bacteria(m-TEC)

    20-60 Colonies are round, raised and smooth with even to lobatemargins; 1 to 6 mm di; light to dark blue in whole or part.Some may have brown or cream colored centers.

    Fecal coliform bacteria(m-TEC)

    20-60 Colonies are round, raised and smooth with even to lobatemargins; 1 to 6 mm di; light to dark blue in whole or part.Some may have brown or cream colored centers.

    Escherichia coli(m-TEC)

    20-80 Colonies are round, raised and smooth; 1 to 4 mm di;yellow to yellow brown; many have darker raised centers.

    Fecal streptococci(KF media)

    20-100 Colonies are small, raised, and spherical; about 0.5 to 3mm di; glossy pink or red in color.

    Enterococci(m-E and EIA)

    20-60 Colonies are round, smooth and raised; 1 to 6 mm di; pinkto red with a black or red dish brown precipitate onunderside.

  • Uneven; not mixedwell; low volume

    Fecal coliforms troubleshooting

    No matter which assay is used, after incubation there should be ~20-60 colonies evenlydistributed across the Petri dish

    poor seal around theedges; poorly seated

    with air bubble Dry spot frompoor seating

    Uneven; not mixedwell; low volume

  • Fecal coliforms troubleshooting(cont.)

    Too many useless sample Too few use more sample

    Looks good