EPA 910-R-96-001
              United States
              Environmental Protection
              Agency
Region 10
1200 Sixth Avenue
Seattle WA 98101
Alaska
Idaho
Oregon
Washington
              Office of Environmental Assessment
                                                   April 1996
              Microscopic Paniculate
              Analysis (MPA) for
              Filtration Plant Optimization

                                   •J-
                                   /i
                                   /1

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                    Microscopic Participate Analysis (MPA)
                       For Filtration Plant Optimization

                                    prepared by
                 Stephanie Harris1, Carrie Hancock1, & Jay Vasconcelos1

                                     April 1996
1U.S. EPA Manchester Laboratory, 7411 Beach Drive East, Port Orchard, WA 98366


2CH Diagnostics & Consulting Service, 214 S.E. 19th Street, Loveland, CO 80537

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                                       A cknowledgements
We wish to thank the many microbiologists and others who actively participated in the development and review of the
USEPA consensus method entitled "Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization".
Dr Susan Boutros
Environmental Associates
1185 East Main
Bradford, PA 16701

Rick Brinkman
250 North 5th St
Grand Junction, CO 81503

Carrie Howe-Carlson
Microsearch Laboratory
2783 Webster Drive
Grand Junction, CO 81503

Dr Jennifer Clancy (Randi McCuin)
Clancy Environmental Consultants
PO Bos 314
St. Albans,VT 05478

Jill Cunningham (Deborah Wayman, Deanna Crump)
Grants Pass Water Lab
558 ME "F"
Grants Pass, OR 07527
Rick Danielson (Craig Johnson)
BioVir Laboratories, Inc.
685 Stone Rd. # 6
Benicia, CA  94510

Michelle Eisenstein
Johnson State College
Chemical Hygiene Office
Johnson, VT 05656

Scott Tighe (Sharon Hallock, Brad Eldred)
Analytical Services, Inc.
PO box 515
Williston, VT 05495

John Rae
2855 Mesa Rd.
Colorado Springs, CO 80904

Dr. Frank Schaefer, III
USEPA/NERL/HERD/BARB
26 W. Martin Luther King Way
Cincinnati, OH 45268
      We also wish to thank those who contributed materials and talent to design the cover page.

                              U.S. EPA Region 10 Graphics Department
                              BioVir Laboratories, Inc.
                              Eugene Water and Electric Board
                                            Disclaimer

        Mention of any trade names of commercial products does not constitute endorsement or recommendation for
use by the U.S. Environmental Protection Agency.

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         Microscopic Participate Analysis (MPA) for Filtration Plant Optimization

                                        Table of Contents

Introduction  	 1
Sample Collection	'.	 2
        1.0     General Overview 	 2
        2.0     Sample Equipment and Materials  	 2
        3.0     Sample Collection Parameters	 2
        4.0     Sample Collection Procedure	 3
        5.0     Sample Volumes and Water Quality Parameters	 4
Filter Processing and Analysis  	, 6
        6.0     Equipment	 6
        7.0     Supplies	 6
        8.0     Processing Reagents 	 7
        9.0     Paniculate Extraction	 7
Palmer-Maloney Counting Cell	 8
        10.0    Subsample Examination 	 8
        11.0    Microscopic Analysis 	 9
        12.0    Whipple Grid Calibration	  12
        13.0    Centrifugate Pellet Measurement	  12
        14.0    Recording of Results & Procedural Parameters	  13
        15.0    Interpretation of Results 	  13
        16.0    Analyst Qualifications	  15
        17.0    Standards of Identity 	  16
        18.0    Quality Assurance	  17
References for Internal Document 	  20
References for Microscopic Identification	  20
Appendix 1     Microscope Alignment and Adjustment	  22
Appendix 2     Use of Electronic Particle Counter	  26
Appendix 3     Sample Data Forms and Report Forms	  29
Appendix 4     Formulation ofMcFarland Standards	  33
Appendix 5     Figures for Document   	  34
Appendix 6     Sample Calculation	  39

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                                            Microscopic Particulate A nalysis (MPA) for Filtration Plant Optimization
                                              Introduction
        With enactment of the 1986 Amendment to the
Safe  Drinking Water Act  (SDWA),  the  EPA has
promulgated   new  regulations   for  filtration  and
disinfection of public water systems using surface water
or groundwater under the direct influence of surface
water (GWUDI).  Those systems identified as surface
water or GWUDI must demonstrate a 3 log (99.9 %)
removal of Giardia and 4 log removal of virus particles
through a combination of filtration and disinfection.
Detection of Giardia  cysts and Cryptosporidium
oocysts in surface  water cannot  be  used  to assess
treatment  plant  performance because of their low
concentrations, intermittent occurrence, and limitations
of the currently available technology. Because Giardia
and  Cryptosporidium may  occur at  concentrations
sufficient  to  cause  disease but not  consistently
numerous enough to assess filtration performance,
surrogates for filtration efficiency have been and
continue to be developed.
         Performance of water treatment plants can be
evaluated by a number of methods, including turbidity,
particle counts, and Microscopic Particulate Analysis
(MPA). Simultaneous use of more than one evaluation
technique may be appropriate, not only for research but
also for plant operation. Particle counting and turbidity
data must be  used with caution because flocculated
particles could give false values compared with cyst
reduction  and overall plant performance.   Several
investigators   have  found   that  MPA  can  provide
information on the effectiveness of water treatment
processes for removing  paniculate  matter  (1, 2).
Electronic particle counting, by itself, does not provide
the operator with critical information about the type and
number of organisms encountered.
        MPA, including particle sizing, is performed
on drinking  water systems where  some  form of
treatment, chemical or physical,  exists between the
natural water source and its distribution to the public.
This analysis compares the type, size and quantities of
bioindicators and particles found  in the raw water to
those found in the  finished, or treated, water.  This
method can be used to evaluate filtration efficiencies, as
log reduction, of conventional filtration  systems, as
well as the on-site evaluation of alternate  filtration
technologies.
        This method can be used to  identify certain
groups of microorganisms, 1 to 600 micrometer (/urn) in
size, which normally only occur  in raw water as
opposed to finished  waters and whose  presence, in the
finished water, may indicate  some breakthrough or
growth in   the  filter  beds.    These   important
microorganisms,  also called  bioindicators,  include
diatoms, algae, Giardia. coccidia,  plant debris, pollen,
rotifers,   crustaceans,   ameba,    nematodes    and
insects/larvae. Comparison of the quantitative numbers
of these bioindicators in raw and finished water  can
also assist  in  the  over-all evaluation of  filtration
efficiency and may  provide information critical to the
optimization of the filtration plant  beyond simple
turbidity   reduction   or   particle   counting   by
instrumentation.
        Historically, water treatment professionals
have relied on chemical and physical measurements to
assess  water treatment plant performance; obviously
these assessments are not adequate because numerous
outbreaks   of Giardia  and  Cryptosporidium  have
occurred during  periods where  the  plant met  all
federally required performance criteria.  An eclectic
approach using several tools, all of  which measure
different  aspects   of  plant  performance,  for  the
assessment  of filtration  efficiency  along  with  a
thorough understanding of the particular plant design
and operation helps  avoid the inadequacy of simplistic
solutions for explaining the complex  interactions of
water treatment.

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Microscopic Paniculate Analysis (MPA) for Filtration Plant Optimization
                                           Sample Collection

 1.0     General Overview: High Volume Filter (HVF) samples for surface water MPA are collected from the raw
        water before it enters any phase of water treatment and from the finished water just prior to disinfection and
        distribution. Evaluation of each filter bed or a composite of the effluent water is optional, and the chosen option
        should be noted in the final report.  However, blending of each filter bed into a composite  many prevent
        identification of individual filter bed inadequacies. The collection site should be selected to avoid stratification
        of the pipes.

2.0     Sample Equipment and Materials

        2.1     Sampling device consists of the following parts (refer to Figure 1 and 2)
                2.1.1     Six foot inlet hose, preferable disposable, with backflow preventer (Watts No. 8)
                2.1.2     Pressure regulator (Watts 26A), or equivalent, plus pressure gauge, 0-100 psi
                2.1.3     Proportionating injector (for chlorinated water), Model 203 B.T. injector, 100-15P-87, or
                         equivalent. (Dema Engineering) (For chlorinated samples only).
                2.1.4     Commercial Filter model LT-10 filter housing (9499-5015)
                2.1.5     Water flow meter, readable in gallons or liters.
                2.1.6     Flow control valve (limiting flow orifice) rated at 1.0 gallon per minute (gpm) for finished
                         water; 0.5 to  1.0 gpm for raw water. (Rationale for this modification is to allow collection
                         for a longer period prior to plugging of the filter in high turbidity waters)
                2.1.7     Discharge hose
                2.1.8     Pump, for non-pressurized sources
                2.1.9     Miscellaneous brass, or PVC, fittings for unit assembly
                2.1.10   Optional peto tube installed at sampling port is recommended to reduce problems caused by
                         flow dynamics in the pipe
        2.2     Sampling Materials
                2.2.1     Ten inch, 1 ^m nominal porosity, polypropylene, yam-wound, cartridge filter, Commercial
                         Honeycomb filter tube (M39R10A).
                2.2.2     Whirl pak plastic bags (5.5" x 14") or zip loc heavy duty quality freezer bags
                2.2.3     Sanitary gloves

3.0     Sample Collection Parameters

        Note: Below are recommendations for typical treatment systems. Large, or atypical, systems where retention
        occurs, may require alteration of these recommendations to provide accurate log reduction values. Moreover,
        electronic particle count collection sites may vary from MPA collection points.  If performing only electronic
        particle counts, the sample  might be  more appropriately collected directly from the source, particularly if
        presedimentation basins are an integral part of the treatment plant.  The  holding time associated with
        presedimentation basins, may allow for settling of participates and may adversely influence  log reduction
        values.

        3.1     Raw  surface water should be sampled prior to chemical  addition and after any presedimentation
                basins (if no chemicals were added prior to presedimentation).  The main objective in raw water
                sampling is to collect a sample representative of the water entering the treatment system; therefore,
                if recycling operations are practiced, the raw water should be sampled after the recycling input. Such
                sampling should allow adequate time for mixing of recycling input prior to sampling. If collection
                at the source is not possible, final report must "qualify" sample

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                                            Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
        3.2     Finished water should be sampled after the filtration system and prior to chlorine addition, if possible.
                Sodium thiosulfate (final concentration 50 mg/1) is injected into samples that cannot be collected prior
                to chlorination.  Samples are collected prior to post treatment storage to provide a more accurate
                evaluation of the filtration system.  Evaluation of log reduction in large treatment plants with post
                filtration holding tanks, may be difficult, given the propensity for algal growth in these circumstances.

        3.3     Treatment plant evaluation. The raw water sampling should be initiated before the finished water
                sampling.  The amount of time elapsed between the beginning of raw sampling and the beginning of
                finished sampling should be equivalent to the detention time of the system.  To accurately assess
                treatment efficiency, finished water sampling should encompass a full cycle run or for a 24 hour
                sampling, including at least one backwash in the sampling.

        3.4     Pressure over the filter face should be set at 10 psi, using the in-line pressure gauge and meter

4.0     Sample Collection Procedure (Flow chart, Figure 3)

        4.1     Cleanliness- before each sample collection the hose and filter housing must be washed with hot water
                containing a mild detergent and bleach solution; rinse with hot water followed by particle free water
                (see 7.12). If this cannot be done, run a minimum of 50 gallons of sample water through the sampling
                equipment prior inserting a new filter.  Do not touch the filter with bare hands, use sanitary gloves or
                the plastic cover the filter is wrapped in.

        4.2     Connect sampling unit to pressure source and pump in the direction of flow indicated on filter housing.
                Flush the  unit without a filter for 3 - 5 minutes with the source water to be sampled.
                4.2.1   Non-Pressurized Sources:  Small 1-5 gallon per minute battery operated bilge pumps or
                        electric or gas powered centrifugal pumps may be used. Be sure to put the sample intake in
                        a location where the least  amount of  bottom sediment will enter into the sampling filter
                        giving a. distorted view of the sample.  If possible, install the pump downstream (on the
                        effluent end) of the filter to eliminate the potential for cross-contamination of samples. Note:
                        Collect sample as near to intake site as possible.  If intake is near the bottom and in fact
                        draws in bottom sediments, then collection here is appropriate.

        4.3     Record the date, time of day and gallon reading from the water  meter before and after sampling.
                Document the name and location of each sample point, sampling site (raw or finished) and type of
                treatment.

        4.4     Insert filter in the housing and tighten housing. Make sure "0" ring is in place. Turn water on slowly
                with the unit in an upright position. Invert unit to make sure all the air within the housing is expelled.
                When the housing is full of water, return unit to upright position and increase flow up to 3.81pm (1
                gpm).  Measure flow rate by either timing the  meter rate or by timing flow rate into a calibrated
                bucket. Maintain 1 gpm (3.8 Lpm) throughout the sampling period. Exceptions to the 1 gpm rate are
                given in 5.1.  If 0.5 gpm flow control valve is being used follow the directions except that the flow
                rate will be less and meter may not register so measuring the flow using a calibrated container may
                be necessary.

        4.5     Check reading on pressure gauge. Adjust pressure gauge to 10 psi, if needed..

        4.6     Information on the sample volume and water quality parameters should be included on data sheet (See
                section 5.0)

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Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
        4.7      When sampling is complete, shut off flow, record stop time and final meter reading or time flow rate
                 into calibrated bucket and average with flow rate measured in 4.4.  Subtract the initial reading from
                 the final reading and record the total volume collected.

        4.8      Turn off the faucet or pump and disconnect the hose from incoming water source. Maintain the inlet
                 hose level above level of opening on outlet hose to prevent backwashing and loss of paniculate matter
                 from the filter.  Pour residual water from filter holder into the ziploc (whirlpac) bag.

        4.9      Remove the sampling cartridge with the plastic cover or sanitary latex gloves. Do not touch with bare
                 hands.  Place filter in the heavy duty quality ziploc (whirlpac)  bag and seal.

        4.10     With permanent marker record the sample identification, gallons sampled, collection dates and times,
                 collector's name and water quality parameters directly on the bag or on a waterproof label. Place the
                 first bag containing the filter into the labeled ziploc bag. Make sure both bags are sealed to prevent
                 leakage.

        4.11     If immediate shipping is not possible, the sample should  be stored in a 1  - 5° C refrigerator, until
                 shipping, within limits set forth in 4.14.

        4.12     Place freezer cold packs  in the shipping container.  Place  insulating material between the filter and
                 cold packs to prevent HVF sample freezing. Samples that arrive at the laboratory frozen, should be
                 rejected, discarded and resampling requested. Place data sheet  containing recorded information in a
                 sealed plastic bag and ship with the filters.

        4.13     Ship by overnight delivery service to the analytical laboratory.

        4.14     Samples must be processed within 96 hours of initiation of sampling.

5.0     Sample Volumes and Water Quality Parameters

        5.1      Raw water: Sampling unit should be allowed to run for a 12 to  24 hour period in which time  a
                 minimum volume of 100 liters (27 gallons) should be filtered. The ideal volume is the amount
                 equivalent to a complete  day of production. If the filter becomes clogged or plugged due to highly
                 turbid waters, terminate sampling and record the volume collected to this point.  If the raw water
                 source is known to have high turbidity, the sampling flow rate may be lowered to < 1 gpm to collect
                 a sample over a longer time period thus obtaining a sample more  representative of the raw water
                 quality.

        5.2      Finished water:  Minimum 1000 liters or 264 gallons. Collection period should encompass  a full
                 cycle run, or for 24 hours, including at least one backwash cycle.  Backwash cycles can occur at the
                 initiation of the sampling  period. If multiple filter beds are  present in the filtration plant, a composite
                 sampling is recommended  initially, although later evaluation of individual filter beds is an option.
                 Additionally, if requested, discrete sampling points at ripening, middle of cycle and after backwash
                 may be added to assist with plant operation.

        5.3      Water Quality Parameters: Measurement of certain water quality parameters should be included
                 in the sample data form  for both raw and finished water. Among these should be total and free
                 chlorine residual, temperature, pH,  turbidity, and operational parameters of the WTP (pretreatment,
                 filtration, disinfection) and water source. Microbiological testing, such as total and fecal coliform and
                 heterotrophic plate count, are optional.

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                                   Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
5.4     Chlorinated Samples: Try to sample water prior to any chlorination. If chlorinated water must be
        sampled, an injector system will need to be installed to add a sodium thiosulfate solution to denature
        the chlorine. Add sodium thiosulfate solution via the injector system to produce a final concentration
        of 50 mg/L. Setting the injector system to produce a  1:100 dilution of 0.5% sodium thiosulfate stock
        solution  will  result in a final concentration of 50 mg/L.   Details on the operation  and use of
        proportioner pumps and injectors can be found in Standard Methods for the Examination of Water
        and Wastewater. Section 95IOC, "Virus Concentration from Large Volumes by Adsorption to and
        Elution from Microporous Filters (Proposed)," 18th ed., 1989, pp. 9-105 to 9-109. Model 203 B.T.
        injector,  100-15P-87 special tip, Dema Engineering, or equivalent, may be used. Alternatively,  a
        peristaltic pump or electric pump can be used to inject the sodium thiosulfate.

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Microscopic Particulate A nalysis (MPA) for Filtration Plant Optimization
                              Filter Processing and Analysis (Fig 3,4)

6.0     Equipment
        6.1     Large capacity centrifuge, refrigerated recommended
        6.2     Large capacity swing-bucket rotor (90 °), 1-6 liter/run
        6.3     250 mL conical bottom bottles with screw caps or 490 mL glass conical bottles
        6.4     15 mL conical graduated centrifugate tubes
        6.5     graduated cylinder
        6.6     1 - 5° C refrigerator
        6.7     50 mL conical graduated centrifuge tubes
        6.8     Stomacher lab blender- model 3500 (optional)
        6.9     Vortex tube mixer
        6.10    Aspiration flask and vacuum source with 0-30 psi gauge
        6.11    Pipet aid, syringe or bulb
        6.12    Motorized  multivolume microliter pipet (Rainin edp plus) or manual equivalent
        6.13    Hollow glass tubes (ca 1/4" bore)
        6.14    Brightfield, phase contrast, differential interference contrast (DIG) or Hoffman modulation optics
                (HMO) capable microscope equipped with 10,40 and  100 X objectives. A 35 mm camera or video
                camera attached, is optional
        6.15    Manual or  electronic differential counter (10 gang)
        6.16    Non-drying immersion oil
        6.17    Single place hand held counter
        6.18    Palmer-Maloney counting chamber available from Wildlife Supply Company, catalog # 1803-B20,
                specify glass model. (301 Cass St. Saginaw, MI, 48602)
        6.19    Sedgewick-Rafter Counting Chamber (optional)
7.0     Supplies
        7.1     Whirl pac bags, 5.5 x 15", sterile, or heavy duty ziploc bags. For filter transportation
        7.2     Polypropylene yarn woven filter tubes (M39R10A, Commercial Filter, Lebanon, IN)
        7.3     Sanitary gloves
        7.4     Pan or tray, stainless steel  or glass, autoclavable
        7.5     4 liter beakers, autoclavable (glass or plastic)
        7.6     Scalpel handles, autoclavable
        7.7     Scalpel blades, sterile
        7.8     Disposable glass pipets, sterile
        7.9     Pasteur pipets, sterile
        7.10    10 % buffered formaldehyde, pH 7.0
        7.11    Polysorbate 80
        7.12    Particle-free water (deionized, distilled or reverse osmosis water, passed through a 0.22 urn filter)
                should contain less than 100 particles/ml (2 ^m or larger)
        7.13    Clear fingernail polish
        7.14    3.5 L capacity Stomacher bags (Seward medical, Tekmar Co)
        7.15    2 L beakers
        7.16    Non-drying immersion oil

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                                            Microscopic Paniculate Analysis (MPA) for Filtration Plant Optimization
8.0     Processing Reagents
        8.1      Wash water (non-sterile)
                 8.1.1    Sterile Erlenmeyer flask (1,2 or 4 L)
                 8.1.2    Particle free water (7.12)
                 8.1.3    Sodium citrate (optional, if iron present)
                 8.1.4    0.01 % polysorbate 20, add and mix immediately prior to use (optional)
                 8.1.5    Mix these in the following proportions:

                                          Wash-Water Proportions

                                                                             LL       2L       4L
                         Sodium citrate (Optional)                             5.0 g    lO.Og     20.0g
                         0.01 % polysorbate (Optional)                        10.0 ml   20.0 ml   40.0 ml
                         Particle-free water (Quantity Sufficient to make)         l.OL     2.0 L     4.0 L

                         Final pH to 5.5 - 7.5

                 8.1.6    Use of 0.85 % NaCl or other buffering agent is optional

                 8.1.7    Record constituents of wash water on laboratory bench sheet

9.0     Participate Extraction:  The filter cartridge is handled aseptically.  All glassware and other equipment is
        mechanically scrubbed, rinsed in particle-free water and autoclaved or chemically sanitized.  Sanitary gloves
        are worn during processing.

        9.1      Remove filter from the ziploc/whirl pac bag and place in pan.
        9.2      Record the color of the filter and any other notable physical characteristics.
        9.3      Rinse the bags with particle-free water. The rinse water is retained in a beaker.
        9.4      The filter fibers are cut length-wise to the filter core and separated into a minimum of 6 equal portions.
                 Each portion is washed sequentially in 3 consecutive 1.0 liter volumes of wash water. Begin with the
                 cleanest fibers and proceed to the dirtiest.  Washing consists of vigorous kneading and  swirling
                 motion. Minimum total wash time of fibers should be 30 minutes. The fibers  are wrung out into a
                 collection beaker by placing them in individual interlocking bags which have one comer snipped off
                 to  allow for drainage.
        9.5      Alternatively, a Stomacher lab blender (model 3500) may be substituted for handwashing. The filter
                 is  cut length-wise to the  core and  after loosening the fibers, place all of the fibers into  a single
                 stomacher bag. To insure against bag breakage and sample loss, place the filter fibers in  the first
                 stomacher bag into a second stomacher bag. Add 1.75 L of wash water to the fibers.  Homogenize
                 for 2- five  minute intervals. Between each homogenization period, hand knead the filter material to
                 redistribute the fibers in the bag. Wring the fibers out to express as much of the liquid as possible and
                 place them in another stomacher bag containing 1.25 liters of wash water. Repeat for 2- five minute
                 homogenization periods.   Wring the fibers to express as much of the  liquid as  possible before
                 discarding.
        9.6      The 2 aliquots of wash water, bag rinse water and residual filter water obtained from the filter housing
                 are combined in one 4 liter beaker.
        9.7      Record the volume of the total paniculate solution.

Note:   The use of Immunofluorescent Assay for Giardia & Cryptosporidium is optional at this  time, but is required
        if Giardia & Cryptosporidium reporting is included in the analysis.  If IFA is chosen, follow the latest method
        publicized in the federal register. The fibers from those samples for which IFA is being done  in addition to
        MPA will be washed a second time in  the IFA prescribed eluting solution (Federal Register 1994). An FA
        eluting solution was developed because more Giardia cysts and Cryptosporidium oocysts could be recovered

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Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
        during the washing process than by using particle-free water alone.  However, many non-encysted organisms
        subjected to the FA eluent deteriorate and cannot be recognized or identified for MPA. Therefore, a paniculate
        extraction scheme will need to be followed so that both MPA and FA can be done from the same sample. The
        particle-free water/particulate solution from the initial wash is halved for MPA and FA. The half retained for
        IFA is combined with half of the secondary wash solution to provide a known quantity of IFA/particulate
        solution.

                                     Using Palmer-Maloney Cell

        The Sedgwick Rafter (S-R) may be used in addition to the PMCC for enumeration of larger zooplankton if long
working distance 20x or 40x objectives are available and if written records of calculation and methodology following
Standard Methods are kept.

10.0    Subsample Examination:  The total particulate solution represents a solution of the participates recovered
        from the number of gallons sampled;  therefore, an accurate record of the number of gallons sampled is a
        prerequisite for the calculations needed to do any further processing.  Convert the  number of gallons to liters.

        10.1    Thoroughly mix the total particulate solution by pouring it back and forth in glass containers  of
                sufficient size to hold the whole sample, or alternatively, use a sterile stir bar and magnetic plate and
                mix for 10 minutes. After mixing, immediately remove  a 200 ml aliquot and place into a 250 ml
                conical centrifuge bottle. Vortex for IS seconds.

        10.2    Immediately after mixing, withdraw a 0.1 mL subsample with an calibrated Eppendorf, or equivalent,
                pipette.

        10.3    Inject the subsample into the Palmer-Maloney counting chamber by introducing the sample with the
                pipette into one of the two 5-mm channels on the sides of the chamber with the cover slip in place.

                10.3.1  Calculate and record the liter equivalent of the withdrawn subsample using this proportion
                        ratio:

                        Total Volume in 250 ml conical centrifuge bottle   =    0.1 mL
                                Liter equivalent in centrifuge bottle             X

                        where: X = liters equivalent of 0.1 ml subsample
                        Liter equivalent in centrifuge bottle is calculated from this proportion ratio:

                        Total volume of particulate solution (in ml)        =    200 ml aliquot
                                   Total # of Liters sampled          Liter equivalent in centrifuge bottle

        Note: If returning to this step for a second time, skip the above calculation; double the previously calculated
        Liter equivalent in centrifuge bottle (triple if a third 200 ml aliquot is added), then subtract the liter equivalent
        of previously withdrawn 0.1  ml subsample (10.7.1).

        Note: If previous history of  this treatment plant warrants it, centrifugation of the entire wash water may be
        performed.

        10.4    View the subsample  at  lOOx magnification.  If >10 plankters (plural for individual plankton
                organisms) are present per field of view, proceed to section 11.0.

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                                            Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
        10.5    If <10 plankters per field of view are present, centrifuge the bottle at 1050 x g for 10 minutes.
                Alternatively, if the turbidity of the paniculate solution examined is comparable to a  1.0 McFarland
                standard, this will provide an acceptable density of particulates in most instances.  (See McFarland
                Standards in Appendix 4.)

        10.6    Aspirate the supernatant down to 4cm above the bottom sediment or the bottom of the bottle, this
                should provide approximately 50 ml.  Measure and record the volume of combined cenlrifugate and
                supernatant.

        10.7    Thoroughly mix the solution by vortexing  for 15 seconds  and  immediately withdraw a 0.1 ml
                subsample for charging the Palmer -Maloney counting chamber.

                10.7.1  Calculate and record the liter equivalency of the 0.1 mL aliquot using this proportion ratio:
                        (For example of calculations see Appendix 6.)

                          Volume of Remaining Particulate Solution (in mL)  =    0.1 mL
                                    Remaining Liter Equivalency                   X

                                 where:
                                 X = liter equivalent of 0.1 ml subsample

                                 Vol. of Remaining Particulate Solution = Vol.
                                 recorded in 10.6.

                                 Remaining Liter Equivalency = Liter equivalent in centrifuge bottle calculated in
                                 10.3.1 less the liter equivalency of previously withdrawn 0.1 mL subsample(s).

        10.8    If >10 plankters are present per lOOx magnification field of view, proceed to section 11.0.

        10.9    If <10 plankters per field of view are present, add an additional 200 ml aliquot of the total paniculate
                solution by  repeating steps 10.1 to 10.9 until the plankter density is correct. Alternatively, if the
                turbidity of the paniculate solution examined is comparable to a 1.0 McFarland standard, this will
                provide an  acceptable density  of particulates in  most instances.   (See McFarland Standards in
                Appendix 4)

                NOTE: Frequently, raw surface water may be examined without centrifugation after filter washing;
                whereas, finished water often requires several centrifugation steps.  Sometimes samples are over-
                concentrated (too dense for microscopic visualization due to overlapping) and need to be diluted. If
                further  dilution is necessary, remember to  include this in the calculations in  10.3.1 or 10.7.1.
                Occasionally, interfering amorphous debris (ex: detritus or flocculent from a conventional treatment
                plant) prohibits a density of 10 plankters per field and these samples must be examined as described
                in section 11.0 at a particle density as dense as possible but without overlapping particulates that could
                obscure visualization of the plankters.

11.0 Microscopic Analysis

        11.1    After application of sample, allow a  10 minute settling period before counting. The entire Palmer-
                Maloney counting chamber is systematically examined at a minimum of lOOx magnification using
                phase optics, brightfield, DIC or HMO.  Begin scanning the chamber at one edge and  use an up-and-
                down or a side-to-side scanning pattern (see Figure 5).  If the distribution of organisms is random and
                the population  fits a Poisson distribution, the counting error may be estimated.  If 100 units are
                counted, the 95 %  confidence limits approximate ± 20 %. (3,4).

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Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
                11.1.1   Separate counts are made for each of the following size ranges: < 10 um, 10 -25 um, 25 -
                         100 um, 100 - 200 um, > 200 um and total number and should be recorded on the data sheet.
                         An ocular micrometer calibrated against a stage micrometer should be used when performing
                         particle sizing. All categories from 11.1.4, as well as amorphous debris and unclassified
                         biological material will be included in this count. Using a calibrated whipple grid (Appendix
                         1), count all particles, at a minimum of 100X magnification, present in a total of 20 - 30
                         whipple grid fields chosen randomly from the PMCC. Averaging methods are  acceptable
                         with densities too high for accurate counts.  Record the number of particles found in each
                         size range in each of the grid fields. Formula for calculation of # per 100 1 is in section
                         11.2.3 and 11.2.5.

                11.1.2   Any microbiota seen that are not too numerous to count at a minimum magnification of lOOx
                         are identified following the Standards of Identity section.

                11.1.3   Identification to the lowest level of taxonomic resolution known by the analyst is recorded.

                11.1.4   Separate counts are made for each of the following categories: nondiatomaceous algae,
                         diatoms, plant  debris, rotifers, nematodes,  pollen, ameba, ciliates, colorless flagellates,
                         crustaceans, other arthropods and "other" (see Standards  of Identity section).  Counts are
                         made by the natural unit (clump count enumeration) method defined as follows:  any
                         unicellular organism or natural colony is counted as one organism. Alternatively,  total count
                         method can be used, defined as follows: each cell is counted as 1 organism.  Making a total
                         cell count is time-consuming and tedious, especially when colonies consist of thousands of
                         individual cells. The natural unit is the most easily used system, however it is not necessarily
                         the most accurate because sample handling, collection or water treatment may result in
                         breakdown of organisms leading to inaccurate removal rates between raw and finished water
                         samples.  Analyst must report the enumeration method used. Counts  are made for those
                         categories of organisms that are not too numerous to count.

                11.1.5   The counts for each category are extrapolated from the 0.1 mL aliquot used in the Palmer
                         Cell to numbers per 100 Liters as follows:

                         11.1.5.1 Calculate X using this proportion ratio: (For calculation example see Appendix 6.)

                                # of organisms   =   X
                                     O.lmL          V

                                where:  # of organisms = the count from one category
                                        V = volume from which 0.1 ml aliquot was withdrawn
                                        (derived from 10.3.1 or 10.7)

                         11.1.5.2 Calculate the number of organisms per 100 liters for each category  using this
                                proportion ratio:

                                        	X	  =    number of organisms
                                    liters equiv. of soln from       100 liters
                               which 0.1 ml aliquot was withdrawn

                                where:
                                      • X = calculation from 11.1.5.1
                                                   10

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                                   Microscopic Paniculate A nalysis (MPA) for Filtration Plant Optimization
11.2    The common organisms, or particles that are too numerous to count at lOOx are counted in calibrated
        Whipple grid fields at 400x. Specific calibration of each microscope used is essential (see 12.0).

        11.2.1   As many Whipple grid fields as necessary to obtain a minimum of a 100 organism count are
                observed. (95 % CL of approximately ± 20 %.)  Use a single place hand held counter to
                count the number of fields observed. Use a manual differential counter (10 gang) or 10 place
                electronic tabulator to count the number of organisms observed in each of the categories
                listed in 11.1.4. Count only those categories that were not counted in section 11.1.4.

        11.2.2   Record the number of organisms or particles found in each category as well as the identity
                of the organisms observed at the lowest level of taxonomic resolution known by the analyst.
        11.2.3   Calculate the number of organisms or particles per mL in each category using the following
                formula:

                        No./mL   = C x  1000mm
                                    A  x D  x  F

                        where:
                                C =     number of organisms counted for each category from 11.2.2
                                A =     area of a field (Whipple grid image), mm2 (width2 - see 12.0)
                                D =     depth of a field (P-M chamber depth = 0.4mm)
                                F =     number of fields counted

        11.2.4   Calculate the number of organisms in the remaining paniculate solution from which 0.1 mL
                of solution with > 10 plankters  per field was withdrawn as follows:

                                 N_     =          X
                                ImL            mLs of remaining paniculate solution
                        where:
                                N =     number of organisms per mL (calculated in section 11.2.3)
                                X =     number of organisms in the remaining paniculate solution

        11.2.5   Calculate the number of organisms per 100 liters using this proportion ratio:

                                X      =       No. of organisms
                                L              100 liters

                        where:
                        X =     the number of organisms in the remaining paniculate solution calculated
                                in section 11.2.4.
                        L =     liters equivalency of the remaining paniculate solution (Total number of
                                liters sampled less the liter equivalence of withdrawn subsamples).
                                           11

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Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
12.0    Whipple Grid and Ocular Micrometer Calibration:  Place a Whipple grid in an eyepiece of the microscope
        and a stage micrometer that has a standardized, accurately ruled scale on a glass slide. The whipple disk has
        an accurately ruled grid subdivided into 100 squares. One square near the center is subdivided further into 25
        smaller squares. The outer dimensions of the grid are such that with a lOx objective and  a lOx  ocular, it
        delimits an area of approximately 1 mm2 on the microscope stage.  At 40x and with the ocular and stage
        micrometers parallel and in part superimposed, match the line at the left edge of the Whipple grid with the zero
        mark on the state micrometer scale.  Determine the width of the Whipple grid image to the nearest 0.001 mm
        from the stage micrometer scale.  (APHA 1995)

13.0    Centrifugate Pellet measurement (Figure 4) Centrifugate pellet measurement may provide information about
        the overall plant performance. However, it is not intended to be used as a sole method for determining filtration
        efficiency.

        13.1    If the liter equivalent in the 250 ml centrifuge bottle (10.3.1) is > 100 liters proceed to 13.1.1.  If not,
                proceed to 13.2.1.
                13.1.1.  Centrifuge the 250 ml bottle at 1050 x g for 10 minutes. Measure the Centrifugate  pellet
                        volume. If volumes are below lowest graduation, mark a "dummy" set of tubes using water
                        injected from calibrated pipettes and compare to sample.

                        13.1.1.1.        Calculate the  volume of Centrifugate  pellet per 100 liters  using the
                                        following proportion ratio:

                                Total Centrifufiate Pellet                     X
                        Liter equivalent in 250 ml centrifuge bottle    =      100 liters

                                where:
                                        Total Centrifugate pellet = volume of Centrifugate from 13.1.1
                                        X = The volume of Centrifugate pellet per 100 liters

                13.2.1   Remove a subsample from the remaining paniculate solution equivalent to 100 liters. For
                        example: if the total paniculate solution is 4,000 mL representing 400 liters sampled, 1,000
                        mL would be removed.

                        13.2.1.1 Pour the subsample into 50 mL tubes.  Centrifuge at 1050 x g for 10 minutes.
                                Aspirate the supernatant down to 5 mL above the bottom sediment. The remaining
                                subsample may be poured  on top of the Centrifugate pellet  and remaining
                                supernatant in the 50 mL tubes. Centrifuge again at 1050 x g for 10 minutes. This
                                may be repeated until all of the 100 liter equivalent subsample has been centrifuged.
                        13.2.1.2 Aspirate the supernatant down to 5 mL above the Centrifugate pellet. Combine the
                                pellets and the remaining 5 mL of supernatant into one 50 mL tube. Centrifuge at
                                1050 x g for 10 minutes.

                        13.2.1.3 Record the Centrifugate pellet volume.
                                                   12

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                                            Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
14.0    Recording of Results and Procedural Parameters

        14.1    Field data should include the following:

                14.1.1   Total water volume filtered in gallons

                14.1.2   Water source identified as to type and location

                14.1.3   Record the type of filtration, any pretreatment and kind of disinfection

                14.1.4   Record both address and exact location of water source being evaluated

                14.1.5   Date and time of sample device installation and removal

                14.1.6   Name, address and phone numbers) of sampler(s)

                14.1.7   Field measurements, such as turbidity, pH, conductivity, chlorine residual

                14.1.8   Record use of sodium thiosulfate if applicable

        14.2    Laboratory data should include the following:

                14.2.1   Total volume of packed pellet .(centrifugate volume)

                14.2.2   Number of each bioindicator from 0.1 ml sample aliquot.  Proper significant figures should
                         be used in calculations. Refer to Standard Methods for Water and Wastewater. 19th ed for
                         details.

                14.2.3   Number of particulates from each slide for the size  ranges described in 11.1.1

                14.2.4   Type of microscopy employed
                                 - brightfield
                                 - phase contrast
                                 - other

                14.2.5   Magnification of objective(s) used

                14.2.6   Number of whipple fields per PMCC at 400 X or other magnification

                14.2.7   Wash  Solution constituents (e.g. Tween 20, Sodium citrate)

                14.2.8   Sedgewick Rafter or Palmer Maloney Counting Cell used

                14.2.9   Kind of count used; total or natural unit
15.0    Interpretation of Results: MPA for filtration plant optimization identifies and enumerates a subsample of the
        organisms/particles eluted from a HVF waterborne paniculate sample collected through a  1 um nominal
        porosity filter cartridge. Other paniculate measurements and observations are also reported. If raw and finished
        samples are analyzed, an estimate of filtration plant efficiency can be determined.  In addition, since biological
        organisms are identified, potential filtration plant problems may be identified and can lead to optimization of
        plant operation.

                                                    13

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Microscopic Paniculate Analysis (MPA) for Filtration Plant Optimization
        15.1    Filter Color.  The one micrometer filter cartridge changes color during sampling depending on the
                water's paniculate composition and color as well as the amount of water sampled. The cartridge color
                can provide useful information about the general quality of water and can be used to make some
                process control decisions.  For example: an efficient water treatment plant will often have a brown raw
                water sampling cartridge and a white finished water sampling cartridge. The presence of a green tinge
                on only the finished filter cartridge may indicate the presence of algae growth within the filter beds.
        15.2    Centrifugate  pellet  volume.  The centrifugate pellet volume in  ml per  100 liters  is a direct
                measurement of the final pellet of paniculate matter recovered from the sampling cartridge after
                paniculate elution and centrifugation.  The percent reduction or log removal between raw and finished
                centrifugate pellet volume  can be useful in interpretation of overall filtration plant efficiency.
                However, it is important to realize that the volume of pellet can be strongly influenced by sampling
                technique and other factors and therefore should not be used as the sole factor in determining filtration
                efficiency.  Treatment problems may be identified when finished sediment is greater in volume than
                the raw sediment.  Situations such as this may occur when excess treatment chemicals are used.
                Percent reduction of centrifugate volume through the treatment system is calculated as follows:

                15.2.1   % reduction = (raw centrifugate - finished centrifugate) x 100
                                                Raw centrifugate
                        Log removal = Log (Raw Centrifugate) - Log (Fin. Centrifugate)

                Example:
                            Raw Water   =  3.0 mL per 100 liters
                         Finished Water   =  0 J mL per 100 liters

                % Reduction = 3.0 mL (in) - 0J mL (out) x  100 = 90%
                                       3.0mL  (in)

                The above example would equal 1.0 log removal.

        15.3    Amorphous Debris.  This category lists the types of inorganic matter and non-living organic matter
                (detritus) in order of predominance. Also included is the size range of these particulates observed
                under the microscope.  Particulates are reported in numbers per 100 liters, using proper significant
                numbers (scientific notation is optional) as outlined in Standard Methods for the Examination of Water
                and Wastewater (19 ed, 1-17)

        15.4    Nondiatomaceous Algae through Other (refer to data sheet). These categories signify the  different
                types of organisms found and their respective numbers. Organisms are reported in numbers per 100
                liters, using proper significant numbers. A qualitative approach can be taken for each category of
                organisms or a more objective approach can be taken by examining the total  organisms percent
                reduction or log removal by the treatment system.

        15.5    The percent reduction and log removal of organisms is calculated as follows:

                15.5.1   Calculate the total number of organisms found in the raw water and in the finished water.

                15.5.2   % Reduction  = (Raw total - Finished total) x 100
                                                 Raw total

                                Log Removal  = Log,0 (Raw total) - Log10 (Finished total)

                                                   14

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                                            Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
                15.5.3  Example:
                                 Raw Water Total Organisms = 40,000,000/100 liters
                                 Finished Water Total Organisms = 400,000/100 liters
                                 % Reduction = 99 %, or 2 log removal. 99.9 % reduction is equivalent to 3 log
                                 removal.

                15.5.4  Percent reduction and log removal can be approximated for plants with more than one raw
                        water source if MOD or percent use records are kept for each source. Separate counts from
                        each source are weighted by the appropriate percentage to calculate a total influent count.

                                 For example: A water utility filters 21 MOD; it is composed of 14 MOD (67 % of
                                 total) of reservoir water and 7 MOD (33 % of total) from a river. Analysis of both
                                 influent sources provides the following information on algal content:

                                 1) Reservoir contains 50,000 per 100 liter
                                 2) River contains 100,000 per 100 liter
                                 The total influent count to be used in percent log removal (river and reservoir
                                 combined) would be calculated as follows:

                                 (50,000 x 0.67) + (100,000 x 0.33) =  60,000 algae per 100 liter.

                15.5.5  . Occasionally organism numbers may increase in the finished water, and it is suspected that
                        either reproduction is occurring in the filter beds or another source of raw water is being
                        introduced that has not been accounted for such as backwash return water or some other in-
                        plant recycle operation. In such instances, information about the kind of organisms present
                        in the finished water may assist the plant operator in improving plant operation. Likewise,
                        the total centrifugate volume may increase in the finished water, or may demonstrate less log
                        removal than the organism removal. For example, flocculent may be passing through the
                        system.  Identification of this substance microscopically, may provide useful information to
                        the plant operator.

16.0    Analyst Qualifications
        Interpretation of results derived from the consensus method will depend upon numerous factors, the most
        important of which will be the level of training and experience of the analyst(s) employing this technique.

        16.1    Analyst should have a strong background in limnology and freshwater biology as well as an academic
                background and/or training in parasitology, protozoology, phycology,  invertebrate zoology and
                bacteriology.

        16.2    Analyst should have extensive experience with a light microscope with skills in brightfield, phase
                contrast and DIC or HMC microscopy.

        16.3    Analyst should have experience in examining a sufficiently large number of Surface Water MPA
                samples.

        16.4    A working knowledge of conventional treatment plants, slow and rapid sand filters, and alternative
                filtration methods is essential to providing adequate interpretation of the results and recommendations
                for controlling treatment plant conditions.

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Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
17.0    Standards of Identity
        Diatoms:
        Other Algae
        Rotifers:
        Plant debris:
        Nematodes:
        Pollen:
Diatoms are a group of algae which are distinctive because their cell wall is composed of
silica. This contributes to their ability to resist environmental, mechanical and chemical
insults. There are numerous species found in surface waters.  They contain chlorophyll and
need  sunlight to live and reproduce.  The size of these organisms is dependent on the
nutritional quality of the water. Some species are known to be nuisance organisms because
they clog filtration systems.  A preponderance of 1 or 2 species in the finished water
indicates possible reproduction or retention in the filter beds rather than the actual passage
through the filtration plant.  It is important to categorize the diatoms presence as living
(containing internal structures) or dead (empty silica skeletal remains). The number of empty
or dead diatoms may be of interest in certain types of filtration plants (e.g. effluent from DE
filters frequently contains large numbers of empty diatoms).

This category is comprised of a large number of chlorophyll containing filamentous, colonial
and unicellular divisions of algae. Like diatoms, these genera of chlorophyll-bearing algae
require sunlight for their metabolism.  Surface water contains more than 10,000 known
species with about 100 different species being commonly found. Diversity, abundance and
organism size are dependent on available nutrients, water temperature, time of year, and
other environmental and biological factors.  Some  species are known to be nuisance
organisms causing taste and odor problems.  Some cause filters to clog and add color to the
water. Some species will reproduce in the filtration system and be present in the effluent.
This can usually be detected because there is an overall decrease in the variety of species and
an increase of only a few species between the raw and finished water.

A major taxonomic group with over 2500 species, of which more than 2375 species are of
fresh water origin.  They are associated with a variety of habitats including small puddles,
damp soils and vegetable debris. They are also found associated with mosses, which can
often be found in or around ground water sources. The vast majority of rotifers encountered
are females ranging  in size from 70-500 urn. Rotifer growth in filtration beds has been
suggested.

This group may be defined as either unidentifiable plant material containing chlorophyll or
undigested fecal detritus from herbivorous animals, usually muskrat and beaver.  Plant debris
is very light weight material which is  large in size (50 - 100 um).  If the plant material is
fecal detritus, it can suggest that animals are present in the watershed and may shed cysts or
oocysts.

These include some 2,000 known free-living species found in fresh water.  Some  species
show an amazing ability to survive and thrive  in aquatic habitats  under a wide range of
ecological conditions.  Benthic sediments of lakes and rivers can contain high numbers of
nematodes, as can sewage effluent.   The top layer  of soil can contain over 1  million
nematodes per square meter. Soil runoff is a major source of nematodes in source waters for
treatment plants.  Nematodes and/or  their  eggs are  common in healthy water sources.
Nematodes found in finished potable water do not portray a quality product to the public and
may also compromise the microbiological integrity of the drinking water.  These organisms
seem to grow or reproduce in filter beds and distribution systems, so proper backwashing and
super chlorination of the filter beds, as well as, proper maintenance of the distribution system
should be conducted  routinely.

This includes all microspores produced by plants.   In the  spring  and fall, pollen  is
everywhere, both airborne and waterborne. Because pollen can become trapped in the filter
                                                    16

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                                            Microscopic Paniculate Analysis (MPA) for Filtration Plant Optimization
                        cartridge during insertion of the filter or in the laboratory while the filter is being processed
                        for examination, it is only rarely useful for assessing filtration efficiency, by itself.

        Ameba:         These include the ameboid, flagellated and cyst stages ranging in size from 10 to 600 um.
                        This group is characterized by the formation of pseudopodia of one type or another.  The
                        external surfaces of these ameba  are usually very thin compared to the cell coverings of
                        ciliates and most flagellates. Most species are free-living and feed on bacteria, algae, other
                        protozoa and debris. Ameba are common in surface waters and proper filtration removes
                        them, but reproduction may occur in filter beds.

        Ciliates:         These free-living protozoa are very common.  Ciliates are distinguished from other protozoa
                        by the presence of a macronucleus.  Like amoeba, they feed on bacteria, algae, small
                        metazoa, other protozoa and debris. Proper filtration removes ciliates, but a few species may
                        reproduce in filter beds.

        Colorless flagellates:
                        Although many flagellates are phototrophic,  there are many colorless species that grow in
                        the absence of light if sufficient dissolved nutrients are available. They are common in
                        surface water and can be removed by filtration; however, some species may reproduce in
                        filter beds. Flagellates possessing chlorophyll are included in the algae category.

        Crustaceans:     These include all aquatic arthropods which have two pairs of antennae and are fundamentally
                        biramous. The vast majority of known species (>35,000) are marine, but approximately 1,200
                        are found in freshwater. Adults range in size from 250 to 500 um, with eggs from 50 to 150
                        um.  Several species occur in healthy surface and  ground water.  Daphnia and Bosmina
                        species have been known to reproduce in very high numbers under the right environmental
                        conditions and cause filter clogging problems for  water  treatment plants.  Finished and
                        distribution waters can contain large numbers of crustaceans.  It is suspected that eggs will
                        hatch in the filter beds or pass through the filters and hatch in the distribution system.
                        Identification of crustaceans is often difficult because of fragmentation and observation of
                        only small portions of the organism.

        Other Arthropods:
                        There are a large  number of organisms, all  with jointed  appendages, in  the phylum
                        Arthropoda. This category includes only the arthropods that are not classified as crustaceans
                        or those  which are identifiable only to the phylum  level due to the  decomposed and
                        fragmented condition of the organism. Chironomid (insect) larvae and eggs are commonly
                        reported in surface waters as are arthropod pieces.  Seen less frequently are other insects,
                        water mites and seed ticks.

        Other:          This category includes any organism seen  that does not fit into the above  categories.
                        Examples include, iron bacteria, fungal spores, gastrotichs and/or tardigardes.


18.0    Quality Assurance. Listed below is the minimum recommended QC to be followed to under a laboratory
        QA/QC program. Documentation of testing is extremely important and careful records need to be maintained
        at all stages of analysis. Additionally, users of this method should develop their own internal QA/QC, and
        attempt to determine precision and bias at least at the analyst level for paniculate counts.

        18.1.   QC on equipment and supplies.

                 18.1.1  Large capacity high/low speed centrifuge (preferably refrigerated).

                                                    17

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Microscopic Paniculate Analysis (MPA) for Filtration Plant Optimization
                                 18.1.1.1 Equipped with swing bucket rotors.  Records maintained on rotor(s) usage
                                          at designated RPM. Post Manufacturer recommendation with regard to
                                          life time hours on rotor.

                                 18.1.1.2 Rotor speed checked with tachometer on a yearly basis.

                                 18.1.1.3 Determine and record RPM necessary for each rotor to attain desired g
                                          force. Post near centrifuge.

                                 18.1.1.4 Annual PM agreement in force or internal maintenance protocol/records
                                          in place.

                 18.1.2   Brightfield/phase-contrast/DIC/HMO microscope (Appendix 1)

                                 18.1.2.1 Phase rings checked for each objective before each use period.  Kohler
                                          illumination adjusted for each objective for DIC/HMO.

                                 18.1.2.2 Ocular  micrometer (reticle) in place and  calibrated against a stage
                                          micrometer for  each objective  in  use.   Calibration data posted near
                                          microscope.  Re-check on an annual basis.

                                 18.1.2.3 Microscope must be cleaned  and optics realigned  and adjusted on a
                                          frequent schedule.

                                 18.1.2.4 Annual PM agreement in force or internal maintenance protocol/records
                                          in place.

                                 18.1.2.5 Whipple grid used must be designed for installation into the laboratory's
                                          microscope ocular and must be calibrated against a stage micrometer.
                18.1.3   Stomacher brand (model 3500) laboratory blender.

                                 18.1.3.1 Operated according to manufacturers recommendations. The use of the
                                         blender is carefully timed to insure consistent washing of filter fibers.
                                         Stomacher is properly adjusted, with set screws, to accept entire filter.

                                 18.1.3.2 Stomacher  unit  is   maintained  and  internal  paddle  cover  is
                                         cleaned/disinfected after each use with dilute detergent/bleach solution.
                18.1.4   MPA sampling apparatus

                                 18.1.4.1 Apparatus is cleaned with dilute detergent and bleach solution, rinsed
                                         thoroughly with hot tap water, followed by a particle-free water rinse in
                                         the lab. Apparatus is flushed with water in the field prior to inserting the
                                         filter.

                                 18.1.4.2 Water meter is periodically checked for accuracy by timing the rate of
                                         flow into a measured gallon container.
                                                    18

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                                     Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
        18.1.5   Sample processing
                         18.1.5.1 All laboratory supplies used during sample processing are autoclaved or
                                  chemically sanitized.
                         18.1.5.2 Particle-free water has been tested and shown to contain less than 100
                                  particles per 100 ml.
18.2    Analytical QC
        18.2.1   Analyst has available identification keys and pictorial atlases to assist in classification of
                 microbiota.  (see reference list).

        18.2.2   Strict adherence to the Consensus Method and the definition of Standards of Identity will aid
                 in maintaining intralaboratory and interlaboratory consistency.
                                             19

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Microscopic Paniculate A Italy sis (MPA) for Filtration Plant Optimization
                                References for Internal Document

        1.      Vasconcelos, GJ. and S.I. Harris. EPA in-house document. Microscopic Particulate Analysis and
                Particle Size Analysis for Determination of Filtration Efficiencies. EPA Manchester Environmental
                Laboratory.

        2.      Hancock, C.M., J.V. Ward, K.W. Hancock, P.T. Klonicki and G.D. Sturbaum. Assessing Water
                Treatment Plant Performance Using Microscopic Analysis (MPA). Proc AWWA WQTC Nov.
                1994, San Francisco.

        3.      Palmer, C.M. and T.E. Maloney. 1954. A New Counting Slide for Nannoplankton. Amer. Soc. of
                Limn, and Oceano. special pub. no. 21, Environ. Health Center, Public Health Service, Cincinnati,
                Ohio.

        4.      Rashash, D. and D.L. Gallagher. 1995.  An Evaluation of Algal Enumeration.  Journal AWWA,
                April 1995. 127-132.

                           References for Microscopic Identification

American  Public Health Association, American Water Works Association, Water Environment Federation. 1995.
Standard  Methods for the Examination of Water and  Wastewater. Section  10200 F. Phytoplankton Counting
Techniques & color plates.  19th Edition, A.P.H.A., Washington, D.C.

AWWA (1995) Selected Problem Organisms in Water Treatment. M7 Operator's Identification Guide.

A;P.H.A., A.W.W.A., W.E.F. 1994. Section 9711 B. Immunofluorescence Method for Giardia and Cryptosporidium
spp.  Standard Methods  for the Examination of Water and Wastewater (Proposed). 19th Edition, A.P.H.A.,
Washington, DC, pp. 9-111.

Belehery, Hilary, 1979. An Illustrated Guide to Phvtoplankton. H.M. Stationery Office, London.

Boutros, S.N.  1993. Microscopic Particulate Analysis (MPA) in Studies of Ground water. Proc. AWWA WQTC,
Nov. 15-19,1992, Toronto, Ontario.

Brady, Nyle C. 1974. The Nature and Property of Soils. MacMillan Publishing, New York, 637 pp.

Federal Register, Monitoring Requirements for Public Drinking Water Supplies; Proposed Rule, February 10,1994.
40 CFR Part 141. Vol. 59, No. 28 Proposed Rules.

Foged, Niels, 1981.   Diatoms in  Alaska.  Bibliotecha phvcoligica.   J.Cramer  Inder  A.R.  Gantner  Verlag
Kommaniditgesellschaft.

Garnett, WJ.,  1965. Freshwater Microscopy in U.S. Dover Publishing, Inc., N.Y., N.Y.

Lee, JJ., Hunter, S.H. and E.C. Bovee, editors. 1985. An Illustrated Guide to the Protozoa. Society of Protozoologists,
Lawrence, KS.

Lund, JWG and H. Canter-Lund, 1995.  Freshwater Algae; Their Microscopic World Explored. Biopress Ltd.,
Bristol, England.
                                                 20

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                                          Microscopic Paniculate Analysis (MPA) for Filtration Plant Optimization
Palmer, C. Mervin, 1962.  Algae in Water Supplies. U.S. Dept. of Health, Education and Welfare, Public Health
Publication No. 657, Washington, D.C.

Pennak, R.W. 1989. Freshwater Invertebrates of the United States • Protozoa to Mollusca. 3rd edition. John Wiley
and Sons, Inc., New York.

Pentecost, A. 1984. Introduction to Freshwater Algae. Richmond Publishing Co. Ltd., Richmond, England.

Pesez, Gaston, 1977. Atlas de Microscopic des Eaux Douces. 19 rue Augereau, Paris.

Prescott, G.W., 1954.  How to Know the Freshwater Algae. In: Pictured Key Nature series, University of Montana
Press, Montana.

Smith, G.M., 1950. 2nd ed. Freshwater Algae of the United States.  McGraw Hill
Book Co., Inc., N.Y.

United States Environmental Protection Agency. 1992. Consensus Method for Determining Groundwaters Under
the Direct Influence of Surface Water Using Microscopic Particulate Analysis (MPA). Manchester Environmental
Laboratory Report #910/9-92-029.53 p.

Vinyard, W.C. 1979. Diatoms of North America. Mad River Press, Inc., Eureka, CA.
                                                 21

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Microscopic Paniculate Analysis (MPA) for Filtration Plant Optimization
                                              Appendix 1.

The microscope portion of this procedure depends upon very sophisticated optics.  Without proper alignment and
adjustment of the microscope the instrument will not function at maximal efficiency and the probability of obtaining the
desired image will not be possible. Consequently, it is imperative that all portions of the microscope from the light
sources to the oculars are properly adjusted.

While microscopes from various vendors are configured somewhat differently, they all operate on the same general
principles.  Therefore, slight deviations or adjustments may be required to make these guidelines work for the particular
instrument at hand.

1)      Transmitted Light Adjustment.  This section assumes that you have successfully replaced the transmitted
        bulb in your particular lamp socket and reconnect the lamp socket to the lamp house. Make sure that you have
        not touched any glass portion of the transmitted light bulb with your bare fingers while installing it. These
        instructions also assume the condenser has been adjusted to produce Kohler illumination.

        Step 1  Usually there is a diffuser lens between the lamp and the microscope which either must be removed
                or swung out of the  light path. Reattach the lamp house to the microscope.

        Step 2  Using a prepared microscope slide and a 40 X objective, adjust the focus so the image in the oculars
                is sharply defined.

        Step 3  Without the ocular or Bertrand optics in place the pupil and filament image inside can be seen at the
                bottom of the tube.

        Step 4  Focus the lamp filament image with the appropriate  adjustment on your lamp house.

        Step 5  Similarly, center the lamp filament image within the pupil with the appropriate adjustment(s) on your
                lamp house.

        Step 6  Insert the diffuser lens into the light path between the transmitted lamp house and the microscope.

2)      Adjustment of Interpupillary Distance and Oculars for Each Eye. These adjustments are necessary, so eye
        strain is reduced to a minimum.  These adjustments must be made for each individual using the microscope.
        this section assumes the use of a binocular microscope.

        A)     Interpupillary Distance. The spacing between the  eyes varies from person to person and must be
                adjusted for each individual using the microscope.

                Step 1.  Place a prepared slide on the microscope stage, turn on the transmitted light, and focus the
                        specimen image using the coarse and fine adjustment knobs.

                Step 2.  Using both hands, adjust the oculars in and out until a single circle of light is observed while
                        looking through the two oculars with both eyes.

        B)      Ocular Adjustment for Each Eye. This section assumes a focusing ocular(s). This adjustment can
                be made two ways, depending upon whether or not the microscope is capable of photomicrography
                and whether it is equipped with a photographic frame which can be seen through the  binoculars.
                Precaution: Persons with astigmatic eyes should always wear their contact lenses or glasses when
                using the microscope.
                                                   22

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                                 Microscopic Particulate A nalysis (MPA) for Filtration Plant Optimization
      1)       For microscopes not capable of photomicrography. This section assumes only the right
              ocular is capable of adjustment.

              Step 1.  Place a prepared slide on the microscope stage, turn on the transmitted light, and
                      focus the specimen image using the coarse and fine adjustment knobs.

              Step 2.  Place a card between the  right ocular and eye keeping both eyes open.  Using the
                      fine adjustment, focus the image for the left eye to its sharpest point.

              Step 3.  Now transfer the card to  between the left eye and ocular.  Without touching the
                      coarse or fine adjustment  and with keeping both eyes open, bring the image for the
                      left eye into sharp focus by adjusting the ocular collar at the top of the ocular.

      2)       For microscopes  capable of viewing a  photographic  frame  through the viewing
              binoculars. This section assumes both oculars are adjustable.

              Step 1.  Place a prepared slide on the microscope stage, turn on the transmitted light, and
                      focus the specimen image using the coarse and fine adjustment knobs.

              Step 2.  After activating the photographic frame, place a card between the right ocular and
                      eye keeping both eyes open. Using the correction (focusing) collar on the  left
                      ocular focus the left ocular until the double lines in the center of the frame are as
                      sharply focused as possible.

              Step 3.  Now transfer the card to between the left eye and ocular. Again keeping both eyes
                      open, bring the image of  the double lines in the center of the photographic frame
                      into as sharp a focus for the right eye as possible by adjusting the ocular correction
                      (focusing) collar at the top of the right ocular.

      3)       Calibration of an Ocular Micrometer (for Whipple grid also) This section assumes that
              an ocular reticle has been installed in one of the ocular by a microscopy specialist and that
              a stage micrometer is available for calibrating the ocular micrometer (reticle).  Once installed
              the ocular reticle should be left in place.  The more an ocular is manipulated, the greater the
              probability is for it to become contaminated with dust particles. This calibration should be
              done for each objective in use on the microscope.  If there is an optivar3 on the microscope,
              then the calibration procedure must be done for  the respective objective at each optivar
              setting.

              Step 1.  Place the stage micrometer on the microscope stage, turn on the transmitted light,
                      and focus the micrometer  image using the coarse and fine adjustment knobs for the
                      objective to be calibrated.  Continue adjusting the focus on the stage micrometer so
                      you can distinguish between the large (0.1 mm) and the small (0.01 mm) divisions.

              Step 2.  Adjust the stage and ocular with  the micrometer so the 0 line on the ocular is
                      exactly superimposed on  the 0 line on the stage micrometer.

              Step 3.  Without changing the stage adjustment, find a point as distant as possible from the
                      two 0 lines where two other lines are exactly superimposed.
Registered trademark product of the Zeiss company. A device between the objectives and the oculars that is
capable of adjusting the total magnification.

                                         23

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Microscopic Paniculate A nalysis (MPA) for Filtration Plant Optimization
                        Step 4.  Determine the number of ocular micrometer spaces as well as the number of
                                 millimeters on the stage micrometer between the two points of superimposition.

                        For example: Suppose 48 ocular micrometer space equal 0.6 mm.

                        Step 5.  Calculate the number of mm/ocular micrometer space.

                        For example:
                                                 0.6mm
                  0.0125 mm
                                        48 ocular micrometer spaces   ocular micrometer space

                        Step 6.  Since most measurements of microorganisms are given in um rather than mm, the
                                value calculated above must be converted to um by multiplying it by 1000 um/mm.
                        For example:

                               0.0125 mm       >
                         Ocular Micrometer Space
1.000 um
12.5 um
   mm      Ocular Micrometer Space
                        Step 7.  Follow steps 1 through 6 for each objective.  It is helpful to record this information
                                in a tabular format, like the example, which can be kept near the microscope.
Item
#


1
2
3
4
Obj.
Power


10 X
20 X
40 X
100 X
Description



N.A.C =
N.A. =
N.A. =
N.A.=
No of
Ocular
Microm.
Spaces




No. of
Stage
Microm.
mm"




urn/Ocular
Micrometer
Space"





        1000 um/mm
"       (Stage Micrometer length in mm x (1000 um/mm) -r No. ocular micrometer spaces
c       N.A. = Numerical aperture.  The numerical aperture value is engraved on the barrel of the objective.

        4)      Kohler Illumination. This section assumes that Kohler illumination will be established for each DIC
                or HMO objective.  Each time the objective is changed, Kohler illumination must be reestablished for
                the new objective lens. Previous sections have adjusted oculars and light sources. This section aligns
                and focuses the light going through the condenser underneath the stage at the specimen to be observed.
                If Kohler illumination is not properly established, then DIC or HMO woptics will not work to their
                maximal potential.  These steps need to become second nature and must be practiced regularly until
                they are a matter of reflex rather than a chore.

                Step 1.  Place a prepared slide on the microscope stage, move the required objective into place, turn
                        on the transmitted light, focus the specimen image using the coarse  and  fine adjustment
                        knobs.
                                                   24

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                            Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
Step 2.  At this point, both the radiant field diaphragm in the microscope base and the aperture
        diaphragm in the condenser should be wide  open.  Now close down the  radiant field
        diaphragm in the microscope base until the lighted field is reduced to a small opening.

Step 3.  Using the condenser centering screws on the front right and left of the condenser, move the
        small lighted portion of the filed to the center of the visual field.

Step 4.  Now look to see whether the leaves of the iris field diaphragm are sharply defined (focused)
        or not.  If they are not sharply defined, then they can be focused distinctly by changing the
        height of the condenser up or down while you are looking through the binoculars.  Once you
        have accomplished the precise focusing of the iris field diaphragm leaves, open the radiant
        field diaphragm until the leaves just disappear from view.

Step 5.  The  aperture diaphragm of the condenser is  adjusted now to make it compatible with the total
        numerical aperture of the optical system. This is done by removing an ocular, looking into
        the tube at the rear focal plane of the objective, and stopping down the aperture diaphragm
        iris leaves until they are visible just inside  the rear plane of the objective.

Step 6.  After completing the adjustment of the aperture diaphragm in the condenser, return the ocular
        to its tube and proceed with the adjustments required to establish either DIG or HMO optics.

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Microscopic Paniculate Analysis (MPA) for Filtration Plant Optimization
                                              Appendix 2.

                                 Use of Electronic Particle Counter

                 Electronic Particle Counting for Filtration Plant Optimization


                                              Introduction

Alternative methods for determining filtration efficiency, such as particle size analysis, have been recommended in the
"Guidance Manual for Compliance with Filtration and Disinfection Requirements for Public Water Systems Using
Surface Water Sources", USEPA, March 91.  The data accumulated from particle size analysis should be used in
conjunction with microscopic participate analysis data to determine over-all filtration efficiency.  The Federal Register,
Vol. 59 number 28, Feb. 10,  1994, states that particle counting  data could be used as a tool  for treatment process
efficiencies and could possibly be used as a surrogate for Giardia  and Cryptosporidium monitoring.  Either electronic
particle counters or treatment plant in-line installations that measure continuously can be used. These instruments give
particle size ranges and the number of particles per size range.  A comparison of raw water particle counts verses the
finished water particle counts can be used to calculate percent removal or log reduction and an estimate of filtration
efficiency established.

                                          Sample Collection

        1.0     The samples collected for electronic particle counting can be grab samples or composite samples. If
                a composite sample is collected,  then the procedure used to collect  and process the sample is
                described in the MPA for Filtration Plant Optimization procedure. A subsample from this composite
                sample will need to be diluted with particle free water before analysis. However, there are potential
                problems with composite sampling that need research and depend greatly on the type of water being
                analyzed.

        2.0     Grab samples require little  processing and may be useful for this reason. The main concern with the
                grab sample is that collection occurs over a very discrete amount of time and therefore may not be
                representative of the water  supply.  This problem can be resolved by either showing repeatability or
                by comparing the grab sample results to a composite sample which is more representative.

        3.0     Grab sampling: "dedicated" glass containers are to be used.  A dedicated container is one which is
                used exclusively for a certain  type of water (one set  for raw waters and another set for finished
                waters). These containers should be cylindrical glass bottles that have been scrupulously  cleaned with
                a mild laboratory detergent and then rinsed a minimum of three times  in particle-free water. Prior to
                sampling, the container should be rinsed a minimum of three times with the water being sampled.
                Plastic bottles should be avoided because they may shed particles into the sample.

        4.0     Collection: Collect grab samples as close to the source as possible.  A continuously flowing tap is
                recommended, if not available, flush the sample tap for 5 minutes prior to sampling.  Run the water
                down the inside of the bottle  to lessen  air  entrapment.  Make sure to  label the bottle:  sample
                identification, date, time, and sampler name.

        5.0     Holding time:  Analyze as soon as possible after collection.  Raw samples are especially prone to
                organic growth, adsorption of particles to the bottle walls and decay of original sample, all resulting
                in alteration of the particles. Filtered, or otherwise treated samples, are not quite as critical, but should
                be analyzed assoon as possible. If analysis can not happen immediately samples should be stored at
                4°C and sealed with a teflon screw bottle cap. Do not expose the sample to sunlight or let it freeze.

                                                   26

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                                    Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
                                   Particle Analysis

6.0     Analysis by electronic counter for grab and composite samples: Electronic counting can be performed
        by  one of two types of counters;  a light blockage device (HIAC, Met One, Hach or Particle
        Monitoring System to name a few) or an electrical sensing zone device (Coulter Counter or Elzone).

7.0     Prior to analysis, verify the instrument's sizing capabilities according to manufacturer's instructions
        using latex beads representing the particle sizes of interest.  If a light blockage device is used, the
        calibration is done during installation and on a routine basis recommended by the manufacturer. The
        most widely accepted dimensions of Giardia cysts are 7-12 um. Cryptosporidium oocysts are in the
        dimensional range of 3 - 7 um.  Individual electronic particle counters measure these  organisms
        differently. Testing of individual instruments will be needed to determine the actual size measured
        by a specific particle counter.

8.0     If sample has been refrigerated bring it to room temperature very slowly. Mix samples gently, by
        swirling just prior to analysis.  Minimize bubble formation, do not shake.

9.0     Run at least 3 rinse samples with particle free water to stabilize the instrument.

10.0    After running the sample in triplicate, run a rinse with particle free water or electrolyte to re-stabilize
        the instrument.

11.0    Always run the cleanest sample first and proceed to the most concentrated.

12.0    Between analyses, keep  particle-free solution in the instrument chamber.  The sample is run in
        triplicate to assess the instrument precision. The individual results should not vary more than 10 %
        from the average of the three runs, except in low particle count water (less than 10 particles per mL).

13.0    To avoid coincidence errors, in concentrated samples (raw samples), dilution of the final sample will
        undoubtedly be required. Coincidence occurs when more than one particle passes through the detector
        at a time, causing inaccurate counting and diameter measurements. This dilution should be done with
        the particle-free solution recommended by the manufacturer.  Generally speaking, a dilution between
        1:5 to 1:20 with particle-free solution will suffice.  It is best to use a dilution as close to tolerance for
        coincidence error as possible to decrease the number of background counts. It may also be necessary
        to screen filter the sample if large debris repeatedly block the orifice tube. For composite samples
        1:1000 dilutions are not uncommon.  Any pipettes or glassware used to make dilutions should be
        calibrated.

14.0    The average of the three values obtained are recorded in the  corresponding box on the data sheet.

15.0    Calculation of the percent removal and  log reductions can be done for both the total number of
        particles or for each size range. See example data sheet.
                                            27

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Microscopic Paniculate Analysis (MPA) for Filtration Plant Optimization
                               References for Particle Counting:

1.      Vasconcelos, GJ. and S.I. Harris. Procedure draft. Microscopic Particulate Analysis and Particle Size
        Analysis for Determination of Filtration Efficiencies. EPA Manchester Environmental Laboratory.

2.      A.P.H.A., A.W.W.A, W.E.F. 1995. Section 2560 Particle Counting and Size Distribution (Proposed) in:  19th
        Edition Standard Methods, A.P.H.A., Washington, DC, pp 2-60.

3.      Federal Register.  1994.  Proposed Monitoring Requirements for Public Drinking Water Supplies:
        Cryptosporidium, Giardia, Viruses, Disinfection Byproducts, Water Treatment Plant Data and Other
        Information Requirements; Proposed Rule, February 10,1994. 40 CFR Part 141. Vol. 59, No. 28 Proposed
        Rules, pp 6336.

4.      Hargesheimer, E.E., C.M. Lewis, and C.M Yentsch 1992.  Evaluation of Particle Counting as a Measure
        of Treatment Plant Performance. A.W.W.A. Research Foundation Denver.  pp 319.

5.      USEPA. Science and Technology Branch. 1991. Guidance Manual for Compliance with the Filtration and
        Disinfection Requirements for  Public Water Systems  using  Surface Water Sources.  Criteria and
        Standards Division. Office of Drinking Water. Washington. D.C.
                                                28

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                                   Microscopic Paniculate Analysis (MPA) for Filtration Plant Optimization
                                     Appendix 3
                        Sample Data Forms and Report Forms
                   Microscopic Participate and Particle Size Analysis
                          For Water Treatment Evaluations
Lab # Raw	
Lab # Finished	
Water Treatment Plant
Sample sites: Raw	
         Sampler's name
         Agency	
        Finished
Raw water source:  (circle one)
              River
        Infiltration gallery

Field Measurements:
      Creek
Horizontal Collector
    Spring well
Other	

Raw
Finished
T. Cl


F. Cl


Turb
(MTU)


PH


Temp
(C)


TC/
100ml


FC/
100ml


Operational Parameters: circle appropriate choice
Pre-treatment
Filtration
Disinfection
alum
R. sand
Cl
lime
S.sand
Cl-A
Polymer
Press.
Filter
Ozone
carbon
Cartr
Other
other
other

Processing Information;

Total volume filtered (Liters)
Total filter sediment collected
(Packed pellet) (g)
Centrifugate volume/ 100 liters



Raw






Finished






                                          29

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Microscopic Paniculate Analysis (MPA) for Filtration Plant Optimization
Particle Size Distribution and Percent Removal Using Microscope.
Average of 20 - 30 fields @ 100 X magnification
Particle Size category
< 10 urn
10 - 25 urn
25 - 100 um
100 - 200 um
>200um
Total
Number in
Raw Water






Number in
Finished Water






Percent
Removal






Particle Size Distribution and Percent Removal Using Electronic Particle Counter.
  Channels
Number in
Raw Water
Number in
Finished Water
Percent
Removal
                                          30

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                                        Microscopic Paniculate Analysis (MPA) for Filtration Plant Optimization
                                  Particle Counting Example

                                    Filtration Efficiency
                                Using an Electronic Particle Counter*
Date:
Raw Water:
Finished Water:
May 20, 1994
Raw River Water
Finished Water
Samples:
Code number:
Code number:
Composite or Grab
2
1
Particle size (urn)**
2
3
5
10
12
>15
Total
Raw Particles/mL •**
40,556.3
8,225.6
20,985.5
12,358.8
4,569.8
2345.6
89,041.6
Finished Particles/ml •••
4563
100
125
96.6
5.2
8
791
% Reduction
98.8749 %
98.7843
99.4044
99.2184
99.8862
99.6589
99.1115
Log Reduction
1.9488
1.9152
2.225
2.107
2.9439
2.4672
2.0514
* All limitations of the analytical methods, laboratory dilutions and instrument apply.
** Measured in equivalent spherical diameter.
**'Numbers represent the average from 3 subsample counts.
                                      Raw vs. Finished
                                                     10          12
                                               Particle Size (urn)
                                 Total
                        Raw Particles/ml*
Finished Particles/ml***
                                                31

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Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
             Analysis for Waterborne Participates

Customer  
          
          
PWSID* Sample Information: Date/Start: Mrs Date/Stop: Mrs Sampler: Gallons: Filter Colon Centrifugate: tnL/100 gals Results of Microscopic Particulate Analysis': Amorphous Debris: /uM diameter Nondiatomaceous Algae: Diatoms: Plant Debris: Rotifers: Nematodes: Pollen: Ameba: Ciliates: Coloriess Flagellates: Crustaceans: Other Arthropods: Othen Comments: Type of Wash water used; total or natural count used, type of counting chamber used Laboratory Information: ; ; Mrs; ; ; Results submitted by: 32

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                                           Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
                                             Appendix 4
                              Formulation of McFarland Standards
McFarland standards provide laboratory guidnace for the standardization of numbers of bacteria for susceptibility testing
or other procedures requiring a standardization of inoculum. They are devised to replace the counting of individual cells
are are designed to correspond to appropriximate cell densities.

1)      Make solution 1  1 % H,SO4
        Make solution 2  1.175 % BaCl2

Dispense in the following amounts of desired standard totaling 10 ml in 16 X 125 mm tubes. Cap and label. Should be
replaced every six months.
Standard Concentration
0.5
1
2
3
4
5
BaCU Volume in ml
0.05 ml
O.lml
0.2ml
0.3ml
0.4ml
0.5 ml
H,SO4 volume in ml
9.95 ml
9.9 ml
9.8ml
9.7ml
9.6ml
9.5ml
                                                   33

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Microscopic Partieulate A nalysis (MPA) for Filtration Plant Optimization
                                         Appendix 5

                                   Figures for Document
Figure 1
Raw Water Sampling Apparatus
                        pressure gauge
                                       backflow preventer
                                                                  >-• •- WATER SOURCE
               pressure regulator
                            Inlet hose      T
                                            I (pump optional at these points)
                         quick connects  \  —


                                       v
                                 filter
                                 holder
                                             34

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                                          Microscopic Paniculate Analysis (MPA) for Filtration Plant Optimization
Figure 2        Finished Water Sampling Apparatus
                (For chlorinated water only)
                       pressure regulator
                                       backflow preventer
                                                               «^ . —   WATER SOURCE


                                                          |   (pump optional at these points)
                                          quick connects  \

                                                         V
    proportloner
 (for disinfected water)
                                                  35

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Microscopic Paniculate Analysis (MPA) for Filtration Plant Optimization
Figure 3
Processing Flow Chart for Palmer Maloney Counting Chamber
                                                     EPA sampler
                                                       10p.s.i.
                                               Minimum of 100 liters raw;
                                                  1,000 liters finished
                                                 Paniculate extraction
                                   Handwash
                                       I	
                                                   Mechanical wash
                                                  (Stomacher* 3500)
                                                   Record volume
                                           of total paniculate solution in liters
                                                   Thoroughly mix
                                                      solution
                                           Transfer 200 ml to 250 ml conical
                                                   centrifuge bottle
                                                   Vortex 15 sec.
                                          Withdraw 0.10 ml, inoculate PMCC
                                        Examine at lOOx magnification minimum
                         plankters/field
                     Microscopic examination
                 (100x phase contrast, DIC, HMO)
                                                   plankters/field
                  Microbiota
                   TNTC
                Microbiota nor
                   TNTC
T
Use Whipple grid
@400x
magnification
i
Total of 100
organisms are
counted

Entire
PMCC
counted
^
w
                                                            plankters/field
                                                          Centrifuge 10min.
                                                              @1050g
                                                           aspirate to pellet
                     Withdraw 0.1 ml
                     inoculate PMCC
                                                                            < 10 plankters/field
                                                                       Additional 200 ml subsample
                                   Count particles in size
                                         ranges
                                   (<10,10-25, 25-100,
                                      100-200, >200)
                                     in 20-30 random
                                    Whipple grid fields
                                        on PMCC
                                                        36

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                                            Microscopic Paniculate A nalysis (MPA) for Filtration Plant Optimization
Figure 4         Centrifugate Pellet Volume Determination
                                       Pellet Centrifugate Measurement
                                                   ±
                                      Remove aliquot equivalent to 1001.
                                       Place in Conical Centrifuge Tubes
                                         Centrifuge 10 min. @ 1050g
                                            Aspirate supernatant
                                       Using calibrated tube set; measure
                                                pellet volume
                                       Record Centrifugate pellet volume
                                                     37

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Microscopic Particulate Analysis (MPA) for Filtration Plant Optimization
Figure 5        Scanning Cover Slip Method

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                                                  pic Paniculate Analysts (MPA) for Filtration Plant Optimization
                                             Appendix 6
                                  Example of Sample Calculation

        Assume that a 1000 Liter (380 gal) water sample was collected. The sample was eluted resulting in 3 L of
paniculate solution. The solution was thoroughly mixed and a 200 mL aliquot was placed in a centrifuge bottle. A
0.1 mL aliquot from the centrifuge bottle was examined in a Palmer-Maloney Chamber but only 5 plankters per field were
observed, so the 200 mL subsample was centrifuged and 150 mL of the supernatant was aspirated. The remaining 50
mL of supernatant and pellet was thoroughly mixed before 0.1 mL was examined in a  Palmer-Maloney Chamber.
Because 20 plankters per field were observed, the plankters were identified and counted.

        The liter equivalent-in the centrifuge bottle was calculated based upon these facts:

                   3000 mL of particulate solution      =           200 mL aliquot  in centrifuge bottle
                          1000 L sampled                      66.667 Liter equivalent in centrifuge bottle

        The liter equivalent in the first 0.1 mL subsample (x), where 5 plankters per field were observed, is calculated
using the proportion ratio inJQJLL———., - —
                                         r*"~ *
                     200 mL in centrifuge bottle        =     0.1 mL [observed in Palmer-Maloney Chamber]
              66.667 Liter equivalent in centrifuge bottle                      x = 0.033 Liter

                                         i  i
        The liter equivalent of the remaining  50 mL of supernatant and pellet is 66.634 which is the 66.667 Liter
equivalent in the centrifuge bottle.prior to centrifugation minus the 0.033 L equivalent withdrawn in the first 0.1  mL
subsample (calculated  in 10.3.1).

        The following organisms were identified and counted in the second O.lmL subsample when the entire Palmer-
Maloney Cell is scanned at lOOx:            r-
                                10 Keratella (rotifers)
                                3 Vonicella (ciliates)
                                1 Nematode ..M—.. -*.—•
        The numbers per 100 Liter are calculated using the proportion ratios in 11.1.5.1 and 11.1.5.2:
                                     10 Keratella    =      x = 5.000
                                      O.lmL              50 mL

Because the 50 mL of paniculate solution in the centrifuge bottle is equivalent to 66.634 L:

                                     5.000          =    7503 Keratella
                              66.634 L equivalent            100 L
        Similarly,

                                      3 Vonicella   =      1500
                                        O.lmL           50 mL

                                            1500   =    2254 Vonicella
                                          66.634 L          100 L
                                                   39

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Microscopic Participate Analysis (MPA) for Ftitratiom:ei**Qlttimiatitm
        and
                                      1 Nematode   =
                                        0.1 mL             50 mL

                                         500      =    TSONematodes
                                       66.634 L              10ft L

        The algae were too numerous to count at lOOx so 100 algal cells were counted in 5 whipple grid fields at 400x.
The number of algae is calculated as in 11.2.3,11.2.4, and  11.2.5:

       100 alga counted	x	1000mm      *                 No. algae/mJL
                A x 0.4  x 5 fields

        A = the area of the whipple grid field, which must t» calculated for the microscope, used for the counts (section
12.0). For example A = .074. (0.4 is the depth of the Palmer-Maloney chamber.)

        then,
                          100     x      1000     =     675,67y.67algrtcells/oiL
                            .074  x  .4  x 5
        then,
                                   675.675.67      *       33.783,783
                                     mL50                    mLs
        and because 50 mL is equivalent to 66.634 L:
                                   33.387.783      =    50.700300 Algae
                                    66.634L               100 Liters
Report the organism values in significant figures using the guidelines in Standard Methods. For this qxajnple the values
would be reported in #'s/100 L as follows:    8,000 Keratella
                                         2,000 Vorticella
                                         800 Nematodes
                                         50,000,000 Algae
                                                  40

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