PB-242 008
MARCH 1975
                            DISTRIBUTED BY:
                            National Technical Information Service
                            U. S. DEPARTMENT  OF COMMERCE


1  Kcport No
PB   242   008
4 1 itlr and Subtitle
  Plankton Analysis (141) training manual
                                                5. Report Date
                                                  March 1975
7 A in hurts)

   t.  M.  Sinr.lmr, IVlnnij.il  Coordinator
                                                8. Performing Organisation Kept
9 !'• ifornuiif <>rg mi/moil N.imt mil Aildr
   U. S. Environmental Protection Agency, OWPO
   MPOD, National Training Center
   Cincinnati, OH 45268
                                                                     10. Project/Task/Work Unit No
                                                11. Contract/Grant No.
12  Sponsoring Org.ini/at ion Name and Address
   Same as #9 above.
                                                13. Type of Report & Period
15. Supplementary Notts
16  Abstracts
  A manual which covers the broad field  of plankton analysis,  including reference
  outlines on classification and identification of algae and zooplankton, limnology of
  plankton, techniques of collection,  and laboratory methods of analysis.
17  Key Words and Document Analysis   17o. Descriptors

   Biomass; Ecology; Plankton; Zooplankton
7b. Identif icrs/Upen-l ndcd I crms

   Plankton Analysis
7c. < O^AI I I icld/droup
                        06  F
8 Availiibilily Sum mi nt

   Release to the  public
                                    19. Security Class (This
        |21. No. of Pages
                                                         20. Security Class (This
          tev  107H  I NIXWSI I) HY ANSI AND UNI SU>
                                                   THIS FOKM MAY BE RFPRODUCED
                                                                              USCOMM-DC 6295-P74

EPA-430/ 1-75-004
March 1975
This course is offered for professional personnel in
the fielda of water pollution control, hmnolo ’, and
water supply Primary empiasis is given to practice
in the identification and enumeration of organisms
v hich may be observed in the microscopic examination
of wat r Methods for Ihe chemical and instrumental
evalua’ ion of plankton are compared with the reaults
of microscopic examination in an extensive practical
exercise Problems of significance and control are
also considered
Office of Water Program Operations

Title or Description Outline Number
Limnology and Ecology of Plankton 1
Biology of Zooplankton Communities 2
Optics and the Microscope 3
Structure and Function of Cells 4
Types of Algae 5
Blue-green Algae 6
Green and Other Pigmented Flagellates 7
Filamentous Green Algae 1 8
Coccoid Green Algae 1 9
Diatoms 10
Filamentous Bacteria 11
Fungi and the “Sewage Fungus” Community 12
Protozoa, Nematodes, and Botifers 13
Free-living Amoebae and Nematodes 14
Animal Plankton 15
Laboratory: Identification of Diatoms 16
Preparation of Permanent Diatom Mounts 17
Laboratory: Identification of Animal Plankton 18
Techniques of Plankton Sampling Programs 19
Preparation and Enumeration of Plankton in the Laboratory 20
Calibration and Use of Plankton Counting Equipment 21
Determination of Odors 22
Determination of Plankton Productivity 23
141. 3.75

2 Contents
‘ Iitle or Description Outline Number
Laboratory Proportional Counting of Plankton 24
Laboratory Calibration of Plankton Counting Equipment 25
Laboratory. Fundamentals of Quantitative Counting 26
Algal Growth Potential Test 27
Algae and Actinomycetes in Water Supplies 28
Algae as Indicators of Pollution 29
Odor Production by Algae and Other OrganIsms 30
Plankton in Oligotrophic 31
The Effects of Pollution on Lakes 32
Application of Biological Data 33
The Problem of Synthetic Organic Wastes 34
Significance of “Limiting Factors’ to Population Variation 35
Nutrients: The Basis of Productivity 36
Algae and Cultural Eutrophication 37
Control of Plankton in Surface Waters 38
Control of Interference Organisms in Water Supplies 39
Case Preparation and Courtroom Procedure 40
Key to Selected Groups of Freshwater Animals 41
Key to Algae of Importance In Water Pollution 42
Classification-Finder (Part I and Part II) 43

A Most Interference Organisms are
Sm all.
B Small Organisms generally have
Short Life Histories.
C Populations of Organisms with
Short Life Histories may Fluctuate
Rapidly in Response to Key Environ-
mental Changes.
D Small Organisms are Relatively
at the Mercy of the Elements
E The Following Discussion will
naiyze the Nature of These Ele-
ments with Reference to the Res-
ponse of Important Organisms.
A Light is a Fundamental Source of
Energy for Life and Heat.
1 Insolation is affected by geo-
graphical location and mete-
orological factors.
2 Light penetration in water is
affected by angle of incidence
(geographical), turbidity, and
colør. The proportion of light
reflected depends on the angle
of incidence, the temperature,
color, and other qualibes of
the water. In general, as the
depth increases arithmetically,
the light tends to decrease geo-
metrically. Blues, greens, and
yellows tend to penetrate most
deeply while ultra violet, vio-
lets, and orange-reds are most
quickly absorbed. On the order
of 90% of the total illumination
which penetrates the surface
film is absorbed in the first
10 meters of even the clearest
‘ Turbidity may originate within
or outside of a lake.
a That which comes in from
outside (aflochthonous) is
predominately thert solids
b That of internal origin (auto-
chthonous) tends to be bio-
logical in nature.
B Heat and Temperature Phenomena
are Important th Aquatic Ecology.
The total quantity of heat avail-
able to a body of water per year
can be calculated and is known
as the heat budget.
2 Heat is derived directly from in-
solation; also by transfer from
air, internal friction, and other
C Density Phenomena
Density and viscosity affect the
floatation and locomotion of
a Pure fresh water achieveg
its maximum density at 4 C
and its maximum viscosity
at 0 C.
b The rate of change of density
increases with the temperature.
2 Density stratification affects
aquatic life and water uses.
a In summer, a mass of warm
surface water, the epilimnion ,
is usually present and separated
from a cool deeper mass, the
hypolimnion , by a relatively
thin layer known as the
the rmoclme .
b Ice cover and annual spring
and fall overturns are due to
successive seasonal changes
in the relative densities of
the epthmmon and the hypo-
RI. MIC. ecO. 4c 8.72

LImn9 and I cology of Plankton
limnion, profoundly influ-
enced by prevailing meteoro-
logical conditions.
c The sudden exchange of
water masses having differ-
ent chemical characteris-
tics may have catastrophic
effects on certain biota, may
cause others to bloom.
d Silt laden waters may seek
certain levels 1 depending
on their own specific gravity
in relation to existing layers
already present.
e Saline waters will also
stratify according to the
relative densities of the
various layers.
3 The viscosity of water Is greater
at lower temperatures.
a This is important not only
In situations involving the
control of flowing water as in
a sand filter, but also since
overcoming resistance to
flow generates heat, it is
significant In the heating
of water by internal friction
from wave and current ac-
tion and many delay the
establishment of anchor
ice under critical conditions.
b It is easier for plankton
to remain suspended in cold
viscous (and also dense)
water than in less viscous
warm water. This Is re-
flected in differences in the
appearance of winter vs
summer forms of life (also
arctic vs tropical).
D Shore development, depth, Inflow -
outflow pattern, and topographic
features affect the behavior of the water.
E Water movements that may affect organ-
isms include such phenomena as waves,
currents, tides, seiches, floods, and
Waves or rhythmic movement
a The best known are traveling
waves . These are effective
only against objects near
the surface. They have little
effect on the movement of
large masses of water.
b Standing waves or seiches
occur in all lakes but are
seldom large enough to be
observed. An “internal seich’
is an osciflation in a density
mass within a lake with no
surface manifestation may
cause considerable water
2 Langmuire spirals (or Langmu.tre
circulation are a relatively mass-
ive cylindrical motion Imparted
to surface waters under the
influence of wind. The axes of
the cylinders are parallel to the
direction of the wind, and their
depth and velocity depend on the
depth of the water, the velocity
and duration of the wind, and other
factors. The net result is that
adjacent cylinders tend to rotate
In opposite directions like meshing
cog wheels. Thus the water betwedn
two given spirals may be meeting
and sinking, while that between
spirals on either side will be meet-
ing and rising. Water over the
sinking areas tends to accumulate
flotsam and jetsam on the surface
in long conspicuous lines. Masses
of microcrustacea attemping to
stay near the surface may impart
a reddish color to this water, and
i is thus often referred to as the
“red dance.” The rising water on
the other hand, having recently
come from some depth, may (at
least in the oceans or large lakes)
have a bluish appearance, and is
Imown as the “bl dance.”
a This phenomenon is of consider-
able importance to those sampling
for plankton (or even chemicals)
near the surface when the wind
is blowing. Grab samples from

Limnolozy and Ecology of Plankton
either dance might obviously
differ consideral.ily. and 1.1
a plankton tow is contemplat-
ed, it should be made across
the wind In order that the
net may pass through a
succession of both dances.
b Langmuire spirals are not
usually established until the
wind has either been blowing
for an extended period, or
else is blowing rather hard.
Their presence can be detect-
ed by the lines of foam and
other floating material which
coincide with the direction
of the wind.
3 Currents
a Currents are arhythrnic
water movements which have
had major study only in ocean-
ography. They primarily
are concerned with the trans-
location of water masses.
Th ’ may be generated inter-
nal]y by virtue of density
changes, or externally by
wind or runoff.
b Turbulence phenomena or
eddy currents are largely re-
sponsible for lateral mixing
in a current. These are of
far more importance in the
economy of a body of water
than mere laminar flow.
c Tides 1 or rather tidal
currents, are reversible
(or oscillatory) on a relative-
ly long and predictable period.
They are closely allied to
seiches. For all practical
purposes, they are restricted
to oceanic (especially coastal)
If there is no freshwater
inflow involved, tidal currents
are basically “In and out.”
$1 a significant amount of
freshwater is added to the
system at a constant rate, the
outflowing current will in general
exceed the inflow by the amount
of freshwater input.
There are typically two tidal
cycles per lunar day (approx-
irnately 25 hours), but there is
continuous gradation from this
to only one cycle per (lunar) day
in some places.
Estuarine plankton populations
are extremely influenced by local
tidal patterns.
d Flood waters range from torren-
tial velocities which tear away
and transport vast masses of
substrate to quiet backwaters
which may inundate normally dry
land areas for extended periods
of time, in the former case,
planktonic life is flushed away
completely; in the latter, a local
plankton bloom may develop which
may be of immediate significance,
or which may serve as an inoculum
for receding waters.
F Surface Tension and the Surface Film
1 The surface film is the habitat of
the “neuston”, a group of particular
importance in water supplies.
2 Surface tension lowered by surfactants
may eliminate the neuston. This can
be a significant biological observation.
A Carbon dioxide is released by plants and
animals in respiration, but taken in by
plants in photosynthesis.
B Oxygen is the biological complement of
carbon dioxide, and necessary for all
animal life.
C Nitrogen and phosphorus are fundamental
nutrients for plant life.
1 Occurm great dilution, concentrated
by plants.

Umnology and Ecol 4 gyof Plankton
The distribution of nitrogen
compounds is generally corre]at-
ed with the oxygen curve, espe-
cially In oceans.
D Iron, manganese, sulphur, and silicon
are other minerals important to aquatic
life which exhibit biological stratification.
E Many other minerals are present but their
biological distribution in waters is less
well known, fluorine, tin, and vanadium
have recently been added to the “essential”
list, and more may well follow.
F Dissolved organic matter Is present in
even the purest of lakes.
A Nutritional Classification of Organisms
1 Ho].ophyt ic or independent or-
ganisms, like green plants, pro-
duce their own basic food elements
from the physical environment.
2 Holozoic or dependent organisms,
like animals, ingest and digest
solid food particles of organtc
3 Saprophytic or carrion eating
organisms, like many fungi and
bacteria, digest and assimilate
the dead bodies of other organ-
l ms or their products.
B The Prey-Predator Relationship is
Simply one Organism Eating Another.
C Toxic and Hormonic Relationships
1 Some organisms such as certain
blue green algae and some ar-
mored f]agellages produce sub-
stances poisonous to others.
2 AntibIotic action in nature Is
not well understood but has been
shown to play a very influential
role In the economy of nature.
A A biotic community will be defined here
as an assemblage of organisms living in
a given ecological niche (as defined
below). Producer (plant-like), consumer
(animal-like) and reducer (bacteria and
fungi) organisms are usually Included.
A source of energy (nutrient, food) must
also be present. The essential concept
In that each so-cafled community is a
relatively independent entity. Actually
this position is only tenable at any given
instant, as individuals are constantly
shifting from one community to another in
response to stages in their life cycles,
physical conditions, etc. The only one
to be considered in detail here is the
B Plankton are the macroscopic and
microscopic animals, plants, bacteria,
etc. floating free in the open water.
Many clog filters, cause tastes, odors,
and other troubles In water supplies.
1 Those that pass through a plankton
net (No. 25 sIlk bolting cloth or
equivalent) or sand filter are often
known as nannoplankton (they
usually greatly exceed the “net”
plankton in actual quantity).
2 Those less than four microns in
length are sometimes called
ultraplankton .
3 There are many ways in which
plankton may be classified: taxo-
nomic, ecological, industrial.
4 The concentration of plankton varies
markedly In space and time.
a Depth, light, currents, and
water quality profoundly affect
plankton distribution.
b The relative abundance of
plankton in the various sea-
sons is generally
1 spring, 2 fail, 3 summer,
4 winter

L1xnnolo v and Ecology of Plnnkton
5 Marine plankton include many
larger animal forms than are
found in fresh waters.
C The benthic community is generally
considered to be the macroscopic life
living In or on the bottom.
D The perip yton community might be
defined as the microscopic benthos,
except that they are by no means confined
to the bottom. Any surface, floating, or
not, is usuaUy covered by film of living
organisms. There is frequent exchange
between the periphyton and plankton
E The nekton is the community of larger,
free- swimming animals (fishes, shrimps,
etc.), and so is dependent on the other
communities for basic plant foods.
A The history of a body of water determines
its present condition. Natural waters have
evolved in the course of geologic time
to what we Imow today.
B In the course of their evolution, streams
in general pass through four general
stages of de relopment which may be called:
birth, youth, maturity, and old age.
1 Establishment of birth. In an
extant stream, this might be
a ‘dry run” or headwater
streambed, before it had eroded
down to the level of ground water.
2 Youthful streerns; when the
strear.i bed is eroded below the
ground water level, spring water
enters and the stream becomes
3 Mature streams; havc wIde
valleys, a developed flood plain,
deeper, more turbid, and usua]i r
warmer water, sand, mud, silt,
or clay bottom materials which
shift with increase In flow.
4 In old age, streams have approa-
ched base level. During flo3d
stage they scour their bed and de-
posit materials on the flood plain
which may be very broad and fiat.
During normal flow the channel is
refilled and many shifting bars are
(Under the influence of man this
pattern may be broken up. or tem-
porarily interrupted. Thus as essen-
tially “youthful” stream might take
on some of the characteristics of a
“mature” stream following soil
erosion, organic enrichment, and
increased surface runoff. Correction
of these conditions might likewise be
followed by at least a partial rever-
sion to the “original” condition.)
C Lakes have a developmental history
which somewhat parallels that of streams.
The method of formation greatly
influences the character and sub-
sequent history of lakes.
2 Maturing or natural eutrophication
of lakes
a If not already preseot, shoal
areas are developed through
erosion of the shore by wave
action and undertow.
b Currents produce bars across
bays and thus cut off irregulars
c Silt brought in by tributary
streams settles out In the quiet
lake water.
d Rooted aquatics grow on shoals
and bars, and In doing so cut
off bays and contribute to the
filling of the lake.
e Dissolved carbonates and other
materials are precipitated In the
deeper portions of the lake in
part throdgh the action of plants.

Umno1o y and Ecolouv of Plankton
I When filling is well advanced
sphagnum mats extend out-
ward from the shore. These
mats are followed by sedges
and grasses which finally
Convert the lake into a
3 Extinction of lakes. After lakes
reach maturity their progress
toward filling up is accelerated.
They become extinct through:
a The downcutting of the out-
b Filling with detritus eroded
from the shores or brought
in by tributary streams.
c Filling by the accumulation of
the remains of vegetable
materials growing in the
lake itself.
(Often two or three pro-
cesses may act concurrently)
When man hastens the above
process, it is often called
“cultural eutrophication.”
A The biological resultant of all physical
and chemical factors is the quantity of
life that may actually be present. The
ability to produce this “biomass” is
often referred to as the “productivity”
of a body of water. This is neither good
nor bad per se. A water of low producti-
vity is a “poor” water biologically, and
also a relatively “pure” or “clean” water;
hence desirable as a water supply. A
productive water on the other hand may
be a nuisance to man or highly desirable.
Some of the factors which influence the
productivity of waters are as follows:
B Factors affecting stream productivity.
To be productive of plankton, a stream
must provide adequate nutrients, light,
a suitabic temperature, and time for
growth to take place.
Youthful streams, especially on
rock or sand substrates are low
in essential nutrients. Tempera-
tures in mountainous regions are
usually low, and due to the steep
gradient, time for growth is short.
Although ample light is available,
growth of true plankton is thus
greatly limited.
2 As the stream flows toward a
more Imatur condition nutrients
tend to accumulate, and gradient
diminishes and so time of flow
increases, temperature tends to
increase, and plankton flourish.
Should a heavy load of Inert silt
develop on the other hand, the
turbidity would reduce the light
penetration and consequently the
general plankton production would
3 As the stream approaches base
level (old age) and the time avail-
able for plankton growth increases,
the balance between turbidity,
nutrient levels, and temperature
and other seasonal conditions,
determines the overall produc-
C Factors Affecting the Productivity
of Lakes
The size, shape, and depth
of the lake basin. Shallow water
is more productive than deeper
water since more light will reach
the bottom to stimulate rooted
plant growth. As a corolla ry,
lakes with more shoreline, having
more shallow water, are in general
more productive. Broad shallow
lakes and reservoirs have the
greatest production potential (and
hence should be avoided for water
2 Hard waters are generally more
productive than soft waters as
there are more plant nutrient
minerals available. This is often

Geographic Location
Human Geological Latitude
Influence Formation Longitude
Sewage Climate
A griculture
Nature o LIUIUW ui Trans- -Light
Bottom A llochthonous parency Penetration
Depos2t$ Materials
Ba sin
I - .
Trophic Nature of a
ration O2 Penetra Seasonal Cycle
and Stratification and Littoral Circulat. Stagnation
Utilization Region Growing Season

L1mnolo y and Ecology of Plankton
greatly influenced by the
character of the Boll and rocks
in the watershed, and the quality
and quantity of ground water
entering the lake. In general,
pH ranges of 6.8 to 8.2 appear
to be most productive.
3 Turbidity reduces productivity
as light penetration is reduced.
4 The presence or absence of
thermal stratification with its
semi-annual turnovers affect
productivity by distributing
nutrients throughout the water
mas 9.
5 Climate, temperature, pre-
valance of ice and snow, are
also important.
D Factors Affecting the Productivity of
The productivity of reservoirs
is governed by much the same
principles as that of lakes, with
the difference that the water
level is much more under the
control of man. Fluctuations
in water level can be used to
deliberately increase of decrease
productivity. This can be dem-
onstrated by a comparison of
the TVA reservoirs which practice
a summer drawdown with some of
those in the west where a winter
drawdown Is the rule.
2 The level at which water is re-
moved from the reservoir is also
important. The upper epilimnion
may have a high plankton turbi-
dity while lower down the plankton
count may be less, but a taste
and odor causer (such as Maiio-
monas)may be present. There
may be two thermoclines, with
a mass of muddy water flowing
between a clear upper epilimnion
and a clear hypolimnion. Other
combinations ad infinitum may
3 Reservoir discharges also pro-
foundly affect the DO, temperature,
and turbidity In the stream below
a dam. Too much fluctuation in
flow may permit sections of the
stream to dry periodically.
A The productivity of lakes and impound-
ments is such a conspicuous feature
that it is often used as a means of
1 Ol1gotro c lakes are the
geologically younger, less produc-
tive lakes, which are deep, have
clear water, and usually support
Salmonoid fishes.
2 Mesotropic lakes are generally
intermediate between oligotrophic
and eutrophic lakes. They are
moderately productive, yet
pleasant to be around.
3 Eutrophic lakes are more mature,
more turbid, and richer. They
are usually shallower. They are
richer In dissolved solids; N, P.
and Ca are ab nidant. Plankton is
abundant and there is often a
rich bottom fauna. Nuisance
conditions often appear.
4 Dystrophic lakes - bog lakes -
low in pH, water yellow to brown,
dissolved solids; N, P. and Ca
scanty but humic materials abun-
dant; bottom fauna and plankton
poor, and fish species are limited.
B Reservoirs may be classified as storage,
or run of the river.
1 Storage reservoirs have a large
volume in relation to their Inflow.
2 Run of the river reservoirs have
a large flow through In relation
to their storage value.

Limnology and Ecology of Plankton
C According to lot .ation, lakes and
regervolrq may be classified as polar,
temperate, or tropical. Differences
in climatic and geographic conditions
result in differences in their biology.
A Liebig’s Law of the Minimum states
that productivity is limited by the
nutrient present in the least amoung
at any given time relative to the
assimilative capacity of the organism.
B SheLford’s Law of Toleration:
C The artificial introduction of nutrients
(sewage pollution or fertilizer) thus
tends to eliminate existing limiting
minimums for some species and create
intolerable maximums for other species.
1 Kn.Dwn limiting minimums may
sometimes be deliberately
2 As the total available energy
supply is Increased, productivity
tends to increase.
3 As productivity increases, the
whole character of the water
may be changed from a meagerly
productive clear water lake
(oligotrophic) to a highly pro-
ductive and usually turbid lake
4 I.utrophication leads to treatment
D Control of eutrophication may be accom-
plished by carious means
1 Watershed management, ade-
quate preparation of reservoir
sites, and pollution control tend
to maintain minimum limiting nu-
tritional factors.
2 Shading out the energy of insola-
tion by roofing or inert turbidity;
Suppresses photosynthesis.
3 Introduction of substances toxic
to some fundamental part of the
food chain (such as copper sul-
phate) tends to temporarily inhibit
A A body of water such as a lake rep-
resents an intricately balanced system
in a state of dynamic equilibrium.
Modification imposed at one point in
the system automatically results m
compensatory adjustments at associated
B The more thorough our knowledge of
the entire system, the better we can
judge where to impose control measures
to achieve a desired result.
1 Chamberlin, Thomas C., and Salisburg,
RoUin P., Geology Vol. 1, “Geolo-
gical Processes and Their Results”,
pp i-xix, and 1-654, Henry Holt and
Company, New York 1 1904.
2 Dorsey, N. Ernest. Properties of
Ordinary Water - Substance.
Reinhold Publ. Corp., New York.
pp. 1-673. 1940.
3 Hut cheson, George E. A Treatise on
Limnology. Jonn Wiley Company.
4 Ruttner, Franz. Funlamentals of
Umnzlogy. University o Toronto
Press. pp. 1-242. 1953.
5 Tarzwell, Clarence M. xperimental
Evidence on the Value of Trout 1937
Stream Improvement in Michigan.
American Fisheries Society Trans.
66.177-187. 1936.
MInIm ,m I I oII
of IoI.r.tIOn
Ab.,n( •. —— —
..ogo of Op%Imum M.,In om IIn,lt of
of f.. br _________________
(fr.aI.,.t .buod.nc. Abjent
Oerret. Iflg

Limnology and Ecology of Plankton
6 Us DHEW, PHS. Algae and Metropolitan
Wastes, transactions of a sefnlnar
held April 27-29, 1960 at the
Robert A. Taft Sanitary Engineer-
ing Center, Cincinnati, Ohio.
No. SEC TRW61-3.
7 Ward and Whipple. Freshwater Biology
(Introduction). John Wiley
Company. 1918
8 Whittaker, R. H. Communities and
Ecosystems. Macmillan,
New York. 162 pp. 1970.
9 Zakdln, V. I. and Gerd, Sr. Fauna
and Flora of the Lakcs and
Reservoirs of the USSR. Avail-
able from the Office of Technical
Services, U. S. Dept. Commerce,
Washington. DC.
This outline was prepared by H. W.Jackson,
chief Biologist. National Training Center,
DTTB, MDS, OWP, EPA. Cincinnati, OH

A The planktonic community is composed of
organisms that are relatively independent
of the bottom to complete their life history.
They inhabit the open water of lakes
(pelagic zone). Some species have inactive
or resting stages that lie on the bottom
and carry the species through periods of
stress, e. g. • winter. A few burrow in
the mud and enter the pelagic zone at night.
but most live in the open water all the
time that the species is present in an active
13 Compared to the bottom fauna and flora,
the plankton consists of relatively few
kinds of organisms that are consistently
and abundantly present. Two major cat-
egories arc often culled phytoplankton
(plants) and zooplankton (animals), but
this is based on an outmoded classification
of living things. The modern tendency is
to identify groupings according to their
function In the ecosystem: Primary pro-
ducers (photosynthetic organisms), consumers
(zooplankton). and decomposers (hetero-
trophic bacteria and fungi).
C The primary difference then is nutritional,
phytoplankton use inorganic nutrient
elements and solar radiation. Zooplankton
feed on particles, much of which can be
phytoplankton cells, but can be bacteria or
particles of dead organisms (detritus)
originating in the plankton, the shore
region, or the land surrounding the lake.
0 The swimming powers of planktonlc
organisms is 80 LimIted that their hori-
zontal distribution is determined mostly
by movements of water. Some of the
animals are able to swim fast enough that
they can migrate vertically tens of meters
each day, but they are capable of little
horizontal navigation. At most, some
species of crustaceans show a general
avoidance of the shore areas during caln
weather when the water is moving more
slowly than the animals can swim, By
definition, animals that are able to control
their hori7ontal location are nekton, not
E In this presentation,a.minimum of clas-
sification and taxonomy is used, but it
should be realized that each group is
typified by adaptations of structure on
physiology that are related to the plank-
tonic mode of existence. These adapta-
tions are reflected in the classification.
A The freshwater zooplankton is dominated
by representatives of three groups of
animals, two of them crustaceans’
Copepoda, Cladocera, Rotifera. All have
feeding mechanisms that permit a high
degree of selectivity of food, and two can
produce resting eggs that can withstand
severe environmental conditions. In
general the food of usual zooplankton pop-
ulations ranges from bacteria and small
algae to small animals.
B The Copepoda reproduce by a normal
biparental process, and the females lay
fertilized eggs in groups which are carried
around in sacs until they hatch. The
immature animals go through an elaborate
development with many stages. The later
stages have mouthparts that permit them
to collect particles. In many cases, these
are in the form of combs which remove
small particles by a sort of filtration
process. In others, they are modified to
form grasping organs by which small
animals or large algae are captured
C The Cladocera (represented by Daphnia )
reproduce much of the time by partheno-
genesis. so that only females are present.
Eggs are held by the mother in a brood
chamber until the young are developed far
enough to fend for themselves. The newborn
animals look like miniature adults, and do
not go through an elaborate series of
developmental stages in the water as do
the copepods. Daphnia has comb-formed
filtering structures on some of its legs
that act as filters.
HI. AQ. 29.5.71

Biology of Zooplankton Communities
1) Under some environmental conditions the
development of eggs is affected and males
are produced. l ertilized eggs are produced
that can resist freezing and drying, and
these carry the population through
unsatisfactory conditions.
E The Rotifera are small animals with a
ciliated area on the head which creates
currents used both for locomotion and for
bringing food particles to the mouth. They
too reproduce by parthenogenesis during
much of the year. but production of males
results in fertilized, resistant resting eggs.
Most rotifers lay eggs one at a time and
carry them until they hatch.
A In general, zooplankton populations are at
a minimum Lfl the cold seasons, although
some species flourish in cold water. Species
with similar food requirements seem to
reproduce at different times of the year or
are segregated in different layers of lakes.
B There is no single, simple measurement
of activity for the zooplankton as a whole
that can be used as an index of production
as can the uptake of radioactive carbon for
the phytoplankton. However, it is possible
to find the rate of reproduction of the species
that carry their eggs. The basis of the
method is that the number of eggs in a
sample taken at a given time represents
the number of animals that will be added
to the population during an Interval that
is equal to the length of time it takes the
eggs to develop. Thus the potential growth
rate of the populations can be determined.
The actual growth rate, determined by
successive samplings and counting, is less
than the potential, and the difference is a
measure of the death rate.
C Such measurements of birth and death rates
permits a more penetrating analysis to be
made of the causes of population change
than tf data were available for population
sue alone.
I) Following is an indication of the major
environmental factors in the control of
I ooplankton.
I Temperature has an obvious effect Ln
its general control of rates. In addition,
the production and hatching of resting
eggs may be affected.
2 Inorganic materials
Freshwater lakes vary in the content
of dissolved solids according to the
geological situation. The totaL salinity
and proportion of different dissolved
materials in water can affect the pop-
ulation. Some species are limited to
soft water, others to saline waters, as
the brine shrimp. The maximum pop-
ulation size developed may be related
to salinity, but this is probably an
indirect effect working through the
abundance of nutrients and production
of food.
3 Food supply
Very strong correlations have been
found between reproduction and food
supply as measured by abundance of
phytoplankton. The rate of food supply
can affect almost all aspects of pop-
ulation biology including rate of mdi-
vidual growth, time of maturity, rate
of reproduction and length of life.
4 Apparently in freshwater, dissolved
organic materials are of little nutri-
tional significance, although some
species can be kept if the concentration
of dissolved material is high enough.
Some species require definite vitamins
in the food.
5 Effect of predation on populations
The kind, quantity and relative pro-
portions of species strongly affected
l y grazing by vertebrate and inverte-
brate predators. The death rate of
Daphnia is correlated with the abun-
dance of a predator. Planktivorous
fish (alewives) selectively feed on
larger species, so a lake with alewives
is dominated by the smaller species of
crustaceans and large ones are scarce
or absent.
6 Other aspects of zooplankton
Many species migrate vertically con-
siderable distances each day. Typically.
migrating species spend the daylight
hours deep in the lake and rise toward
the surface in late afternoon and early
Some species go through a seasonal
change of form (cyclomorphosis) which
is not fully understood. It may have an
effect in reducing predation.
2 -.2

Biology of Zooplarikton Communities
1 Baker, A. 1. An Inexpensive Micro-
sampler. Limnol. and Oceanogr.
15(5): 158—160. 1970.
2 Brooks. .J. L. and Dodson, S. I.
Predation. Body. Size, and Corn-
posItion of Plankton. Science 150:
28-35. 1965.
3 Dodson, Stanley I. Complementary
Feeding Niches Sustained by Size-
Selective Predation, Lirrinology
and Oceanography 15(1): 131-137.
4 Hutchlnson, G. E. 1967. A Treatise
on lininology. Vol. II. Introduction
to Lake Biology arid the Llmnoplanktou.
xl+ 1115. JohnWtley& Sons, Inc.,
New York.
5 Jossl, Jack W. Annotated Bibliography
of Zooplankton Sampling Devices.
USFWS. Spec. Sd.: Rep. -Fisheries.
609. 90 pp. 1970.
7 Lund, J. W. G. 1965. The Ecology of
the Freshwater Plankton. Biological
Reviews, 40:231-293.
8 UNESCO. Zoolplankton Sampling.
UNESCO Monogr. Oceanogr. Methodol.
2. 174 pp. 1968. (UNESCO. Place
de Fortenoy, 75, Paris 7e France).
6 LIkens, Gene E. and Gilbert, John J.
Notes on Quantitative Sampling of
Natural Populations of P lanktonic
Rotifers. Limnol. arid Oceanogr.
15(5): 816—820.
ThiS outline was prepared Dy W. ‘:. c1mondson,
Professor of Zoology, University of
Washington, Seattle, Waehington.

Biology of Zooplankton (omm initics
Each panifl shows the abundance of a species of animal. Each
mark en die vertical axis represents 10 tndividuala/liter.
r4auwerck, A. 1963. Die Beziehungen zwischen Zooplankton end
Phytoplankton im See Erken. Symbolae Botanicae Upsaliensis, 17:1—163.
. 1 P K A K J J A S 0 N U

Biology of Zooplankton Communities
Difflu gia

1U U Y 01 L.OOPJ.aniaofl ‘.ommunitie
Crust cea
Nauplius ]arva c( copepod
I neecta - Cheoborus
y chaeta Polygarthra

[ 3io1o r of Zooplankton Communities
spined (fin attached)
staple (gill attached)
Glochidia (Unionidae) Fish Parasites
Veliger Larvae (Corbiculidae) Free Living Planktonic
Pediveliger atlachcs byssus lines)
38O j

An understanding of elementary optics is
essential to the proper use of the microscope.
The microacopist will find that unusual pro-
bleme in Illumination and photomicrography
can be handled much more effectively once
the underlying ideas In physical optics are
A Reflection
A good place to begin is with reflection at
a surface or interface. Specular (or
regular) reflection results when a beam
of light leaves a surface at the same angle
at which it reached it. This type of
reflection occurs with hi ily polished
smooth surfaces. It is stated more pre-
cisely as Sriell’s Law, i.e., the angle of
incidence, 1. is equal i IFie angle of
reflection, r (Figure 1). Diffuse (or
scattered) reflection results when a beam
of light strikes a rough or Irregular sur-
face and different portions of the incident
light are reflected from the surface at
different angles. The light reflected from
a piece of white paper or a ground glass is
an example of diffuse reflection.
Figure 1
BI. MIC. 18.6.68
Strictly speaking, of course, all reflected
light, even diffuse, obeys Snell’s Law.
Diffuse reflected light is-made up of many
specularly reflected rays, each from a
a tiny element of surface, and appears
diffuse when the reflecting elements are
very numerous and very small. The terms
diffuse and specular , referring to reflection,
describe n ot so much a difference in the
nature of the reflection but rather a differ-
ence In the type of surface. A polished sur-
face gives specular reflection, a rough
surface gives diffuse reflection.
It Is also important to note and remember
that specularly reflected light tends to be
strongly polarized In the plane of the reflect-
ing surface. This is due to the fact that
those ra whose vibration directions lie
closest to the plane of the reflection surface
are most strongly reflected. This effect is
strongest when the angle of incidence is
such that the tangent of the angle is equal
to the refractive index of the reflecting sur-
face. This particular angle of Incidence is
called the Brewster angle .
B Image Formation on Reflection
Considering reflection by mirrors, we find
(FIgure 2) that a plane mirror forms a
virtual image behind the mirror, reversed
right to left but of the same size as the
object. The word virtual means that the
image appears to be In a given plane but
that a ground glass screen or a photographic
film placed in that plane would show no
image. The converse of a virtual image is
a real image.
Spherical mirrors are either convex or con-
cave with the surface of the mirror repre-
senting a portion of the surface of a sphere.
The center of curvature is the center of the
sphere, part of whose surface forms the
mirror. The focus lies halfway between the
center of curvature and the mirror surface.

Ontics and the Microacooe
Q Ic1
Figure 2
Construction of an Image by a concave
mirror follows from the two premises
given below (Figure 3)
I A ray of light parallel to the axis of
the mirror must pass through the
focus after reflection.
2 A ray of light which passes through the
center of curvature m t return along
the same path.
A coronary of the first premise Is;
1 A ray of light which passes through the
focus is reflected paraUel to the axis
of the mirror.
The image from an object can be located
using tJ familiar lens formula
1 1
— + —
p q
where p distance from the object to
the mirror
q distance from the image to
the mirror
f focal length
C Spherical Aberration
No spherical surface can be perfect in its
image-forming ability. The moat serious
of the imperfections, spherical aberrations
occurs In spherical mirrors of large
aperture (Figure 4). The rays of light
making up an image point from the outer
zone of a spherical mirror do not pass
through the same point as the more central
rays. This type of aberration is reduced by
blocking tl outer zone rays from the Image
area or by using aspheric surfaces.
Figure 4
Figure 3

Opti s and the Microscone
D Refra tion of light
Turning now to li ’i . . , ‘ i .itls ’ I thait iiiiu’roi
we find that thc nio ’ t i iiipoi Lint Ii.i ii li i —
istic is refi a turn. R’fi .ii I ion i ’. ’Ii ’ i Lu
the change ol cIire tioji .iiidlui v.’k.i ity iii
light as it pasece Ii urn iiiii’ iii ‘diii iii Lu
another. The ratio of tlit’ v&’lot lt HI . 1 1 1
(or more corre tl in .i v.i tin nil to Lii .
velocity in the In ed in in i .tl led Lii.
refractive ind ’ . Sons’ t)pI . .l v.ilu, ’ ol
refractive inde Inca ‘,ui’t’d with .floiio—
chrornatii light (sodiu i ii I) lint’) ,i r.’ I ii,tt’d
in Table I.
Refrac tion c’auei’s un oh n’s t mum r ed in
a n ccliim flu of lnghc’r refra tive indi’x than
air to appear loser to the surface than it
actually Is (l ’igure 5). This eth’. t may
Figure 5
be used to determine the refractive index
of a liquid with the microscope. A flat
vial with a scratch on the bottom (inside)
18 placed on the stage of the microscope.
The microscope is focused on the scratch
and the fine adjustment micrometer reading
is noted. A small amount of the unknown
liquid is added, the scratch is again brought
into In. u’ and the ne micrometer reading
i ’s talut ii. l”inally , thi mu rosr.ope is re-
fui usi’d until th . s .urfai e of the liquid appears
i ii ‘..h.u r p fo. us, The micrometer reading
is t.ilo’n agdin and, with this information,
the rifra tivi index m y be calculated from
the ‘ iniplifi .d t’qua Lion
actual depth
ii has tive m.ndx
apparent depth
Vzn uum
Crown glass
1.48 to 1. 61
Rock salt
CO 2
I. 3.340
Lead sulfide
When the situation is reversed, and a ray
of light from a medium of high refractive
index passes through the interface of a
medium of lower index, the ray is refracted
until a critical angle is reached beyond which
all of the light is reflected from the interface
(Figure 6). This critical angle, C, has the
following relationship to the refractive i dices
of the two media
sin c .! 2 where
W”ien the second medium is air, the formula
be Omes
sin C
Figure 6

Optics and the Microscope
E Di8pcrsion
Dispersion is another important property
of transparent materials. This is the
variation of refractive index with color
(or wavelength) of light. When white light
passes through a glass prism, the light
rays are retracted by different amounts
and separated into the colors of the
spectrum. This spreading of light into
its component colors is due to di8pcrs ion
which, in turn, is due to the fact that the
refractive index of transparent substances.
liquids and solids, is lower for long wave-
lengths than for short wavelengths.
Because of dispersion, determination of
the refractive index of a substance re-
quires designation of the particular wave-
length used. Light from a sodium lamp
has a strong, closely spaced doublet with
an average wavelength of 5893A, called
the D line , which is commonly used as a
reference wavelength. Table 2 illustrates
the change of refractive index with wave-
length for a few common substances.
Refractive index
D line C line
(yellow) (red)
5893A 6563k
F line
Carbon disulfide
Flint glass
1. 6270
1. 6221
The dispersion of a material can be defined
quantitatively as
n (yellow) -
n (blue) - n (red)
- n (593m i) -
- n (486m i) - n(656m 1 )
where n is the refractive index of the
materiai at the particular wavelength
noted In the parentheses.
There are two classes of lenses, con-
verging and diverging, called also convex
and concave , respectively. The focal
point of a converging lens is defined as
the point at which a bundle of light rays
parallel to the axis of the lens appears to
onverge after passing through the lens.
The fo al length of the lens is the distance
from the lens to the focal point (Figure 7).
Figure 7
G Image Formation by Refraction
Image formation by lenses (Figure 8)
follows rules analogous to those already
given above for mirrors:
I Light traveling parallel to the axis of
the itlns will be refracted so as to pass
through the focus of the lens.
2 Light traveling through the geometrical
center of the lens will be unrefracted.
The position of the image can be determined
by remembering that a light ray passing
through the focus. F, will be parallel to
the axis of the lens on the opposite siae of
the lens and that a ray passing through the
geometrical center of the lens will be
F Lenses

Optics and the Microscope
l .’iguri’ 8
The magnification. M, of an image of an
object produced by a leno is given by the
M image size image distance q
object size object distance p
where q distance from image to lens
and p distance from Dbject to lens.
H Aberrations of Lenses
Lenses have aberrations of several types
which, unless corrected, cause loss of
detail in the image. Spherical aberration
appears in lenses with spherical surfaces.
Reduction of spherical aberration can be
accomplished by diaphragmlng the outer
zones of the lens or by designing special
asphern al surfa es in the lens system.
Chromatic dberratlOn is a phenomenon
caused by the variation of refractive index
with wavelength (dispersion). Thus a lens
receiving white light from an obje4 t will
form a violet Image closer to the lens and
a red one farther away. Achromatic
lenses are employed to minimize this
effect. The lenses are combinations of
two or more lens elements made up of
materials hay uig different dispersive
powers The use of monochromatic light
is another obvious way of eliminating
chromati aberration.
Astigmatism Is a third aberration of
spherical lens systems It occurs when
obje t point ’ are not locatcd on the optical
ixJs of the lens and results in the formation
of an indistuu t image. The simplest
remedy for astigmatism is to place the
objet t lose to the axis of the lens system.
Interferern e Phenomena
Interferen e and diffraction arc two phe-
nomena whit h are due to the wave haracter-
istit s of light, The superposition of two
light rays arriving simultaneously at a given
point will give rise to interference effects,
whereby the intensity at that point will vary
from dark to bright depending on the phase
diffcren es between the two light rzi
The first requirement for interference is
that the light must come from a single
source. The light may be split into any
number of paths but must originate from
the same point (or coherent source). Two
light waves from a coherent source arriv-
ing at a point in phase agreement will
reinforce each other (Figure 9a). Two
light waves from a coherent source arriv-
ing at a point in opposite phase will cancel
each other (Figure 9b).
Figure 9a. Two light rays, 1 arxl 2, of
the same frequency but dif-
ferent amplitudes, are in phase
in the upper diagram. In the
lower diagram, rays 1 and 2
interfere constructively to give
a single wave of the same fre-
quency and with an amplitude
equal to the summation of the
two former waves.
r i

Qp_tiçs and the Microscope

Optics and the Microscone
Each dark band represents an equivalent
air thickness of an odd number of half
wavelengths. Conversely, each bright
band is the result of an even number of
half wavelengths.
With interference illumination, the effect
of a transparent object of different re-
fractive index than the medium in the
microscope field is
I a change of light intensity of the object
if the bat kground is uniformly illumi-
nated (parallel cover slip), or
2 a shift of the interference bands within
the object if the background consists
of bands (tilted cover slip)
The relationship of refractive indices of
the surrounding medium and the object Is
as follows
ns= n ( 1 + 360t
where n 8 refractive index of the
nm refractive index of the
surrounding medium
0 phase shift of the two
beams, degrees
K wavelength of the light
thickness of the specimen.
J Diffraction
In geometrical optics, it is assumed that
light travels in straight lines. This is not
always true We note that a beam passing
through a slit toward a screen creates a
bright band wider than the slit with alter-
nate bright and dark bands appearing on
either side of the central bright band,
decreasing in intensity as a function of
the distarxe from the center. Diffraction
describes this phenomenon and, as one of
its practical consequences, limits the
lens in its ability to reproduce an image.
For example, the image of a pm point of
light produced by a lens is not a pm point
but is revealed to be a somewhat larger
patch of light surrounded by dark and
bright rings. The diameter, d, of this
diffraction disc (to the first dark ring)
is given as
- 2.44fx
d D
where f is the focal length of the lens,
K the wavelength, and D the diameter
of the lens.
It is seen that in order to maintain a
small diffraction disc at a given wave-
length, the diameter of the lens should
be as large as possible with respect to
the focal length. It should be noted,
also, that a shorter wavelength produces
a smaller disc.
If two pm points of light are to be distin-
guished in an image, their diffraction discs
must not overlap more than one half their
diameters. The ability to distinguish such
image points is called resolving power and
is expressed as one half of the preceding
resolving power 1. f K
The compound microscope is an extension in
principle of the simple magnifying glass.
hence it is essential to understand fully the
properties of this simple lens system.
A Image Formation by the Simple Magnifier
The apparent size of an object is determined
by the angle that is formed at the eye by the
extreme rays of the object. By bringing the
object closer to the eye, that angle (called
the visual angle ) is increased. This also
increases the apparent size. However a
limit of accommodation of the eye is reached,
at which distance the eye can no longer focus.
This limiting distance is about 10 inches or 25
centimeters. It is at this distance that the
magnification of an object observed by the
unaided eye is said to be unity. The eye can,
of course, be focused at shorter distances but
not usually in a relaxed condition.
A positive, or converging, lens can be used
to permit placing an object closer than 10
inches to the eye (Figure 12). By this means
the visual angle of the object is increased
(as is its apparent size) while the image of

Optics and the Microscope
Figure 12
the object appears to be 10 inches from
the eye, where It 18 be8t accommodated.
B Magnification by a Single Lens System
The magnification, M, of a simple magni-
tying glass is given by
M + 1
where f focal length of the lens in
Theoretically the magnification can be
increased with shorter focal length lenses.
However such lenses require placing the
eye very close to the lens surface and
have much image distorUon and other
optical aberrations. The practical limit
for a simole magnifying glass is about
In order to go to magnifications higher
than 20X, the compound microscope is
required. Two lens systems are used
to form an enlarged image of an object
(Figure 13). This is accomplished in
two steps, the first by a lens called the
obiective and the second by a lens known
as the eyepiece (or ocular) .
C The Objective
The objective is the lens (or lens system)
closest to the object. Its function is to
reproduce an enlarged image of the object
in the body tube of the microscope.
Objectives are available in various focal
VirtuS ituagi
Figure 13
lengths to give different magnifications
(Table 3). The magnification is calculated
from the focal length by dividing the latter
into the tube length, usually 160 mm.
The numerical aperture (N. A.) is a measure
of the ability of an objective to resolve detail.
This is more fully discussed in the next
section. The working distance is in the free
space between the objective and the cover
slip and varies slightly for objectives of the
same focal length depending upon the degree
of correction and the manufacturer.
There are three basic classifications of
objectives achromats, fluorites and
apochromats. listed in the order of their
complexity. The achromats are good for
routine work while the fluorites and apo-
chromats offer additional optical corrections
to compensate for spherical, chromatic and
other aberrations.
Ey 1p4t 1
Obh,c tiuS

Op tics and the Mi ’ roscopi
focal length
2.5X 0.08 40 SO
5 0.10 25 16 5
10 0.25
20 0.50 1.3
43 0.66
45 0.85
90 1.30
80X 30X
.3.9 90X
Another system of objectives employs
reflecting surfaces in the shape of concave
and convex mirrors. Reflection optics,
because they have no refractmg elements.
do not suffer from chromatic aberrations
as ordinary refraction objectives do. Based
entirely on reflection, reflecting objectives
are extremely useful in the inlrared and
ultraviolet regions of the spectrum. They
also have a much longer working distance
than the retracting objectives.
The body tube of the microscope supports
the objective at the bottom (over the object)
and the eyepiece at the top. The tube
length is maintained at 160 mm except for
Leita instruments, which have a 170-mm
tube length.
The objective support may be of two kinds,
an objective clutch changer or a rotating
I The objective clutch changer (“quick-
change” holder) permits the mbunting
of only one objective at a time on the
microscope. It has a centering arrange-
ment. so that each objective need be
centered only once with rcspcct to the
stage rotation The changing of objec-
lives with this system is somewhat
awkward compared with the rotating
nog up ieee
2 The revolving nosepice allows mounting
three or four objectives on the microscope
at one time (there are some nosepieces
that accept five and even six objectives).
In this system, the objectives are
usually noncenterable and the stage is
centerable. Several manufacturers pro-
vide centerable objective mounts so that
each objective on the noseplece need be
centered only once to the fixed rotating
stage. The insides of objectives are
better protected from dust by the rotating
nosepiece. This, as well as the incon-
venience of the so-called “quick-change”
objective holder, makes it worthwhile
to have one’s microscope fitted with
rotating nosepiece.
D The Ocular
The eyepiece, or ocular, is necessary in
the second step of the magnification process.
The eyepiece functions as a simple magni-
fier viewing the image formed by the
There are three classes of eyepieces in
common use huyghenian. compensating
and flat-field. The huyghenian (or huyghens)
eyepiece is designed to be used with
achromats while the compensating type is
used with fluorite and apochromatic
objectives. Flat-field eyepieces, as the
name implies, are employed in photo.
micrography or projection and can be used
with most objectives. It is best to follow
the recommendations of the manufacturer
as to the proper combination of objective
and eyepiece.
Diam. of
N. A distanec
8. 5
4. 4
0. 7
0. 5
0. 4
0. 5
0. 4
0. 35

Optics and the Microscope
The usual magnifications available in
oculara run from about 6X up to 25 or
30X. The 6X is generally too low to be of
any real value while the 25 and 30X oculars
have slightly poorer imagery than medium
powers and have a very low eyepoint. The
most useful eyepieces lie in the 10 to 20X
magnification range.
E Magnification of the Microscope
The total magnification of the objective-
eyepiece combination is simply the product
of the two individual magnifications. A
convenient working rule to assist in the
proper choice of eyepieces states that the
maximum useful magnification (MUM) for
Uae miLroscopt? in 1 • 000 times the numeri-
cal aperture (N.A.) of the objective.
The MUM is related to resolving power
in that magnification In excess of MUM
gives little or no additional resolving
power and results in what is termed empty
magnification . Table 4 shows the results
of such combinations and a comparison
with the 1000X N.A. rale. The under-
lined figure shows the magnification near-
est to the MUM and the eyepiece required
with each objective to achieve the MUM.
From this table it is apparent that only
higher power eyepieces can give full use
of the resolving power of the objectives.
It is obvious that a lOX, or even a 15X,
eyepiece gives insufficient magnification
for the eye to see detail actually resolved
by the objective.
F Focusing the Microscope
The coarse adjustment is used to roughly
position the body tube (in some newer
microscopes, the stage) to bring the image
into focus. The fine adjustment is used
after the coarse adjustment to bring the
image into perfect focus and to maintain
the focus as the slide is moved across the
stage. Most microscope objectives are
parfocal so that once they are focused any
other objective can be swung into position
without the necessity of refocusing except
with the fine adjustment.
The student of the microscope 8hould first
learn to focus In the following fashion, to
prevent damage to a specimen or objective:
1 Raise the body tube and place the speci
men on the stage.
2 Never focus the body tube down (or the
s.tage up) while observing the field
through the eyepiece.
3 Lower the body tube (or raise the stage)
with the coarse adjustment while care-
fully observing the space between the
Focal Magni-
length fication
(1000 NA)
aMUM maximum useful magnification

Optics and the Microscope
objec’tivc’ and slid, and permitting the
two to come close together without
4 Looking through the microscope and
turning the fanc adjustment in such a
way as to move the objective away from
the specimen, bring the image into
sharp focus.
The fine adjustment is usually calibrated
in one- or two-mit ron steps to indicate
the vertical movement of the body tube.
This feature is useful in making depth
measurements but should not be relied
upon for accuracy
G The Substage Condenser
The substage holds the condenser and
polarizer. It (an usually be focused in a
vertical direction so that the condenser can
be brought into the correct position with
respect to the specimen for proper
illumination. In some models, the conden-
ser is centerable so that it may be set
exactly in the axis of rotation of the stage,
otherwise it will have been precentered at
the factory and should be permanent.
H The Microscope Stage
The stage of the microscope supports the
specimen between the condenser and
objective, and may offer a mechanical stage
as an attachment to provide a means of
moving the slide methodically during obser-
vation. The polai izing microscope is
fittcd with a circular rotating stage to
which a mechanical stage may be added.
The rotating stage, which is used for object
oi ientation to observe optical effects, will
have centering screws if the objectives are
not centerable, or vice versa It Is tin-
desirable to have both objectives and stage
centerable as this does not provide a fixed
reference axis.
The Polarizing Elements
A polarizer is fitted to the condenser of all
polarizing microscopes. In routine instru-
ments, the polarizer is fixed with its
vibration direction oriented north-south
(east-west for most European Instruments)
while in research microscopes, the
polarizer can be rotated. Modern instru-
ments have polarizing filters (such as
Polaroid) replacing the older calcite
prisms. Polarizing filters are preferred
beause they
I are low-cost,
2 require no maintenance,
.1 permit use of the full condenser
An analyzer, of the same construction as
the polarizer, is fitted in the body tube of
the microscope on a slider so that it may
be easily removed from the optical path.
It is oriented with its plane of vibration
perpendicular to the corresponding direction
of the polarizer.
J The Bertrand Lene
The Bertrand lens is usually found only on
the polarizing microscope although some
manufacturers are beginning to include it
on phase microscopes. It is located In the
body tube above the analyzer on a slider
(or pivot) to permit quick removal from
the optical path. The Bertrand lens is used
to observe the back focal plane of the objective.
It i convenient for checking quickly the type
and quality of illumination, for observing
Interference figures of crystals, for adjust-
ing the phase annull In phase microscopy
and for adjusting the annular and central
stops in dispersion staining.
K The Compensator Slot
The compensator slot receives cornpensators
(quarter-wave, first-order red and quartz-
wedge) for observation of the optical prop-
erties of crystalline materials. It is usually
placed at the lower end of the body tube just
above the objective mount, and is oriented
450 from the vibration directions of the
polarizer and analyzer.
L The Stereoscopic Microscope
The stereoscopic microscope, also called
the binocular, wide-field, dissecting or

Optics ana the Microscope
Greenough binocular rnkroscope , is in
reality a combination of two separate
compound microscopes. The two micro-
scopes, usually mounted in one body, have
their optical axes inclined from the vertical
by about 70 and from each other by twice
this angle. When an obje t is placed on the
stage of a stereos opac microsope, the
optical systems view it from slightly
different angles, presenting a stereoscopic
pair of images to the eyes, which fuse the
two Into a single three-dimensional image.
The objectives are supplied in pairs, either
as separate units to he mounted on the
microscope or, as in the new instruments,
built into a rotating drum. Bausch and
Lomb was the first manufacturer to have a
zoom lens system which gives a continuous
change in magnification over the lull range.
Objectives for the stereomicroscope run
from about 0. 4X to 12X, well below the
magnification range of objectives available
for single - objective microscopes.
The eyepieces supplied with stereoscopic
microscopes run from 10 to 25X and have
wider fields than their counterparts in the
single-objective microscopes.
Because of mechanical limitations, the
stereomicroscope is limited to about 200X
magnification and usually does not permit
more than about 120X. It is most useful
at relatively low powers in observing
shape and surface texture, relegating the
study of greater detnU to the monocular
microscope. The stereomicroscope is
also helpful in manipulating small samples,
separating ingredients of mixtures, pre-
paring specimens for detailed study at
higher magnifications a rxl performing
various mechanical operations under micro-
scopical observation, e. g. mic rornanipulation.
Good resolving power and optimum specimen
contrast are prerequisites for good microscopy.
Assuming the availability of suitable optics
(ocular, objectives and substage condenser)
it is still of paramount importance to use
proper Illumination. The requirement for a
good illumination system for the microscope
is to have uniform Intensity of illumination
over the entire field of view with independent
control of intensity and of the angular aperture
of the illuminating cone.
A Basic Types of Illumination
There are three types of illumination
(Table 5) used generally
I Critical. This is used when high levels
of illumination intensity are necessary
for oil immersion, darkfjeld, fluores-
cence, low birefrmgence or photo-
micrographic studies. Since the lamp
filament is imaged in the plane of the
spec imen, a ribbon filament or arc
lamp is required. The lamp must be
focusable and have an iris diaphragm,
the position of the filament must also
be adjustable in all directions.
2 K hler. Also useful for intense illumi-
nation, K hler illumination may be
obtained with any lamp not fitted with a
ground glass. The illuminator must,
however, be focusable, it must have an
adjustable field diaphragm (iris) and the
lamp filament position must be adjust-
able in all directions.
3 “Poor man’s”. So-called because a low-
priced illuminator may be used, this
method gives illumination of high quality
although of lower intensity because of the
presence of a ground glass In the system.
No adjustments are necessary on the
illuminator or lamp filament although
an adjustable diaphragm on the illuminator
is helpful.
All three types of illumination require that
the microscope substage condenser focus
the image of the illuminator aperture in the
plane of the specimen. In each case, then,
the lamp iris acts as a field diaphragm and
should be closed to just illuminate the field
of view. The differences in these three
types of illumination lie in the adjustment
of the lamp condensing lens. With poor
man’s illumination there is no lamp conden-
ser, hence no adjustment. The lamp should
be placed close to the microscope so that

Optics and the Micros
Poor mants
Lamp fijament
ribbon filament
any type
any type
Lamp condensing lens
Lamp iris
Ground glass at lamp
Image of light source
in object plane
at substage
Image of field iris
near object
in object
near object
Image of substage iris
back focal plane
of objective
back focal plane
of objective
back focal plane
of objective
the entire field of view is always
illuminated. If the surface structure of the
ground glass becomes apparent in the field
of view the substage condenser is
slightly defocused.
Critical Illumination
With critical iflumuiation the lamp conden-
ser is focused to give parallel rays, focus-
ing the lamp filament on a far wall is
sufficient. Aimed, then, at the substage
mirror, the substage condenser will focus
the lamp filament in the object plane. The
substage condenser iris will now be found
imaged in the back focal plane of the ob-
jective, it serves as a control over con-
vergence of the illumination. Although the
substage iris also affects the light intensity
over the field of view it should most decid-
edly not be used for this purpose The
intensity of illumination may be varied by
the use of neutral density filters and, unless
color photomicrography is anticipated, by
the use of variable voltage on the lamp
Ko’ilcr illumination (Figure 14) differs
fiom ritical illumination in the use of the
lamp ondenqer. With critical illumination
the lamp condenser focuse the lamp
filament at infinity, with Kohier illumination
the lamp filament is focused in the plane of
the substage condenser iris (also coincident
with the anterior focal plane of the substage
condenser). The functions of the lamp
conderts er iris and the substage condenser
iris in controlling, respectively, the area
of the illuminated field of view and the
angular aperture of the illuminating cone
are precisely alike for all three types of
Critical illumination is seldom used because
it requires a special lamp filament and be-
cause, when u8ed, it hows no advantage
over well-adjusted Kohler illumination.
Kohier Illumination
To ar ange the microscope and illuminator
for Kohler illumination it is well to proceed
through the following steps
a Remove the diffusers and filters
from the lamp
b Turn the lamp on and aim at a con-
venient wall or vertical screen about
19 inches away. Open the lamp
c By moving the lamp condenser, focus
a sharp image of the filament. It
ahould be of such a size as to fill,
not necessarily evenly, the microscope

OptIc. and the M crosLope
E yepoint
Focal plane ‘
Focal plane
Light source

- — - Opti and the’ Microscope
8Ul tJ g i 011 (1 ’ iist’I Opeiiiiig. I i it
dot’ . i t , 1110¼’ i tile i.i nip W I \ Ii ‘iii
tht v.’ i i i It) eiila I I hit I iI.i l iii ill I 1 1 1. 1 141,
rt’fui ii
d l’ui n the lamp .iiicl . 1 liii it IL Lii , liii it)—
St ‘ i ’’ Ill I ci (ii t I .1 It) lilt II it.., ill iii
‘,j Ills III LIlt II I ’ S (Ill .itt ti t.s ,I Ii
dist.in ’ r)
t l’Izi . .i ‘ , )I 1111111 UIi ttit ill Is I I).,s lI Iq
stage U i I fos ti ha rpi v. ith
(I PX) objt ’¼ Live. Open I u II) tii i’
apel Liii e diaphragm in (1w sub 1.igi
condi’nsc’ If the light too bright,
tcnporarli place a neutral density
filter or a diffuser in the lamp.
f (‘lose the lamp diaphragm, or field
diaphragm, to about a I-cm opening.
Rack the mit roscope substage con-
denser up and down to focus the
field diaphragm sharply in the same
plane as the specimen.
g Adjust the mirror to center the field
diaphragm in the field of view.
h Remove the 16-mm objective and
replace with a 4-mm objective. Move
the specimen so that a clear area is
under observation. Place the
Bertrarxl lens in the optical path, or
remove the eyepiece and insert an
auxiliary telescope (sold with phase
contrast accessories) in its place,
or remove the eyepiece and observe
the back aperture of the objective
directly. Remove any ground glass
diffusers from the lamp. Now
OI)Serve the lamp filament through
the necroscope.
Jf the filament does not appear to be
centered, swing the lamp housing in
a horiiuntal arc whose center is at
the field diaphragm. The purpose
is to niaintaLn the field diaphragm on
the lamp in its centered position. If
a vertical movement of the filament
is required, loosen the bulb base and
slide it up or down. If the base is
fixed, tilt the lamp housing in a
vertit al arc with the field diaphragm
is the inter of n i3vein nt (again
rntle.ivt ,, ing to keep tlit ’ lamp din—
phi., got in tht iitcrt ’d position).
II you h vc in i’,tet i’d this step. you
11.1 VI ui t onip! i .Iii d th iii .s t d iffit ult
put lion (Ilettil uiiu i o i ope lamps
Ii ,IVI iii IU’,tfll(flt ’, to niave the bulb
iiiilept i l, ntiy ol Pit Ia ma housing to
‘.iiiiplify this ‘,ti p.
p Put th ‘ ,pit men in phi’ i, I iplac i ’
tI i y ,pit , t’ and (hi di’ ’, ir , ’d ob pet —
tivi .intl I (‘lOt US
k Open or lose th field diaphragm
until it just disappears from the field.
1 Ob’.erve the back aperture of the
objctive, preferably with the Bertrand
lens or the auxiliary telescope, and
close the aperture diaphragm on the
substage condenser until it is about
four-fifths the diameter of the back
aperture. This is the best position
for the aperture diaphragm, a posi-
tion which minimizes glare and maxi-
mizes the resolving power. It is
instructive to vary the aperture dia-
phragm and observe the image criti-
cally during the manipulation.
m If the illumination is too great,
insert an appropriate neutral density
filter between the illuminator and
the condenser. Donotuse the con-
denser aperture diaphragm or the
lamp field diaphragm to control the
intensity of illumination
Poor Man’s Illumination
Both critical and K hler illumination re-
quire expensive illuminators with adjust-
able focus, lamp iris and adjustable lamp
mounts. Poor man’s illumination requires
a cheap illuminator although an expensive
illuminator may be used if its expensive
features are negated by inserting a ground
glass diffuser or by using a frosted bulb.
Admittedly an iris diaphragm on the lamp
would be a help though it is not necessary.
a The illuminator must havp frosted
bulb or a ground glass difiuser.

Optics and the Microsi ope
It should lii pos . ibli to (lint t it iii
the genera I ii iret tion of tlii 1il14tJ gi
mirror, vi r y • loi..e Liii ’ ii’ to 01 iii
plaL e the I (Of.
b [ ‘ocus on .iiiy prupix .1 LiOn .iIL i r
tilting th( iiiirror to illu otitia Li the
Remove the top li ’ii of the on il i’ii i,i ’r
and, by r It king Liii t ondeiisi r up or,
moi often, down, hi ing into lot us
(in the same plum’ js the s;wt iin.n)
a finger, i’m ii or othe i Olijet t pLo id
in the same general region is the
ground glass diffuser on the lamp.
The glass surfa itself an then hi
focused in the plane of the spe im ‘n.
d Ideally the ground glass surfa e will
Just fill the field of view when centered
by the substage mirror, adjustment
may be made by moving the lamp
closer to or farther from the mi ro-
scope (the position might be marked
for each objective used) or by cutting
paper diaphragms of fixed aperture
(one for each objective used). In this
Instance a lamp iris would be useful.
e Lower the condenser Just sufficiently
to defocus the ground glass surface
and render the field of illumination
f Observe the back aperture of the
objective and open the substage con-
denser iris about 75 percent of the
way. The final adjustment of the
substage iris is made while observing
the preparation, the iris should be
open as far as possible, still giving
good contrast.
g The intensity of illumination should
be adjusted only with neutral density
filters or by changing the lamp voltage.
Proper iUummation is one of the most im-
portant operations in microscopy. It is
easy to judge a microscopist’s ability by
a glance at his field of view and the olijec-
tive back lens.
U i eso1viiig Power
Tlii riMilving power of the mitroscope is
tt.. ihility Lu distinguish separate details
of ii i ly .p i. ‘d mo roscopic structures.
i’hi Lliior i Lit al limit of resolving two
ili’.i riLi pointi., a distan t’ X apart, is
I. 22 )
whi ii wavi I ngth of light used to
iltuminjte the specimen
N. A. - numer icil aperture of the
objc c ti v (‘
Substituting a wavelength of 4, 500
Angi.troms and a numerical aperture of
I. 3, ibout the best that can be done with
visible light, we find that two points about
2, 000A (or 0. 2 micron) apart can be seen
as two separate points. Further increase
in resolving power can be achieved for the
light ma roscope by using light or shorter
wavelength. Ultraviolet light near 2, 000
Angstroms lowers the limit to about 0. 1
micron, the lower limit for the light
The numerical aperture of an objective is
usually engraved on the objective and is
related to the angular aperture, AA
(Figure 15). by the formula’
where n = the lowest index in the space
between the object and the
Figure 15
Mgular opertize

Optics a i the Microscope
I Maximum useful magnification
A helpful rule of thumb lb that the use-
ful magnification will not exceed 1.000
times the numerical aperture of the
objective (see Tables 3 and 4). Although
somewhat higher magnifiLatton may be
used in specific cases, no additional
detail will be resolved
It is curious, considering the figures
in the table, that most, if not all, manu-
facturers of microscopes furnish a lOX
eyepiece as the highest power. A lOX
eyepiece is useful but anyone interested
in critical work should use a l5-25X eye-
piece. the 5- lox eyepieces are best for
scanning purposes.
2 Abbe’s theory of resolution
One of the most cogent theories of
resolution is due to Ernst Abbe, who
suggested that microscopic objects act
like diffraction gratin (Figure 16) and
that the angle of diffraction, therefore,
increases with the fineness of the detail.
He proposed that a given microscope
objective would resolve a particular
detail if at least two or three transmitted
rays (one direct and two diffracted rays)
entered the objective. In Figure 16 the
detail shown would be resolved in A and
C but not In B. This theory, which can
be borne out by simple experiment, is
useful in showiig how to improve resolu-
tion. Since shorter wavelengths will
give a smaller diffraction angle, there
is more chance of resolving fine detail
with short wavelengths. Also, since
only two of the transmitted rays are
needed, oblique light and a high N.A.
condenser will aid in resolving fine detail.
3 Improving resolving power
The following list summarizes the
practical approaches to higher resolu-
tion with the light mi roscope
a The speLimen ‘,hould be illuminated
by either critical or Kohler
Eigure 16
b The condenser should be well-
corrected and have a numerical
aperture as high as the objective to
be used.
c An apochroraa tic oil-immersion
objective should be used with a corn-
pensatuig eyepiece of at least 15X
magnification. The immersion oil
should have an index close to 1. 515
and have proper dispersion for the
objective being used.
d Immersion oil should be placed
between the condenser and slide and
between cover slip ar objective.
The preparation itself should be
surrounded by a liquid having a
refractive index of 1. 515 or more.
e The illumination should be reasonably
monochromatic and as short in wave-
length as possible. An interference
filter transmitting a wavelength of
about 480-500 millimicrons is a
suitable answer to this problem.
Ideally, of course, ultraviolet light
should be used to decrease the wave-
length still further.
The practical effect of many of these
factors is critically discussed by
Loveland( 2 ) in a paper on the optics of
object space.

Optics and the Microscope
A Introduction
Photomicrography, a ’i distinct from micro-
photography. is the art of taking pictureb
through the microscope. A mit rophoto-
graph is a small photograph, a photomicro-
graph Is a photograph of a small object.
Photomicrography 18 a valuable tool in
recording the results of microstopi al
study. It enables the microseopist to
1 describe a microscopic field objectively
without resorting to written descriptions.
2 rccord a particulac field for IUIUI ’L
3 make particle size counts and counting
analyses easily and without tying up a
4 enhance or exaggerate the visual micro-
scopic field to bring out or emphasize
certain details not readily apparent
5 record images in ultraviolet arid infra-
red microscopy which are otherwise
invisible to the unaided eye.
There are two general approaches to photo-
rntcrography, one requires only a plate or
film holder supported above the eyepiece
of the microscope with a light-tight bellows.
the other utilizes any ordinary camera with
its own lens system, supported with a light-
tight adaptor above the eyepiece. It is
best, in the latter case, to use a reflex
camera so that the image can b carefully
focused on the ground glass. Photonu-
crography of this type can be regarded
simply as replacing the eye with the camera
lens system. The camera should be focused
at infinity, just as the eye 18 for visual
observation, and it should be positioned
close to and over the eyepiece.
The require ments for photomic rography,
however, are more rigorous than those
for visual work. The eye can normally
compensate for varying light intensities,
curvature of field and depth of field. The
photographic plate, however, lies in one
plane, hence the greatest care must be
used to focus sharply on the subject plane
of interest and to select optics to give
minimum amounts of field curvature and
chromatic aberrations.
With black and white film, color filters
may be used to enhance the contrast of
some portions of the specimen while mini-
mizing chromatic aberrations of the lenses.
In color work, however, filters cannot
usually be used for this purpose and better
optics may be required.
Photornicrographic cameras which fit
directly Onto the microscope are available
in35 -mrnorupto3-114X4-1!4 inch sizes.
Others are made which accommodate larger
film sizes and which have their own support
independent of the microscope. The former,
however, are preferred for ease of handling
and lower coat. The latter system is pre-
ferred for greater flexibility and versatility
and lack of vibration. The Polaroid camera
has many applications In microscopy and
can be used on the microscope directly but,
because of its weight, only when the micro-
scope has a vertically moving stage for
focusing rather than a focusing body tube.
B Determination of Correct Exposure
Corruct exposure determination can be
accomplished by trial and error, by relating
new conditions to previously used successful
onditions and by photometry.
With the trial and error method a series of
trial exposures is made, noting the type of
subject, illumination, filters, objective,
eyepiece, magnification, film and shutter
speed. The best exposure is selected. The
following parameters can be changed and
the exposure time adjusted accordingly:
I Magnification. ExpOsure time varies
as the square of the magnification.
Example Good exposure was obtained
with a 1/10-second exposure
and a magnification of IOOX
If the magnification is now

ptics and the Microscope
200X, the correct expobure
is calculated as follows
new exposure time old exposure time
> ( new magnificauon)2 i/ (200)2
old magnification 100
4/10 or, say, 1/2 second.
It should be noted, however, that th
above calculation can he made only when
there has been no change in the illumi-
nation system including the condrnser
or the objective. Only changes in magni-
fication due to changing eyepiece’i or
bellows extension distance can be hand-
led in the above manner.
2 Numerical aperture. Exposure time
varies inversely as the square of the
smallest working flume rical aperture
of the condenser and objective.
Example Good exposure was olitarned
at 1/10 second with the lOX
objective, N. A. 0. 25. at
full aperture. With a 20X
objective, N. A. 0. 25. at
full aperture and the same
final magnification, what is
the correct exposure time?
new exposure time old exposure time
X ( Old N.A . )2 1/10 (2.:_ )2 1/40 or,
newN A. 0.50
say, 1/50 second.
It is seen that more light reaches the
photographic film with higher numeri-
cal apertures at the same magnification.
3 Film. Exposure time varies inversely
with the American Standards Association
speed index of the film.
Example A good picture was obtained
with Eastman Tri-X film at
1/100 second. What is the
correct exposure for
Eastman Kodachrome II
Type A. The A.S.A. speed
for Tri-X is 400 and for
Kodachrome II Type A
Professional is 40.
new exposure time old exposure time
A. S. A. of old film 1/100(400/40)
A. S. A. of new film
10/100 or 1/10 second.
4 Other parameters may be varied but the
prediction of exposure time cannot be
made readily. Experience and photo-
electric devices are the best guides to
the proper exposure.
Photoelectric devices are excellent for
determining correct exposure. Since
ordinary photographic exposure meters
are not sensitive enough for photomi-
crography, more sensitive instruments,
having a galvanometer or electronic
mph.fying circuit, are required. Some
photosensitive cells are inserted in the
body tube in place of the eyepiece for
light intensity readings. This has the
advantage of detecting the light level at a
point of high intensity but does not take
into account the eyepiece, the distance to
the film or the film speed.
The cell may be placed just above the eye-
piece so that it registers the total amount
of light leaving the eyepiece. Again, the
effects of film speed and the projection
distance are not accounted for. The prin-
cipal drawback with the total light
measuring photometer is the difficulty of
taking into account the area of field covered.
Take, for example, a bright field in which
only a few crystals appear, perhaps 1 per-
cent of the light entering the field of view is
scattered by the crystals and the photometer
shows close to a maximum reading. Now
assume that everything remains constant
except the number of crystals and, conse-
quently, the amount of light scattered.
The photometer reading could easily drop
by 50 percent, yet the proper exposure is
unchanged. The situation is similar for
photornicrography with crossed polars since
the photometer reading depends on the
intensity of illumination, on the bire-
fringence and thickness of the crystals and

9p ç S
on the number and size of the crystals in
the field or, alternatively, on the area of
the field covered by birefringent rystalb.
One of the best solutions to this problem
is to measure the photometer reading with
no preparation on the stage. A first-order
red compensator or a quartz wedge is in-
serted when crossed polara are being used
to Illuminate the entire field.
An alternative is to place the cell on the
ground glass where the film will be
located. However, although all variables
except film speed are now taken into
account, measurements in the image plane
have the disadvantage of requiring a more
sensitive electronic’ photoelectric apparatus.
No matter what method is used for placing
the photocell, the exposure time can be
determined by the general formula
exposure time
meter reading
The constant k will depend on the physical
arrangement and film used. To determine
k for any particular system, first set up
the microscope to take a picture. Record
the meter reading and take a series of
trial exposures. Pick out the best exposure
and calculate k. Then the k which was
determined holds as long as no change is
made in the light path beyond the photocell.
!..k changing to a faster film or changing
the projection distance. Thus the objective.
condenser position or illuminator may be
changed without affecting k if the cell is
used as described above.
Example With one particular arrange-
ment of photocell and film,
the meter reading is found to
be 40. A series of photographs
are taken at 1/2, 1/5, 1/10,
if 25 and 1150 seconds. The
photomicrograph taken at 1/5
second is judged to be the beat,
hence k Is calculated as follows
k meter reading X exposure
time 40X 1/5 c 8.
Assume now that a new picture
is to be taken at another
magnification (but with the
same film and projection
distance) and that the new
meter reading is 16, therefore.
exposure time k/meter
reading 8/16 = 1/2 second.
A Particle Size Determination
Linear distances and areas can be
measured with the microscope. This
permits determination of particle size
arid quantitative analysis of physical
mixtures. The usual unit of length for
microscopical measurements 18 the micron
(1 )< 10 3 mm or about 4 X 10 5 inch).
Measuring particles in electron microscopy
requires an even smaller unit, the ml iii-
micron (1 X l0 micron or 10 Angstrom
units). Table 6 shows the approximate
average size of a few common airborne
Ragweed pollen
25 micronB
Fog droplets
20 microns
Power plant flyash
(after precipitators)
2-5 microns
Tobacco smoke
0. 2 micron
(200 mjllimicroris)
Foundry fumes
0. 1 - 1 micron
(100-1000 mlllimicrons)
The practical lower hmit of accurate
particle size measurement with the light
microscope is about 0. 5 micron. The
measurement of a particle smaUer than
this with the light microscope leads to
errors which, under the best circurn-
stances, increase to about + 100 percent
(usually i ).
One of the principal uses of high resolving
power is in the precise measurement of

Ontics and the Microscone
particle size There are, however, 8
variety of approximate and useful pro e-
dures as well.
1 Methods of particle size measurement
a Knowing the magnification of the
microscope (product of the magni-
fication of objective and eyepioLe),
the size of particles can be esti-
mated. For example, with a lox
eyepiece and a 16-mm (or lOX)
objective, the total magnification
is 100X. A particle that appears to
be 10-mm at 10 inches from the eye
has an actual size of 10 mm divided
by 100 or 0. 10 mm or 100 microns
This is in no sense an accurate
method, but it does permit quick
estimation of particle size, the error
in this estimation is usually 10-25
b Another approximate method is also
based on the use of known data. If
we know approximately the diameter
of the microscope field, we can
estimate the percentage of the
diameter occupied by the object to
be measured and calculate from
these figures the approximate size
of the object. The size of the micro-
scope field depends on both the objec-
tive and the ocular although the latter
is a minor influence. The size of
the field should be determined with
a millimeter scale for each objective
and ocular. If this is done, esti-
mation of sizes by comparison with
the entire field diameter can be quite
accurate (5-10%).
c The movement of a graduated mechan-
ical stage can also be used for rough
measurement of diameter8 of large
particles. Stages are usually gradu-
ated (with vernier) to read to 0. 1
millimeter, or 100 microns. In
practice, the leading edge of the
particle Is brought to one of the lines
of the cross hair in the eyepiece and
a reading is taken of the stage position.
Then the particle is moved across the
field by moving the mechanical stage
in an appropriate direction until the
second trailing edge just touches the
cross-hair line. A second reading is
taken and the difference in the two
readings is the distance moved or the
size of the particle. This method is
especially useful when the particle
is larger than the field, or when the
optics give a distorted image near the
edge of the field.
d The above method can be extended to
projection or photography. The image
of the particles can be projected on a
screen with a suitable light source or
they may be photographed. The final
magnification, M, on the projection
surface (or film plane) is given approxi-
mately by
M DX 0. M. X E. M. /25
where 0. M. objective magnification
E. M. eyepiece magnification
D = projection distance
from the eyepiece in
The image detail can then be measured
in centimeters and the actual size com-
puted by dividing by M. This method
is usually accurate to within 2-5 percent
depending on the size range of the detail
e The stated magnifications and/or focal
lengths of the microscope Optics are
nominal and vary a bit from objective
to objective or eyepiece to eyepiece.
To obtain accurate measurements, a
stage micrometer is used to calibrate
each combination of eyepiece and
objective. The stage micrometer is
a glass microscope slide that has,
accurately engraved in the center, a
scale, usually 2 millimeters long,
divided into 200 parts, each part repre-
senting 0. 01 millimeter. Thus when
this scale is observed, projected or
photographed, the exact image magni-
fication can be determined. For
example, if S spaces of the stage micro-
meter measure 6 millimeters when
projected, the actual niagrn.fication is

Optics and the Microscope
5 (0. 01) 120 times.
This magnification figure can be
used to improve the accuracy of
method 4 above.
f The simplest procedure and the most
accurate is based on the use of a
micrometer eyepiece. Since the
eyepiece magnifies a real image
from the objective, it is possible
to place a transparent scale in the
same plane as the image from the
objective and thus have a scale
superimposed over the image. This
Is done by first placing an eyepiece
miciometer sale dibc in the eyepiece.
The eyepiece micrometer has an
arbitrary scale and must be cali-
bra ted with each objective used. The
simplest way to do this is to place
tJ stage micrometer on the stage
and note a convenient whole number
of eyepiece micrometer divisions.
The value in microns for each eye-
piece micrometer division Is then
easily computed. When the stage
micrometer is removed and replaced
by the specimen, the superimposed
eyepiece scale can be used for accu-
rate measurement of any feature in
the specimen by direct observation,
photography or projection.
2 Calibration of eyepiece micrometer
Each micrometer stage scale has
divisions lOOu (0. 1 mm) apart, one
or two of these are usually subdivided
into lO (0. 01-mm) divisions. These
form the starr:lard against which the
arbitrary divisions in the micrometer
eyepiece are to be calibrated. Each
objective must be calibrated separately
by noting the correspondence between
the stage scale and the eyepiece scale.
Starting with the lowest power objective
focus on the stage scale, arrange the
two scales parallel and in good focus.
It should be possible to determine the
number of eyepiece divisions exact.ly
equal to some whole number of
divisions of the stage scale, a distance
readily expressed in microns.
The calibration consists, then, of
calculating the number of microns per
eyepiece scale division. To make the
com!arison as accurate as possible, a
large part of each scale must be used
(see Figure 17). Let’s assume that
with the low power 16-mm objective
6 large divisions of the stage scale
(s. m. d.) are equal to 38 divisions of
the eyepiece scale. This means that
38 eyepiece micrometer divisions (e.rn. d.)
are equivalent to 600 microns. Hence
1 e.m.d. 600/38
l5.8 .
Thus when that micrometer eyepiece
is used with that 16-mm objective each
division of the eyepiece scale is equivalent
to 15. 8 a, and it can be used to make an
accurate measurement of any object on
the microscope stage. A particle, for
example, observed with the 16-mm objec-
tive and measuring 8. 5 divisions on the
eyepiece scale is 8. 5 (15. 8) or l35 in
Each objective on your microscope must
be calibrated an this manner.
A convenient way to record the necessary
data and to calculate ,j!emd Is by means
of a table.
FIgure 17

Optics and the Microscope
Table 7
No smd
no. emd
no emd
I emd
= 38
15 8
= 30
100 =
3 33
3 Determination of particle size
The measurement of particle size can
vary in complexity depending on parti-
cle shape. The size of a 8phere may be
denoted by its diameter. The size of a
cube may be expressed by the length of
an edge or diagonal. Beyond these two
configurations, the particle ‘size” must
include information about the shape of
the particle in question, and the
expression of this shape takes a more
comi,llcated form.
Martin’s diameter is the simplest means
of measuring and expressing the dia-
meters of irregular particles and is
sufficiently accurate when averaged for
a large number of particles. In this
method, the horizontal or east-west
dimension of each particle which divides
the projected area thto halves is taken as
Martin’s diameter (Figure 18).
The more particles counted, the more
accurate will be the average particle
size. Platelike and needlelike particles
should have a correction factor applied
to account for the third dimension since
all such particles are restricted in their
orientation on the microscope slide.
When particle size is reported, the
general shape of the particles as well as
the method used to determine the
“diameter” should be noted.
Particle size distribution is determined
routinely by moving a preparation of
particles past an eyepiece micrometer
scale in such a way that their Martm’
diameter can be tallied. All particles
whose centers fall within two fixed
divisions on the scale are tallied. Move-
ment of the preparation is usually
accomplished by means of a mechanical
stage but may be carried out by rotation
of an off-center rotating stage. A sample
tabulation appears in Table 8. The eye-
piece and objective are chosen so that
at least six, but not more than twelve,
size classes are required and sufficient
particles are counted to give a smooth
curve. The actual number tallied (200 -
2, 000) depends on particle shape
regularity and the range of sizes. The
size tallied for each particle is that
number of eyepiece micrometer divisions
most closely approximating Mirtin’s
diameter for that particle.
4 Calculation of size averages
The size data may be treated in a variety
of ways, one simple, straightforward
treatment is shown in Table 9. For a
more complete discussion of the treat-
ment of particle size data see Chamot
and Mason’s l 1andbooic of Chemical
Microscopy( 3 ’ , page 26.
The averages with respect to number,
d 1 , surface, d 3 . arid weight or volume,
d 4 , are calculated as follows for the
data in Table 9.
I—p —I
Figure 18

Op ics and the Micro8cop4
fl ”
Nu s
r of partici..
p1 —4 -a
11 - 44
r e- I - I
t -*-4..1
r a
ri - 1J


rI- I - I
r i— I-I
1 1-I4
1#-4 .j
11 -34
ri—i -a
2 144
1 I—I.I
‘md •
C ieci . mtc
d IaIon.
r nd/Ln r 1758/470
3.74 emd X 2.82* - 10. 5 i
d 3 End 3 /End2 - 37440/7662
4.89 emdx 2.82 13.
- End 4 /Znd 199194/37440
= 5.32 emd X 2.82 - 15. 0
*2. 82 microns per emd
(determ med by calibration of the
eyepiece-objective combination
used for the determination).
Cumulative percents by number,
surface and weight (or volume) may be
plotted from the data in Table 9 The
calculated percentages, L
: A 4X 100
for the cumulative weight or volume
curve, are plotted against d. Finally.
the specific surface, Sm, in square
meters per gram, m, may be calculated
if the density, D, is known, the surface
average d 3 , is used.
if D 1. 1, Sm — 6Jd 3 D - 6/13. 8(1. 1)
- 0. 395m 2 1g.

Optics arxl the Microscop
(Aver diam. n nd nd 2 nd 3
98 196 392 784 1568
110 330 990 2970 8910
107 428 1712 6848 27392
71 355 1775 8875 44375
45 270 1620 9720 58320
21 147 1029 7203 50421
2 16 128 1024 8192
470 1758 7662 37440
B Counting Analysis
Mixtures of particulates can often be
quantitatively analyzed by caantuig the
total number of particulates from each
component in a representative sample.
The calculations are, however, compli-
cated by three factors average particle
size, particle shape and the density
of the components. If all of the compon-
ents were equivalent in particle size,
shape and density then the weight per-
centage would be identical to the number
percentage. Usually, however, it is
necessary to determine correction factors
to account for the differences.
When properly applied, this method can
be accurate to within + 1 percent and.
in special cases, even better. It is often
applied to the analysis of fiber mixtures
and is then usually called a dot-count
because the tally of fibers is kept as the
preparation is moved past a point or dot
in the eyepiece.
A variety of methods can be used La
simplify recognition of the different
components. These include chemical
stains or dyes and enhancement of optical
differences such as refractive indices,
dispersion or color. Often, however, one
relies on the differences in morphology.
!..L counting the percent of rayon fibers
in a samole of “silk”.
Example 1 A dot-count of a mixture of
fiberglass and nylon shows
% nylon = 262/(262 + 168)X 100
= 60. 9To by number.
However, although both fibers are smooth
cylinders, they do have different densities
and usually different diameters. To
correct for diameter one must measure
the average diameter of each type of fiber
and calculate the volume of a unit length
of each.
aver. diam. volume of
l- slice,
18. 5 268
fiberglass 13. 2
The percent by volume is, then.
262 X 268
% nylon (262 X 268)+(168 X 117) X 100
78. 1% by volume.
Still we must take into account the density of
each in order to calculate the weight percent.

Optics and the Microscope
If the ii ,i’,itj or I I f i iiykiiu ,ig I . 2
mr glos’, then thc p (’r (nt by w(’ight i
262 X 2(8 x I I
% nylon
(262 x 268 X 1. h)+(lb8 x 17 X 2.2) X 100
= 72% by weight.
Example 2 A count of quortz and
gypsum shows
To ol ulate the percent by weight we must
take into a ount the average partu I c size,
the shape and the density of (‘a h
The avcrogc parti( Ic size with rcspcLt to
weight. d 4 , must be measured for each
and the shape factor must be determined.
Since gypsum is more plutelike than quartz
each particle of gypsum is thinner. The
shape factor (an be approxima ted or can be
roughly calculated by measuring the actual
thickness of a number of particles. We
might find, for example, that gypsum parti-
cles average 80% of the volume of the aver-
age quartz particle, this is our shape factor.
The final equation for the weight percent is
283X lTd 4 /6XDq
%quartz 238X wd 4 /6XDq+467Xi /
where Dq and Dg are the densities of quartz
and gypsum respectively. 0. 80 is tha shape
factor and d 4 and d are the average parti-
cle sizes with respect to weight for quartz
and gypsum respectively.
A CKNOW LEDGM ENT This outline was
prepared by the II. S. Public Health Service,
Department of Health, Education and Welfare,
foi use in its Training Program.
1 flunn, C.W. (‘rystal Giowtli from Solution.
I)iscussionc. nf the [ “araday Society No. 5.
12. Gunit y ond .Jackson. l ndon. (1949).
6 X 0.80 X Dg X 100
2 Loveland, R.P., J. Roy. Micros. Soc.
79, 59. (1960).
3 Chamot, Emile Monnin, and Mason,
Clyde Walter. Handbook of Chemical
Microscopy, Vol. 1, third ed. John
Wiley and Sons, New York (1959).

What are cells? Cells may be defined as the
basic structural units of life. The ceU has
many different parts which carry on the
various functions of cell life. These are
called organelles (“little organs”).
A The branch of biology which deals with the
form and structure of plants and animals
is called “Morphology.” The study of the
arrangement of their several parts is
called “anatomy”, and the study of cells
is called “cytology”.
B There is no “typical” cell, for cells differ
from each other in detail, and these
differences are in part responsible for the
variety of ll.fe that exists on the earth.
A How do we recognize a structure as a cell?
We must look for certain characteristics
and/or structures which have been found
to occur in cells. The cell Is composed
of a variety of substances and structures,
some of which result from cellular
activities. These include both living and
non-living materials.
1 Non-living components thclude
a A “cell wall” composed of cellulose
may be found as the outermost
covering of many plant cells.
b ‘Vacuoles” are chambers In the
protoplasm which contain fluids of
diifcrent densities (i.e., different
from the surrounding protoplasm).
2 The “living” parts of the cell are called
“protoplasm.” The following structures
are included:
a A thm “cell membrane” is located
just inside the cell wall. This
membrane may be thought of as the
outermost layer of protoplasm.
b In plant cells the most conspicuous
protoplasmic structures are the
“chioroplasts”, which contain
highly organized membrane systems
bearing the photosynthetic pigments
(chlorophylls, carotenoids, and
xanthophylls) and enzymes.
c The “nucleus” is a spherical body
which regulates cell function by
controlling enzyme synthesis.
d “Granules” are structures of small
size and may be “living” or
non -living” material.
e “Flagella” are whip-like structures
found in both plant and animal cells.
The flagella are used for locomotion,
or to circulate the surrounding
f “Cilia” resemble short flagella, found
almost exclusively on animal cells.
In the lower animals, cilia are used
for locomotion and food gathering.
g The “pseudopod”, or false foot, is
an extension of the protoplasm of
certain protozoa, in which the
colloidal state of the protoplasm
alternates from a “sol” to a
condition from time to time to
facilitate cell movement.
h “Ribosomes” are protoplasmic bodies
which are the site of protein
synthgsls. They are too small
(150 A lndiameter)to be seen with
a light microscope.
i “Mitochondria” are small mem-
branous structures containing
enzymes that oxidize food to produce
energy transfer compounds (ATP).
BI.CEL. la. 3. 70

Structure and Function of Cells
B How basic structure is expressed in some
major types of orgamams.
We can better visualize the variety of cell
structure by considering several specific
1 BacterIa have few organelles, and are
so minute that under the light
microscope only general morphological
types (i.e.. the three basic shapes;
rods, spheres, and spirals) can be
recognized. The following structures
have been defined:
a The “capsule” is a thick protective
covering of the cell exterior, con-
sisting of polysaccharide or
polyp eptide.
b The cell wall and plasma membrane
are present.
c Although no well defined nucleus is
visible in bacterial cells, the
electron microscope has revealed
areas of deoxyribose nucleic acid
(DNA) concentration. This ath-
stance is present within the nucleus of
of higher cells, and is the genetic
or hereditary material.
d Some types of bacteria contain a
special type of chlorophyll
(bacterlochiorophyll) and carry on
2 The blue-green algae are similar to the
bacteria in structure, but contain the
photosynthetic pigment chlorophyU a.
a Like the bacteria, these forms also
lack an organized nucleus (the
nuclear region Is not bounded by a
b The chlorophyll-bearing membranes
are not localized in distinct bodies
(chioroplasts), but are dispersed
throughout the cell.
c Gas-filled structures called
“pseudiovacuoles” are found in some
types of blue-greens.
3 The green algae as a group include a
great variety of structural typeB,
ranging from single-celled non-motile
forms to large motile colonies. Some
types are large enough to resemble
higher aquatic plants.
a The chloroplasts are modified into
a variety of shapes and are located
In different positions. Examples
of ch]orop]ast shape and position are:
1) Parietal - located on the
periphery of the cell; usually
cup-shaped and may extend
completely around the inner
surface of the plasma membrai e.
2) Discoid - also located on the
periphery of the cell, but are
plate-shaped; usually many per
3) AxIal - lying In the central axis
of the cell, may be rthbon-lIice
or star-shaped.
4) Radial - have arms or processes
that extend outward from the
center of the cell (radiate),
reaching the plasma membrane.
5) Reticulate - a mesh-like network
that extends throughout volume
of the cell.
b Located In the chlorop]asts may be
dense, protelnaceous, starch-
forming bodies called “pyrenoids”.
4 The flagellated algae possess one-to-
eight flagella per cell. The chloro-
plasts may contain brown and/or red
pigments In addition to chlorophyll.
a Reserve food may be stored as
starch ( Chlamydomonas ) paralny]on
or as oil.
5 The protozoa are single-celled
animals which exhibit a variety of
cell structure.

Structure and Function of Cells
a The amoebae move by means of
pseudopodia, as described
b The flagellated protozoa
(Mastigophora) possess one or more
c The ciliates are the most highly
modified protozoans. The cilia may
be more or less evenly distributed
over the entire surface of the cell,
or may be localized.
What are the functions of cells and their
structural components? Cellular function
is called “life”, and life is difficult to define.
Life is characterized by processes commonly
referred to as reproduction, growth, photo-
synthesis, etc.
A Mlcroorganism8 living in surface waters
are subjected to constant fluctuations in
the physical and chemical characteristic
of the environment, and must constantly
modify their activities.
1 The cell requires a cource of chemical
energy to carry on life processes and
successfully compete with other
organisms. Plant cells may obtain
this energy from light, which is
absorbed by chlorophyll and converted
into ATP or food reserves, or from
the oxidation of food stuffs. Animal
cells obtain energy only from the
oxidation of food.
2 Cells mu8t obtain raw materials from
the environment in order to grow and
carry out other Life functions. Inorganic
and organic materials may be taken up
by passive diffusion or by “active
transport”. In the later process,
energy is used to build up and maintain
a higher concentration of a substance
(such as phosphate) inside the cell than
is found outside. Algae are able to
synthesis organic matter from inorganic
raw materials (carbon dioxide and
water), with the aid of energy derived
from light, whereas animal cells must
obtain their organic matter “ready-
made” by consuming other organisms.
organic debris, or dissolved organics.
The cell is made up of many highly special-
ized substructures. The types of sub-
structures present, and their appearance
(shape, color, etc.) are very important in
understanding the role of the organism in
the aquatic community, and in classification.
1 Bold, H.C. Cytology of algae. In: G.M.
Smith, (ed.), Manual of Phycology.
Ronald Press. 1951.
2 Bourne, Geoffry H., ed. Cytology and
Cell Physiology. 3rd ed. Academic
Press. 1964.
3 Brachet, Jean. The Living Cell.
Scientific American. 205(3). 1961.
4 Corliss, John 0. Ciliated Protozoa.
Peganion. 1961.
5 Fritsch. F. E. The structure and
reproduction of the algae. Cambridge
Univ. Press. 1965.
6 Frobisher, M. Fundamentals of
microbiology. 7th edition. W. B.
Saunders Co., Philadelphia. 1962.
7 Round, F.E. The biology of the algae.
St. Martin’s Press. New York. 1965.
This outline origthaUy prepared by Michael
E. Bender, Biologist, formerly with
Training Activities, FWPCA, SEC. and
revised by Cornelius I. Weber, March 1970.

A Algae in general may be defined as
small pigmented plant-like organisms
of relatively simple structure. Actually
the size range is extremeS from only a
few microns to over three hundred feet
in length Commonly observed examples
include the greenish pond scum or frog
8pittle of freshwater ponds, much of the
golden brown slime covering rocks In a
trout stream, and the great marine kelps
and seaweeds Large freshwater forms
as Nitefla and Chara or stonewort are
also included
B Algae approach ubiquity in distribution.
In addition to the commonly observed
bodies of water, certain algae also live
in such unlikely places as thermal springs,
the surface of melting snow, on the hair
of the three toed sloth in Central America,
and in con)unctton with certain fungi to
form lichens.
A Blue-greens (See plate: Blue-Green
Algae, Cyanophyceae). This is a valid
technical group. The size range is not
very great, some being so small as to
approach the size range of the bacteria
1 These are the only algae in which
the pigments are not localized in
definite bodies but dissolved through-
out the cell Blue, red, or other
pigments are present in addition to
chlorophyll thus giving the cells a
bluish green, yellow, or r d color,
at least enmasec
2 ‘]‘ht. nucleus lacks a nuclear membrane.
3 Tend to achieve nuisance concentrations
more frequently in the warm summer
months and in the richer waters.
4 Vegetative reproduction, in addition
to cell division, Includes the forma-
tion of ‘hormogones,” or short specif-
ically delimited sections of trichomes
5 Spores of three types are encountered:
a Akinetes are usually larger, thick
walled resting spores.
b Heterocysts appear like empty
cell walls, but are actually filled
with protoplasm, have occasionally
been observed to germinate
c Endospores, also called “gonidia”
or conidia, are formed by repeated
division of the protoplast within a
given cell wall. Present in only a
few genera.
6 Some common examples of blue-
green algae are:
Anacyatis ( Microcystis or
Polycystis), Anabaena, Aphani-
zomenon , and Oscillatorta
B The Pigmented flagellates (in contrast
to the non-pigmented or animal-like
flagellates) are a heterogeneous
collection of motile forms from several
different algal groups (See plateS
Flagellated algae).
1 There may be one, two, four, or
more flagella per cell.
2 There is a well organized nucleus
3 A light-sensitive red eyespot usually
4 The chlorophyU is contained in one
or more distinctive bodies called
RI MIC.cla. 19a 8 69
5- 1

Types of Algae
5 Two or more cells may be associated
In a colony
6 Non-motile life history stages may
be encountered
7 Masses of stored starch called
pyrenotd bodies are often conspicuous.
8 Some examples of pigmented flag-
ellates are: Euglena, Phacus,
Chiamydomonas , Gonium, Volvox,
Peridinium, Ceratium Mallomonas,
nura and Dinobryon .
C The Non-motile green algae constitute
another heterogeneous assembly of Un-
related forms (See p’ate: Non-Motile
Green Algae)
1 Like the flagellates they have well
organized nuclei and chloroplasts.
The shape of the chloroplast is often
2 They lack flagella or any other loco-
motor device.
3 There is extreme structural variation
among the group.
4 Some types tend to occur as a general
planktonic mass or bloom,” often in
combinations of two or more species.
Some examples are Sphaerocystts,
Pedlastrum, Scenedesmus , and the
desmid Cosmarium .
5 Threadlike (filamentous) green algae
may form masses or blankets, cutting
off light, and reducing water circula-
tion. They also add considerably to
the total mass of organic matter.
Some examples of this type are
Spirogyra, Hydrodictyon, Cladophora,
Oedogonlum , and Chara .
D The Diatoms constitute another valid
technical group (See plate Diatoms-
1 In appearance, they are geometrically
regular in shape The presence of a
brownish pigment in addition to the
chlorophyfl gives them a golden to
greenish color.
2 Motile forms have a distinctive
hesitating progression.
3 The most distinctive structural
feature is the two-part shell
(frustule) composed of silicon
dioxide (glass).
a One part fits inside the other as
the two halves of a pill box, or a
petri dish.
b The surface of these shells are
sculptured with minute pits and
lines arranged with geometrical
c The view from the side is called
the “girdle view,” that from above
or below, the “valve view.”
4 There are two general shapes of
diatoms, circular (centric) and
elongate (pennate). The elongate
forms may be motile, the circular
ones are not.
5 Diatoms may associate in colonies
in various ways
6 Examples of diatoms frequently en-
countered areS Stephanodiscus
Cyclotelia, Astertonella, Fragilaria,
Tabellaria, Synedra , and Nitzschta .
This outline was prepared by H.W. Jackson,
Chief Biologist, National Training Center,
Water Programs Operations, EPA, Cincinnati,
OH 45268

Types of Algae
Beginning with “in” and “lb”, choose one of the two contrasting
statements and follow this procedure with the “a” and “b” state-
ments of the number given at the end of the chosen statement.
Continue until the name of the algal group is given instead of
another key number.
is. P].astld (separate color body) absent, complete protoplast
pigmented, generally blue-green; Iodine starch test*
negative Blue-green algae
lb. Piastid or plastids present; parts of protop]ast free of some
or all pigments; generally green, brown, red, etc., but not
blue-green; iodine starch test* positive or negative 2
2a. Cell wail permanently rigid (never showmg evidence of
collapse), and with regular pattern of fine markings
(striations, etc.), plastids brown to green, Iodine starch test*
negative; flagella absent; wall of two essentially similar halves,
one placed over the other as a cover Diatoms
2b. Cell wall, if present, capable of sagging, wrinkling, bulging
or rigi ty, depending on existing turgor pressure of cell
protoplast; regular pattern of fine markings on wall generally
absent; plastids green, red, brown, etc.; Iodine starch test*
positive or negative; flagella present or absent; cell wall
continuous and generally not of two part 3
3a. Cell or colony motile, flagella present (often not readily visible);
anterior and posterior ends of cell different from one another in
contents and often in shape Flagellate algae
3b. Non-motile, true flagella absent; ends of cells often not
differentiated Green algae and associated forms
*Add one drop Lugol’s (Iodine) solution, diluted i-i with distilled water, in about 1 minute,
if positive, starch i 9tained blue and, later black. Other structures (such as nucleus,
plastids, cell wall) may also stain, but turn brown to yellow.

Typea of Algae
of pigment
In plastida
In piastids
In plastids
Present or
in most
in moat
in most
Cell Wall
from slimy
Thin or
smooth or
with spines
Very rigid,
with regular
“Eye” spot

The blue -green algae (Myxophyceae) comprise
that large group of microscopic orgazusms
living in aquatic or moist habitats, carrying
on photosynthesis and having differentiation
of cells which is a little more complex than
bacteria, and simpler than aU of the other
plants called algae
In addition to the green photosynthetic pigment
(chlorophyll -a) they always have a blue pig-
ment (phyocyanin-c) which tends to give the
cushions or mats they may form a blue-green
Some are free floating (pelagic and planktonic),
others grow from submerged or moist soil.
rocks, wood and other objects in both fresh-
water and marine habitats.
Some are gelatinous masses of various shapes
floating in water. Others, microscopic in
size, grow in great numbers so as to color
the water in which they live. Structurally
their cells are similar to bacteria. Their
protoplasts may be sheathed or imbedded in
gelatin, making them slimy. Cells of blue-
green algae are without organized nuclei,
central vacuoles, or cilia and flagella.
No sexual reproduction Is known. Asexual
reproduction may be effected by fragmentation,
in which case special separation devices are
formed (dead cells, and heterocysts). Some
species arc preserved over unfavorable
periods by special spores (akinetes an’i endo-
They have both positive and negative economic
significance. Because they can convert
radient energy into chemical energy, they
are producers forming a first link at the base
of the food chain. Because many very in-
tricate nutritional relationships exist among
the niyraids of organisms it is difficult to
know the value of the blue-greens. However,
people who know what the blue-greens can do
to drinking and recreational water classify
them as o! negative economic importance,
because they are often nuisances when they
impart color, bad odors, and fishy tastes,
or toxins. Some of them can foul pipes
and clog filters.
They are widely distributed in time and space,
but tend to reach nuisance concentrations more
frequently in the late summer and in eutrophic
The pioneer-forms are of great ecological
importance because they live m habitats fre-
quented by few other forms of live, synthesiz-
ing organic substances and building substrata
that can support other kinds of life.
A Some blue-greens live in association
with other organisms as symbionts.
Still others are found i.n polluted
waters, because they are able to
exist in habitats poor in oxygen. The
growth of these kinds of algae under
such conditions tends to make a pol-
luted condition worse.
B On the other hand some species
should be promoted because they
provide oxygen and food through photo-
synthesis. The first evident product
of photosynthesis is glycogen, and
is the cause of the brown coloration
with the iodine test. Some of the
glycogen is used to produce glycopro-
teins. The gelatinous sheath is com-
posed of pectic substances, cellulose
and related compounds.
BI. MIC. cia. iCa. H. 69

Blue-Green Algae
C When blue-green8 mat at the surface
of the water the increased lighting
may oe too strong, resulting in a
kill. At this time they may turn
from a blue-green to a yellow-green
color. Here they decompose In
mass. The resulting Intermediate
products of decomposition may be
highly undesirable, because of bad
looks, four odors, bad tastes and
toxins. Under these conditions the
BOD may produce conditions not
unlike raw sewage.
A A cross section of a typical cell
would show an outside nonliving
gelatinous layer surrounding a woody
cell wall, which is bulging from
turgor pressure from the cell (plasma)
membrane, pushing the wall outward-
ly. The protoplasm, contained with-
in the plasma membrane, is divided
into two regions. The peripheral
pigmented portion called chroma-
toplasm, and an inner centroplasm,
the centroplasm contains chromatins,
which is also known as in incipient
nucleus or central body, containing
chromosomes and genes. Structures
(chromatophores or plastids) con-
taining pigments have not been found
in the blue-greens. The photosyn-
thetic pigments are dissolved in the
peripheral cytoplasm, which is known
as the chromatoplasm.
B A simple way to understand the cross
section would be to compare it with
a doughnut, with the hole represent-
ing the colorless central body or
incipient nucleus, which houses the
chrornatoplasm, having the charac-
teristic blue-green color from its
dissolved photosynthetic pigments.
When the protoplasts become aick or old they
may develop a great number of “pseudovac-
uoles’ filled with gas. These gas bubbles make
the algae buoyant in such a way that they may
“flower” or bloom by rising to the surface
(planktonic, healthy blue-greens normally
possess pseudovacuoles, which are here
excepted). Soon they begin to stink because
of the odors produced from putrefaction.
The lack of dissolved oxygen during this
period may effect other organisms.
No. Healthy blooms are produced by myraids
of cells living near the surface of the water
at times when cnvironmental conditions are
especially favorable for them. Putrefactive
blooms are usually from masses of algae
undergoing degradation.
Most species of blue-greens may be placed
into two major groups: the nonfilamentoug
(coccoid) forms, and the filamentous forms.
See the set of drawings following this treat-
ment to get a graphic concept of the two
A In comparing the blue-greens with
other algae it is easier to tell what
they do not possess than what they
do. They do not have chromatophores
or plastids, cilia, flageUa, organized
nuclei, gametes, central vacuoles,
chlorophyll-b. or true starch.
B Many of the filamentous forms, es-
pecially the Oscillatorlaceae, exhibit
an unexplained movement. When the
filamentous forms are surrounded
by a gelatinous sheath the row of cells
inside is called a trichome , and the
trichome with its enclosing sheath is
called a filament. There may be more
than one trichome within a sheath.

Blue-Green Algae
Tru branching occurs when a cell
of t}u series divides lengthwise and
the oiit€ r-forrned cell add cells to
form a true branch. However, two
or more trichomes within a single
sheath may be so arranged that though
they appear to be branches, their cells
actually have all divided in the same
plane, and the trichomes have pushed
out from growth to form false branch-
as in Tolypothrix .
C An occasional reticulated or bubbly
appearance is referred to as pseudov-
acuolation, and en mass imparts a
pale, yellowish color to the algae.
Under low powers these vacuoles
appear dark, under higher magnifi-
cations they are reddish.
D Vegetative reproduction in addition
to cell division for the unicellular
forms, is by special kinds of frag-
mentation. This includes the for-
mation of hormogones , which are
specifically delimited sections of
trichomes, and are characteristic
of some taxonomic entities.
E Spores of three types are encountered.
1 Akinetes are usuaUy larger, non-
motile, thick-walled resting spores.
2 Heterocysts appear like empty cell
walls, but are filled with colorle8s
protoplasm and have been occasion-
ally observed to germinate.
3 Endospores, also called gonidla,
are formed by a repeated division
of the protoplast within a cell wall
A Anacystis ( Microcystis ) is common
in hard waters.
I Colonies are always free floating.
2 ‘I ’heir shapes may be roughly
.phcrical or irregular, micro-
scopic or macroscopic.
3 ‘ [ ‘he gelatinous matrix may be
extremely transparent, easily
broken up on preservation.
4 They frequently contain pseudov-
B Anabaena is an example of a fila-
rnentous form.
1 Filaments may occur singly or
in irregular colonies, and free
floating or in a delicate nucous
2 Trichomes have practically
the sanie diameter throughout,
may be straight, spiral, or
irregularly contorted.
3 Cells are usually spherical,
or barrel shaped, rarely cy-
lindrical and never discoid.
4 Heterocysts are usually the same
shape but are slightly larger
than the vegetative cells.
5 Akinetes are always larger than
the vegetative cells, roughly
cylindrical, and with rounded
6 It may be readily distinguished
from Nostoc by the lack of a
firm gelatinous envelope.
7 It may produce an undesirable
grassy, moldy or other odor.
C Aphanizomenon is a strictly plank-
tonic filamentous form.
1 Trichomes are relatively straight,
and laterally joined into loose
macroscopic free-floating flake-
like colonies.
2 Cells are cylindrical or barrel
shaped, longer than broad.
3 Heterocysts occur within the
filament (i. e., not terminal).
4 Akinetes are cylindrical and
relatively long.

Blue-Green Algae
1. NP ittamontoi1i(cOccoid) hum Green Al ae:
I :
Anacy (Chr4 occus) X600.
fl. Fitam.ntous blue-green algal:
Trichornes of p! rulina . (X600).
Trichomes of Arthrospi
.‘ akinete
J (spore)
i 1 Ti
Phornildium (with sheath)
False branching
Tolypothrlx (X375)
Oscillatoria (without sheath)
Hapalosiphon Prepared by Louis C. Williams
(X375J Aquatic Biologist. Basic I ta, SEC.
Coccochioris (Closocapsa)
Microcystis (x600). Polycysti .

Blue-Green Algae
5 Often imparts grassy or nastur-
tium-like odors to wa’ter.
D Oscillatoria is a large and ubiquitous
1 Filaments may occur singly or
interwoven to form mats of
indefinite extent.
2 Trichomes are practically the
same diameter throughout.
3 Sheaths are usually distinct,
fairly firm, and with a single
2 Trichomes are unbranched, cy-
lindrical, and practically with-
out sheaths.
3 Species with narrow trichomes
have long cylindrical cells
while those with broader tn-
chomes have short broad cells.
4 No heterocysts or akinetes are
known in Oscillatoria . It re-
produces by fragmentation from
hormongonia only.
5 Live species exhibit “osciliatorta”
movements, which are oscillating.
6 Species of Oscillatoria may be
readily distinguished from
Lyngbya by the absence of a
E Nodularia is an occasional producer
of blooms.
1 Vegetative cells, heterocysts,
and even the akinetes are broader
than long.
1 Bartach, A. F. (ed.) En rironmental
Requirements of Blue-Green Algae.
FWPCA. Pacific Northwest Water
Laboratory, Corvallis, Oregon.
111 pp. 1967.
2 Desikachary, T. V. Cyanophyta, Indian
Council Agric. Res. New Delhi. 1G 9.
3 Drouet, Francis. Mxyophyceae. Chapter
5 in Edmondson. Freshwater Biology.
p. 95-114. Wiley. 1959.
4 Drouet, Francis. Revision of the Classifi-
cation of the Oscillariaceae. Monograph
15. Acad, Nat. Sci. Phil. 370 pp. 1968.
5 Jackson, Daniel F. (ed.) Algae, Man, and
the Environment. Univ. Syracuse Press.
554 pp. 1968.
This outline was prepared by L. G. Williams,
Formerly Aquatic Biologist, Aquatic Biology
Activities, Research and Development,
Cincinnati Water Research Laboratory, FWPCA.

A A flagellate is a free swimming cell
(or colony) with one or more flagella.
B Motile flagellated cells occur in most
(not all) great groups of plants and animals.
C Out main concern will be with “mature”
flagellated algae.
A There is a well organized nucleus.
B The flagellum is a long whip-like process
which acts as a propeller.
1 It has a distinctive structure.
2 There may be one or several per cell.
C The chlorophyll is contained In one or
more chioroplasts.
D Two or more cells may be associated In
a colony.
E Non-Motile Life history stages may be
F Size is of little use in identification.
G Pyrenoid bodies are often conspicuous.
m The Euglenophyta or Euglena-Like algae
(Figures 1-4) are almost exclusively Single
celled free swimming flagellates. Nutrition
may be holophytic, holozoic, or saprophytic,
even within the same species. Referred to
by zoologists as mastigophora, many animal
like forms arc parasitic or commensalistic.
Food reserves of plant-like forms are as
paraniylin (an insoluble carbohydrate) and
fats (do not respond to starch test). Thick
walled resting stages (cysts) are common.
“Metabolic movement” characteristics of
some genera ( Euglena) .
Eyespot usually present In anterior end,
rarely more than one flagellum.
A Euglena is a large genus with pronounced
metabolic movement (Figure 1).
1 Cells spindle shaped
2 Single flagellum
3 Eye8pot usually present
4 Chioroplasts numerous, discoid
to band shaped
5 E. sanguinea has red pigment.
6 E. viridis generally favors water
rich lii organic matter.
7 E. gracilis 18 less tolerant of pollution.
B Phacus cells maintain a rigid shape
(Figure 2).
1 Often flattened and twisted, with
pointed tip or tail end.
2 Cell wall (periplast) often marked
with fine ridges.
3 P. pyrurn favored by polluted water.
4 P. pleuronectes relatively intolerant
of pollution.
C Trachelomonas cells surrounded by a
distinct shell (lorica) with flagellum
sticking through hole or collar (Figure 4).
1 Surface may be smooth or rough
2 Usually brown in color
3 Some species such as T. cerebea
known to clog filters
BI.MIC.cla.6c. 3.70

Green and Other Pigmented Flagellates
D Lepocincils has rigid naked cells with
longitudinal or spiral ridges (Figure 3).
1 Cells uncompressed, elipsoidal to oval
(in contrast to phacus)
2 Only two species with pointed tails
3 L te,cta often associated with waters
of high organic content
IV The Qilorophyta or grass green algae
(Figures 5-9) are the largest and most varied
group. Non-flagellated forms predominate but
many conspicuous flagellates are included.
Food reserves are usually stored as starch
which is readily Identified lth Iodine.
Usually two flagella of equal length are
present. More planktonic forms are included
than ui any other group, predominating In the
late spring and early autumn.
The cell Is typically surrounded by a definite
wall and usually has a definite shape. Cell
pigments closely resemble those of higher
plants, but some have accessory pigments
and a few forms have little or none. The
ch]oroplaats always have a shape charac-
teristic of the genus.
The flagellated chiorophyta are contained in
the Order Volvocales, the Volcocine algae.
All are actively motile during vegetative
phases. May be unicellular or colonial. AU
have an eyespot near the base of the flagella.
Colonies may range from a simple plate
( Gonium sociale ) to a complete hollow sphere
( Volvox spp ).
A Chiamydomonas is a solitary free swimming
genus (Figure 5).
1 SpecIes range from cylindrical to
2 Some apecle8 have a gelatinous sheath.
3 There are two flagella inserted close
4 Generally favored by polluted watera.
B Carteria resembles Chiamydomonas very
closely except that it has four flagella
Instead of two. Generally favored by
polluted water (FIgure 7).
C Phacotus usually has free swimming
blflageflate cells surrounded by biconcave
envelopes resembling two clam shells.
These are usually sculptured, dark
colored, and Impregnated with calcium
1 The eyespot ranges from anterior
to posterior.
2 Several daughter cells may be retained
within the old envelopes of the parent
3 A clean water indicator.
D Chiorogonjum Is a distinctive genus In
which the cell Is fusiform, the tail end
pointed, and the anterior end slightly
blunt (FIgure 6).
1 The two flagella only about half as
long as the cell.
2 The cell wall is rather delicate.
3 An eyespot usually present near the
anterior end.
4 Favored by pollution.
E Gonium colonies typically have 4 to 32
cells arranged in a plate (FIgure 8).
1 The cells are Imbedded In a gelatinous
2 Sixteen celled colonies move through
the water with a somersault-like
3 Four and eight celled colonies swim
flagella end first.
4 Gonium pectorale is typically a
plankton form.
F Pandorina colonies range up to 32 celia,
usually roughly spherical (Figure 9).

Green and Other Pigmented Flagellates
1 Cells arranged in a hoLlow sphere
within a gelatinous matrix.
2 Often encountered especially in hard-
water lakes, but seldom abundant.
3 P. morurn may cause a faintly fishy
G Eudorina has up to 64 ceUs in roughly
spherical colonies.
1 The cells may be deeply imbedded in
a gelatinous matrix.
2 Common In the p]ankton of soft water
3 E. elegans is widely distributed.
4 May cause faintly fishy odor.
H Pleodorina has up to 128 celle located
near the surface of the gelatinous matrix.
It is widespread in the United States.
I Volvox rarely has less than 500 cells
per colony.
1 Central portion of the mature colony
may contain only water.
2 Daughter colonies form inBide the
parent colony.
3 V. aureus imparts a fishy odor to the
water when present In abundance.
J Chiamydobotrys has “mulberry shaped”
colonies, with biflagellate cells alternately
arranged in tiers of four each.
( Spon iylomnorum has quadriflagellate cells).
1 There is no enveloping sheath.
2 c. stellata is favored by pollution.
V The Pyrrhophyta includes principally the
armored or dinoflagellates (Dlnophyceae)
(Figures 14-16). This group is almost
exclusively flagellated and is characterized
by chromnatophores which are yellow-brown
in color. Food reserves are stored as
starch or oil. Naked, holozoic, and
saprozoic representatives are found.
Both “unarmored”, and “armored” forms
with chromatophores are found to ingest
solid food readily, and holozoic nutrition
may be as important as holophytic.
The great majority have walls of cellulose
consisting of a definite number of articulated
plates which may be very elaborate In
structure. There is always a groove
girdling the cell In which one flagellum
operates, the other extends backward from
the point of origin.
Most of the dm0-flagellates are marine and
some are parasitic. There are six fresh
water genera of importance in this country.
A Gymnodinum species are generally naked
except for a few freshwater species.
0. brevis (marine) is a toxic form
considered to be responsible for the
“red tide” episodes in Florida and
B Species of Gonyau].ax ( catanel]a and
tamarensis ) are responsible for the
paralytic shellfish poisoning.
C Ceratlum is distinctive in that the
anterior and posterior ends are con-
tinued as long horns (Figure 16).
1 Seasonal temperature changes have a
pronounced effect on the shape of the
cells of this species.
2 c. hlrudinel]a in high concentration is
reported to produce a “vile stench”.
Protect ,ø, Agency
Corvalh Enviyo 90 1 Raseard L ab
2003 W 35thStr
Cnr ia j ,g Oregon 9733Q

Green and Other Pigmented Flagellates
I) I’urlcllriiuixi is a circular, oval, or
..tngular form, depending on the view
(Figure 15).
1 Cell wall is thick and heavy.
2 Plates are usually much ornamented.
3 P. cinctum has been charged with a
fishy odor.
VI The Division Chrysophyta contains two
classes which include flageUates, the
Xanthophyceae or Heterokontae (yellow-
green algae) and the Chrysophyceae (golden-
green algae) (Figures 10-13). The third
class, the diatoms (Bacillarieae or
BaciUariophyceae), is not flagellated.
A None of the Xanthophyceae are included
in the present discussion.
B The Chrysophyceae possess chroma-
tophores of a golden brown color, usually
without pyrenoids. Food reserves are
stored as fats and leucosin. One or two
flagella, if two, they may be of equal or
unequal length. Internal silicious cysts
may be formed. Tend to occur In
relatively pure water. Both holozoic and
holophytic types of nutrition are found.
CertaIn minute forms considered to be
highly sensitive to pollution.
1 Mallomonas is a solitary, free
swimming genus with one flagellum
(Figuze 13).
a Covered with silicious plates, many
of which bear long sillcious spines.
b Tends to inhabit clear water lakes
at moderate depths.
c M. caudata imparts a fishy odor
to the water.
2 Chrysococcue cells are minute, with
two yellowish brown chromatophores
and one flagellum.
a Droplets of stored oil present
b Lorica distinct
c C. rules ceus a clean water form
3 C’iromullna has a single flagellum,
may accumulate single large granule
of leucosin at posterior end of cell
(Figure 10).
C. rosanoffil is a clean water indicator.
4 Synura is a bif]agellate form growing
in radially arranged, naked colonies
(Figure 11).
a Flagella equal in length
b Cells pyriform or egg shaped
c S. uvella produces a cucumber or
muskmelon odor
5 Uroglenopsia forms free swimming
colonies of approximately spherical
biflagellate cells embedded near the
periphery of a roughly spherical
gelatinous matrix.
a Flagella are unequal in length.
b U. americana may range up to
.5 mm in diameter, and contain
1000 or more cells.
c U. am. also causes strong fishy
6 Dinobryon may be solitary or colonial,
free floating or attached. Colonie8
are arborescent (Figure 12).
a Cells attached to bottom of open
roughly cylindrical lorica or sheath.
b Two flagella of unequal length.
c Conspicuous eyespot usually present.
d Taxonomy of the group is involved.
e D. sertularla may clog filters.
f D. divergens may cause a fishy odor.

ireen anu titner k’1 zinented k’Ia ellateS
(fIg 1 - 13 !‘rom Lackey and Callaway)
Lepoc inc 1 is
Ch lorogonium
Chromul ma
DInob yon Mallosonas
Massart in
Cerat ium
Per idinium
Carter Ia

Green and Other Pigmented Flagellates
Figure 17 Phylogenetic Family Tree of the Flagellates
(from CaJaway and Lackey)
V I I There are two distinctive groups whose
systematic position is uncertain, the chioro-
monads and the cryptomonads. Only one
genus of the latter group is included here.
A Rhodomonas may range from bright red
through pale brown to olive green.
1 Celia compressed, narrow at the
posterior end
I Calaway, Wilson T. and Lackey, James
B. Waste Treatment Protozoa
Flage].lata. Series No. 3. Univ. Fla.
140 pp. 1962.
2 Gojdlcs, M. The Genus Euglena.
Univ. of Wisconsin Press, Madison.
2 Two flagella of unequal length
3 R. lacustris a small form intolerant
of pollution
This outline was prepared by H. W. Jackson.
Chief Biologist, National Training Center,
MDS, Water Programs Operations, EPA,
Cincinnati, OH 45268.

I MANY OF THESE FORMS ARE VISIBLE C Specialized structures are present in
TO THE UNAIDED EYE Borne filaments.
A They may be several inches or even a foot 1 Some filaments break up into PIHU
or more in length. In many cases they arc sections.
not found as isolated filaments but develop
in large aggregations to form floating or 2 Apical caps are present In others.
attached mats or tufts. The attached
forms are generally capable of remaIning 3 Replicate end walls are present In
alive after being broken away from the some.
4 Some filaments are overgrown with a
B Included in the group are some of the most cortex.
common and most conspicuous algae In
freshwater habitats. A few of them have 5 Attached filaments have the basal cell
been given common names such as pond developed into a “hold fast cell”
silk, green felt, frog-spawn algae, and (hapteron).
A Cell division may occur in all cells or
A These algae are In the form of cylindrical in certain selected ones.
cells held together as a thread (“filament”),
which may be In large clusters or growing B Spores called akinetes may be formed.
separately. Some are attached to rocks
or other materials while others are free. C Zoospores (motile) and aplanospores
They may be unbranched (“simple”) or (non-motile) are common.
branched, the tips are gradually narrowed
(“attenuated”) to a point. Some are D Fragmentation of filaments may occur.
surrounded by a mucilaginous envelope.
E Many kinds reproduce sexually, often
B Each cell is a short or long cylinder with with specialized gamete forming cells.
a distinct wall. The protoplast contains
a nucleus which is generally inconspicuous.
1 ‘I’he plastid or chloroplast is the ALGAE ARE
prominent structure. It contains
chlorophyll and starch centers A Unbranched forms
(“pyrenoids’), and varies in size,
5 Spirogyra
shape, and number per cell. It may
be pressed against the wall (“parietal”) 5 Mougeotia
or extend through the central axis of
the cell (“axial”). Ulothrix
2 Clear areas of cell sap (“vacuoles”) are Trthonema
generally present In the cell. Desmidium
1 Including a few yellow-brown and red algae. *Planktonic or occasionally planktonlc
BI.MIC.cla. 14b.3.70 8-1

Fi]amentous Green Algae
H Branched forms E Together with other algae, they release
oxygen required by fish, and for sell-
Cladophora purification of streams.
Stigeoclonjum F They may produce a slime which inter-
Chaetophora feres with some Industrial uses of water
Draparnaldia such as In paper manufacture and in
Rhizoclonium cooling towers.
C Specialized and related forms A Ulotrichaceae
Schizomerjs Ulothrix, Microspora, Horniidjum
Batzacitospermurn B Cladophoracese
Lemariea C]adophora Pithophora, Rhizoclonjum
C Chaetophoraceae
V Habitats include the planktonic growths as Chaetoph , Stigeoclonjum, Draparnalcjja
well as surface mats or blankets and benthic
attached forms on rocks in riffles of streams, D Oedogeniaceae
at the shoreline of lakes and reservoirs,
concrete walls, etc. Oedogonjurn, Buibochaste
A Attached forms may break ]oose to E Schizomeridaceae
become mixed with plankton or to form
floating mats. 1 Schizomerjs
B Cladophora mats are a nuisant e on many F Ulvaceae
beaches on the Great Lakes.
Enteromorpha, Monostroma
Z nema. pirogyra, Mougeotla
A They may cause clogging of sand filters,
intake screens, and canals. H Desmidiaceae
B They may produce tastes and odors In Desmidium i yalotheca
water or putrid odor (also producing
H 2 S which damage painted surfaces) when I Tribonemataceae
washed ashore around lakes and reservoirs.
Tribonema, BumUlerja
C They may cause unsightly growths or
interfere with fishing and swimming in J Characeae
recreation areas.
Chara, Nitella, To ypeUa
D Some are useful as indicators of water
quality in relation to pollution.

1 i. GkthJ N ,
,i • i /(. j ?Ip l

Fi]amentoua Green Algae
A Branching and attenuation are of primary
B P]astids shape, location and number per
cell are essential.
C Other characteristics include grouping
of filaments, gelatinous envelope and
special features such as “H” shaped
4 Pal, B. P., Kundu, B.C., Sundarathagam,
V. S., and Venicataraman, G. S.
Charophyta. Indian Coun. Agric.
Res., New Delhi. 1962.
5 Soderatrom, J. Studies in Cladophora .
Almquist, Uppsala. 1963.
6 Tilden. J. The Myxophyceae of North
America. Minn. Geol. Surv.
(Reprinted 1967, J. Cramer, Lehre,
Germany) 1910.
7 Transeau, E.N. The Zygnernataceae.
Ohio State Univ. Press. 1951.
1. Collins, F.S. 1909. The green algae
of North America. Tufts College
Studies, Scientific Series 2:79—480.
Reprinted Hafner Pubi. Co., 1928
(Reprinted. 1968) Lew’s Books,
San Francisco.
2 Farldi, M. A monograph of the fresh-
water species of Dadophora and
Rhizoclonlum . Ph.D. Thesis.
University Microfilms, Ann Arbor.
3 Him, K. E. Monograph of the
Oedogonlaceae. Hafner Pubi.,
New York. 1960.
8 Van der Hoek, C. Revision of the
European species of Cladophora .
Brili Publ, Leiden, Netherlands. 1963.
9 Wood, R. D. and Imahari, K. A revision
of the Characeae. Volume I.
Monograph (by Wood). Vol. II,
Ionograph (by Wood & Imahari). 1964.
This outline was prepared by C. M. Palmer,
Former Aquatic Biologist, In C2iarge ,
Interference Organisms Studies, Micro-
biology Activities, Research and
Development, Cincinnati Water Research
Laboratory, FWPCA.

For the sake of convenience, the non-motile
green algae are to be discussed in two
sectiona’ those that tend to live as relatively
discrete or free floating planktonic units,
and those that tend to grow in masses or
mats of material, often fi]amentous in nature,
attached or free floating.
II The green or “grass green” algae is one
of the most varied and conspicuous groups
with which we have to deal. The forms
mentioned below have been artificially grouped
for convenience according to cell shape.
Botanists would list these genera in several
different categories In the family “Chioro-
These algae typically have a relatively high
chlorophyll content, and the food reserves
accumulated are typically starch. Thus
these forms will usually give a typical black
or deep purple color when treated with iodine.
A Individual cells of the following genera are
perfectly round, or nearly so. The first
does not form organized colonies. In the
next two the colonies themselves tend to
be round, and in the last, the colonies are
triangular or irregular, and the cells bear
long slender spines.
1 Chiordlla cells are small and spherical
to broadly elliptical. They have a
single parietal chlorop]ast. This is a
very large genus with an unknown
number of similar appearing species.
living In a great variety of habitats.
Although often accumulating In great
numbers, organized colonies are not
1 Including miscellaneous yellow-brown algae.
a Chiorella e].lipsoides in reported to
be a common plankton form.
b Chiorella pyrenoidosa and Chiorella
vulgaris are often found in
organically enriched waters.
Indeed a dominance of Chiorella
species is considered In some
placçs to be an indication that a
sewage stabilization pond is func-
tioning to maximum capacity.
c Chlore]la pyrenoidosa is reported
as a filter clogger in water treat-
ment plants.
2 Sphaerocystis colonies are free floating
and almost always with a perfectly
spherical, homogeneous gelatinous
envelope. Up to 32 spherical cells
may be included. Sphaerocystis
scheoeteri , the only species, is of
wide occurrence In the plankton of
lakes and reservoirs.
3 Coe]astrum forms coenobial* colonies
of up to 128 cells. Generally spherical
or polygonal in shape--both cells and
colony. Cells connected by protoplasmic
processes of varying length.
Coe]astrum microporurn is often
reported in the plankton of water
supplies. Not surrounded by gelatinous
envelope as in Sphaerocystis .
4 Micractinium . The cells of this alga
are spherical to broadly ellipsoidal and
are usually united In irregular 4-celled
coenobes. These in turn are almost
always united with other coenobes to
form multiple associations of up to
100 or more cells. The free face of
*A coenobe is a colony in which the number of cells does not increase during the life of the
colony. It was e8tablished by the union of several independent swimming cells which simply
stick together and Increase in size.
B!. MIC. cia. 1k. 3.70
9- 1

Coccoid Green Algae
each cell in a coenobe bear8 from one
to seven very long slender setae or
Micractinium pusillum. This is a
strictly planktonic genus.
B Individual cells of the following genera
are elongate. In the first two they are
relatively straight or Irregular and pointed.
The next two are also long and pointed.
but bent into a tight “C” shape (one in a
gelatinous envelope, one naked). The last
one ( Actinastrum ) La long and straight,
but with blunt ends, and with the cells of
a coenobe attached at a point.
1 Ankistrodesinus cells are usually long
and slender, tapering to sharp point at
both ends. They may be straight,
curved, or twisted into loose aggregations.
Anicistrodesmus falcatus is often found
in the plankton In water supplies and is
considered to be one of the forms
Indicative of clean water.
2 Schroederia is a solitary, free floating
alga. Cells are long and pointed at
both ends. May be bent in various ways.
Terminal points are continued as long
slender spines which may be forked and
bent back, or end as a plate. Of the
three species reported in this country,
Schroederia setigera has been reported
in water supplies.
3 Selenautrum cells are pointed at both
ends, and bent so that their tips approach
each other. They tend to occur in groups
of 4, 8, or 16, which may be associated
with other groups to form masses of a
hundred or more cells. There is no
gelatinous envelope. Selenastrum
gracile occurs in the plankton of water
4 KirchncrieLla . The cells of this genus
are gcncraUy relatively broad, tapering
to a sharp or rounded point at each end,
and the whole cell bent into a C-shape.
They usually occur In groups of four
to eight in a broad, homogeneous,
gelatinous matrix. Klrchneriella
lunaris is known principally from the
5 Actlnastrum colonies or “coenobes”
are composed of 4, 8, or 16 elongate
cells that radiate in all directions from
a common center.
Actinastrum is a widely distributed
plankton organism. There are two
Actinastrum graciflirnum and
Actthastrwn Hantzschil differ only
in the sharpness of the taper toward
the tips of the cells. The former has
relatively little taper, and the latter,
C Cells of the following genera are
associated in simple naked colonies.
The first has elongate cells arranged
with their long axes parallel (although
some cells may be curved). The last
two are flat plate-like coenobes.
Crucigenla has four-celled coenobes
while Pedlastruni coenobes may be
larger, appear plate-like, and are much
more ornate.
1 Scenedesmus is a flat plate of elliptical
to double ended pointed cells arranged
with their long axes parallel. Coenobe
consist of up to 32, but usually 4 to 8
cells. The number of cells in a
coenobe may vary from mother to
daughter colony. The appearance of
cells may vary considerably with the
a Scenedesmus blj iga, S. dimorphu 1
and S. guadricauda are common
planktonic forms.
b Scenedesinus guadricauda is also
common in organicaUy enriched
water, and may become dominant.
c Scenedemus abundans is reported
to impart a grassy odor to drinking

Coccoid Green Algae
2 Crucigenia forms free f1oatin g four-
celled coenobes that are solitary or
joined to one another to form p]ate-
like multiple coenobes of 16 or more
cells. The cells may be elliptical.
triangular, trapezoidal, or semi-
circular in surface view. Cruci enia
guadrata is a species often reported
from water supplies.
3 Pediastrum . Colonies are free floating
with up to 128 polygonal cells arranged
in a single plane. There may or may
not be open spaces between the cells.
The exact arrangement of the cells
seems to depend largely on the chance
distribution of the original motile
swarming zoaspores at the time the
coenobe was formed. Peripheral cells
may differ in shape from interior cells.
a Pediastruni boryanum and , duplex
are frequently found in the plankton,
but seldom dominate.
b Pedlastrum tetras has been reported
to impart a grassy odor to water
D Cells of the following Genera are slightly
1 Oocystis . The cells of Oocystis may
be solitary, or up to 16 cells may be
surrounded by a partially gellatinized
and greatly expanded mother cell wall.
Cells may be ellipsoidal or almost
cylindrical, ceLl wall thai, no spines
or other ornamentation. Oocystis
borgei , for example, is of frequent
occurrence ui the.plankton.
2 Dimorphococcus cells are arranged in
groups of four, and these tetrads are
united to one another in irregularly
shaped free floating colonies by the
branching remains of old mother-cell
walls. Two shapes of cell are normally
found in each tetrad (hence the name), two
longer ovate cells end to end, and a
pair of slightly shorter, C-shaped cells
on either side. Dimorphococcus
lunatus is a widely distributed plankton
organism, sometimes reported In
considerable numbers.
E A distinctive group of green algae
characterized by a median constriction
dividing the cell into two geometrically
similar halves is known generally as the
“desmids.” ( Closterlum and Penium do
not have this construction). Each half
of the cell is known as a “semicell.”
The nucleus lies in the “isthmus.”
Extremes of ornamentation and structural,
variety exist. Most are unicellular, but
a few are filamentous or have the cells
associated In shapeless colonies. They
are found sparingly In the plankton almost
everywhere, but predominate In acid
1 Closterium is one of the exceptional
genera without a median constriction.
The cells are elongate, attenuated
toward the tips but not sharply pointed,
usually somewhat bent.
a Cloeterium aciculare is a p]anktonlc
b Closterium moniliforme is reported
as a filter clogging organism.
2 Cosmarium is a large, poorly defined
genus of over 280 species, many of
which apparently lntergrade with other
genera such as Staurastrum . In
general, it can be said that Cosmarium
species are relatively small, with a
length only slightly greater than the
width, and with a deep median con-
striction. Shapes of the semicells
may vary greatly. Although shallow
surface ornamentation may occur,
long spines do not occur.
a Cosmarium botrytis is reported in
plankton from water supply
b Cosmarium portlanum is said to
impart a grassy odor to water.
c Other species have been reported
to be sufficiently resistant to
chlorine to penetrate rapid sand
filters and occur in distribution
systems in considerable numbers.

Coccoid Green Algae
3 Mlcr eterias is relatively common,
4 Euastrum cells tend to be at least twice
as long as broad, with a deeply con-
stricted isthmus, and a dip or incision
at the tip of each semicell. The cell
wall may be smooth, granulate, or
Euastrum oblongum is reported as a
planktonic species from water reser-
voirs. It has also been noted as
intolerant of pollution, and hence an
indicator of clean water.
5 Staurastrum is the commonest of the
desmids in the plankton of fresh waters;
the genus contains upwards of 245 species
in the United States alone. Inter-
gradation with other genera 8uch as
Cosmarium make it a difficult group
to define. Most of the species are
radially symmetrical, and almost all
have a deeply constricted isthmus.
The cell wall may be smooth, orna-
mented, or splned In a variety of ways.
Relatively long truncated processes
extending from the cell body in
symmetrical patterns are common.
a Staurastrum polymorphum 18 a
typical planktonic form.
b Staurastrum punctu]atum is reported
as an Indicator of clean water.
c Staurastrum paradoxicum causes a
grassy odor in water.
111 A type of “green” alga known as “golden
green” (Xanthophyceae) is represented in the
plankton by two genera. In these a]gae there
is a predominance of yellow over green pig-
ments, hence frequently imparting a yellowish
or golden tint to the cell. Reserve food
material is stored as oil and leucosin, rather
than as starch, hence giving a negative test
with iodine In most cases.
1 The plant body is a free floating colony
of indefinite shape, with a cartiiag-
inous and hyaline or orange-colored
envelope; surface greatly wrinkled
and folded.
2 Individual cells lie close together, in
several aggregates connected in
reticu]ar fashion by strands of the
colonial envelope.
3 The envelope structure tends to
obscure cell structure. Considerable
deep orange colored oil may collect
within the envelope, outside of the
cells, obscuring cell structure.
B Qphiocytjum capitatum like Botryococcus, ,
is widely distributed, but seldom abundant.
1 Both ends of cylindrical ceU are
rounded, with a sharp spine extending
2 Many nuclei and several chioroplasts
are present.
1 Palmer, C. M. Algae in Water Supplies.
Government Printing Office. PHS
Publication No. 657. 1959.
2 Smith, G. S. Phytoplankton of the
Inland Lakes of Wisconsin. Part I.
Bulletin No. 57, Scientific Series
No. 12. 1920.
A Botryococcus braunli is a widely dis-
tributed plankton alga, though it is
rarely abundant.
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
Water Programs Operations, EPA, Cincinnati,
OH 45268.

I GENERAL CHARACTERISTICS 4 Internal shelves (“septae”) extending
longitudinally or transversely.
A Diatoms have cells of very rigid form due
to the presence of silica in the waU. They
contain a brown pigment in addition to the II REPRODUCTION
chlorophyll. Their walls are ornamented
with markings which have a specific pattern A The common method is by cell division.
for each kind Two new half cells are formed between the
halves of the parent cell.
I The ce1l often arc isolated but others
are in filaments or other shapes of B Awcospores and gametes may also be
colonies, formed.
2 The protoplast contains normal cell
parts, the moat conspicuous being the Ill EXAMPLES OF COMMON DIATOMS:
plastids. No starch is present.
A Pennate, symmetrical:
B Cell shapes include the elongate (“pennate”) Navicula
and the short cylindric (“centric”) one view Pinnularia
of which is circular, ynedra
I Pennate diatoms may be symmetrical, Diatoma
transversely unsymetrical, or longitudi- Fragilaria
nally unsymmetrical, Tabeilaria
C Wall is formed like a box with a flanged
cover fitting over it. B Pennate, unsymmetrical:
I “Valve” view is that of the top of the Gothphonema
cover or the bottom of the box. Surirella
2 “Girdle” view is that of the side where Achnanthes
flange of cover fits over the box. Asterionella
3 End view is also possible for pennate
C Centric:
D (‘oil markings include çyc lotella
§ phanodiscus
I Raphe or false raphe extending Sira
lotigit iid inally.
2 Striations whieh arc linec of pores IV Habitats include fresh ar salt water. Both
xtending fiow the area of the raphe to planktonic and attached forms occur, the latter
the margin. Coarse ones are “costae”, often are broken loose. They may be attached
by stalks or by their slimy surface.
3 Nodules which may be terminal and
B!. MIC.cl,i. lOa 8.69 10—1

A Many diatoms are more abundant in late
autumn, winter, and early 8pring than in Synedra
the warmer sea8on. Asterionelj,a
B The walls of dead diatoms generally remain 2 Achnanthjneae, Group with cells
undecomposed and may be common in water. having one false and one true raphe.
Many deposits of fossil diatoms exist.
a Representative genera:
V Importance of diatoms is in part due to Cocconeis
their great abundance and their rigid walls. Achnanthes
A They are the most Important group of
orgarnsms causing clogging of sand filters. 3 Naviculineae. True raphe group with
raphe in center of valve.
B Several produce tastes and odors in water,
including the obnoxious fishy flavor, a Representative genera:
C Mats of growth may cause floors or steps Pinnularia
of swimming poois to be slippery. Stauroneis
PIe u rosigma
D They may be significant in determining Ampluprora
water quality in relation to poUution. Gomphonema
E They release oxygen into the water. Epithemia
VI Classification. There are several thou- 4 Surirelllneae. True raphe group with
sand species of diatoms. Only the most corn- raphe near one aide of valve.
mon of the freshwater forms are considered
here, a Representative genera:
A Centrales Group Nitzsclua
1 Representative genera’ Surirella
Stephanodisc us
Bidduiphia A Some genera are easily recognized by their
distinctive shape.
B Pennals Group B Many genera and most species can be
determined only after diatoms are freed
I Fragilarineae. The false raphe group, of their contents and observed under the
high magnification of an oil immersion
Representative genera lens of the compound microscope.
Tabellaria C Contents of the cell are generally not
Meridion used in identification. Only the char-
Diatoma aracterjstjcs of the wall are used,

D For identification of genera, mQSt im-
portant features include
I Cell shape, and form of colony
2 Raphe and false raphe
3 Striations
4 Septa
E For identification of species, measure-
ments involving the number of striae per
10 microns, the direction of the striae
and many other characteristics may be
I Boycr, C.S. The Diatomaceae of
Philadelphia and Vicinity. J. B. Lippin-
cott Co. Philadelphia. 1916. p 143.
2 Boyer, C. S. Synop8iS of North America
Diatomaceae. Part8 I (1927) and II
(1928). Proceedings of the Academy
of Natural Sciences. Philadelphia.
3 Elmore, C. J. The Diatoms of Nebraska.
University of Nebraska Studies. 21:
22-215. 1921.
4 Hohn, M. H. A Study of the Distribution
of Diatoms in Westcrn New York
State. Cornell University Agricultural
Experimental Station. Memoir 308.
pp 1-39. 1951.
7 Patrick, Ruth and Reimer, Charles W.
The Diatoms of the United States.
Vol. 1 Fragilariaceae, Eunotlaceae,
Achnanthaceae, Na’v-iculaceae.
Monog. 13. Acad. Nat. Sd.
Philadelphia. 888 pp. 1966.
8 Smith, G.M. Class Bacillariophyceae.
Freshwater Algae of the United
States. pp 440-5 10, 2nd Edition.
McGraw Hill Book Co. New York.
9 Tiffany 1 L. H. and Britton, M. E. Class
BaciU.ariophyceae. The Algae of
illinois. pp 2 14-296. University
of Chicago Press. 1952.
10 Ward, H. B. and Whipple. G. C. Class
I, Bacillariaceae (Diatoms). Fresh-
water Biology. pp 17 1-189. John
Wiley & Sons. New York. 1948.
1 Weber, C. I. A Guide to the Common
Diatoms at Water Pollution
SurvejUance System Stations.
FWPCA. CincinnatI. 101 pp.
12 Whipple, G. C.. Fair, G. M., and
Whipple, M.C. Diatomacene.
Microscopy of Drinking Water.
Chapter 21. 4th Edition. John Wiley
& Sons. New York. 1948.
5 Pascher, A. BaciUariophyta (Diatomeae).
Heft 10 in Die Susswasser-FlOra
Mitteleuropas, Jena. 1930. p 466.
6 Patrick, R. A Taxonomic and Ecological
Study of Some Diatoms from the
Pocono Plateau and Adjacent Regions.
Farlowia. 2 143—22 1. 1945.
This outline was prepared by C. M. Palmer,
Former Aquatic Biologist, In Charge,
Interference Organisms Studies, Microbiology
Activities, Research and Development,
Cincinnati Water Research Laboratory,

‘I’here are a number of types of filamentous
hacteria that occur in the aquatic environment.
They inc]uth the sheathed sulfur and Iron
bacteria such as Begg [ atoa. Crenothrix and
Sphaeroiilus , the actinomycetes which are
inic Uu1ar microorganisms that form chains
of cells with .pccIal branchings, and
( aUioneUa , a unicellular organism that
secret s a long twisted ribbon-like stalk.
These tilamentous forms have at times
treated serious problems in rivers,
reservoirs, wells, and water distribution
fleggiatoa is a sheathed bacterium that grows
as a long filamentous form. The flexible
filaments may be as large as 25 microns wide
and 100 microns long (Figure 1)
Begglatos Iba
2-iSp X
up to 1.000p
Transverse separations within the sheath
indicate that a row of cells is included in
one sheath The sheath may be clearly
visible or so slight that only special staining
will indicate that it is present.
The organism grows as a white slimy or
felted cover on the surface of various objects
undergoing decomposition or on the surface
of stagnant areas of a stream receiving
sewage It has also been observed on the
base of a trickling filter and in contact
It is most commonly found in sulfur springs
or polluted waters where H 2 S is present.
I3eggiatoa is distinguished by its ability to
deposit sulfur within its cells; the sulfur
deposits appear as large refractile globules.
(Figure 2)
amente of Beg la1oa
containing granules of
Vhen H S is no longer present in the environ-
ment, t e sulfur deposits disappear.
Dr. Pringsheim of Germany has recently
proved that the organism can grow as a true
autotroph obtaining all its energy from the
oxidation of H 2 S and using this energy to fI.x
CO into all material It can also use
cer ain organic materials If they are present
along with the H 2 S
Faust and Wolfe. and Scotten and Stokes
have grown the organism in pure culture in
this country Beggiatoa exhibits a motility
that is quite different from the typical
flagellated motility of most bacteria, the
filaments have a flexible gliding motion.
FIgure 1
)tA.Ba 12.72

Vi]amentous Bacteria
The only major nuisance effect of Beggtatoa
known has been overgrowth on trickling filters
i eceiving waste waters rich in H 2 S. The
normal microflora of the filter was suppressed
and the filter failed to give good treatment.
Removal of the H 2 S from the water by blowing
air through the water before It reached the
filters caused the slow decline of the
i3eggtatoa and a recovery of the normal
microflora. Begglatoa usually indicates
polluted conditions with the presence of H 2 S
rather than being a direct nuisance.
A. tinomycetes are unicellular microorganisms
1 micron In diameter, filamentous, non-
sheathed, branching monopodlaUy, and
reproduced by fission or by means of special
coriidia. (Figure 3)
Their filamentous habit and method of
sporulation is reminiscent of fungi However,
their size, chemical composition, and other
characteristics are more similar to bacteria.
(Figure 4)
dull end powdery smooth and mucoid
Plgur. 4
Egg albumin leolation plate
A’ an actinomycete colony.
and B’ a bacterial colony
These organisms may be considered as a
group intermediate between the fungi and
the bacteria. They require organic matter
for growth but can use a wide variety of
substances and are widely distributed.
Actinomycetes have been implicated as the
caune of earthy odors in some drinking
waters (Romano and Safferman, Silvey and
Roach) and in earthy smnel]lng substance has
been isolated from several members of the
group by Gerber and Lechevalier. Safferman
and Morris have reported on a method for the
“Isolation and Enumeration of Actinomycetes
Related to Water Supplies.” But the actino-
mycetes are primarily soil microorganisms
and often grow in fields or on the banks of a
river or Jake used for the water supply.
Although residual chlorination will kill the
organisms in the treatment plant or distribution
Appesranc. Appearance
Figurs 3 PlIsminte of Actlnomycet•s
1 1..2

ruamentous tiacteria
ysteni, the odors often are present before
the watt r enters the plant. Use of perman-
gana e oxidation and activated carbon filters
have been most successful of the methods
tried to remove the odors from the water.
Control procedures to prevent the odorous
material from being washed into the water
supply by rains or to prevent possible develop-
ment of the actinomycetes in water rich in
decaying organic matter is still needed.
The filamentous iron bacteria of the
Sphaerotilus- Leptothrlx group, Crenothrlx ,
and Ga]lionel]a have the ability to either
oxidize rnanganous or ferrous ions to manganic
or ferric salts or are able to accumulate
precipitates of these compounds within the
sheaths of the organisms. Extensive growth.
or accumulations of the empty, metallic
encrusted sheaths devoid of cells, have
created much trouble in welle or water dis-
tribution system.. Pumps and back surge
valves have been clogged with muses of
material, taste and odor problem . hive
occurred, and rust colored masses of
material have spoiled products In contact
with water.
Crenothrix polyspora has only been examined
under the microscope as we have never been
able to grow it in the laboratory. The orga-
nism is easily recognized by it. special
morphology. Dr. Wolfe of the University of
Illinois has published photomicrographs of
the organism. (Figure 5)
Organisms of the Sphaerotilus- L.eptothrix
group have been extensively studied by many
investigators (Donderoet. al., Dondero,
Stokes, Waltz and Lackey, Mulder and van
Veen, and Ambcrg and Cormack.) Under
different environmental conditions the mor-
phological appearance of the organism varies.
1’he usual form found in polluted streams or
bulked activated sludge ii Sphaerotilus natans .
(I”igure 6)
Pigurs 5
Chr.mothrix polyspora
cells are very vsriabl in
size trorn small cocci or
polyspores to cells 3x12 a
3-8 X 1.2 - i.ep
Figur. S
Sphaerotlius natans

F’ilavncntous Bacteria
l hib is a sheathcl bacterium consisting of
long, unhranched Illaments, whereby individual
od-shaped bacterial cells are enclosed in a
linear order within the sheath. The individual
cells arc 3-8 microns long and 1.2-1.8
microns wide Sphaerotilus grows in great
masses, at times in streams or rivers that
receive wastes from pulp mills, sugar
refineries, distilleries, slaughterhouses,
or milk processing plants. In these conditions,
it appears as large masses or tufts attached
to rocks, twigs, or other projections and the
masses may vary in color from light grey to
reddish brown In some rivers large masses
of Sphaerotilus break loose and clog water
Intake pipes or foul fishing nets. When the
cells die, taste and odor problems may also
occur in the water
Amberg, Cormack, and Rivers and McKeown
have reported on methods to try to limit the
development of Sphaerotilus in rivers by
intermittant discharge of wastes. Adequate
control will probably only be achieved once
the wastes are treated before discharge to
such an extent that the growth of Sphaerotilus
is no longer favored in the river. Sphaerotflus
grows well at cool temperatures and slightly
low DO levels in streams receiving these
wastes and domestic sewage. Growth is slow
where the only nitrogen present is inorganic
nitrogen, peptones and proteins are utilized
GaLlioneUa is an iron bacterium which appears
as a kidney-shaped cell with a twisted ribbon-
like stalk emanating from the concavity of the
cell. Gallionefla obtains its energy by
oxidizing ferrous iron to ferric Iron and uses
only CO 2 and inorganic salts to form all of
the cell material, it is an autotroph. Large
masses of Gallionella may cause problems
in wells or accumulate in low-flow low-
pressure water mains. Super chlorination
(up to 100 ppm of sodium hypochiorite for
48 hours) followed by flushing will often
remove the masses of growth and periodic
treatment will prevent the nuisance effects
of the extensive masses of Gallionella .
(Figure 7)
1 Faust, L. and Wolfe, R. S. Enrichment
and Cultivation of Begglatoa Ala.
Jour. Bact , 81:99-106. 1961.
2 Scotten, H. L. and Stokes, J. L.
Isolation and Properties of Begglatoa .
Arch Fur Microbiol. 42:353-368.
3 Kowallik, U. and Pringsheim, E.G.
The Oxidation of Hydrogen Sulfide by
Beggiatoa . Amer. Jour of Botany.
53•80l805 1986.
Actlcwmycetes and Earthy Odors
4 Silvey, J.K.G. et.al. Actinomycetes and
Common Tastes and Od3rs. JAWWA,
42•1018-1026, 1950
5 Safferman, R. S. and Morris, M E.
A Method for the Isolation and
Enumeration of Actinomycetes Related
to Water Supplies. Robert A. Taft
Sanitary Engineering Center Tech.
Report W-62-10. 1962.
Figure 7
GellonellA forrugIr ea
05 X 07 -1

Filamentous Bacteria
6 Gerber, N.N. and Lechevaller, H.A.
Geosmin, an Earthy-Smelling Substance
Isolated from Actinomycetes. Appi
Microbiol. l3 135-938. 1965.
Filamentous Iron Bacteria
7 Wolfe. R.S. Cultivation, Morphology, and
Classification of the Iron Bacteria.
JAWWA. 501241-1249. 1958.
8 Kucera, S. and Wolfe, R. S. A Selective
Enrichment Method for GaU.tonella
ferruginca . Jour. f3acteriol. 74344-
349. 1957.
9 Wolfe, I L S. Observations and Studies
of Crenothrix polyspora . JAWWA,
52915-918. 1960.
10 Wolfe, R. S. Micrqbiol. Concentration
of Iron and Manganese in Water with
Low Concentrations of these Elements.
JAWWA. 52 1335- 1337. 1960.
11 Stokes, J. L. Studies on the Fi]amentous
Sheathed Iron Bacterium Sphaerotilus
natans. Jour. Bacteriol. 67:278-291.
12 Waita, S. and Lackey, J. B. Morphological
and Biochemical Studies on the
Organism Sphaerotilus natans . Quart.
Jour. Fin. Acad. Sci. 21(4):335-340.
19 ’ 8.
14 Don:Iero, N. C. Sphaerotilus , Its Nature
and Economic Significance. Advances
Appl. Microbiol. 377-107. 1961.
15 Mulder, E.G. and van Veen, W. L.
Investigations on the Sphaerotilus-
Leptothrix Group. Antonie van
Leewenhoek. 29:121-153. 1963.
16 Amberg, H.R. and Cormack, J.F.
Factors Affecting Slime Growth in
the Lower Columbia River and
Evaluation of Some Possible Control
Measures. Pulp and Paper Mag. of
Canada. 6l:T70-T80. 1960.
17 Amberg, H.R., Cormack, J.F. and
Rivers, M.R. Slime Growth Control
by Intermittarit Discharge of Spent
Sulfite Liquor. Tappi. 45:770-779.
18 McKeown, J.J. The Control of
Sphaerotilus natans . md. Water
and Wastes. 8:(3) 19-22 and
8:(4)30-33. 1963.
13 Dondero, N.C., Philips, R.A. and
Itenkelkian, H. Isolation and
Preservation of Cultures of Sphaerotilus .
Appi. Microbiol. 9:219-227. 1961.
This outline was prepared by R. F. Lewis,
Bacteriologist, Advanced Waste Treatment
Research Laboratory, NERC, EPA,
Cincinnati, OH 45268.

A Description
Fungi are heterotrophic achylorophyllous
plant-like organisms which possess true
nuclei with nuclear membranes and nu-
cleoli. Dependent upon the species and
in some instances the environmental
conditions, the body of the fungus, the
thallus, varies from a microscopic
single cell to an extensive plasrnodium
or mycolium. Numerous forms produce
macroscopic fruiting bodies.
B Life Cycle
The life cycles of fungi vary from simple
to complex and may include sexual and
asexual stages with varying spore types
as the reproductive units.
C Classification
Traditionally, true fungi are classified
within the Division Eumycotina of the
Phylum Mycota of the plant kingdom.
Some authorities consider the fungi an
essentially monophyletic group distinct
from the classical plant and animal
In general, fungi possess broad enzymatic
caparities. Various species are able to
actively degrade such compounds as
complex polysaccharides (e. g., cellulose,
chitin, and glycogen), proteins (casein,
albumin, keratin), hydrocarbons (kerosene)
and pesticides. Most species possess an
oxidative or microaerophilic metabolism,
but anaerobic catabolism is not uncommon.
A few species show anaerobic metabolism
and growth.
A Distribution
Fungi are ubiquitous in nature and mem-
bers of all classes may occur in large
numbers In aquatic habitats. Sparrow
(1968) has briefly reviewed the ecology
of fungi in freshwaters with particular
emphasis on the zoosporic phycomycetes.
The occurrence and ecology of fungi in
marine and estuarine waters has been
examined recently by a number of in-
vestigators (Johnson and Sparrow, 1961;
Johnson, 1968; Myers, 1968; van Uden
and Fell, 1968).
Wm. Bridge Cooke, in a series of in-
vestigations (Cooke, 1965). has estab-
lished that fungi other than phycomycetes
occur in high numbers in sewage and
polbitedwaters. His reports on organic
pollution of streams (Cooke, 1961; 196?)
show that the variety of the Deuteromy-
cete flora is decreased at the immediate
sites of pollution, but dramatically in-
creased downstream from these regions.
Yeasts, in particular, have been found
in large numbers in organically enriched
waters (Cooke, et al. , 1960, Cooke and
Matsuura , 1963; Cooke, 1965b; Ahearn.
et a!. . 1968). Certain yeasts are of
special interest due to their potential
use as “Indicator” organisms and their
abihty to degrade or utilize proteins,
various hydrocarbons, straight and
branch chained alkyl-benzene sulfonates,
fats, metaphosphates, and wood sugars.
B Relation to Pollution
BI. FLJ.6a.5.71

C “Sewage FUngUS Community (Plate I)
A few microorganlbrns have long been
termed sewage fungi. “ The most
common microorganisms inc’uded In
this group are the iron bacterium
Sphaerotilus natans and the phycomy-
cete Leptomitus lacteus.
1 Sphaerotllus natans is not a fungus;
rather it is a sheath bacterium of
the order chiamydobactertales.
This polymorphic bacterium occurs
commonly in organically enriched
streams where It may produce
extensive slimes.
a Morphology
Characteristically. S. natans
forma chains of rod shaped
cells (1. 1 -2. x 2.5 - l? )
within a clear sheath or tn-
chome composed of a protein-
polysacchartdae-lipid complex.
The rod cells are frequently
motile upon release from the
sheath; the flagella are lopho-
trichous. Occasionally two
rows of cells may be present
in a single sheath. Single tn-
chomes may be several mm
in length and bent at various
angles. Empty sheaths, ap-
pearing like thin cellophane
straws, may be present.
b Attached growths
The trichomea are cemented
at one end to solid substrata
such as stone or metal, and
their cross attachment and
bending gives a superficial
similarity to true fungal hyphae.
The ability to attach firmly to
solid substrates gives S. natans
a selective advantage in the
population of flowing streams.
For more thorough reviews of
S.natans see Prigsheim(1949)
arid Stokes (1954).
2 Leptomttus lacteus also produces
extensive a limes and fouling flocs
in fresh waters. This species forms
thalli typified by regular constrictions.
a Morphology
Cellulin plugs may be present
near the constrictions and there
may be numerous granules in
the cytoplasm. The basal cell
of the thallus may possess
b Reproduction
The segments delimited by the
partial constrictions are con-
verted basipetally to aporangia.
The zoospores are dip]anetic
(t. e., dimorphic) and each
possesses one whiplash and one
tinsel flagellum. No sexual
stage has been demonstrated
for this species.
c Distrthution
For further information on the
distribution and systematics
of L. lacteus see Sparrow(1960),
Yerkes (1966) and Emerson and
Weston (1967). Both S. riatans
and L. lacteus appear to thrive
in organically enriched cold
waters (5°-22°C) and both seem
incapable of extensive growth at
temperatures of about 30°C.
d Gross morphology
Their metabolism is oxidative
and growth of both species may
appear as reddish brown flocs
or stringy slimes of 30 cm or
more in length.
e Nutritive requirements
Sphaerotilus natans is able to
utilize a wide variety of organic
compounds, whereas L. lacteus
does not assimilate simple

(Attached “filamentous” and slime growths)
phaeroti1ua natans
Beggiatoa alba
Leptomitus lacteus
/DO ic-
Epistylia 8
ç percu1aria

Figure -
Fusarium equ .aeJ.ic uw ’.
[ LU
(Rluid.er an ’ 1
Ralusiltorat ) Sactailo
Microconidia (A) produced
from phialides a. in CepIsoit ’-
aponism, remaining in slime
ball.. Macroconidis (B). with
to ,.cvrraj cru,.. walk..
produced from collared phial-
idea. I)r.wn from culture.
FIgure 3
C., 0 srtrhiun ,andi4um
link a Persoon
Myrelium with short cells
and artbro.porra. ‘Young by-
pbs (A): and mature .rthro-
spore, (B). Drawn from rul-
FIgure 5
Arhiva ame r i cana humphrey
Ooogonium with three no-
spores (A); young soospor-
.ngtum with ileilmited zoo-
spec. (B); and ,o.aporangia
(C) with released inoapoava
that remain encysted in dun-
tori at the mouth of the dl i.
charge tube. Drawn from cal-
FIgurs Z
Leptomise. Igcteui (Roth)
Calls ad the byphac slow-
ing constrictions with c.llulin
plug.. In one cell large zos
spore. have loan daRseltuL
Redrawn from Color, 192&
F lgit r s4
Zo.pisggaa injidianj
Mycellum with hyphal pegs
(A) on which rotiferi will
become impaled; gcmmae (B)
produced as oceidia on abort
hyphal branches; and roth or
impaled on hyphal peg (C)
from which hypbae have
pown Into the rotifer whose
shell will be discarded after
the contents are consumed.
Drawn from cultuEe.
Ikruiuey, 1i liuin nuiriun to
Fioune 7 I!aplonporidinm costnI . A—mnture .po ,i-;
l3—..——cnrly l)I%St11O(lItIlt.
Figures 1 through 5 from Cooke; F’igures 6 and 7 from Gaitsoif.

Fun f
sugars and grows most luxuriantly in
the presence of organic nitrogenous
3 Ecological roles
Although the “sewage fungi” on
occasion attain visually noticeable
con’entrations , the less obvious
populations of deuteromycete8 may
be more important in the ecology of
the aquatic habitat. Investigations of
the past decade Indicate that numerous
fungi are of primary importance in the
mineralization of organic wastes; the
overall significance and exact roles of
fungi in this process are yet to be
D Predacious Fungi
1 Zoophagus insidians
(Plate II, Figure 4) has been observed
to impair functioning of laboratory
activated sludge units (see Cooke and
2 Arthrobotrys is usually found along
with Zoophagus in laboratory activated
sludge units. This fungus is predaclous
upon nernatodes. Loops rather than
“pegs” are used In snaring nematodes.
In recent classification schemes 1 classes
of fungi are distinguished primarily on the
basis of the morphology of the sexual and
zoosporic stages. In practical schematics,
however, numerous fungi do not dernon8trate
these stages. Classification must therefore
be based on the sum total of the morphological
and / or physiological characteristics. The
extensive review by Cooke (1963) on methods
of isolation and classification of fungi from
sewage and polluted waters precludes the
need herein of extensive keys and species
illustrations. A brief synopsis key of the
fungi adapted in part from Alexopholous
(1962) is presented on the following pages.
This outline was prepared by Dr. Donald G.
Ahearn, Professor of Biology, Georgia State
College, Atlanta, GeorgIa 30303.
PLATE II (Figure 4)

si V 10 liii t 1A (( lit I A\A ur IIJN(,l
I), I no . II alli lacking oi,aio phase a free living Plasmodiunt
‘.uh-phyiurii Myxomycotma (true slime molds) Class Myxomycetes
I tl Is II ‘ma I ly well ii .- lined soniat ic phase n0 a free - living Plaimodium
(true ungi) Sub-phylum Eumycotina
Hyphal filaments usually coenoclytic. rarely septate, sex cells when present forming
nosporeli or /ygospores. aquatic species propagating asexually by zoospores. terrestrial
speci es by Loospores, sporangiospores conidia or conidia-like sporangia ‘Phycomycetes” 3
I he phycomycetes Ire generally considered to include the most primitive of the true
fungi As a whole they encompass a wide dIversity of forms with some showing relation-
ships t . lb. flagellates, while others tiosely resemble colorless algae, and still others
ar , t rue molds 1 he vegetative body Phallus) may be non-specialized and entirely con-
V. rh .1 intO a reproductive organ (holocarpic) or it may bear tapering rhizoids, or be
itivi dial and very extensive The outstanding characteristics of the thallus is a tendency
Ii, Ii . nonseptate and in most groups. multinucliate, cross walls are laid down in vigorously
ing material only to delimit the reporductive organs. The spore unit of nonsexual re-
ductaon 16 borne in a sporangium, and, in aquatic and semiaquatic orders, is provided
iih a single post. rii,r or anterior flagellum or two laterally attached ones Sexual activity
in the phycotnyretce characteristically results in the formation of resting spores
I) ilyplial filaments i hen present septate, o Ithout Loospores, with or without sporangla.
usually iIh conida. sexual reproduction absent or culminating in the formation of asci
or hasiitia . 8
( l l lagettat, it .. its characteristically produced 4
P Flag. bird .-, (Is lacking or rarely produced 7
4 (3) Motile ells tiniliagellat. 5
4’ Motil, elI.. t)iflagellate 6
S 4) /o.ispi.res post. riorly uniflagellate. formed inside the sporangiurn class . Chytridiomycetes
The Chyti idiomycetee produce asexual zoospores with a single posterior whiplash
flagellum The thallue Is highly variable the most primitive forms are unicellular and
holocarpic anti in their early stages of development are plasmodial (lack cell walls), more
advanced forms develop rhizoids and with further evolutionary progress develop mycelium
The principle chemical component of the cell wall is chum, but cellulose is also present
(‘hytrids a. typically aquatic organisms but may be found in other habitats Some species
are i hitinolytii- and/or keratinolytic Chytridi may be isolated from nature by baiting (e g
h,’in 1 , 6, mIs or pine pollen) Chytrids occur both in marine and fresh water habitats and are
of ifliflC c coiiolFiic importance due to their parasitism of algae and animals The genus
D.riiiuey..tt,liiirn niny he provisionally grouped with the chytrids Species of this genus
cause si I i O i i’ i I pideinks of oysters and marine and fresh water fish
5’ Loospores anteriorly uniftagellate, formed inside or outside the sporangium , class
Hyphochyt ridiomycetes
These fungi are aquatic (fresh water or marine) chytrid.like fungi whose motile cells
possess a single anterior flagellum of the tinsel type (feather-like) They are parasitic on
algae and fungi or may be saprobte Cell walls contain chitin with some species also demon-
sI rating cellulose etintent Little information is available on the biology of this class and
at present it is muted to less than LO species
4’) Flagella flea ny • qual. one whiplash the other tin..el class Oomycetes
A numb. r of representatives of the Oomycetes have been shown to have cellulosic cell
walls 1 he n.yceliom is coenocytic. branched and well developed in most cases I he sexual
pro. cas i esuli .i in the formation of a resting spore of the oogamous type i e . a type of
fe rtm Ii, at ion in who h two hete rogametangia come in contact and fuse their contents through
a pore or tub. I he thalli in this class range from unicellular to profusely branched
fil.,mrntous types Must forms are eucarpic, oospores are produced throughout the class
ear. p1 in the more highly advanced species Certain species are of economic Importance due
to their destruction of food crops (potatoes and grapes) while others cause serious diseases of
fish I c g Saprol gina parasitica ) Members of the family Saprolegniaceae are the common

taMer m,,tda anal are .ainuui th, no.et ubiquitous fungi in nature Th order Lagentdiales
ma tail, only a less sp cia ’s Satlia Ii arc parasitic- ian algae small animals and other aquatic
tile I Ii, a .oaI l .llic St ructcit c ’s tat llama acon arc holocarpic and c’ndobmotir rh sewage fungi
nra’ classitmed in the order Leptomitales Fungi of this order are characte rized by the
formation ‘1 r, fractile constrictions a .ellulin plugs occur throughout the thalli or. at least
at iii. ha’o ‘ ‘I hyplmac or to cut oil reproductive structures Lcptomltun lacteus may
produce rather extensive fouling (Inca or slime. in organically enriched tcateru.
Flagella of unequal size 1)0th uhaplash class Plasmodiophoromycetes
Members of this class are obligate t’ndoparasites of vascular plants. algae, and fungi
The thallu. onsists of a plasmodmum ahmch deva lops taithmn the host cclii Nuclear division
at some stages ol the life cycle is of a type found in no other fungi but known to occur in
protn,oa /ouspcarangia which arise directly from the plasmodium bear zoospores with two
unequal .ini, nor lalgella The ci II walls of the fungi apparently lack cellulose
7 II’ ) Mainly saprohic sex cell when present a zygospore class 7 gatmycetes
‘1 his a laip has acell d vcloped mycelium with septa developed in portion, of th
older hyphaa actively growing hyphae are normally non-aeptate The aiexual spores am
non’ntotilc sporangiospores taplanosporesl Such spores lack flagella and are usually
aeriaty allas,’mlnated S xuaI reproduction is initiated by the fusion of two gametangia
with r.’qultant loritcatlon of a thick-wailed, resting spore. the zygospore In the more
advani a at species the sporangia or the sporangiospores arc conidia-like Many of the
Zygornycetes are of economic Importance due to their ability to synthesize commercially
valuable organic acid, and alcohol,, to transform steroids such as cortisone, and to
para.itiee and destroy food crops A few species are capable of causing disease in man
and animals zygiamycosl,l
7’ Obligate conmmensal. of arthropods. zygo.pores usually lacking . . class Trichornycetes
TIta Trichomyceta’. are an Ill -studied group of fungi which appear to be obligate
comma nsal. of arthrupods The trichomycete. are associated with a wide variety of Insects
dipiopods, and crustacea of terrestrial and aquatic (fresh and marine) habitats None of
the members of this class have been cultured in vitro for continued period, of times with any
success Asexual reproduction a by means of eporangiosporea Zygoapores have been
observed in species of scv.ral order,.
8 (LI Sexual spores borne In asci . . ciass . Ascomycetes
In the Ascomycetes the products of meiosis, the aacospores. are borne in sac
like structures te mcccl aset 1 he a.cus usually contains eight ascospores, but the number
produced may vary with the species or strain. Moat species produce extensive septate
nayca, huna 1 hi’ large class is divided into two subclasses on the presence or absence
of an .ascacarp [ he Ilemiascomycetidae lack an ascocarp and do not produce ascogenous
hyph.i. , this ‘cubclasi includes the true yeasts The Euascon,ycetidae usually are divided
Into three serIes (Plertomycetes, Pyrenomycetes, and Diecomycetes) on the basis of
ascoc-arp ‘.t ructure
14 Sexual spores borne on basidia . class Basidiomycetes
The Ilasidiomycetes gene raliy are considered the most highly evolved of the fungi
Karyoganay and nceiosls occur in the baaidium which bears sexual exogenous spores.
hasidiocipores The mushrooms toadstool., rust., and smuts are included in this class
It ,esu,,l stage lacking .Form class Imperfecti ) Deuteromycetes
I h.’ U. Ut, romycetea is a form class for those fungi (with morphological affinities
to the Ascomyceies or Basidiomycete.) which have not demonstrated a sexual stage
The gent-rally employed classification scheme for the.e fungi is based on the morphology
and color of the asexual reproductive stages This scheme ,s briefly outlined below
Newer oncepts of the classification based on conidium development after the classical
acork of S I Ilughes (1953) may eventually replace the gross morphology system (see
Itairron 191,111

Reproduction by mean. of conidia, oidia. or by budding 2
I ’ No reproductive structure, present . Mycelia Sterilia
2 (I) Reproduction by mean, of conidia borne in pycnidla Spharrop .idale .
2’ Conldi&, when formed, net in cycnidia. 3
3 (2) Conldla borne in acervulj . . . . Melanconiales
3’ Conidia borne otherwise, or reproduction by oldie or by budding. . .. Monihales
Reproduction mainly by unicellular budding, yeast-like; mycelial phase. if present.
secondary. srthro.pores occasionally produced. manifest melanin pigmentation lacking 2
I’ Thallus mainly filarnentous, dark melanin pigments sometime, produced . .. 3
2 W liallistospore. produced Sporobolomyceteccac
2’ No ballistospores Cryptococcaceae
3 Conidiophores, it present, not united Into sporodochia or synnemata 4
3 Sporoctochia present Tuberculariaceae
3’ Synnemata present Stilbellaeeae
4 (3) ConidIa and conidiopbore. or oidia hyaline or brightly colored Monhliaceae
4’ Conidia and/or conidiophores, containing dark melanin pigment Dematiaceac

Ahearn. D.G., Roth,F.J. Jr , Meyers. S.P.
Ecology and Charact erization of Yeasts
from Aquatic Regions of South Florida.
Marine Biology 1 291-308. 1968
Barron, G. L. The Genera of Hyphomycetes
from Soil. Williams and Wilkins Co.,
Baltimore. 364 pp. 1968
Cooke, W. H. Population Effects on the
Fungus Population of a Stream.
Ecology 421-18. 1961
__________ A Laboratory Guide to Fungi in
Polluted Waters, Sewage, and Sewage
Treatment Systems. U. S. Dept. of
Health, Education and Welfare, Cincinnati,
132 pp. 1963
__________ Fungi in Sludge Digesters.
Purdue Univ. Proc. 20th Industrial
Waste Conference, pp 6-17. 1965a
__________ The Enumeration of Yeast
Populations in a Sewage Treatment Plant.
Mycologia 57 696-703. 1965b
__________ Fungul Populations in Relation
to Pollution of the Bear River, Idaho-Utah.
Utah Acad. Proc. 44(l) 298-3l5. 1967
__________ and Matsuura, George S. A Study
of Yeast Populations in a Waste Stabilization
Pond System. Protoplasma 57:163-187.
___________ Phaff, H. J., Miller, M. W.,
Shifrine, M , and 1 iapp, E. Yeasts
in Polluted Water and Sewage.
Mycologia 52 210-230. 1960
Emerson, Ralph .ind Weston, W. H.
Aguahndereli.a fermentans Gen. et Sp.
Nov., A Phycomycete Adapted to
Stagnant waters. I. Morphology and
Occurrence in Nature. Amer. J.
Botany 54:702-719. 1967
Hughes, S. J. Conidiophores, Conidia and
Classification. Can. J. Bot.31:577-
659. 1953
Johnson, T. W., Jr. Saprobic Marine Fungi.
pp. 95-104. InAinsworth, G.C. and
Sussman, A . S. The Fungi. III.
Academic Press, New York. 1968
and Sparrow, F.K., Jr. Fungi
in Oceans and Estuaries. Weuul&eiiu,
Germany. 668 pp. 1961
Meyers, S. P. Observations on the Physio-
logical Ecology of Marine Fungi. Bull.
Misaki Mr. Biol. Inst. 12:207-225. 1968
Prigsheim, E.G. Iron Bacteria. Biol. Revs.
Cambridge Phil. Soc. 24:200-245. 1949
Sparrow, F. K., Jr. Aquatic Phycomycetes.
2nd ed. Univ. Mich. Press, AnnArbor.
1187 pp. 1960.
__________ Ecology of Freshwater Fungi
pp. 41-93. InAinsworth, G.C. and
Suegman, A.S. The Fungi, III. Acad.
Press, New York. 1968
Stokes, J. L. Studies on the Filamentous
Sheathed Iron Bacterium SphaerotiLlus
natans . J. Bacteriol. 67:278-291. 1954
van Uden, N. and Fell, J.W. Marine Yeasts.
pp. 167-201. In Droop, M.R. and Wood,
E. J. F. Advances in Microbiology of
the Sea, I, Academic Press, New York.
Yerkes, W. D. Observations on an Occurrence
of Leptomitus lacteus in Wisconsin.
Mycologia 58976-978. 1966
Cooke, William B. and Ludzack, F. J.
Predacious Fungus Behavior in
Activated Sludge Systems. Jour. Water
Poll. Cont. Fed. 30(12)’1490-1495, 1958.
A lexopoulos,
2nd ed.
613 pp
J. C. Introductory Mycology.
John Wileyand Sons, New York,

A Microbial quality constitutes only one
aspect of water sanitation; microchemicals
and radionudlides are attracting increasing
amount of attenticn lately.
B Microbes considered here include bacteria,
protozoa, and microscopic metazoa; algae
and fungi excluded.
C Of the free- Uvlng forms, some are
members of the flora and fauna of surface
waters; others was ied into the water from
air and soil; still others of wastewater
origin, nematodes most commonly from
sewage effluent.
D Hard to separate ‘native” from “foreign”
free-living microbes, due to close
association of water with soil and other
environments; generally speaking, bacteria
adapted to water are tho8e that can grow
on very low concentrations of nutrient
and zoomicrobes adapted to water are
those that feed on algae, and nematodes,
especially bacteria eaters, are uncommon
in water but In large numbers In sewage
E More species and lower densities of
microbes in clean water and fewer species
and higher densities in polluted water.
F Pollution-tolerance or nontolerance of
microbes closely related to the DO level
required in respiration.
C From pollution viewpoint, the following
groups of microbes are of importance:
Bacteria, Protozoa, Nematoda, and
A No ideal method for studying distribution
and ecolo ’ of bacteria in freshwater.
B According to Coil ns, 9 Pseudomonas,
Achrombacter , Alcaligenes, Chromobac-
terium, Flavobacterium , and Micrococcus
are the most widely distributed i i ay be
considered as indigenous to natural
waters. Sulfur and iron bacteria are
more common In the bottom mud.
C Actinomycetes, Bacillus ap. Aerogenes
ap., and nitrogen-fixation bacteria are
primarily soil dwellere and may be washed
into the water by runoffs.
E Nematodes are usually of aerobic sewage
treatment origin.
D E. coil, streptococci, and Cl. perfrlngens
are true indicators of fecal pollution.
A Classification
1 Single-cell animals In the most
primitive phylum (Protozoa) In the
animal kingdom.
2 A separate kingdom, Protista, to in-
clude protozoa, algae, fungi, and
bacteria proposed in the 2nd edition
of Ward-W ipple’s Fresh-Water
Biolo r 0 ’
3 Four subphyla or classes:
a Mastigophora (flagellates)- Subclass
phytomastigtha dealt with under
algae; only subclass Zoomastigina
included here; 4 orders:
1) Rhizomastiglna - with flagellum
or flagella and pseudopodla
2) Protomonadjna - with 1 to 2
fl lj most free-living many
3) Polymastiglna - with 3 to 8
flagella;’ mostly parasitic In
elementary tract of animals
and man
4) Hypermastigtha - all inhabitants
of alimentary tract of insects.
W. BA. 45c. 10.72

Protozoa,_Nernatodes, and Rotifers
b Ciliopliora or infusoria (ciliates) —
no pigmented members, 2 classes:
1) Ciliata - cilia present during the
whole trophic life, containing
majority of the ciliatee
2) Suctoria - cilia present while
young and tentacles during trophic
c Sarcodina (amoebae) - Pseudopodia
(false feet) for locomotion and food—
capturing, 2 subclasses
1) Rhizopoda - Pseudopodia without
axial filaments. 5 orders:
a) Proteomyxa - with radiating
pee udopodia, without test or
b) Mycetozon - forming plasmodium;
resembling fungi in sporangium
c) Amoebina - true amoeba -
forming lobopodia
d Teetacea - amoeba with single
teat or shell of chitinous
e) Foraminifera - amoeba with 1
or more shells of calcareous
nature, practically all marine
d Sporozoa - no organ of locomotion,
ainoeboid in asexual phase, all
B General Morphology
1 Zoomastigina.
Relatively small size (5 to 40 ), with
the exception of Rhizomastigina, the
body has a definite shape (oval, leaf-
like, pear-like, etc.), common members
with 1 or 2 flagella and some with 3, 4,
or more; few forming colonies, ej .:tome
present in many for feeding.
2 Ciliophora:
Most highly developed protozoa; with
few exceptions, a macro and a micro-
nucleus; adoral zone of membranellae,
mouth, and groove usually present in
swimming and crawling forms, some
with conspicuous ciliation of a disc-like
anterior region and little or no body
cilia (stalked and shelled forms);
Suctoria nonmotile (attached) and with-
out cytostome cysts formed in most.
3 Sarcodina:
Cytoplasmic membrane but no cell wall,
endoplasm and ectoplasm distinct or in-
distinct, nucleus with small or large
nucleolus, some with test or shell,
moving by protruding pseudopodia, few
capable of flagella transformation, fresh-
water actinopods usually sperical with
many radiating a.xopodia, some Testacea
containing symbiotic algae and mistaken
for pigmented amoebae; cysts with Biflgle
or double wall and 1 or 2 nuclei.
4 Sporozoa: to be mentioned later.
C General Physiology
1 Zoomastiglna:
Free-living forms normally holozic,
food supply mostly bacteria in growth
film on surfaces or clumps relatively
aerobic, therefore the first protozoa to
disappear in anaerobic conditions and
re-appearing at recovery, reproduction
by simple fission or occasionally by
2 Ciliophora:
Holozoic; true ciliates concentrating
food particles by ciliary movement
around the mouth part, suctoria sucking
through tenacles; bacteria and small

Protozoa, Nematodes, and Roti.fers
algae and protozoa constitute main
food under natural conditions, some
shown in laboratory to thrive on dead
organic matter and serum protein, not as
aerobic as flagellates — some surviving
under highly anaerobic conditions, such
as Metopus , reproduction by simple
lisa ton, conjugation or encyatment.
3 Sarcodina:
Holozoic, feeding through engulfing by
pseudopodia; food essentially same as
for ciliates, DO requirement somewhat
similar to cillates - the small amoebae
and Testacea frequently present in large
numbers in sewage effluent and polluted
water, reproduction by simple fission
and encystation.
A Classification
1 All in the phylum Nemata (nonsegment-
ed round worms); subdivided by s e
authors into two classes:
Secernentea - 3 orders:
Tylenchida, Rhabditida, Strongylida,
and Teratocephalida, with papillae on
male tail, caudal glands absent.
Adenophora - 6 orders:
Araeolaimida, Dorylaimida,
Chromdorida, Monhysterida, Enoplida,
and Trichosyringida no papillae on
male caudal glands absent.
2 Orders encountered in water and sewage
treatment - Free-living forms inhabitat-
ing sewage treatment plants are usually
bacteria-feeders and those feeding on
other nematodes; those inhabitating clean
waters feeding on plant matters; they
fall into the following orders:
3 Tyleachida - Stylet in mouth; mostly
plant parasites; some feed on
nematode such as Aphelenchoides .
4 Rhabthtida - No stylet in mouth or caudal
glands In tail; mostly bacteria-feeders;
common genera: Rhabiditia, D plogaster,
Diplogasteroides, Monochoidéi , 1 elodera,
Panagrellus , and Turbatrix .
5 Dorylaimida - Relatively large nematodes;
stylet in mouth; feeding on other nematodes,
algae and probably zoomlcrvbes; Dorylaimus
common genus.
6 Chromadorida - Many marine forms,
some freshwater dwellers feeding on
algae, characterized by strong orna-
mentation of knobs, bristles or
punetations in cuticle.
7 Monhysterida - Freshwater d l1ers,
esophago-intestinal valve spherical to
elongated; ovaries single or paired,
usually straight; common genus in
water - Monhyatera .
8 Enoplida - Head usually with a number
of setae; Cobb reported one genus,
Mononchulus , in sand filters in
Washington, D. C.
B General Morphology
Round, slender, nonsegmented (transverse
markings in cuticle of some) worms,
some small (about mm long, as Ti -i-
cephalobus) , many ito 2 mm long
( Rhabditis, Dipjo aater . and Diplogasteriodes
i or instance), and some large (2 to 7 mm,
such as Dorylalmus) , sex separated but few
parthenogenetic, complete alimentary canal;
with elaborate mouth parts with or without
stylet, complete reproductive system in
each sex, no circulatory or respiratory
system, complex nervous system with
conspicuous nerve ring across oesophagus.
C General Physiology
1 Feeding - Most sewage treatment plant
dwellers feeding on bacteria, others
preying on protozoa, nematodes, rotifers,

Protozoa, Nematodes, and Ftotifers
etc., clean-water species apparently
vegetarians, those with stylet in mouth
use the Latter to pierce the body of aqimal
or plant and suck contents, metabolic
waste mostly liquid containing ammonium
carbonate or bicarbonate, enteric
pathogens swallowed randomly with
suspending fluid, hence remote possi-
bility of sewage effluent-borne nematodes
being pathogen-carriers.
2 Oxygen requirement - DO apparently
diffused through cuticle into body. DO
requirement somewhat similar to
protozoa. Rhabditis tolerating reduced
DO better than other Rhabditida members;
all disappear under sepsis in liquid; some
thrive in drying sludge
3 Reproduction - Normal life cycle requires
mating, egg with embryo formation,
hatching of eggs inside or outside femal8,
4 larval stages, and adult, few repro-
duce in the absence of males.
A Classification
1 Classified either as a class of the phylum
Aschelminthes (various forms of worms)
or as a separate phylum (Rotifera); com-
monly called wheel animalcules, on
account of apparent circu]ar movement of
cilia around head (corona); corona con-
tracted when crawling or swimming and
expanded when attached to catch food.
2 Of the 3 classes, 2 (Seisonidea and
Bdelloidea) grouped by some authors
under Digononta (2 ovaries) and the
other being Monogononta(lovary);
Seisonidea containing mostly marine
3 Class Digononta containing 1 order
(Bdelloida) with 4 families, Ph.ilodinedae
being the most important.
4 Class Monogononta comprising 3 orders:
Notommatida (mouth not near center of
corona) with 14 families, Floscularida
Me]icertida(corona with two wreaths of
cilia and furrow between them) with 3
families, most import genera included
in the order Notommatida: Brachionus ,
Keratella, Monostyla, Trichocerca,
A splanchna, Polyarthra, Synchaeta,
Microcodon ; common genera under the
order Flosculariaceae Floscularia ,
and Atrochus . Common genera under
order Melicertida: Ltmnias and
5 Unfortunately orders and families of
rotifers partly based on character of
corona and trophi(chewing organ),
which are difficult to study, esp. the
latter; the foot and cuticle much easier
to study.
B General Morphology and Physiology
1 Body weakly differentiated into head,
neck, trunk, and foot, separated by
folds, in some, these regions are
merely gradual changes in diameter
of body and without a separate neck,
segmentation external only.
2 Head with corona, dosal antenna, and
ventral mouth; mastax, a chewing organ,
located in head and neck, connected to
mouth anteriorly by a ciliated guilet and
posteriorly to a large stomach occupying
much of the trunk.
3 Common rotifers reproducing partheno-
genetically by diploid eggs; eggs laid in
water, cemented to plants, or carried
on female until hatching.
4 Foot, a prolongation of body, usually
with 2 toes, some with one toe, some
with one toe and an extra toe-like
structure (dorsal spur).
5 Some, like Philodina , concentrating
bacteria and other microbes and minute
particulate organic matter by ciliary
movement on corona larger microbes
chewed by mastax; some such as
Monostyla feeding on clumped matter,
such as bacterial growth, fungal masses,
etc. at bottom; virus generally not
ingested - appar ntly undetected by
6 DO requirement somewhat similar to
protozoa, some disappearing under
reduced DO, others, like PluLodina ,
surviving at as little as 2 ppm DO.

Protozoa, Nematodes , and Rotifers
A Pollution tolerant and pollution non-
tolerant species - hard to differentiate -
requiring specialist training in protozoa,
nematodes, and rotifers.
B SAgnificant quantitative difference in clean
and polluted waters - clean waters con-
taining large variety of genera and species
but quite low in densities.
C Aerobic sewage treatment processes
(trickling filters and activated sludge
processes, even primary settling) ideal
breeding grounds for those that feed on
bacteria, fungi, and minute protozoa and
present In very large numbers; effluents
from such processes carrying large nurn-
bers of these zoomicrobes; natural waters
receiving such effluents showing significant
increase in all 3 categories.
D Possible Pathogen and Pathogen Carriers
1 Na ia causing swimming associated
menlngto encephalitis and Acanthameoba
causing nonswimmlng associated cases.
2 Amoebae and nematodea grown on
pathogenic enteric bacteria in lab; none
aUve in amoebic cysts; very few alive
in nematodes after 2 days after ingestion;
virus demonstrated in nematodes only
when very high virus concentrations
present, some freeliving amoebae
parasitizing humans.
3 Swimming cLliatea and some rotifers
(concentrating food by corona) ingesting
large numbers of pathogenic enertic
bacteria, but digestion rapid; no
evidence or concentrating virus; crawling
dillates and flagellates feeding on clumped
4 Nematodes concentrated from sewage
effluent in Cin lnnati area showing
liveE. coil and streptococci, but no
human eiiertic pathogens.
A Bacteria - not dealt here.
B Protozoa and rotifers - should be included
in examination for planktonic microbes.
C Nematodes
D Laboratory Apparatus 3
1 Sample_Bottles - One-gallon glass or
plastic bottles with metal or plastic
screw caps, thoroughly washed and
rinsed three times with distilled water.
2 C ptilary Pipettes and Rubber Bulbs -
t ng (9 In.) Pasteur capillary pipettes
and rubber bulbs of 2 ml capacity.
3 Filtration Unit - Any filter holder
assembly use cbin bacteriological
examination. The funnel should be
at least 650 ml and the filter flask at
least 2 liter capacity.
4 Filter Membranes - Millepore SS ‘SS
047 MMltype membranes or equivalent.
5 Micro e - Binocular microscope
with lox eyepiece, 4X, lOX, and 43X
objectives, and mechanical stage.
E Collection of Water Samples
Samples are collected in the same mannef 1 )
as those for bacteriological examination,
except that a dechiorinating agent is not
needed. One-half to one gallon samples are
collected from raw water and oae-gallon
samples from tap water. Refrigeration is
not essential and samples may be transported
without it unless examination is to be delayed
for more than five days.
F Concentration of Samples
1 One gallon of tap water can usually be
filtered through a single 8-u membrane
withIn 15 minutes unless the water has
high turbidity. At least one gallon of
sample should be used in a single examina-
tion. Immediately after the last of the
water Is disappearing from the membrane,
the suction line is disconnected and the
membrane placed on the wall of a clean
50 to 100 ml beaker and flushed repeatedly
with about 2-5 ml of sterile distilled water

Protozoa, N’matodes, and itotifers
with the aid of a capillary pipette and a
rubber bulb. The concentrate Is then
pipetted into a clean Sedgewick-Rafter
Counting Cell and is ready for examina-
2 In concentration of raw water samples
having visible turbidity, two to four
8-micron membranes may be required
per sample, with filtration through each
membrane being limited to not more
than 30 minutes. Samples ranging from
500 ml to 2 lIters may be filtered with
one membrane, depending on degree of
turbidity. After filtration the membranes
are placed on the walls of separated
beakers and washed as above. To
prevent the particulates from obscuring
the nematodes, the washing from each
filter is examined in a separate counting
G Direct Microscopic Examination
Each counting chamber containing the
filter concentrate is first examined under
a 4X objective. Unless the concentrate
contains more than 100 worms, the whole
cell area is surveyed for neinatodes, with
respect to number, developmental stage,
and motility. When an object having an
outline resembling that of a nematode is
observed, it is re-examined under a lox
objective for anatomical structures, unless
the object exhibits typical nematode move-
ment, which is sufficient for identifying the
object as a neniatode. When the concentrate
contains more than 100 worms, the worm
density can be estimated by counting the
number of worms in representative micro-
scopic fields and multiplying the average
number of worms per field by the number
of fields In the cell area. The nematode
density may be expressed as number of
worms per gallon with or without differenti-
ation as to adult or larval stages or as to
H General Identification of Nematodea
I While actively motile nematodes can be
readily recognized by any person who
has some general concept of micro-
scopic animals, the nonmotile or
sluggishly motile nematodes may be
confused with root fibers, plant fila-
ments of various types, elongated
ciliates such as Homajpzppn vermi-
ia1are , or segments of appendages of
small cruetacea. To facilitate a
general identification of nematodes, the
gross morphology of three of the free-
living nematodes that are frequently
found in water supplies is shown in the
attached drawing. The drawing provides
not only the general anatomy for recogni-
tion of neinatodes but also most of the
essential structures for guidance to those
who want to use the “Key to Genera” in
chapter No. 15 on Nemata by B. G.
Chitwood and M. W. Allen in the book,
Fresh Water Biology . (10)
2 Under normal conditions, practically
all nematodes seen in samples of
finished water are in various larval
stages and will range from 100 to 500
microns in length and 10 to 40 microns
in width. Except in the fourth (last)
stage, the larvae have no sexual organs
but show other structural characteristics.
3 If identification of genera is desired,
the filter washings are centrifuged at
500 rpm for a few minutes. The
supernate is discarded, except a few
drops, and the sediment is resuspended
in the remaining water. A drop of the
final suspension is examined under both
lox and 43X objectives for anatomical
characteristics without staining, and for
supplementary study of structures the
rest is fixed in 5% formahn or other
fixation fluid and stained according to
iistructions given in Chitwood and
Allen’s Chapter on Nemata,( )
Goodey’s Soil and Freshwater Nema-
todes(l1) or other books on nematology.
A Idea not new, protozoa suggested long ago,
many considered impractical because of
the need of identifying pollution-intolerant
and pollution-tolerant species - proto-
zoologist required. Method also time
1 —6

Protozoa, Nematodes, and Roti.fers
B ( ‘ iri tuti Lit nt Ott a lu.trbLittLtiv . battis —
n4 mt1to(I4 II. rtnd nrinplgm’ntr d
In ()to7.oa prFHlnt In Hn a11 numbei s in
tI ..tn w iti. Nunthi i H grtatly increased
whri polluh with filuent from aerobic
tteatment plant or recovering from sewage
pollution, no significant error introduced
when clean-water members Included in the
enumeration if a suitable method of com-
puting the pollution index developed.
C Most practical method involves the
equation: A+B + 1000 C Z. P.!.,
A number of pigmented protozoa,
B non pigmented protozoa, and
C nematodes in a unit volume o sample,
arid Z. P.1. • zoological pollution index.
For relatively clean water, the value of
Z.P.I. close to 1, the larger the value
above 1, the greater the pollution by aerobic
effluent (see attached report on zoomicrobial
indicator of water pollution).
A Chlorination of effluent
B Prolongation of detention time of effluent
C Elimination of slow sand filters in
nematode control.
Sarcodina - Amoebae
Amoeba p teus, Aradiosa
Hartm anne 1 Ia
Arcella Vu garis
Noegleria gruberi
Actinoph —
Hodo caudatus
Pleuromonas j _ aculans
Oikomonas terrno
Cercomonas longicauda
Peranema trichophorium
Swimming type
Colpidium colpoda
poda cuculus
Glaucoma pyriformis
Paran-iecium candatum , P bursaria
Stalked type
Qpercularia ep. (short stalk dichotomous)
Vorticella sp. (stalk single and contractile)
‘ jpUcati.us (like opercularla, more
colonial, stalk not contractile)
Carcheejuin sp. (like vorticel]a but colonial,
— individual zoolds contractile)
Zoothamniuin sp. (entire colony contracts)
Crawling type
Euplotes ateUa
Sty chia mylitus
! ! 2 t 8p.
Diplogaster sp. Doryl mus sp.
Monocholdes sp. Chlindrocorpussp.
g teroldes sp. Cephalobus sp.
Rhabditia ep. Rhabditolaimug sp.
p hyst sp.
Aphelenchoides sp.
Trilobus sp.

Protozoa. Nematodes. and Rotifers
i2 & r
r th_
Phi lodtha
OLIGOCHAETA (bristle worms)
he cnp rich!
Aul phorus limos a
Tubifex tubifex
Lumbricillus Uneatua
Psychoda ep. (trickling filter fly)
Lessertia sp.
Po:rhomma sp.
Achoratus subuiaticus (collemb3la)
Foleornia sp. (collembola)
Tomocerus ap. (collemb.,la)
1 American Public Health Association 1
American Water Works Association and
Water Pollution Control Federation.
Standard Methods for the Examination
of Water and Wastewater , 13th ed.
New York. 1971.
2 Chang, S. L., et al. Survey of Free-
Living Nematodes and Amoebas In
Municipal Supplies. J.A.W.W.A. 52:
3 Chang, S. L. Interactions between Animal
Viruses and Higher Forms of Microbes.
Proc. Am. Soc. Civ. Eng. .fl. San. Eng.
96:151. 1970
4 Chang, S. L. Zoomicrobiai Indicators
of Water Pollution presented at the
Annual Meeting of Am. Soc. Microbial 1
Philadelphia, April 23-28, 1972.
5 Chang, S. L. Pathogenic Free-Living
Amoebae and Recreational Waters.
Presented at 6th International Confer-
ence of Water Pollution Research
Association, Jerusalem, Israel,
June 19-24, 1972.
6 Chang, S. L. Proposed Method for
Examination of Water for Free-Living
NematodeB. J.A.W.W.A. 52:695-698.
7 Chang, S. L., et al. Survival and Protection
Against Chlorination of Human Enteric
Pathogt ns in Free- Living Nematodes
Isolated from Water Supplies. Am. Jour.
Trop. Med. and Hyg. 9:136-142. 1960.
8 Chang, S. L. Growth of SmaU Free- Living
Amoebae in Bacterial and Bacteria-Free
Cultures. Can. J. Microbial.
6:397-405. 1960.
9 Chang, S. L. and Kabler, P. W. Free- Living
Nematodes In Aerobic Treatment Plant
Effluents. J.W.P.C.F. 34:1256-2161.
19 3.
10 Chitwood, B. G. and Chitwood, M. B. An
Introduction to Nemato . Section I:
Anatomy. 1st ed. Monumental Printing
Co. Baltirmre. 1950. pp 8-9.
11 Cobb, N. A. Contributions to the Science of
Nematciogy VII. Williams and Willdns Co.
BaltImore. 1918.
12 CollIns, V. G. The Distribution and Ecology
of Bacteria In Freshwater, Pts. I & II,
Proc. Soc. for Water Treatment and Exam.
12:40-73. 1963. (England)
13 Edmondson, W.T., et al. Ward-Wh pp1e’s
Fresh Water B1o y. 2nd ed. Joon
Wiley & Sons, New York. 1958. pp 368- 401.

______ Protozoa, Nematodes, and Rotifers
14 Goodey, T. Soil and Freshwater
Nematodes . (A Monograph) let ed.
Methuen and Co. Ltd. London. 1951.
This outline was prepared by S. L. Chang,
Chief, Etiology, Criteria Development
Branch, Water Supply Research Laboratory,
NERC, EPA. Cincinnati, OH 45268.
I —3

Protozoa, Nematodes, and Rotifera
Food Chain In Aerobic Sewage Treatment Processes

A Importance of Recognizthg Small,
Free- Living Amoebae In Water
1 Commonly found in soil, aerobic
sewage effluent and natural, fresh
waters - hence, frequently en-
countered In examination of raw
2 Cysts not infrequently found in
municipal supplies - not pathogen
3 Flagellate-amoebae Naegleria
Involved In 50 some cases of
mentngoencepha].ttis, about half
In the U.S.; associated with
swimming in small warm lakes.
Acanthamoeba rhy des parasitizing
hymen throats and causIng (3 cases)
nonewimmlng- associated menthgo-
4 Cysts not to be confused with those
of Endamoebahistoly ica in water-
borne epidemics.
B Classification of Small, Free- Living
1 Recognized classification based
on characte -istics in mitosis.
2 Common species fall into the
following families and genera:
Family Schizopyrenidae: Genera
Naegler1a Dldascalus and
Sch jrenus - first two being
flagellate amoebae.
Family Hartmanndllidae: Genera
a J ( )
3 How to prepare materials for
studying mitosis - Feulgen stain
C Morphological Characteristics of
Small, Free-Living Amoebae
1 Morphology of Trophozoites -
Ectop]asm and endoplasm usually
distinct; nucleus with large nucleolus.
2 Morphology of cysts - Single or
double wall with or without pores
D Cultural Characteristics of SmaU,
Free- Living Amoebae
1 How to cultivate these amoebae -
plates with bacteria; cell cultures,
axenic culture.
2 Growth characteristics on plate,
cell, and axenic culture
3 Complex growth requirements
for most of these amoebae
E Resistance of Amoebic Cysts to
Physical and Chemical Agents
A Classification of Those Commonly
Found in Water Supplies
1 Phasmidia (Secerneutes):
Genera Rhabditis , Di plogaster
Dipl g gteroides , heilob ,
2 Aphasmidia (Aderiop’ioro): Genera
Monhyst he1enchus, Turbatrix
(vinegar eel), Dorylaimus, and
B Morphological Features
1 Phasmlds: paplua on tail of males,
mouth adapted to feed on bacteria,
few exceptions.
2 Aphasmids: no papilla on male tail;
glandular cells in male.
BI. AQ. 14b. 10. 72

Amoebae and Nernatodea in Water SUDDIIeS
C Life Cycle
1 Methods of mating
2 Stages of develop nent
3 Parthenogenesis
D Cultivation
1 Bacteria-fed cultures
2 Axenic cultures
E Occarren e in Water Supplie8
1 Relationship between their
a earance in fthi hed water
and that In raw water.
2 Frequency of occurrence In
different types of raw water
and sources.
3 Szrvival of human enteric path-
ogeriic bacteria and viruses In
4 Protection of human enteric
pathogenic bacteria and viruses
in -iematode-carrjers.
F Control
1 ChlorInation of sewage effluent
2 Flocculation and sedimentation
of water
3 chlorination of water
4 Other methods o destruction
1 SLngh, B. N., “Nuilear Division In Nine
Species of Small, Free- Living Amoe-
bae and its Bearing on the Classifica-
tion of the Order Amoebida”, Ph11o .
Tra’is. Royal Soc. London, Series B,
236:405-461, 1952.
2 Chang, S. L., et al. “Survey of Free-
Living Nematodes and Amoebas In
Mw iclpal Supplies”. J. A. W . W. A.
52:613-618, 1960.
3 Chang, S. L., “Growth of Small Free-
Living Amoebae In Various Bacterial
and In Bacteria-Free Cultures”. Can.
Jour. Mlcrobiol. 6:397-405, 1960.
Ne rnatodes
1 Goodey, T., “Freshwater Nematodes”,
1st. Edition, Methuen & Co., London,
2 Edmondson, W.T., Ed., Ward & Whipple’s
“Fresh-Water Biology” 10th Edition,
page 397, 1955.
3 Chang, S. L., et al., “Occurrence of a
Nematode Worm in a City Water Supply”.
J.A.W.W.A., 51:671-676, 1959.
4 Chang, S. L., et al., “Survival, and
Protection Against Chlorination, of
Human Enteric Pathogens In Free-
Living Nematodes Isolated From Water
Supplies”. Am. Jour. Trop. Medicine
& Hygiene, 9:136-142, 1960.
5 Chang, S. L., et ai., “Survey of Free-
Living Nematodes and Amoebas In
Municipal Supplies”. J.A.W.W.A.,
52:613-618, 1960.
6 Chang, S. L., “Proposed Method for
Exa n1nation of Water for Free- Living
Nematodes”. J. A. W. W. A., 52:695-698,
7 Chang. S. L., “Viruses, Amoebas,
and Nematodea and Public Water
Supplies”. J.A.W.W.A., 53:288-296,
8 Chang, S. L., and Kahier, P. W., “Free-
Living Nematodes In Sewage Effluent
from Aerobic Treatment Plants”. To
— be published . _____ _____
This outline was prepared by Shlh L. Chang,
M. D., Chief, Etiology, Criteria Development
Branch, Water Supply Research Laboratory,
NERC, EPA, Cincinnati. 3H 45268.

Suhphylunv Sarcoclina Hertwig and Lesser
Class Rhizopoda von Siebold
S bc1ass Amoebaea i3utschli
Order: Amoebida Calldns and Ehrenberg
Superfamily Amoebaceae - free-living
(Endamoebaceae - parasitic in animals)
FaJTuly: Schizopyrentdae - active limax form common; transcient
flagellates present or absent; nucleonus-origin of
polar masses, polar caps and interzonal bodies present
or absent
Genus: Sch yrenus - no trariscient flagellates, single-walled
cysts; no polar cape or fnterzonal bodies in mitosis
Species S. thaenusa - reddish orange pigment formed in agar
cultures with gram-negative bacillary bacteria
S. r isselJi - no pigment produced in agar cultures
Genus: Didascalus- morphology and cytology similar to Schiz!pyrenus
but small numbers of transcient flagellates formed at times
Species: D. thorntoni- only species described by Slngh (1952)
Genus: Na 1eria Alexe eff - double-walled cysts; transcient
flagellates formed readily; polar caps and interzonal
bodies present In mitosis
Species N. Lruberi (Schardinger) - only species established;
Slngh (1952) disclaimed the N. solihe described In 1951
Family Hartmannellidae - no transcient flagellate formed; motility
sluggish; no lirnax form, nucleolus dlsappearing probably
forming spindle In mitosis, no polar caps or masses 1 aster
and cehtrosome not known
Genus HartrnanneUa - ectoplasm clear or less granular than
endoplasm, single- walled cysts; single vacuole
Species: H. bae - clear ectoplasm
H. g ico1a - ectoplasm less granular than endoplasm
Genus Acanthamoeba - filamentous processes from ecto- or
endoplasm, growing axenically in flutd bacteriological
n ,edla

S iggc sted Classification of Small Amoebae
Species: A. j sode8
Genus: 1n he],la - double-walled cysts; ecto- and endoplasm
1ndistingu ahable; many vacuoles
Species: Sth ea1eptomemus

A Flanktonic animals or zooplankton are
found in nearly every major group of
I Truly planktonic species (euplankton)
spend all or most of their active life
cycle suspended in the water. Three
groups are predominantly involved in
fresh water; the protoroa, rotifers,
and microcrustacea.
2 Transient planktonic phases such as
floating eggs and cysts, arid larval
stages ocrur in many other groups.
B Many forms arc strictly seasonal in
C C’crtairi rare forms c,ccur in great numbers
at unpredictable Intervals.
D Techniques of collection, preservation,
and identification strongly influence the
species reported.
E In oceanographic work, the zooplankton is
considered to include many relatively large
animals such as siphonophores. ctenophores,
hepteropods, pteropods, nrrowworms, and
euphausid shrimp.
F The plant-like or phytoplankton on the
other hand are essentially similar in all
waters, and are the nutritional foundation
for the animal community.
A The three typically free living classes.
Mastigophora, Rhizopoda, and Ctllophora,
all have planktonic representatives As
a group however, the majority of the phylum
is benthic or bottom-loving. Nearly any
of the benthic forms may occasionally be
washed up into the overlying waters and
thus be collected along with the cuplankton
B Class mastigophora, the nonpigrnented
These have frequently been confused with
the phytomastigina or plant-like flagellates.
The distinction is made here on the basis
of the presence or absence of chlorophyll
as suggested by Palmer and Ingram 1955.
(Note Figure: Nonpigmented, Non-Oxygen
Producing Protozoan Flagellates In the
outline Oxygen Relationships.)
1 Commonly encountered genera
2 Frequently associated with eutrophic
C Class Rhizopoda - arnoeboid protozoans
I Forms commonly encountered as
Pt rcella
E uglypha
Helio oa
2 Cysts of some types may be encountered
in water plants or distribution systems;
rarely in plankton of open lakes or
D Class Cihophora
1 Certain Tattachedh forms often found
floating freely with plankton:
C arc he slum
2 Naked, unattached dilates Halteria
one of commonest in this group. Various
heavily ciliated forms (holotrich ) may
occur from time to time such as
Colpidlum, Enchelys , etc.
3 Ciliates protected by a shell or test
(teataceous) are most often recorded
from preserved samples. Particularly
common in the experience of the National
Water Quality Sampling Network are:
Codonella fluviatile
Codonella cratera
Tintinnidlum (usually with organic matter)
Tintinno ig
BI.AQ.20e.4 70
15 —I

Animal l’lankton
A Some forms such as Anuraea cochlearis
and p anchr1a pridóiita tend to be present
at alTUines oUthe year. Others such as
Notholca striata, N. longispina and Poly-
arthra pIatyp1 Fä are reported to be eases-
ti&LLy wInter forms.
B Specie8 in appro .mate order of descending
frequency currently recorded by National
Water Quality Sampling Network are:
Keratella cochlearis
Polyarthra vulgaris
Synchaeta pectinata
Brachionu8 guadridentata
Trichocerca longiseta
r l a p.
Filinia longiseta
Kellicottia longispina
Pornpholyx sp .
C Benthic species almost without number may
be collected with the plankton from time to
A Class Crustacea
1 The Class Crustacea includes the larger
common freshwater euplankton. They
are also the greatest planktonic consum-
ers of basic nutrients in the form of
phytoplankton, and are themselves the
greatest planktonlc contribution to the
food of fishes. Most of them are herb-
ivorous. Two groups, the cladocera
and the copepods are most conspicuous.
2 Cladocera (Subcla8a Branchiopoda,
Order Cladocera) or Water Fleas
a Life History
1) During most of the year, eggs
which will develop without fertil-
ization (parthenogenetic) are
deposited by the female in a dorsal
brood chamber. Here they hatch
into minature adults which escape
and swim away.
2) As unfavorable conditions develop,
males appear, and thick-walled
sexual eggs are enclosed in egg
cases called ephippia which can
often endure freezing and drying.
3) Sexual reproduction may occur
at different seasons in different
4) Individuals of a great range of
sizes, and even ephippia, are
thus encountered in the plankton,
but there is no “larval” form.
b Seasonal variation - Considerable
variation may occur between winter
and summer forms of the same
species in some cases. Similar
variation also occurs between arctic
and tropical situations.
c Forms commonly encountered as
open water plankton include:
Bosmina_longirostris and others
Daphn.ta galeata and others
Other less common genera are:
Diaphanosoma, Chydorus, Sida,
Acroperus, ceriodaphnla, flio-
trephes, and the cariii örous
L ra and Polyphemus .
d Heavy blooms of Cladocerans may
build up in eutrophic waters.
3 The copepods (order Copepoda) are the
perennial microcrustacea of open waters,
both fresh and marine. They are the
most ubiquitous of animal plankton.
a Cyclops is the genus most often
T und by the National Water QuaUty
Sampling Network activities.
clops, Paracyclops , Diaptomus,
Canthocamptus. F ptschura ,
Limnocatanus are other forms
reporTed to be planktonic.
b Copepods hatch into a minute char-
acteristic larvae called a nauplius
which differs considerably from the
adults. After five or six moults, the
copepodid stage is reached, and after
six more moults, the adult. These
larval stages are often encountered
and are difficult to identify.

Animal Plankton
B Class Inserta
1 Only a single species of insect can be
ranked as a true plankton, this is the
midge fly Chaborus (approx. 8 app,
formerly C orethra) .
2 The larva of this insect has hydrostatic
organs that enable it to remain perman-
ently suspended in the water.
3 It is usually found in the depths during
the daytime, but comes to the surface
at night.
A While the protozoa, t-otlfers, and micro-
crtistacea ninke up the bulk of the plankton.
there are many other groups as mentioned
above that may also occur. Locally or
periodically these may be of major import-
ance. Examples are given below.
B Phylum Coelenterata
1 Polyps of the genus Hydra may become
detached and float about hanging from
the surface film or floating detritus.
2 The freshwater medusa Craspedacusta
occasionally appears in lakes or reser-
voirs in great numbers.
C Phylum Platyhelininthes
1 Minute Turbellaria (relatives of the
well known Planaria ) are sometimes
taken with the plankton in eutrophic
conditions. They are readily confused
with ciliate proto7oa.
2 Cercaria larvae of Trematodes (flukes)
parasitic on certain wild animals,
frequently appear In great numbers
When trapped in the droplets of water
on a swimmer’s skin, they attempt to
bore in. Man not being their natural
host, they fail. The resultant irritation
is called “swimmer’s itch”. Some can
be identified, but many unidentifiable
species may be found.
3 In many areas of the world, cercaria
larvae of human parasites such as the
blood fluke Schistosoma japonicum may
live as plankton, and penetrate the human
skin directly on contact.
D Phylum Nemathelminthes
1 Nematode (or nemas) or rouridworras
approach the bacteria and the blue-green
algae in ubiquity. They are found in
the soil and In the water, and in the air
as dust. In both marine and fresh waters
and from the Arctic to the tropics.
2 Although the majority are free living.
some occur as parasites of plants.
animals, and man, and some of these
parasites are among out most serious.
3 With this distribution, it is obvious that
they will occasionally be encountered as
plankton. A more complete discussion
of nematodes and their public health
implications in water supplies will be
found elsewhere (Chang, S L ).
E Additional crustacean groups sporadically
met with in the plankton include the following:
1 Order Anostraca or fairy shrimps
(formerly included with the two
following orders in the Euphyllopoda)
primarily planktonic in nature.
a Extremely local and sporadic, but
when present, may be dominating.
b Arteniia , the brine shrimp, can
tolerate very high saitnities.
c Very widely distributed, poorly
2 Order Notostraca, the tadpole shrimps.
Essentially southern and western in
3 Order Conchostraca, the clam shrimps.
Widely distributed, sporadic in occur-
rence. lvlany local species.
4 Subclass Ostracoda, the seed shrimps.
Up to 3 in. in length. Essentially
benthic but certain species of Cypris ,
and Notodromas may occur in consid-
erable num Th as plankton at certain
times of the year.
5 Certain members of the large subclass
Malacostraca are limnettc, and thus,
planktonic to some extent.
a The scuds, (order Amphipoda) are
essentially benthic but are sometimes
collected In plankton samples around
US i. vlron ,00fltdI Protection Aqency
Corvallis Environmen i Ressarc 14b.
200 3 W 35th Street
Cor ajh 9 Oregon 97330

Anunal hriktøn
w d hd o: J .ar Hhu . Nc kto—
phnkton c. form s include Pontoporela
and some species of Gamn-iarus .
b The mysid, or opossum shrimp8 are
represented among the plankton by
M _ ysls relicta , which occurs in the
deeper waters, large lakes as far
north as the Arctic Ocean.
F The Class Archnoidea, Order Hydracarina
(or Acari) the mites. Frequent in plankton
tows near shore although Unionlco],a crass-
Ipes has been reported to be virtually
U The phylum Mollusca is but scantily
represented in the freshwater plankton,
in contrast to the marine situation.
Glochldia (ciliated) larvae are occasion-
ally collected, and snails now and then
glide out on a quiet surface film and are
taken in a plankton net. An exotic
bivalve Corbicula has a planktotrophlc
veliger stage.
H Eggs and other reproductive structures
of many forms including fish, insects, and
rotifers may be found in plankton samples.
Special reproductive structures such as
the statoblasts of bryozoa and sponges,
and the ephippia of cladocerans may also
be included.
I Adventltjou and Accidental Planicters
Many shallow water benthic organisms
may become accidentally and temporarily
incorporated into the plankton. Many of
those in the preceding section might be
listed here, In addition to such forms as
certain free living nematodea, small
ollgochaetes, and tardigrades, Collembo]a
and other surface film livers are also
taken at times but should not be mistaken
for plankton. Fragments and molt skins
from a variety of arthropods are usually
Pollen from terrestrial or aquatic plants
Is often unrecognized, or confused with
one of the above. Leaf hairs from
terrestrial plants are also confusing to
the uninitiated, they are sometimes
mistaken for fungi or other organisms
(and vice versa).
In flowing waters, normally benthic
(bottom living) organisms are often found
drifting freely in the stream. This
phenomenon may be conatant or periodic.
When included In plankton collections,
they must be reported, but recognized
for what they are.
1 Edmondson, W.F., ed. Ward and
Whlpples’s Freshwater Biology, 2nd
Edition, Wiley & Sons, Inc., New York.
2 Hutchlnso; G. Evelyn. A Treatise on
Limnology. Vol. 2. Introduction to
Lake Biology and the Liinnoplankton.
Wiley. 1115 pp. 1967.
3 Lackey, J. B. Quality and Quantity of
Plankton In the South End of Lake
Michigan in 1952. JAWWA.
36:689—74. 1944.
4 McGauhey, P.H., Elch, H.F., Jackson,
H.W., and Henderson, C. A Study
of the Stream Pollution Problem In the
Roanoke, Virginia, Metropolitan
District. Virginia Polytech. Inst.,
Engr. Expt. Sta.
5 Needham, J. G. and Lloyd, J. T. The
Life of Inland Waters. Ithaca, New
York, Cornstock Publishing Co., Inc
6 Newell, G. E. and Newell, R. C.
Marine Plankton. Hutchinson Educ.
Ltd. London. 2 2lpp. 1963.
7 Palmer, C.M. and Ingram, W.M.
Suggested Classification of Algae and
Protozoa in Sanitary Science.
Sew. & lad. Wastes. 27:1183-88.

Animal Plankton
8 Pennak, R.W. Freshwater Invertebrates 10 Welch, P.S. Limnology, McGraw-Hill
of the United States. The Ronald Press, Book Co., Inc., New York. 1935.
New York. 1953.
9 Sverdrup, H.W., Johnson, M.W., and
FlemIng, Ft. H. The Oceans, Their _______________________________________
Physics, Chemistry and General This outline was prepared by H. W. Jackson,
Biology. Prentice-Hall, Inc., New York. thief Biologist, National Training Center, OWP,
1942. EPA, Cincinnati, OH 45268.

Prei Livii g Representatives
I. Pla ell*t.d Protozos, Class sstigopbora
A thoDhy ,U
Pollution toll.rsnt
Pollution tollsraftt
19 p
II . Ameboid Protozoa, Class Saroodina

Pollution toll.rsnt
J. 2 Ler.LI zeport.d
to be intollorent of
pollution, 45,11.
III. Ciltatod Protozoa, Class Ciliophora
aolothrva , r.portod
to be intollorant of
pollution, 35 i
LW. Jackson
ao1 of ____________
Poll*tion toll.rant, 35 ,4*
6 o..5ooj
Pollution tollorunt
LL zA&z , pollution
toll.rant. Colonies often

Animal Plankton
vulgar is

Animal Plankton
Various Forms of

Keratella co hleari .
y chaeta
! y arthra
Rotaria ap
15 .8

A Nauplius larva of a Copepod
Copepod; Cyc oj Order Copepoda
2-3 mm
Water Fles
t ptn1a
1-5 mm
2-3 mm
Order Ciadocera
Left: Shell closed
1-2 mm
Appendages extended
15 -9

Animal Plankton
Chaoborus inidge larva In sect
Aspects In the life cycle of the human tapeworm
Diphyllobothrium laturn , class Cestoda. A a< as In human
Intestine; B. procercoid larva In copepod; C. plerocercoid
larva in flesh of pickerel (X-ray view).
H.W. Jackson
A mysid shrimp - crustacean
A water mite - arachnid

A To become familiar with important
structural features of diatoms.
B To learn to recognize some common forms
at eight.
C To learn to identify lees common forms
using technical keys.
A Transfer a drop of the water sample con-
taining diatoms to a microscope slide. Cover
with cover glass and observe under low
power (lOX) of microscope.
1 Do all of the diatom cells appear to
have the same shape? Do Borne have
square ends and some rounded ends?
Touch the cover glass with your pencil
several times as you observe through
the microscope and note the relationship
of the two types of ends to one another.
2 Find a place where a round-ended and a
square-ended cell are close together
and observe these under oil power
(90X). The round-ended view Is that of
the top or bottom of the diatom cell and
is called the “valve” view. The square
ended view is that of the side of the cell
and is called the “girdle” view.
3 In the valve view note the cross lines in
the wall. In this diatom there are many
fine lines and a smaller number of
coarse lines. The former are present
in all diatoms and are called “striae” or
“striations.” The latter are present
only in some genera of diatoms and are
called “septa,” or in other genera,
“costae.” Which of the two types of
lines arc continuous from side to side?
The space left in the center by the in-
terrupted lines is known as a “false
(pseudo) raphe.”
4 What is the predominant color of the
diatom? How many plant id a? In
diatoms, the identification is based al-
most entirely on the characteristics of
the cell wall.
5 Make an outline drawing, at least 3
inches long, of a valve view and a girdle
view of the diatom. Show the markings
in the upper third of each. Label the
striae, septa, and falee-raphe. Make
a drawing of what you image an end
view or cross(transverse) section view
would be like.
6 Using the key, identify your specimen,
listing the alternatives selected.
B Use the key to identify other unknowns as
far as possible, listing the alternatives
selected in the key. Make a sketch of
Navicula and Cyclotella if you identify these
Capitate - having a known-Uke end.
Costae - coarse transverse ribs in wall.
False raphe - (see pseudoraphe)
Frustule - the wall of the diatom.
Girdle view - the side view, in which the
diatom appears to have square or blunt ends.
Nodule - a lump-like swelling in the center or
ends of the valve.
Pseudoraphe - a clear space extending the
length of the diatom and bordered on both
sides by striae.
Punctae - the dots which comprise the
st riae.
SI. MIC. cla. lab.

Laboratory: Identification of Diatoms
Raphe - a longitudinal line (cleft) bordered on
both sides of striae.
Septa - a self-Like partition in the diatom,
appearing often as a coarse line.
Striae - fine transverse lines especially
evident in the valve view.
Valve view - the top or bottom view, in which
the diatom has rounded ends, or is circular
in outline.
1 Boyer. C.S. The Diatomaceae of
Philadelphia and Vicinity. J. B. Lippin-
cott Co. Philadelphia. 1916. p 143.
2 Boyer. C.S. Synopsis of North America
Diatomaceae, Parts 1(1927) and U
(1928). Proceedings of the Academy
of Natural Sciences, Philadelphia.
3 Elmore, C. J. The Diatoms of Nebraska.
University of Nebraska Studies, 2 1:22-
215. 1921.
4 Hohn, M. H. A Study of the Distribution
of Diatoms in Western New York State.
Cornell University Agricultural Experi-
mental Station. Memoir 308, pp 1-39.
5 Pascher, A. Bacillariophyta (Diatomeae).
Heft 10 in Die Suaswasser-Florg
Mitteleuropas, Jena. 1930. p466.
8 Patrick, R. A Taxonomic and Ecological
Study of Some Diatoms from the
Pocono Plateau and Adjucant Regions,
Farlowia. 2:143-221. 1945.
7 Smith, G.M. Class Baci1larioph yceae.
Freshwater Algae of the United States,
McGraw-Hill Book Co. New York.
pp 440-5 10 2nd Ed. 1950.
8 Tiffany, L. H., and Britton, M.E. Class
Baclllariophyceae. The Algae of
Illinois, University of Chicago Press.
pp 214-296. 1952.
9 Ward, H. B., and Whipple, G.C. Class I,
Bacillariaceae (Diatoms). Freehwuier
Biology, J. Wiley & Sons. New York. -
pp 17 1-189. 1948.
10 Weber, C.!. A Guide to the Common
Diatoms at Water Pollution Surveillance
System Stations. USD1. FWPCA,
Cincinnati, OH. 1966.
11 Whipple, G. C., Fair. G.M. • and Whipple,
M.C. Diatomaceae. Microscopy of
Drinking Water. Chapter 21, 4th ed.
J. Wiley and Sons, New York. 1948.
This outline was prepared by L. G. Williams,
Aquatic Biologist, Formerly with Research
and Development, Cincinnati Water Research
Laboratory, FWPCA, SEC.

I The identification of many diatoms to
genus and all diatoms to species requires
that the celia be free of organic contents.
This Ia necessary because the taxonomy of
the diatoms is based on the structure of the
frustule ( helis) of the organisms and many
features are masked by the presence of
organic materials which may remain inside.
It is also necessary that at least 1000X
magnification (oil immersion) be used to
detect the structural features used In
identification. No simple procedure for the
accurate routine counting of diatoms has yet
b een developed.
A Sample Concentration
1 Centrifuge (such as Universal DU)
2 100 ml centrifuge tubes
3 Membrane filter apparatus
4 Vacuum
B Slide Preparation
1 Slides, 1 X 3 inch, frosted-end
2 Cover glasses, circular #1, 18 mm,
0. 13 - . 18 mm thick
3 Resinous mounting medium (such as
Harleco microscope mounting medium)
4 Hot plates
a 180°F
b 700° F
5 DIsposable pipettes
6 3 X6 X 1/4 inch steel plate
A The volume of sample needed will vary
according to the density of diatoms and
alit, and only with experience can the
correct sample size be determined. In
most cases, 100 ml will be sufficient.
1 Spin 100 ml at 1000 G for 20 mInutes.
2 Withdraw the supernatant liquid with
an aspirator, being careful not to
disturb the concentrate at the botton
of the centrifuge tube. (Draw off all
but 2-3 ml.)
3 Transfer the concentrate to a labelled
10 ml disposable vial. Label the vial
with a magic marker, diamond pencil,
or “time” label.
4 If the sample has been preserved with
formalin, or contains more than
1.0 gram per liter dissolved solids,
it will be necessary to wash the
concentrate with distilled water. In
this case, transfer the entire concen-
trate to a 15 ml centrifuge tube.
Dilute to 15 ml with distilled water,
making certain that the sample is well
mixed. Spin for 10 minutes at full
speed in a clinical centrifuge. With-
draw the Bupernatant liquid, and refill
with distilled water. Spin again for 10
minutes. Withdraw the supernatant
liquid as before, return the concentrate
to the rinsed vial in 2-3 ml of distilled
water and proceed with the mounting.
5 II more than 200 ml of sample must be
centrifuged to obtain sufficient material
to prepare a diatom slide, concentrate
the diatoms by filtering the sample
through a 1. 2 micron pore diameter
membrane filter. Transfer the filter
to a 15 ml centrifuge tube, and dissolve
with 90% acetone. Centrifuge 10
minutes (full speed) and decant with an
aspirator. Refill with 90% acetone,
B!. MIC. enu. lab. Sb. 6.68

Preparation of Permanent Diatom Mounts
agitate, and spin again for 10 minutes.
Repeat until three fresh acetone washes
have been used. Replace the acetone
with 2-3 ml of distilled water and
transfer to a labelled vial as described
In #4.
B If the loss of minute forms in supernatant
is suspected, spin 100 ml at 1000 gs in
a batch centrifuge for as long as may be
necessary, then proceed as below.
C Mounting
1 Heat the hot plates to the prescribed
2 Place one cover glass on the steel plate
for each sample.
3 Place the steel plate on the 1800 F hot
4 Transfer a drop of sample to a cover
5 Allow the water to evaporate (caution:
do not allow it to boil.)
6 Continue to add more sample until a
thin layer of material is noticeable on
the dry cover glass, or until all of the
concentrate has been used. This step
is especially critical, and can be
learned only by trial and error.
7 Transfer the steel plate to the 700°F
hot plate for 20-30 minutes. (The
plate should be hot enough to incinerate
8 While the material is on the high
temperature hot plate, label the
microscope slides (use a #2 pencil
or a fine point drawing pen); place
them on the low temperature hot plato,
which now has been reset to approxi-
mately 275°F.
9 Place a drop of mounting resin on the
n icroscope slides and allow the solvent
to evaporate.
10 When the incineration of the material
on the cover glasses is complete,
transfer the cover glasses, while still
hot, to the mounting medium.
11 Allow the resin to penetrate the
frustules (1-2 minutes).
12 Remove the slide, place it on a cool
desk top, and press the cover glass
lightly with a pencil eraser for a few
seconds. The medium will harden In
5-10 seconds.
13 Scrape off the excess resin with a
razor blade.
D The preparation is now ready for exam-
ination under an oil immersion objective.
Certain portions of this outline contains
training material from a prior outline by
M.E. Bender.
This outline was prepared by Dr. C.I.
Weber, Chief, Biological Methods Section,
Analytical Quality Control Laboratory,
1014 Broadway, Cincinnati, OH 45202.

A The great majority of organisms commonly
encountered in plankton analysis work are
plants or at least plant-like (holophytic).
Animals, however, (holozoic or nonchioro-
phyli bearing forms) are an impoctax t part
of the community, and the ability to recog-
nize them may be quite important.
B Many animals are soft bodied and so are
best observed in the living condition, as
Uiey shriiik arid become otherwise distorted
on preservation. There are consequently
many which will not be available in a
suitable form for the following exercise.
Only such forms will be dealt with as can
readily be obtained alive, or which retain
essential characteristics on preservation.
A To Study the nature and use of a key for
identifying organisms
B To Introduce the Beginners to the Use of
thy Microscope
C To Learn to Recognize Basic Animal Types
D To Identify Animal Plankton Species as
Available, and to Become Familiar with
the Literature
A The Use of the Biological Key
1 Obtain a “Basic Invertebrate Collection”
from the instructor
2 Sclect a specimen designated by the
instructor, and t,,rn to the “Key to
Selected Larger Groups of Aquatic
a Examine your specimen carefully,
then read the first couplet of
statements in the key (la and ib).
b Since the specimen is large enough
to see, it obviously could not be the
object of statement Ia. Therefore
due to the nature of the key (as
explained in the second paragraph of
the introduction) the second alternative
(lb) must apply This alternative
instructs us to proceed to couplet 2.
c From here on, follow from couplet
to couplet, considering each couplet
by itself, until a final selection leads
to a name. If this name or couplet
is, followed by another couplet
number, this means that the group
named is further subdivided.
3 Identify the other specimens in the
Basic Invertebrate Collection in the
same way.
4 Carry the identification further, to
genus and species if possible, in one
or more of the more detailed keys
listed at the end of the “Key to Selected
Larger Groups of Aquatic Animals.”
B The Use of the Microscope
1 Obtain preliminary information from
the instructor as to how to set up and
operate the instrument.
2 Place a prepared slide of a printed letter
on the stage and observe it successively
under low (lOOX) and high (45X) powers.
When the letter is right side up to you,
how does it appear through the microscope?
3 Place a prepared slide of a micro-
crustacean on the stage and identify it
using the “Key to Selected Larger Groups
of Aquatic Arumals.” Continue your
BI. MIC. cia. lab. 5c. 10. 72

Laboratory Identification of Animal Plankton
identilication as far as possible using
Eddy and Hodson’s “Taxonomic Keys.”
4 Prepare a “wet mount” under the
direction of the instructor and identify
the organism. Confirm your identifica-
tion in one or more of the technical
reference books available.
C Identify each of the specimens in the
reference collection as to phylum and
class, and then genus and species JJ
possible (do not spend undue time on the
specie8 without assistance).
Make a flash caI-d sketch of at least
one organism of each phylum observed
as an example of a type.
ED Examine the living material provided.
Sketch and identify animal forms
encountered as far as time permits.
Can you draw any conclusions as to the
types of animal Life found In the various
habitats indicated?
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
DTTB, MDS, OWP, EPA, Cincinnati, 0 11

A A plan is necessary. ‘1.1 you fail to plan,
you are planning to fail.” Overall objec-
tives, integration with other survey units,
statistical design.
B A planned program of plankton analysis
should involve periodic sampling at weekly
intervals or more often.
1 Most interference organi8m8 are smaU,
and hence have relatively short-We
2 Pop ]ations of such organisms may
fluctuate rapidly In response to chang-
ing water, weather, or seasons.
3 Seasonal growth patterns of plankton
tend to repeat themselves from year to
year, thus they are relatively predictable.
C A well-planned stucb’ or analysis of the
growth pattern of plankton in one year will
provide a basis for predicting conditions
the following year.
1 SInce the seasons and the years differ,
the more records are accumulated, the
more useful can they become.
2 As the time for an anticipated bloom of
some trouble maker approaches, the
frequency of analysis may be increased.
D Detection of a bloom in its early stages
will facilitate more economical control.
A Two general aspects of sampling are com-
monly recognized quantitative and
1 Qualitative examination tells what is
2 Quantitative tells how much.
3 Either approach is useful, a combination
is best.
B Equipment of collecting samples in the
field is varied.
1 A half—liter bottle will suffice for sur-
face samples of phytoplankton if carefully
taken. If zooplankton also are of interest,
2 or more liters should be collected.
(See be1ov .
2 Plankton nets concentrate the sample
in the act of collecting, and capture
certain larger forms which escape from
the bottles. Only the more elaborate
types are quantitative however.
3 A kernmerer-type sampler is suggested
for depth samples.
4 Other methods such as the Clark-Bumpus
sampler or the Juday plankton trap may
be employed for special purposes.
C The location of sampling points is
I Both shallow and deep samples are
a “Shallow” samples should be taken
at a depth of 6 inches to one foot.
b “Deep” samples should be taken at
such intervals as the depth of the
reservoir permits. There should be
at least one open water sampling
c Each major bay or shoal area should
have at least one sampling point.
d Additional sampling stations should
be established on the basis of ex-
perience and resources.
e Samples may be composited if nec-
essary to give an overall summary of
conditions. Such summaries are not
advised and should be interpreted
with care.
flr ff,n — I ,,

Techniques of Plankton Sampling Programs
A standardized vertical haul however
c n be useful for routine co1nparison .
A Physical Features
1 Temperature
a Lakes are warmed In spring princi-
pally by the action of wind forcing
the warmer water down into the
cooler water against the forces of
b Thermal stratification
2 Turbidity
3 Color
4 Water movement
5 Ugit penetration
a A factor of turbidity, color, biolog-
ical activity, and time of day
(1) EffectIve length of daylight
dimenishes with the depth of
the lake.
6 Wind velocity and direction
7 Bottom materials
8 Size, shape, and slope of lake basin
B Chemical Factors
1 Alkalinity, pH, and dissolved minerals
excluding nitrogen and phosphorus
2 Dissolved oxygen
a From photosynthesis in sunlight
b From contact of lake surface with
the air
3 Nutrients for biological growth -
especially nitrogen and phosphorus
a A given body of water will produce
a given quanity of aquatic life.
Biological production is determ.Ined
primarily by the nutrients in solu-
tion in the water, and an Increase
in basic fertility will increase
biological activity.
b Basic suppliers of nutrients
include tributary streams, precip-
itation from the atmosphere, and
Interchange with lake bottom sedi-
Provision should be made for the field
stabilization of the sample until the laboratory
examination can be made. Techniques and
materials are listed below. No “ideal” pre-
servative or technique has yet been developed.
each has its virtues.
A Refrigeration or icing. The container
containing the sample can be cooled, but
under no circumstances should ice be
dropped into the sample.
B Preservation by 3-5% formaline is time-
tested and widely used. Formaline shrinks
animal tissue, fades colors, and makes all
forms brittle.
C Ultra-violet sterilization is useful in the
laboratory to retard decomposition of
D Lugol’s solution is often used.
E A special merthiolate preservative
developed by the FWPCA Water Pollution
Surveillance System which has proved very
satisfactory and is described in reference
No. 9.
c Fluctuates 8easonally because of
temperature and biological activity,
and diurnaUy because of biological

Techniques of Plankton Sampling Prog am
A The field iampling program should be
carefully planned to evaluate all signifi-
cant locations in the reservoir or stream,
giving due consideration to the capacity
of the laboratory.
8 Adequate records and notes should be
made of field conditions and associated
with the laboratory analyses in a permanent
C Once a procedure for processing plankton
is adopted, it should be used exclusively
by all workers at the plant.
D Such a procedure qhould enable the water
plant operator to prevent plankton troubles
or at least to anticipate them and have
corrective materials or equipment
Portions of this outline were prepared by
K. M. Mackenthun, Bioiogist, formerly with
Technical Advisory and Investigation Activities 1
FWPCA, SEC, Cincinnati 1 Ohio.
1 API-LA. Standard Methods for the
Examination of Water and Wastewater,
13th Ed, pp 726-742, NY, 1971.
2 llutcheson, George E. A Treatise o i
Limnology. John Wiley and Co. New
York. 1957.
3 Jackson, I-I. W. Biological Examination
(of plankton) Part Ill in Simplified
Procedures for Water Examination.
AWWA Manual M12. Am. Water
Works Assoc., N Y. 1964.
4 Lickey, J. B. The Manipulation and
Counting of River Plankton and Changes
in Some Organisms Due to Forma]in
Preservation. Public Health Reports,
53: 2080-93. 1938.
5 Mackenthun, K. M., Ingram, W. M.,
and Ralph Porges. Limnological
Aspects of Recreation Lakes, DHEW,
PHS Publication No. 1167, 1964.
6 Olson, Theodore A. and Burgess, Fred-
erick J. Pollution and Marine Ecology.
Intersclence Publishers. 364 pp. 1967.
7 Palmer, C. M. Algae in Water Supplies.
U. S. Department of Health, Education,
and Welfare, Public Health Service
Publication No. 657, Superintendent of
Documents, Washington 25, D. C.
8 Schwoerbel, J. Methods of Hydrobiology
(Freshwater Biology). Pergamon
Press, 1970.
9 Weber, C. I. Methods of Collection and
Analysis of Plankton and Periphyton
Samples in the Water Pollution
Surveillance System. App. and Devel.
Rep. (AQC Lab., 1014 Broadway,
Cincinnati, OH 45202) 19 pp 1966.
10 Welch, P.S. Limnological Methods.
The Blaldston Co., Phija. Toronto.
11 Williams, L. 0. Plankton Population
Dynamics, in National Water Quality
Network, Supplement 2, U.S. PHS
Pub. No. 663. 1962.
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
DTTB, MDS, OWP, EPA, Cincinnati, OH

A Preliminary samphng and analysis 18 an
essential preliminary to the establish-
ment of a permanent or semi-permanent
B Concentration or sedimentation of pre-
served samples may precede analysis.
1 Batch centrifuge
2 Continuous centrifuge
.3 Sedimentation
C Unpreserved (living) samples should be
analyzed at once or refrigerated for
future analysis.
A The Water Pollution Surveillance System
of the FWPCA has developed a modified
merthiolate preservative’. (Williams.
1967) Sufficient stock to make an approx-
imately 3 5% solution in the bottle when
filled is placed In the sample bottle in
the laboratory The bottle 18 then filled
with water in the field and returned to
the laboratory for analysi8
B Preparation of Merthiolate Preservative
1 Merthlolate is available from many
chemical laboratory supply houses;
one should specify the water soluble
sodium salt
2 Merthiolate stock dissolve approxi-
mately 1 5 gram of sodium borate
(borax and approximately I gram of
merthiolate in 1 liter of distilled water
The amount of sodium borate and
merthiolate may be varied slightly to
adjust to different waters, climates,
and organic contents.
3 Prepare a saturated aqueous LugoV s
solution as followg
a Add 60 grams of potassium iodide
(KI) and 40 grams of iodine crystals
to 1 liter of distilled water
4 Prepare the preservative solution by
adding approximately 1. 0 ml of the
Lugol’s solution to 1 liter of merthi-
olate stock.
A Microscopic examination is most frequently
employed in the laboratory to determine
what plankton organisms are present and
how many there are:
1 Optical equipment need not be elaborate
but should Include.
a Compound microscope with the
following equipment:
1) Mechanical stage
2) Ocular: laX, with Whipple type
counting eyepiece or reticuie
3) Objectives:
1OX( 16mm)
20X(8 mm)
4QX(4 mm)
95X(1. 8 mm)(optional)
A 40X objective with a working
distance of 12. 8 mm and an erect
Image may be obtained as special
equipment A water Immersion
objective (In addition to oil) might
be considered for use with water
Binocular eyepieces are optional,
B!. MIC. enu. l f. 10. 72

Preparation and Enumeration of Plankton
Stage micrometer (this may be
borrowed, if necessary, as it is
usually used only once, when the
equipment is calibrated)
b Inverted microscopes offer certain
advantages but are not widely
available The same is true of
some of the newer optical systems
such as phase contrast microscopy.
These are often excellent but ex-
pensive for routine plant use
2 Precision made counting chambers
are required for quantitative work with
liquid mounts.
a Sedgwick-Rafter cells (hereafter
referred to as S-R cell) are used
for routine counts of medium and
larger forms.
b Extremely small forms or t nanno
plankton’ may be counted by use of
the nannoplankton (or Palmer) cell,
a Fisher- Ltttman cell, a hemacyto-
meter, the Lackey drop method, or
by use of an inverted microscope.
3 Previous to starting serious analytical
work, the microscope should be cali-
brated as described elsewhere Di-
mensions of the S-ft cell should also
be checked, especially the depth.
4 Automatic particle counters may be
useful for coccoid organisms.
B Quantitative Plankton Counts
All quantitative counting techniques
Involve the filling of a standard cell
of known dimensions with either
straight sample or a concentrate or
dilution thereof.
2 The organisms in a predetermined
number of microscope fields or other
known area are then observed, and by
means of a suitable series of multi-
plier factors, projected to a number or
quantLty per ml gallon, etc.
3 Direct counting of the unconcentrated
sample eliminates manipulation, Saves
time, and reduces error. If frequency
of organisms is low, more area may
need to be examined or concentration
of the sample may be in order
4 Conventional techniques employing
concentration of the sample provide more
organisms for observation, but because
they involve more manipulations, intro-
duce additional errors and take more
C Several methods of counting plankton are
in general use.
The numerical or clump count is
regarded as the simplest.
a Every organism observed must be
enumerated. If it cannot be identi-
fied, assign a symbol or number
and make a sketch of it on the back
of the record sheet.
b Filaments, colonies and other
associations of celia are counted
as units, equal to single isolated
ceUs. Their identity as Indicated
on the record sheet is the key to
the significance of such a count
2 IndivIdual cell count . In this method,
every cell of every colony or clump
of organisms is counted, as well as
each individual single- celled organism.
3 The areal standard unit method offers
certain technical advantageB, but also
involves certain inherrent difficulties.
a An areal standard unit Ia 400 square
microns. This is the area of one
of the smallest subdivided squares
in the center of the Shipple eyepiece
at a magnification of IDOX.
b In operation, the number of areal
units of each species is recorded on
the record sheet rather than the
number of individuals. Average
areas of the common species are

Preparation and Enumberation of Plankton
are sometimes printed on record
sheets for a particular plant to
obviate the necessity of estimating
the area of each cell observed
c The advantage of the method lies in
the cognizance taken of the relative
masses of the various species as
indicated by the area presented to
the viewer These areas, however,
are often very difficult to estimate.
4 The cubic standard unit method in a
logical extension of the areal method,
but has achieved less acceptance.
5 Separate field count
a In counting separate fields, the
question always arises as to how
to count organisms touching or
crossed by the edge of the Whipple
field Some workers estimate the
proportion of the organism lying
Inside the field as compared to that
outside. Only those which are over
bali way inside are counted.
b Another system is to select two
adjacent sides of the square for
reference, such as the top and left
boundaries Organisms touching
these lines in any degree, from
outside or inside, are then counted,
while organisms touching the opposite
sides are ignored It is important
to adopt some such system and
adhere to it consistently.
c It is suggested that if separate
microscopic fields are examined,
a standard number of ten be adopted.
These should be evenly spaced In two
rows about one-third of the distance
down from the top and one-third of
the distance up from the bottom of
the S-El cell
6 Multiple area count . This is an ex-
tension of the separate field count.
A considerable increase in accuracy
ha recently been shown to accrue by
emptying and refilling the S-El cell.
after each group of fields are counted
and making up to 5 additional such
counts. This may not be practical with
high counts.
7 The strip count . When a rectangular
slide such as the S-R cell is used, a
strip (or strips) the entire length (or
known portion thereof) of the cell may
be counted instead of separate Isolated
fields Marking the bottom of the cell
by evenly spaced cross lines as ex-
plained elsewhere greatly facilitates
a When the count obtained is multi-
plied by the ratio of the width of
the strip counted to the width of
the cell, the product is the esti-
mated number of organisms in the
cell, or per ml.
b When the material in the cell is
unconcentrated sample water, this
count represents the condition of
the water being evaluated without
further calculation.
8 Survey count . A survey count is an
examination of the entire area of a
volumetric cell using a wide field low
power microscope. The objective is
to locate and record the larger forms,
especially zooplankton such as copepods
or large rotifers which may be present
in size. Special large capacity cells
are often employed for this purpose.
For still larger marine forms, numerous
special devices have been created.
9 Once a procedure for concentration
arid/or counting is adopted by a plant
or other organization It should be
used consistently from then on so
that results from year to year can be
D Differential or qualitative “counts ” are
essentially lists of the kinds of organisms

Preparation and Enumeration of Plankton
E I’roportional or r lattv countl-3 of spIcial
groups are often very useful For cx-
ampk, diatoms It is best to always
count a standard numbers of cells.
F Plankton are sometimes measured by
means other than microscopic counts.
Settled volume of killed plankton in an
Imhoff cone may be observed after a
standard length of time. This will
evaluate primarily only the larger forms.
2 A gravimnetric method employs drying
at 60° C for 24 hours followed by
ashing at 800° C for 30 minutes. This
Is particularly useful for chemical
and radlochemnical analysis.
3 Chemical and physical evaluation of
plankton populations employing various
instrumental techniques are coming to
be widely used. Both biomass and
productivity rates can be measured
Such determinations probably achieve
their greatest utility when coordinated
with microscopic examination.
4 The membrane (molecular) filter has
a great potential, but a generally
acceptable technique has yet to be
a Bacteriological techniques for
coliform determination are
widely accepted
b Nematodes and larger organisms
can readily be washed off of the
membrane after filtration.
c It is also being used to measure
ultraplanktori that pass treatment
plant operations
d Membranes can be cleared and
organisms deposited thereon
observed directly, although
accessory staining is desirable.
e Difficulties include a prediiec-
tion of extremely fine membranes
to clog rapidly with silt or
increase in plankton counts, and
the difficulty of making observations
on individual cells when the
organisms are piled on top of each
other. It is sometimes necessary
to dilute a sample to obtain suitable
A The field sampling program should be
carefully planned to evaluate all significant
locations in the reservoir or stream,
giving due consideration to the capacity
of the laboratory.
B Adequate records and notes should be
made of field conditions and associated
with the laboratory analyses in a
permanent file.
C Optical equipment in the laboratory should
be calibrated.
D Once a procedure for processing plankton
is adopted, it should be used exclusively
by all workers at the plant
E Such a procedure should enable the water
plant operator to prevent plankton troubles
or at least to anticipate them and have
corrective materials or equipment stockpiled.
Ely Lilly Company. Merthiolate as a
Preservative. Ely Lilly & Co.
Indianapolis 6, Indiana.
2 Gardiner, A. C. Measurement of
Phytoplankton Population by the
Pigment Extraction Method. Jour.
Marine Biol. Assoc. 25(4):739-744. 1943.
3 Goldberg, E. D., Baker, M., and Fox, D. L.
Microfiltration in Oceanographic
Research Sears Foundation. Jour.
Mar. Res. 11:194-204. 1952.

Preparation and Enumeration of Plankton
4 Ingram, W. M., and Palmer, C. M.
Simplified Procedures for Collecting,
Examining, and Recording Plankton
in Water. Jour. AWWA. 44(7):
617-624. 1952 —
5 Jackson, H. W. Bilogical Examination
(of plankton) Part III tn Simplified
Procedures for Water Examination.
AWWA Manual M 12. Am. Water
Works Aseoc. N.Y. 1964.
6 Lund, J. W. G.. and Tailing, J. F.
Botanical Limnological Methods with
Special References to the Algae
Botanical Review. 23(8&9):489-583.
October. 1957.
7 Weber, C. I. The Preservation of
Plankton Grab Samples. Water
Pollution Surveillance Systems,
Applications and Development Report
No. 26, USD1, FWPCA, Cincinnati,
Ohio. (1967)
8 Williams, L. G. Plankton Population
Dynamics. National Water Quality
Network Supplement 2. U. S. Public
Health Service Publ. No. 663 (1962)
9 Wohiechag, D. D., and Hasler, A. D.
Some Quantitative Aspects of Algal
Growth in Lake Mendota. Ecology.
32(4):581-593. (1951)
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
DTTB, MDS, OWP, EPA, Cincinnati, OH

A With the exception of factory-set
instruments, no two microscopes can
be counted upon to provide exactly the
same magnification with any given com-
bination of oculars and objectives. For
accurate quantitative studies, it is there-
fore necessary to standardize or ‘calibrate”
each instrument against a known standard
scale. One scale frequently used is a
microscope slide on which two millimeters
are subdivided into tenths, and two addi-
tional tenths are subdivided into hundredths.
FIgure 3.
B In order to provide an accurate measuring
device in the microscope, a Whipple
Plankton Counting Square or reticule
(Figure 2a) Is installed in one ocular
(there are many different types of reticules).
This square is theoretically of such a size
that with a lOX objective, a lox ocular,
and a tube length of 160 mm, the image
of the square covers a square area on the
slide one mm on a slide. Since this
objective is rarely attained however, most
microscopes must be standardized or
“calibrated” as described below in order
to ascertain the actual size of the Whipple
Square as seen through the microscope
(hereinafter referred to as the “Whipple
field”). This process is schematically
represented in Figures 5 and 7. If the
Whipple eyepiece is to be used at more
than one magnification, it must be recali-
brated for each. A basic type of monocular
microscope is shown in Figure 1.
C Microscopes with two eyepieces (binocular)
are a convenience but not essential. Like
modern cars they are not only great
“performers,” but also complicated to
service or, in this instance, calibrate.
On some instruments, changing the inter-
pupillary distance also changes the tube
length, on others it does not. The “zoom”
feature on certain scopes is also essentially
a system for changing the tube length.
The resultant is that in addition to calibra-
tion at each combination of eyepiece and
objective, any other factor which may
affect magnification must also be considered.
In some instances this may mean setting up
a table of calibrations at a series of micro-
scope settings.
Arother procedure is to select a value
for each of the variables involved (inter-
pupillary distance, zoom, etc.) and
calibrate the scope at that combination.
Then each time the scope is to be used for
quantitative work, re-set each variable to
the value selected. A separate multipli-
cation factor must be calculated for each
adjustment which changes the magnification
of the instrument.
Since the Whipple Square can be used to
measure both linear dimensions and
square areas, both should be recorded on
an appropriate form. A suggested format
Is shown in Figure 6.
(Data written in are used as an illustration
and are not intended to apply to any
particular microscope. An unused form
is included as Figure 6-A.)
A In8talling the Whipple Square or Reticule
To install the reticule in the ocular
(usually the right one on a binocular
microscope), carefully unscrew the upper
lens mounting and place the reticule on
the circular diaphragm or shelf which will
be found approximately half way down inside
(Figure 4). Replace the lens mounting and
observe the markings on the reticule. If
they are not in sharp focus, remove and
turn the reticule over.
On reticules with the markings etched on
one side of a glass disc, the etched sur-
face can usually be recognized by shining
the disc at the proper angle in a light.
The markings will usually be in the best
focus with the etched surface down. If the
markings are sandwiched between two glass
discs cemented together, both sides are
alike, and the focus may not be quite as
B Observation of the Stage Micrometer
Replace the ocular in the microscope and
observe the stage micrometer as is illus-
trated schematically in Figure 5: Calibration
of the Whipple Square. On a suitably ruled
form such as the one illustrated, Figure 6,
Calibration Data, record the actual distance
in millimeters subtended by the image of
BI. MET.mic. Id. 4.70

Calibration and Use of Plankton Counting Equipment
the entire Whippic field and also by each
of Its subdivisions. This should be
deternnned for each cigruficant settling of
the interpupillary distance for a binocular
microscope, and also for each combination
of lenses employed. Since oculars and
objectives marked with identical magnifi-
cation, and since microscope frames too
may differ, the serial or other identifying
number of those actually calibrated should
be recorded. It is thus apparent that the
determinations recorded will only be valid
when used with the lenses listed and on that
particular microscope.
C Use of the 20X Objective
Due to the short working distance beneath
a 46X (4mm) objective, it is Impossible
to focus to the bottom of the Sedgewick-
Halter plankton counting cell with this lens.
A lox (16mm) lens on the other hand
“wastes” space between the front of the
lens and the coverglass, even when focused
on the bottom of the cell. In order to make
the most efficient use possible of this cell
then, an objective of intermediate focal
length is desirable. A lens with a focal
length of approximately 8 mm, having a
magnification of 20 or 21X will meet these
requirements. Such lenses are available
from American manufacturers and are
recommended for this type of work.
The internal dimensions of a Sedgewick-Rafter
plankton counting cell should be 50 mm long
by 20 mm wide by 1 mm deep (Figure 8).
The actual horizontal dimensions of each new
cell should be checked with calipers, and the
depth of the cell checked at several points
around the edge using the vertical focusing
scale engraved on the fine adjustment knob of
most microscopes. One complete rotation of
the knob usually raises or lowers the objective
1 mm or 100 microns (and each single mark
equals 1 micron). Thu8, approximately ten
turns of the fine adjustment knob should raise
the focus from the bottom of the cell to the
underside of a coverglass resting on the rim.
Make these measurements on an empty cell.
The use of a No. 1 or 1-1/2, 24 X 60 mm
covergiass is recommended rather than the
heavy coverglass that comes with the S-H
cell, as the thinner glass will somewhat con-
form to any irregularities of the cell rim
(hence, also making a tighter seal and reduc-
ing evaporation when in actual use). Do not
attempt to focus on the upper surface of the
rim of an empty cell for the above depth
measurements, as the covergiasa is supported
by the highest points of the rim only, which
are very difficult to identify. Use the average
of all depth measurements as the “true” depth
of the cell. To simplify calculations below, it
will be assumed that we are dealing with a
cell with an average depth of exactly 1. 0 mm.
A Principles
Since the total area of the c l is 1000 mm 2 ,
the total volume is 1000 mm or 1 ml. A
“strip” the length of the cell thus constitutes
a volume (V 1 ) 50 mm long, 1 mm deep, and
the width oflhe Whipple field.
The volume of such a strip in mm 3 is:
V 1 50 X width of field X depth
= 5OXwX1
= 50w
In the example given below on the plate
entitled Calibration Data, at a magnification
of approximately 200X with an interpupillary
setting of “80”, the width of the Whipple
field is recorded as approximately 0. 55 mm
(or 550 mIcrons). In this case, the volume
of the strip is:
V 1 = 50 w 50 X 0. 55 = 27. 5 (mm 3 )
B Calculation of Multiplier Factor
In oi’der to convert plankton counts per
strip to counts per ml, it is simply
necessary to multiply the count obtained
by a factor (F 1 ) which represents the
number of tim s the volume of the strip
examined (V 1 ) would be contained in 1 ml or
1000 mm 3 . Thus in the example given
F = volume of cell in mm 3
I volume examined In mm
- 1000 - 1000
— —vS— — = 36.36
= approx. 36
If more than one strip is to be counted,
the factor for two, three, etc., strips
could be calculated separately using the
same relationships outlined above, changing
only the measurement for the length of

Calibration and Use 01 1anKtOfl Couzfling guiprneni
A) coarse adjustment; B) fine adjustment;
C) arm or pillar; D) mechanical stage which
holds slides and is movable in two directions
by means of the two knobs; E) pivot or joint.
This should not be used or “broken” while
counting pia kron; F) eyepiece (or ocular cf:
fIgure 4); 0) draw tube. This will be found
on monocular microscopes only (those having
only one eyepiece). Adjustment of this tube
is very helpful in calibrating the micA o cope
for quantitative counting (Sec. 5. 5. 2. 2.).
H) body tube. In some makes of microscopes
this can be replaced with a body tube having
two eyepieces 1 thus making the ‘scope into
a “binocular.’ I) revolving nosepiece on
which the objectives arc niount d; .1) through
M arc objectives, any oiu of which cai be
turned toward the object being studied. In
this case J is a 40X, K is a 100X, L is a 20X,
and M is a lox objective. The product of
the magnification power of the objective being
used times the magnification power of the
eyepiece gives the total magnification of the
microscope. Different makes of microscopes
employ objectives of slightly different powers,
but all are approximately equivalent. N) stage
of the microscope; 0) Sedgwick-Rafter cell In
place for observation; P) substage condenser;
Q) mirror; R) base or atand;.note: for
information on the optical system, consult
reference 3. (Photo by Don Moran.).
I ______________
t 1I1 —G

Calibration and Use of Plankton CounUng Equipment
T1 1llHhi
Types of eyepiece micrometer discs or
reticules (reticulea, graticulee, etc.).
When dimensions are mentioned in the
following description, they refer to the
markings on the reticule diecs and not to
the measurements subtended on the micro-
scope slide. The latter must be determined
by calibration procedures such as those
described elsewhere. (a) Whipple plankton
counting eyepiece. The fine rulings in the
subdivided equare are sometimes extended to
the margin of the large square to facilitate
the estimation of sizes of organisms in
different parts of the field. (b) Quadrant
ruling with 8. 0 mm circle, for countint
bacteria in milk smears for example. (C)
Linear scale 5.0 mm divided into tenths.
For measurement of linear dimensions.
(d) Porton reticule for estimating the size
of particles. The sizes of the series of discs
is based on the square root of two so that the
areas of successive discs double as they
progress in size.
, ) : ) (
Figure 2

Cahbration and Use of Plankton Countthg Equipment
strip counted. Thus for two strips in the
example cited above
V 2 100W 100 X 0.55 55 mni 3
- 1000 - 1000 -
F 2 - -v.--— -— - -18.2
it will however be noted that F 2 =
Likewise a factor F 3 for three strips
F 1
would equal — - or approximately 12. etc.
C An Empirical “Step-Off” Method
A simpler but more empirical procedure
for determining the factor is to consider
that if a strip 20 mm wide were to be
counted the 1en h of the cell, that the
entire 1000 mm would be included since
the cell 18 20 mm wide and 1 mm deep.
This 20 mm strip width can be equated to
1000 mm 3 . If a strip (or the total of 2 or
more strips) is less than 20 mm in width,
the quotient of 20 divided by this width will
be a multiplier factor for converting from
count per strip(s) to count per ml.
Thus in the example cited above where at
an approximate magnification of 200X and
with an interpupillary setting of 60, the
width of the Whipple field is . 55 mm. Then:
F 1 — = 36.36 or approx. 36
(as above)
If two strips are counted:
+ 55 20
1:10 andF 2 =r_l. = 18.2= approx. 18,etc.
This same value could be obtained without
the use of a stage micrometer by carefully
moving the cell sidewise across the field
of vision by the use of a mechanical stage.
Count the number of Whipple fields in the
width of the cell. There should be approxi-
mately 36 in the instance cited above.
A Circumstances of Use
The use of concentrated samples, local
established programs, or other circumstances
The type illustrated has two millimeters divided into tenths, plus two additional
tenths subdivided Into hundredths.
EnI&rgsm t a Mlcrometsr Scile

(alibration and Use of Ulankton Counting E uipmcnt
Figure 4. Method of Mounting the Whipple Disc in an Ocular. Note the upper
lens of the ocular which has been carefuUy unscrewed, held in the left
hand, and the Whipple disc, held in the right hand. (Photo by
Don Moran).

Calibration and Use of Plankton Counting Equipment
as seen with lOX Ocular and 43X Objective
(approximately 430X total m .gni.ftc*tion)
\. __.Lmm -_...\
(lOO i)
Figure 5
The apparent relationship of the Whipple
Square is shown as it Is viewed through a
microscope while looking at a stage
micrometer with a magnification of
approximately 430X (lox ocular and 43X
obj ective).
Whapple Square as
seen through ocu1ac
(“Whipple field’)
“Large square” subtend.
one tenth of entire Whipplo
Square .026 mm or 26i
- ‘1/ --
(iO )

Calibration and Use of Plankton Counting Equipment
Microet opr No 25 79
Apr oximnte
M . ,t1ificntInn
Length, or
Intq.rp , pt1Jnry
Linear dimenelona of Whipple
equni 08 In tiutlinwtcrg*
F’actor for
to count/mi
Whole [ .ar e JSmail
I(JOX, obtained with (2 S-R Stripe)
-—---- - - —-- -___ -_______
Serial No
‘/71a t’dthO%) 50 LIio C /1.3 & 9
and 0, ular
S.rIal No 60 .
h29k7 LáM) 70 f/to 0 ho 9 ,1
200 )1, obtained with
Serial No
t i 92I9f Ix)
and Otular
t 0
/29I,7qL6 14 70
400X, obtained with
0 . S 0
O P5 ,
0/ 1.2
p -g

Serial No

and Ocular
/A96? 2(’o. )
6), 7

&-? 2
6’6’ 3
0o Z ’
/7.2 g,
2. 2e ,o
• mm I 0 1 ) 0 ,,ti , II N
Microscope calibration data. The fui iii
shown is suggested for the recording of
data pertaining to a particular microscope.
Heathngs could be modified to suit local
D I. AQ. p1. 8b. 7.66
Figure 6
situations. For example, ‘Interpupillary
Setting” could be re , laced by “Tube Length”
or the “ZS-R Strips could be replaced by
“per field” or “per 10 fields.

Magnification irneri
Calibration and Use of Plankton Counting Equipment
Microscope No.
100X. obtained with
Serial No.
and Ocular
Serial No
200X, obtained with________
(2 S-R Strips)
Serial No.
and Ocular
Serial No.
I with (cell-20 fields
400X obtaine
Serial No.
and Ocular
Serial No. _______
5 lmm = 1000 microns
SI. AQ. p1 8 10. 60.
Figure 6-A
Suggested work sheet for the calibration of a microscope. Details will need to be adapted
to the particular instrument and situation.
(2 S-R Strips)

Calibration and Use of Plankton Counting Equipment
Figure 7
A cube of water as seen through a Whipple square at bOX magnification in
a Sedgewick-Rafter cell. The figure Is drawn as If the microscope were
focused on the bottom of the cell, making visible only those organisms lying
on the bottom of the cell. The little “bug” (copepod) halfway up, and the
algae filament at the top would be out of focus. The focus must be moved up
and down in order to study (or count) the entire cube.

Calibration and Use of Plankton Counting Equipment
may make it necessary to employ the more
conventional technique of counting one or
more separate Whipple fields instead of
the strip count method. The basic relation-
ships outlined above still hold, namely:
F = volume cell in mm 3
volume examined in mm 3
The volume examined in this case will
consist of one or more squares the dimen-
sions of the Whipple field in area and 1 mm
in depth (Figure 7). Common practice
for routine work is to examine 10 fields,
but exceptionally high or low counts or
other circumstances may indicate that
some other number of fields should be
employed. In this case a “per field”
factor may be determined to be subsequently
divided by the number of fields examined
as with the strip count. The following
description however is based on an assumed
count of 10 fields.
As stated above, the total volume
represented in the fields examined con-
sists of the total area of the Whipple fields
multiplied by the depth.
V 4 (side of Whipple field) 2 X depth
(1 mm) X no. of fields counted)
For example, let us assume an approxi-
mate magnification of bOX (see Figures
6 and 7 and an interpupillary setting of
“50”. The observed length of one side
of the Whipple field in this case is 1. 13
mm. The calculation of V 4 is thus:
V 4 side 2 X depth X no. of fields
l.l3Xl.l3XlXlO 12.8 m m 3
The multiplier factor is obtained as
above (Section IV A):
volume cell in mm 3
volume examined in mm
- 1000
- (approx.) 78
(If one field were counted, the factor
would be 781, for 100 fields it would
be 7. 8.)
and F 5 = —n- = (approx.) 1850
B Principles Involved
For counting nannoplankton using the high
dry power (lOX ocular and 43X objective)
and the “nannoplankton counting cell”
(Figure 9) which is 0. 4 mm deep, a minimum
of 20 separate Whipple fields is suggested.
The same general relationships presented
above (Section IV) can be used to obtain a
multiplier or factor (F 5 ) to convert counts
per 20 fields to counts per ml.
To take another example from Figure 4, at
an approximate magnification of 400X and an
interpupillary setting of 70 (see also Figure 3)
we observe that one side of the Whipple field
measures 0. 260 mm. The volume of the
fields examined is thus obtained as follows:
V 5 = side 2 X depth X no. of fields
0.26 X 0. 26 X 0.4 X 20 = . 54 mm 3
C Calculation of Multiplier Factor
It should be noted that the volume of the
nannoplankton cell, . 1 ml, is of no significance
in this particular calculation.
F 4
1 American Public Health Association, et. al.
Standard Methods for the Examination
of Water, Sewage, and Industrial Waste8.
13th Edition. Am. Public Health Assoc.
New York. 1970.
2 Jackson, H. W. and Williams, L. G.
The Calibration and Use of Certain
Plankton Counting Equipment. Trans.
Am. Mic. Soc. LXXXj(l):96-103. 1962.
3 Ingram, W. M. and Palmer, C. M
Simphfied Procedures f or Collecting,
Examining, and Recording Plankton in
Water. Jour. Am. Water Works.
Assoc. 44(7): 617-724. 1952.
4 Palmer, C M Algae in Water Supplies.
U. S. D. H. E. W. Public Health Service
Pub. No. 657. 1959.
5 Palmer, C. M and Maloney. T E. A
New Counting Slide for Nannoplankton.
American Soc. Limnol. and Oceanog.
Special Publications No. 21. pp. 1-6.
2 1—11

Calibration and Use of Plankton CountIng Eauipment
Sedgewick-Rafter counting cell 8hOwiflg bottom scored across for ease in counting
strips. The “strips” as shown in the illustration simply represent the area counted,
and are not marked on the slide. The conventional dimensions are 50 X 20 X 1 mm, but
these should be checked for accurate work.
Figure 9
Nannoplankton cell. Dimensions of the circular part of the cell are 17. 9 mm diameter
X 0. 4 mm depth. When covered with a covergiass, the volume contained is 0. 1 ml.
The channels for the introduction of sample and the release of air are 2 mm wide and
approximately 5 mm long. This slide Is designed to be used with the 4 mm or 43X
(high dry) objective.
6 Welch, Paul S. Limnological Methods.
Blakiston Company. Phila. Toronto.
Drinking Water. John Wiley aid Sons.
New York. 1948.
7 Whipple, C. C., Fair, 0. M., and
Whipple, M. C. The Microscopy of
This outline was prepared by H W. Jackson,
Chief Biologist, National Training Center,
DTTB, MDS, OWP , EPA, Cincinnati, OH
Figure 8

Odor shall be determined 8Ubstaritially as
prescribed by the 11th edition of ‘Standard
Methods for the Examination of Water and
Sewage”, subject to certain stipulations and
modifications made necessary by the Inter-
national Joint Commission.
The procedure and technique to be followed
are described below
A Odor-free water - prepared by passing
tap water through activated carbon at a
slow rate of speed. Activated carbon
can be placed at the bottom of a 20-liter
glass bottle The bottle can be connected
to the tap by rubber tubing leading to glass
tubing above the water. The outlet from
the bottom of the bottle should be glass
tubing. A trap made of inch glass tubing
filled with activated carbon is placed at
the end of the outlet.
B 500 ml glass-stoppered Erlenmeyer
flasks, each flask with a number. Glass-
ware must be thoroughly cleaned and
rinsed several times with odor-free
water before each use.
C Chemical Thermometer (0-100°C)
D 10 ml Mohr pipettes, 25 ml graduated
cylinders, 50 ml graduated cylinders,
200 m.l graduated cylinders, 500 ml
graduated cylinders. Other pipettes
and cylinders as needed.
E One liter glass-stoppered bottles to hold
samples of water being examined Other
glass bottles and flasks as needed.
A Certain conditions are required to
obtain consistent results. Considerable
practice with the test is desirable to
develop consistent sensitivity to the
sense of smell
1 In view of the perishability of the
odor test, these determinations
should be made immediately after
2 The prepared odor-free water shnuld
be truly free of all detectable odor.
3 All glassware must be free of odor.
This is accomplished by thorough
cleansing followed by several rinses
with odor-free water.
4 All dilutions should be compared with
an odorless standard. This aids the
observer in deciding whether air odor
is present or not.
5 All dilutions when examined for odor
should be of a uniform temperature,
deviation not to exceed 1°C.
6 A sudden change in the character of
the odor during the testing procedure
should be considered as a warning
that there may be interference from
outside odors or that the diluting
water may not be odor-free. The
character of odor should always be
recorded for future consideration.
B To eliminate psychological influences,
the samples should be coded and in-
terni .xed so as not to suggest to the
observer what odor concentration is
being observed.
I Bottles should be colored or covered
with odor-free matezial or the observer
blindfolded to eliminate auto suggestion
WS. TO. lab. la. 1. 66

Determination ol Odors
since many samples may possess
color or turbidity.
2 Test should be conducted in a room
free of outside odors. The observer
should be cautioned to refrain from
smoking or eating for an appreciable
time before taking test. Odors should
be washed from the hands prior to
taking test.
3 The test should not be prolonged to a
point where the sense of smell becomes
A To obtain the approximate range of odor
value take 50 ml, 14 ml and 5 ml of sample
and make each sample up to 200 ml with
odor-free water. Compare the odor of
these three with 200 ml of odor-free
1 Cold odor: Bring dilutions to temper-
ature of 24 - 2 5°C.
2 Shake each flask uniformly before
smelling for odor. Observer should
characterize type of odor.
3 Note which flasks contain odor and
which do not. According to result8
obtained, prepare intermediate di-
lutions, in each case using sufficient
odor-free water to make a total
volume of 200 ml.
4 Include a flask with 200 ml of odor-
free water with each series, as a
blank for comparison.
B Arrange flasks so that their identity is
unknown and bring to desired temperature.
I Observe for odor and make chart with
a “plus” or “zero” for each dilution.
2 The results are reported in “threshold
odor numbers”. The threshold odor
number is calculated from the amount
of sample in the most diluted portion
which gives perceptible odor. The
volume of the d.ilution(200 ml) divided oy
the volume of the sample In the dilution
equals the threa hold odor number. For
example, if 5 ml diluted to 200 ml is
the most dilute portion giving perceptible
40, the threshold odor is
numbered 40.
C The threshold odor number shall not be
confused with the “threshold odor con-
centration”. The threshold odor concentra-
tion is the smailest amount of odor-producing
material in mg/i required to give perceptible
odor. If the threshold odor concentration is
known, that value multiplied by the threshold
odor number will give the concentration of
the odor-producing material in the sample.
This outline was prepared by E. L.
Robinson, Research Aquatic Biologist,
Fish Toxicology Laboratory, 3411 Church
Street, Newtown, OH 45244.

Determinatjoii of Odors
Amount of Culture ________________ ml Exp. No. ________________
Age of Culture___________________ days Temp. Tested at °C
No. Cells per ml Culture Medium________
Mixed, Unialgal, Pure Date___________________
Observer No. 1
4 1 6
Culture Dilution R
Threshold Odor No.
Description of Odor
+ Odor Detected 0 No Odor Detected
Estunated Composite* T. 0. No. ________________________________
*Geometric average of T.O. No. of individual observers E. L. R. 1956

Primary production is the synthesis of organic
matter from inorganic raw materials. The
energy required for this process may come
from light (photosynthesis), or from chemical
sources (chemosynthesls). The primary
synthesis of organic matter in lakes and
streams is carried on by planktonic and ben-
thic algae and bacteria, and aquatic
The productivity of a body of water Is indicated
in a general way, by the density of the plankton
population. The standing crop of plankton is
commonly measured by determining one or
more of the following:
A Dry and Ash-free Weight of Seston
B Cell or Unit Counts
C Cell Volume
The photosynthetic process involves the up-
take of CO 2 and the release of 02. The
reactions are enzyme catalyzed and are af-
fected by the following factors.
A Temperature
B Light Intensity
C Light Quality
D Chlorophyll
E Particulate and Dissolved Carbohydrate
F Particulate and Dissolved Organic Carbon
Increases in the standing crop over a period
of time may be used to determine productivity.
However, this method provides only a rough
approximation of the rate of primary
D pH
E Nutrients
F Trace Elements
Methods employed to measure plankton pro-
ductivity are:
A Standing Crop
B Oxygen
C pH
D Carbon-14
The use of dissolved oxygen to determine
short-term rates of primary production was
introduced by Gaardner and Gran (192?).
Estimates of the amount of carbon fixed are
based on the premise that one molecule of
oxygen is given off for each atom of carbon
CO 2 + H 2 0 -. CH 2 O + 02
A “ Light ” and “ dark ” bottles are filled with
sample and resuspended at various depth8
for 4 - 24 hours.
B The concentration of dissolved oxygen is
determined (using the Wink.ler Method) at
B!. ECO. pro. la. 4.70

Determination of Plankton Productivity
the beginning and end of the incubation
period. The values obtained are as
1 Final “light” bottle 02 — initial 02
net photosynthesis
2 InItial 02 - Final “dark” bottle °2 =
3 Net photosynthesis + respiration
gross photosynthesis
This method has some serious dlsadvantages
A The bottles provide an artificial substrate
for the prohferat on of bacteria which
use up large amounts of °2’ resulting in
erroneously high respiration and low net
photosynthesis values.
B The lower limit of sensitivity of the Winkler
Method is 0.02 mg 02/liter. This is a
serious handicap when working in oligo-
tropic lakes and the open sea.
The use of carbon- 14 for the measurement
of the rate of carbon assimilation by phyto-
plankton was pioneered by E. Steernann
NIelsen (1952). The method is simple and
very sensitive.
A Carbon- 14 labelled sodium bicarbonate
(4 - l0 ic/liter) is added to ‘ g t” and
“ dark ” bottles, which are resuspended in
the water for 4 - 24 hours.
B An aliquot of the sample is passed through
a membrane filter (1.2 p. pore diameter),
and the filters are treated with acid to
remove any inorganic labelled carbon.
C The (beta) activity of the filter is deter-
mined with an end-window Geiger tube,
or with gas flow or liquid scintillation
D The carbon fixed is determined as
There are several important disadvantages
in this method.
A Some of the labelled photosynthesis pro-
ducts will be broken down immediately by
reapiration, and the liberated carbon-14
reused in photosynthesis. Therefore, it
is generally agreed that the method mea-
sures only net photosynthesis.
B It has been found that the algae rapidly ex-
crete up to 50% of the photosynthate In the
form of organic acids, carbohydrates,
and amino acids. Since these labelled
materials are not retained by the filter,
they escape detection.
The uptake of CO 2 by the algae during photo-
syntheses results in an increase in the pH of
the surrounding medium. Periodic pHmeaaure—
ments are made of the body of water being
studied, and the carbon uptake is determined
using published nomographa.
Verduin (1952) used this method in a study
of the productivity of Lake Erie. However
the method has not gained wide acceptance
because it can be used only in waters with
low alkalinity.
1 Allen, M. B. Excretion of Organic Com-
pounds by Chlamydomonas. Arch. f.
Miicrobiol. 24 163-168. 1956.
2 Curl, H. Jr., and Small, L. F. Variations
in Photosynthetic Assimilation in Natural
Marine Photoplankton Communities.
Limnol. Oceanogr. lO(Suppl.):R67-R73.
3 Gaardner, T., and Gran, H. H. Investi-
gations of the Production of the Plankton
in the Oslo Fjord. Rapp. et Proc. -
Verb., Con. Internat. Explor. Mer.
42 1-48. 1927.
Correction for
carbon - activity on filter < available < isotope
fixed - total activity added HCO discrimination
2 . —2

Determination of Plankton Productivity
4 Goldman, C. R. Molybdenum as a Factor
Limiting Primary Productivity in
Castle Lake, California. Science 132:
1016-1017. 1960.
5 Kamen, M. D. Primary Processes in
Photosynthesis. Academic Press,
New York. 1963.
6 Marshall. S. M., and Orr, A. P. Carbo-
hydrate as a Measure of Photoplankton.
J. Mar. Biol. Aesoc. U.K. 42:511-519.
7 Ryther, J. H. Photosynthesis in the Ocean
as a Function of Light intensity.
Limnol. Oceanogr. 1:61-70. 1956.
8 Steemann Nielsen, E. The Use of Radio-
active Carbon (C-14) for Measuring
Organic Production in the Sea. J. Con.
Internat. Explor. Mer. 18 117-14O. 1952.
10 Verduin. J. Photosynthesis and Growth
Rates of Two Diatom Communities in
Western Lake Erie. Ecology 33(2):
163—168. 1952.
11 Vernon, L.P. Bacterial Pbytosyntheala.
Ann. Rev. Plant. Physiol. 15:73-100. 1962
12 Wetzel, R. G. A Comparative Study of the
Primary Productivity of Higher Aquatic
Plants, Per iphyton, and Phytoplankton
in a Large, Shallow Lake. Internat.
Rev. Hydrobiol. 49:1-61. 1964.
13 Yentsch, Charles S. The Measurement
of Chioroplastic Pigments- Thirty
Years of Progress? pp. 255-270 in
Chemical Environment In the Aquatic
Habftat. Proc. IBP Symposium.
Amsterdam. 1967. (N.y. Noord-
Holiandsche Uitgevers Maatschappij.
Amsterdam, Netherlands. 8.95)
9 Strickland, J. D. H. Measuring the
Production of Marine Phytop]ankton.
Bull. Fish. Res. Bd. Can. No. 122:
1-172. 1960.
This outline was prepared by C. I. Weber,
Chief, Biological Methods Branch,
Analytical Quality Control Laboratory,
NERC, EPA, Cincinnati, OH 45268.

I OBJECTIVE b Move the slide at random and repeat
the process. Do this for 5 or 10
To learn and practice the techniques of fields, or for one or two stripe.
proportional counting of mixed plankton
samples. c Tally the results and compute the
percent of each type.
U MATERIALS 2 Five hundred count
A Several plankton samples, each containing a Moving the slide at random count
a number of plankton forms, and tally all the types of plankton
as before until a total of 500 cells
B Class slides, cover slips, and dropping or clumps have been counted.
b TaUy the results and compute the
percentage of each type as before.
A Make an ordinary wet mount of the IV RESULTS
sample provided.
A Record your results for both methods
B Scan the slide. Identify and list all types on the board.
of plankton present.
B Discuss the two methods and the use of
C Proportional Counting (use clump count) the proportional count results.
1 Field count
a Count and tally all individuals of
each type present In a field. The
beat way to do this is to list the _____________________________________
most common types separately and This outline was prepared by M. E. Bender,
record the counts and then enumerate Biologist, formerly with FWPCA Training
the other forms. Activities, SEC.
BI.MIC.enu.]ab. 6a. 8.69

I OBJECTIVES D Record the exact dimensions of the entire
field in the column marked “Whole” on the
plate “Microscope Calibration Data.”
A To Become Familiar with Microscope
Calibration Procedures E Do the same with the 200X and 400X
B To Calibrate the Particular Equipment
Assigned to you F Return the stage micrometer to the supply
II MATERIALS G Values for the “Large” and “Small”
columns may now be calculated
arithmetically. There are ten large
A Whipple, Plankton Counting Reticule squares across the whole field, and 5
small squares across the large square
B Compound Microscope as Assigned which is subdivided 1 in the center of the
C Stage Micrometer
H Calculate the conversion factors to counts
per ml according to the formulae in the
UI PROCEDURE lecture entitled “Calibration and Use of
Plankton Counting Equipment.”
A Adjust the interpupillary distance to the
position most comfortable for your eyes.
and record the setting on the “Microscope
Calibration Data” sheet.
B Install a Whipple plankton counting reticule
in the right eyepiece.
This outline was prepared by H. W. Jackson
C Obtain a stage micrometer and focus on the chief Biologist, National Training Center,
scale at 100X magnification. DTTB, OWP, EPA, Cincinnati, OH 45288.
BI. MET. mic. lab. la. 5. 70 251

Laborstory Calthration of Plankton Counting Equipment
and Ocular
Serial No.
Microscope No.
Length, or
Magnification Interpuptilary
- Setting
Linear dimensions of Whipple
squares in millimeters*
Factor for
tO count/mi
EEte T ube__
whole ge 5mah1
l00X obtained with
(2 S-R Strips)
Serial No.
and Ocular
Serial No
200X, obtained
(2 S-R
Serial No.
and Ocular
Serial No.
400X, obtained
with (Nannoplankton)
(cell-20 fields
Serial No.
•lmm 1000 microns
81. AQ. p1 8 10. 60.

Laboratory: Calibration of Plankton Counting Eq pment
and Ocular
Serial No.
Microscope No.
Length, or
Tube 1 Linear dimensions of Whipple
Interpupulary aquarea in millimeters*
Setting Whole Large Small
Factor for
to count/mi
bOX, obtained with
(2 S-R Strips)
Serial No.
and Ocular
Serial No
200X, obtained with________
Objectiv — (2 S-R Strips)
Serial No.
and Ocular
Serial No.
400X. obtained with (Nannoplankton)
(cell-20 fields
Serial No.
*1mm 1000 microns
BI. AQ. p1. 8 10. 60.

I OBJECTIVE C Starting from one end of the S-R cell and
proceding to the opposite (this is called a
To learn and practice the basic techniques of strip count, begin counting (clump counts)
quantitative plankton counting the plankton forms. The length of the
cell may be traversed in several ways.
U MATERIALS 1 Count all the forms in the Whipple
square or in a portion of the square,
record the count and move the slide so
A Plankton Samples Containing a Variety of that the square covers the adjoining
Plankton Forms area.
B S-R Cells and Covergiasses , Large Bore 2 Move the slide very slowly counting
1 ml Pipettes, Whipple Di8cs. Plankton and recording the various forms as
Record Form they pass the leading edge of the
Whipple disc.
A FLU the S-R cell with sample number 1 as
follows: A Using the conversion factor obtained In
the previous laboratory compute the
Place the covergiass diagonally across number of plankton organisms per ml.
the S-R cell. This leaves the other two
corners uncovered; one for putting in B Record the results on the board.
the sample fluid, the other to allow
air to be driven out as it is replaced c Discussion of Results
by the incoming aliquot. Shake the
sample to disperse the plankton. Before D Refill the slide with a fresh aflquot and
settling occurs in the sample draw about recount the sample. Compare results
1-1/4 ml of the fluid into the pipette with the first count.
and quickly fill the S-R cell by delivering
the aliquot into one of the Open corners E Count the other samples of mixed plankton
of the chamber, as assigned, following the same procedure.
B Using lOOx focus on the sample. After
focus has been obtained switch to 2 00x. This outline was prepared by M. E. Bender,
Scan the slide and list the plankton forms Former Biologist, FWPCA, Water Pollution
present. Training A ctivities, SEC.
BI.MIC.enu.lab.7.6.68 26-1

Laboratory Fundamentals of Quantitative Counting
.ourr DO _________
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7 11 10, nun.

Dense growths of algae in surface waters are
aesthetically undesirable, cause problems In
water treatment, produce changes In the aquatic
environment that are harmful to fish and other
aquatic life, and are symptomatic of pollution.
The density of phytoplankton opulationa is
directly related to the concentration of
nutrients. This relationship has been well
documented, and is now embodied in the
concept of trophic level or trophic status of
surface waters. One or more of the following
parameters are commonly used to descrthe
the trophic status: (a) nutrient concentration -
principally N and P, (b) algal count,
(c) chlorophyll concentration, (d) primary
productivity. (e) particulate organic matter,
(f) oxygen depletion in the hypolimnion, and
(g) phytoplankton species composition or
Indicator species (Rawson 1956; Davis 1964;
Goldman & Carter 1965, Oglesby & Edmondson
1986; Fruh, Steward, Lee & Rohltch 1986).
Three general trophic levels now recognized,
here arranged in ascending order, are:
oligotrophic (low), mesotrophic (intermediate),
and eutrophic (high). The addition of nutrients
to surface waters raises the trophic level and
results in an increase in phytoplankton density
and changes in the species composition.
This process, commonly referred to as
eutrophication, is greatly accelerated by the
discharge of nutrient-laden domestic and
industrial wastes (Hasler 1947), Edznondson
& Anderson 1956).
Although chemical analyses provide Information
on the concentration of nutrients, their
availability to the algae can be determined
only by biological assay. Biological assays
to determine the potential (algal) productivity
of surface water were first used in the late
twenties (Schreiber 1927) and early thirties
(Strom 1933). but until recently had been
used only infrequently (Potash 1956,
Skulberg 1964, 1967; Shelef & Halperin 1970).
In 1987, the Joint Industry-Government Task
Force on Eutrophication took steps to develop
a standardized algal growth potential (AGP)
test. Using this test, one can:
A Evaluate the effectiveness of waste
treatment processes In removing elements
that support or stimulate the growth of
B Determine at what point along the time
scale of progressing eutrophication the
water of a given lake or stream happens
to lie (trophic status).
C Anticipate the effect on algal production
of introducing extraneous nutrients.
D Determine the extent to which nutrient
levels must be reduced in a body of water
to effect an acceptable remedy.
A A surface (test) water sample is collected
and the indigenous microorganisms are
removed by filtration (0. 45 micron
membrane filter at 15 inches of mercury)
or ultracentrifugatlon.
B The surface water and standard medium
(Table 1) are inoculated with 1000 cells/mi
of Selenastrum capricornutum , or 50, 000
cells/mi of Anabaena flos-aquae or
Microcystis aeruginosa .
C The cultures are prepared in triplicate
and incubated 7-10 days at 24° C, 200 fc
(blue-greens) or 400 fc ( Selenastrum )
continuous illumination, with shaking at
100 oscillatIons/mm ( ilturing may be by
flask, chemostat, or in situ technique).
D Algal growth is measured daily by
(1) cell counts, (2) determining the
B!. BlO. alg.Lb. 10.72

AIKaI Growth Potential Test
(This formula consists of 30% of the concentrations of the macroelements
listed in the February, 1969, PAAP Booklet. The Na 2 CO 3 was replaced by
NaHCO 3 , and the EDTA was reduced to 333 Mg/i.)
MACROELEMENTS : (milligrams per liter)
Compound Final Conc . Element Element
Furnished Conc .
NaNO 3 25.500 N 4.200
K HPO 1.044 P 0.186
2 4 K 0.469
MgC1 2 5.700 Mg 1.456
MgSO •7H 2 0 14.700 Mg 1.450
S 1.911
CaC 1 2 2H 2 O 4.410 Ca 1.202
NaHCO 3 15.000 Na 1l.OOj
If the medium is to be filtered, add the following trace-element-tron-EDTA
solution from a single combination stock solution after filtration. With no
filtration, K HPO should be added last to avoid iron precipitation. Stock
solutions of ?nd1vi ua1 salts may be made up In 1000 X’s final conc. or less.
MICROELEMENTS : (micrograms per liter)
H 3 B0 3 185.5 B 32.5
MnCI 2 264.3 Mn 115.4
ZnC 1 2 32.7 Zn 15.7
CoC1 2 0.780 Co 0.354
CuCl 2 0.009 Cu 0.004
Na 2 MoO 4 •2H 2 0 7.26 Mo 2.88
FeCl 3 96.0 Fe 33.05
Na 2 EDTA2H 2 O 300.0

A1 ia1 Grnwth PnfPnflkl Tecit
chlorophyU content, In vivo f1uorescen e,
light scattering or optical density
(600 nm) of the culture, (3) measurIng
the C- 14 uptake, or (4) determining the
dry weight of the algae at the end of the
Incubation period. Regardless of the
parameter used to measure growth
response, the result should always be
expressed in terms of the final dry weight
of the culture.
E The growth response of the alga in the
test water is compared to its growth in
the standard medium.
A Composition of the standard growth
B Effects of ventilation and shaking on the
growth response of batch cultures.
C Techniques of measuring growth response.
D Techniques of removing indigenous
microorganisms from test surface waters.
VI For copies of the Provisional Algal Assay
Procedure and information on the availability
of subcultures of the test organiazn, contact:
Dr. A. F. Bartach, Chairman
JTF Research Program Group
Director, Pacific Northwest
Water Research Laboratory
Corvallis, Oregon 97330
_________ Provisional Algal Assay
Procedure. Joint Industry-Government
Task Force on Eutrophication, P.O.
Box 3011, Grand Central Station,
N.Y. 10017. 1969.
2 Davis, C. C. Evidence for the eutrophication
of Lake Erie from phytoplankton records.
Umnol. Oceanogr. 9:275. 1964.
3 Edmondson, W. T. and Anderson, G. C.
Artificial eutrophication of Lake
Washington. Limnol. Oceanogr.
1 (1):47—53. 1956.
4 Fruh, E.G., Stewart, K.M., Lee, G.F.,
and Rohlich, G.A. Measurements of
Eutrophication and Trends. JWPCF
38(8):1237-1258. 1966.
5 Goldman, C.R. and Carter, R.C.
An investigation by rapid C’ 4
bioassay of factors affecting the
cultural eutrophication of Lake Tahoe,
California. JWPCF 37:1044- 1063.
6 Hasler, A. D. Eutrophication of lakes by
domestic drainage. Ecology 28(4):
383-395. 1947.
7 Oglesby, R. T. and Edmondson, W. T.
Control of Eutrophication. JWPCF
38(9):1452-1460. 1986.
8 Potash, M. A biological test for
determining the potential productivity
of water. Ecology 37(4):631-639.
9 Rawson, D. S. Algal Indicators of lake
types. Limnol. Oceanogr. 1:18-25.
10 Schrelber, W. Der Reinkultur von
rnarinem Phytoplankton und deren
Bedeutung fur die Erforschung der
Produktions-fahigkeit des Meerwasaers.
Wissensch. Meeresunters., N.F.
16:1-34. 1927.
11 Shelef, G. and Halperin. R. 1970. Wastewater
nutrients and algae growth potential.
In: H. I. Shuval, ed., “Developments
in Water Quality Research”, Proc.
Jerusalem Internat’l. Con!, on Water
Quality and Poll. Rca., June, 1969.
Ann Arbor-Humphrey Science Pubi.,
p. 211-228.

Algal Growth Potential Test
12 Sku]berg, O.M. A].galproblems related
to the eutrophication of European water
8uppUes. and a bioassay method to
assess fertilizing Influences of pollution
on th.land waters. In: D.F. Jackson 1
ed., “Algae and Man”, Plenum Press,
N. Y. p. 262-299. 1964.
13 Skulberg, O.M. Algal cultures as a means
to assess the fertilizing Incluence of
pollution. In: Advances in Water
Pollution Research, Volumn 1,
Pergamon Press, Washington, D. C.
14 Strom, K. M. Nutrition of algae. Experi-
ments upon; the feasibility of the
Schrelber method in fresh waters,
the relative inportance of Iron and
manganese In the nutritive medium;
the nutritive substance given off by
lake bottom muds. Arch. Hydrobiol.
25:38-47. 1933.
_______________ Algal Assay Procedure
Bottle Test. 82 pp. Environmental
Protection Agency, National Eutrophica-
tion Research Program, Corvallis,
Oregon. 1971.
-. Inter- Laboratory
Precision Test. An Eight-Laboratory
Evaluation of the Provisional Algal
Assay Procedure Bottle Test. 70 pp.
Environmental Protection Agency,
National Eutrophication Research
Program, Corvallis, Oregon. 1971.
3 Berge, G., Predicted Effects of
Fertilizers Upon the Algae Production
in Fern Lake. Fisk Dir. Skr. Serv,
Hay. Unders., 15:339-355. 1969.
4 Jonnson, J.M., T.O. Odlaug, T.A. Olson,
and O.R. Ruschmeyer. The Potential
Productivity of Freshwater Environ-
ments as Determined by an Algal
Bioassay Technique. Water Resources
Research Center Bulletin No. 20,
University of Minnesota, Minneapolis.
5 Maloney 1 T.E., W.E. Miller, andT.
Shiroyama. Algal Responses to
Nutrient Additions in Natural Waters.
I. Laboratory Assays. In: Special
Symposia 1:134-140. Amer. Soc.
Limnol. Oceanogr. 1972.
6 MIller, W.E., andT.E. Maloney.
Effects of Secondary and Tertiary
Wastewater Effluents on Algal Growth
in a Lake-River System. JWPCF
43(12)2381—2365. 1971.
7 Murray, S., J. Scherfig, and P.S. Dixon.
Evaluation of Algal Assay Procedures-
PAAP Batch Test. .JWPCF 43(10):
1991—2003. 1971.
8 Shapiro, J., and R. Riberiro. Algal
Growth and Sewage Effluent In the
Potomac Estuary. JWPCF 37(7):
1034-1043. 1965.
9 Toerien, D. F., C. H. Huang, J. Radimsky,
E. A. Pearson, and J. Scherfig. Final
Report, Provisional Algal Assay
Procedures. 211 pp. Sanitary Engineer-
ing Research Laboratory Report No.
7 1-6, University of California,
Berkeley. 1971.
This outline has been prepared by Dr. C. I.
Weber, Chief, Biological Methods Section,
Analytical Quality Control Laboratory,
NEEC, EPA, Cincinnati, OH 45268.

I Water treatment always should include
detection and control of microorganisms.
A Two types of microorganisms are involved
1 Pathogenic types include such forms as
the typhoid bacteria, the dysentery
ameba, and the Infectious hepatitis
2 Interference types include taste and
odor organisms, filter-clogging
organisms, pipe-infesting organisms,
and others.
B Water treatment practices are closely
associated with these organisms.
1 For pathogens, practices include
coliform tests, use of chlorine, and
guarding the water supply against fecal
2 For interference organisms, practices
include plankton enumeration, use of
copper sulfate and the covering of
3 Many of the other treatment practices
have significant effects on the organisms.
C This discussion will be limited to the inter-
ference organisms.
A At Chicago, the alga Dinobryon reappears
almost every year, generally In June and
July in numbers sufficient to impart a
prominent fishy odor to the water. In
1951, it required an estimated $70, 500
worth of activated carbon to control the odor
of this organism for a period of two months.
B At Indianapolis, copepods were present in
parts of the distribution system In numbers
sufficient to be visible in the drinking
water. The eggs of the copepode were
found to pass through the filters and to
hatch In the distribution system.
C At Oklahoma City, prominent earthy odors
have appeared frequently. The organisms
blamed for this trouble are the mold-like
D At Peoria, white wigglers up to 3/8” long
were reported in the tap water, during
early March, 1956. These chironomid
larvae had hatched in the city’s open
reservoir, requiring that the reservoir be
drained, cleaned and treated with a larvicide.
E At Chicago, diatoms are a very important
cause of short filter runs. The one diatom
TabeLlarja is considered to be more
responsible than any other organism for
this trouble.
F In Ontario, the alga Cladophora often
grows in large numbers attached to rocks
on the shoreline of lakes. When the alga
is broken loose it collects near the shore-
line and gives rise to very offensive odors.
G In a water supply impoundment in Utah the
plankton algae frequently cause the pH of
the water to increase to 8. 3 or higher,
requiring that the water be treated with
acid to obtain the desired pH of 8 or lower.
H In Texas a water supply from underground
sources was stored in a large open settling
basin. Osciflatoria and unicellular green
algae developed in large numbers in the
stored water, turning it green and pro-
ducing a strong odor.
I Los Angeles has more than 25 open reser-
voirs of various sizes and ranging in
elevation from almost sea level to over
BI.MIC. 12c. 3.70

Algae and Actinomycetes in Water Supplies
7,000 feet. Many tons of copper sulfate
are used every year in these reservoirs
for rigid control of plankton, chiefly
diatoms and occassionally blue-green
algae. This treatment is carried out to
improve the water quality including the
reduction of tastes and odors.
A Tastes and Odors
1 May be caused by algae, actinomycetes,
crustacea, and anaerobic bacteria.
2 Common algal odors imparted to water
are ones described as fish, earthy,
musty, grassy, cucumber, geranium,
nasturtium, and septic.
3 Common actinomycete odor is earthy.
4 Tastes produced in water by algae
include sweet and bitter.
5 Other causative agents of tastes and
odors may be industrial wastes, sludge,
and compounds dissolved from soil and
rock, and chemicals used in treatment.
B Filter Clogging
1 Both rapid and slow sand filters are
2 Diatoms are the organisms most
frequently involved but blue-green
algae, filamentous green algae and
other organisms as well as silt may
cause it.
C Other Pro ems in the Treatment Plant
1 Algae may cause variation in the pH,
hardness, color, and organic content
of the water.
2 Amount of plankton organisms often
influences the rate and effectiveness
of coagulation.
3 Qilorlne dosage may depend upon
amount of plankton organisms present.
4 Growths of algae may reduce the flow
through thfluent channels and screens.
5 Organisms may be responsible for
increasing the quantity of sludge to be
disposed of in sedimentation basins.
6 Miorocrustacea “spot” paper in paper
mill rolls.
D Infestation of Distribution Systems
1 Attached organisms reduce the rate of
flow in the pipes.
2 Iron and sulfur bacteria may initiate
or stimulate corrosion of pipes.
3 Organisms may appear as visible
bodies in tap water.
4 Tastes and odors may result from
presence of organisms.
S Chlorine residual is difficult to main-
tain when organic matter is present.
6 Organisms could theoretically harbor
and protect against chlorine certain
pathogenic bacteria.
E Profuse Growths of Organisms in Raw
Water Supplies
1 A limited and balanced growth of
various organisms is generally an
2 Extensive surface mats, blooms and
marginal growths often cause troubles
along the shoreline and eventually in
the treatment plant.
3 Some fish kills may be caused by
profuse growths of algae by reducing the
DO during the night.
4 Certain massive growths of blue-green
algae are deadly poisonous to animals.
Animal forms include protozoa, rotifers,
crustaceans, worms, bryozoans, fresh water

Algae and Actinomycetes in Water Supplies
sponges, water mites and larval stages of 3 By eliminating shallow marginal areas
various insects.
4 By reducing the amount of fertilizing
B Plant forms include algae, actinomycetes nutrients entering the reservoir.
and other bacteria, molds and larger
aquatic green plants.
VII It is generally more satisfactory to
anticipate and prevent problems due to these
V IMPORTANCE OF BIOLOGICAL organisms than it is to cope with them later.
A Routine biological tests are essential to
A The increased use of surface water supplies detect the initial development or presence
increases the problems cauBed by organ- of interference organisms.
isms. Biological problems are less
common with ground water supplies. 1 Control measures can then be used
before problem becomes acute.
B Standards of water quality requested by
domestic and industrial patrons are rising. 2 These tests should be applied to the
raw treatment plant water supply and
C Procedures for detection, control and distribution system.
prevention of problems caused by organisms
are improving and are receiving more B In the Reservoir or Other Raw Water Supply
extensive use.
1 Routine plankton counts should be made
of water samples from selected loca-
VI A number of methods may be used to tions. Plankton counter should be
control the interference organisms or their
aware of the particular organisms
products: known to be most troublesome.
A Addition to water or an algicide or pesticide 2 During the warmer months routine
such as copper sulfate, chlorine dioxide
surveys of the reservoir, lake or
or copper -chlorine-ammonia.
stream should be made to record any
visible growths of algae and other
B Mechanical cleaning of distribution lines,
settling basins, sand filters, screens, and
reservoir walls.
3 Odor tests of water from several
locations should be made to obtain
C Modification of coagulation, filtration, advance notice of potential trouble at
chemical treatment, or location of intake
the treatment plant.
D Use of absorbent, such as activated
carbon, for taste and odor substances. C In the Treatment Plant
E Modification of Reservoir to Reduce the 1 Records of plankton counts and threshold
Opportunities for Massive Growths of odor between each step in treatment
Algae gives data on effectivenesB of each
1 By covering treated water reservoir to
exclude sunlight 2 Coagulation and filtration can be
adjusted to remove up to 95% or more
2 By increasing the depth of the water in of organisms in water.

Alaae nd Actiriomycetes in Water unnH
i Mi roscnpic analysis of samples of
filter material for organisms can
supply data useful in modifying sand
filtration and treatment of finished
D In the Distribution System With Its
Finished Water
1 Open reservoirs require constant
attention especially during summer.
2 Parts of the system farthest from the
treatment plant or adjacent to dead
ends require most frequent sampling
for organisms and tastes and odors.
A Interference organisms cause problems
in distribution systems, treatment plants.
raw water supplies.
B Organisms involved include algae, actino-
rnycetes, other bacteria, and minute
aquatic animals.
C Control is by special chemicals, mechanical
cleaning, adjustment of chemical or
mechanical treatment and by modification
of reservoirs, intakes, etc., for the raw
water supply.
D Facilities for detection of problems in
their early stages are required for most
efficient and satisfactory control.
1 Palmer, C M. Algae in Water Supplies.
An Illustrated Manual on the Identification,
Significance, and Control of Algae in
Water Supplies. U. S. Public Health
Service Publication No. 657. 1959.
p. 88.
2 Palmer, C.M. and Poston, H.W.
Algae and Other Interference
Organisms in Indiana Water Supplies.
Jour. Amer. Water Works Asen,
48:1335—1346. 1956.
3 Palmer, C.M. Algae and Other Inter-
ference Organisms in New England
Water Supplies. Jour. New England
Water WorkeAsen, 72:27-46. 1958.
4 Palmer, C.M. Algae and Other Orga-
nisms in Waters of the Chesapeake
Area. Jour, Amer. Water Works
Asen. 50:938-950. 1958.
5 Palmer, C. M. Algae and Other Inter-
ference Organisms In the Waters of
the South Central United States. Jour.
Amer. Water Works Asen. 52:897-
914. 1960.
6 Siivey, J.K. and Roach, A.W.
Actinomycetes May Cause Tastes
and Odors in Water Supplies. Public
Works 87. 5:103-106,210,212. 1956.
7 Ingram, W.M. and Bartech, A.F.
Operators Identification Guide to
Animals Associated with Potable
Water Supplies. Jour. Amer. Water
Works As n. 52:1521-1550. 1960.
8 Otto, N.E. and Bartley, T.R. Aquatic
Pests on Irrigation Systems.
Identification Guide. Bur. of
Reclamation. USD1. 72 Pp. 1965.
9 Herbst, Richard P. Ecological Factors
and the Distribution of C]adophera
glomerata in the Great Lakes.
Amer. Midi. Nat. 82:90-98. 1969.
This outline was prepared by C.M. Palmer,
formerly Aquatic Biologist, In Charge,
Interference Organism Studies, Microbiology
Activities, Research & Development,
Cincinnati Water Research Laboratory,

Algae and Actinornycetes in Water Supplies

g e and Actinornycetes in Water Supplies
28 —8

Algae and Actinomycetes in Water Supplies

Algae and Actinomycetes in Water Supplies
IZo 1 ,Uu

Algae and Actinornycetes in Water Supplies
1 I/Ifl JIJiZ’hViJh

Algae and Actinornycetes in Water Supplic
c1To., o *

A Algae are only one of a number of types
of organisms present which could be
B Forms recognized here as algae are
comparatively simple, pigmented, aquatic
organisms, including blue-greens, greens,
diatoms and pigmented flagellates.
C Various pollutants react differently on
algae. Organic pollutants such as house-
hold sewage will be dealt with here.
D No algae are intestinal organisms. They
therefore are not indicators of pollution
in the same way that coliiorm bacteria
A Heavy pollution may tend to limit various
kinds of algae to certain zones in the
affected area.
B These zones are distinguished according
to the degree of change which has
occurred in the organic wastes. One set
of names for these zones includes the
Polysaprobic, alpha -mesosaprobic, beta -
mesosaprobic and oligosaprobic.
C A few “pollution” algae are common in
the first two zones. Many algae are
common in and often limited to one or
both of the last two zones.
D Some workers have listed separately
those algae indicative of each of the four
A Certain components of wastes are chemi-
cain toxic to some algae but not to others.
B Wastes may have physical effects on
certain algae. May cause plasmolysis,
change in rate of absorption of nutrienta,
C Wastes may reduce available light,
increase the water temperature, and
cover up the areas for attachment to
D Wastes may prevent algal respiration at
night by reducing the DO of water.
E Wastes may stimulate other organisms
at the expense of certain algae.
F Products of waste decomposition may
act as powerful growth stimulants for
certain algae.
A Selection of list of “pollution” algae
follows an evaluation of the kinds re-
ported in published reports by numerous
workers as relatively prominent in, or
representative of, the polysaprobic and
alpha-mesosaprobic zones in a stream
poUuted with sewage. It includes also
other conditions or areas approximating
these zones.
B A total list of more than 1000 kinds of
algae has been compiled to date.
1 In order to tabulate the information,
an arbitrary numerical value is
allotted to each author’s record of
each pertinent alga.
2 The algae are then arranged in order
of decreasing emphasis by the
authors as a whole.
BI.IND. l0a.8.69

Alg e as Indicators of Pollution
A Genera’ Oscillatoria, Euglena, Navlcu]a,
Chiorella, Chiamydomonas, Nitzschla,
Stigeoclonium, Phormidium, Scenedesmus,
Ankietrodesmus, Phacus ,
B Speciee: Euglena viridia, Nltzschia
palea, Oscillatoria chiortha,
Osciflatoria limosa, Osciliatoria tenuis
Scenedesmus guadricauda, Stigeoclonium
tenue Synedra ulna and Pandorina morum .
Chry8OCOCcu8 rufeecens, Cocconets
p].acentula, Entophysalis lemaniae , and
Rhodomonas lacustris .
1 Brinley, F. J. Biological Studies. Ohio
River Pollution Survey. I.
Biological Zones in a Polluted Stream.
II. Plankton Algae as Indicators of
the Sanitary Condition of a Stream.
Sewage Works Journal, 14:147-159.
2 Butcher, R.W. Pollution and
Repurification as Indicated by the
Algae. Fourth International
Congress for Microbiolo (held) 1947.
Report of Proceedings. 1949.
3 Fjerdingatad, E. The Microflora of the
River Moelleaa with Special Reference
to the Relation of the Benthal Algae
to Pollution. Folia Limnological
Scandinavia. No. 5. 1950.
4 Fjerdingstad, E. Taxonomy and Saprobic
Valency of Benthic Phytomicro-
Organisms. Intern. Rev. Ges.
Hydrobiol. 50:475-604. 1965.
5 Hawkes, H.A. The Biological Assess-
ment of Pollution in Birmingham
Streams. The Institute of Sewage
Purification, Journal and Proceedings.
177-186. 1956.
6 Kolkwltz, R. Oekologie der Saprobien.
Schriftenrejche des Vereins für
Wasser-, Boden-, und Lufthyglene
Berlin-Dahiem. Piscator - Verlage,
7 Lackey, J. B. The Significance of
Plankton in Relation to the Sanitary
Condition of Streams. Symposium
on Hydrobiology. University of
Wisconsin. 311-328. 1941.
8 Liebmann, H. Handbuch der
Fris hwasser - und Abwasserbiologie.
R. Oldenbourg, Munchen.
9 Palmer, C.M. Algae as Biological
Indicators of Pollution. In
Biological Problems in Water
Pollution. Trans. of 1956 Seminar.
Robert A. Taft Sanitary Engineering
Center. 1957.
10 Palmer, C.M. The Effect of Pollution
on River Algae. Annal. N.Y. Acad.
Sci. 108:389-395. 1963.
11 Palmer, C.M. A Composite Rating of
Algae Tolerating Organic Pollution.
Jour. Phycology 5 (1):78-82. 1969.
Algae in Water Supplies
States. In. Algae and
Plenum Press. N.Y.
13 PatrIck, R. Factors Effecting the
Distribution of Diatoms. Botanical
Review, 14: 473-524. 1948.
14 Whipple, G.C., Fair, G.M. and
Whlpple, M. C. The Microscopy of
Drinking Water, 4th ed. J. Wiley
and Sons. New York. 1948.
This outline was prepared by C. M. Palmer,
Aquatic Biologist, Cincinnati Water Research
Laboratory, FWPCA.
12 Palmer, C.M.
of the United
Man,Ch 12,
pp. 239-261.

\ \\\

Indicators of Pollution.
) Li
49 . t4OT H f Cf
1)1 IRP

I Most biological odors present in our water
supplies are derived from algae, actinomycetes,
and bacteria.
A The odor produced by algae and actino-
mycetes is generally the result of
intracellular metebolic activity while
the odor caused by bacteria usually
results from extracellu]ar enzymatic
activity upon other organisms.
B The odors produced by actinoinycetes are
usually earthy while those produced by the
algae are aromatic, grassy, and fishy.
A Diatoms
1 Asterionella (aromatic, fish)
2 Cyclotella (aromatic)
B Pigmented Flagellates
1 Synura (cucumber)
2 Dinobryon (fishy)
C Blue-green Algae
1 Anabaena (grassy, green corn,
2 Aphanizomenon (grassy, nasturtium)
D Green Algae
1 Chlorocoecum (grassy)
A Growing Algae for Odor Research
1 ObtainIng unialgal bacteria-free
a Plating out on semi-solid medium
b Single cell isolation
c Use of antibiotics
d Exposure to ultra -violet light
2 Determining nutritional requirements
a Inorganic salts
b Organic growth factors
B Methods of e ctract1ng odoriferous
material from algal cultures
1 Distillation - steam and vacuum
2 Solvent extraction
3 Use of ion exchange resins
4 Freeze out methods
C Some Results of Research
1 Effect of culture age upon odor
2 Effect of pH on odor intensity
3 Comparison of odor intensity in intact
and broken cells
4 Groups of chemicals which may be
responsible for causing algal odors
A A number of actinomycetes were isolated
from water and muds of rivers and lakes.
BI.MIC.to. lOc. 3.70

Odor Production by Algae and Other Organisms
1 Large numbers were o nd to
be present in muds, while there
were relatively few in the water.
2 Most species belonged to the
Streptorn es and a few to the
B Extraction of Odoriferous Material
1 Streptornjces oluteus was
used in this work.
a Cultured in a defined niedium
(1) Cultures have threahhold
ddor of 20, 000 to 53, 000
2 Primary eictraction was by 0
dlatl]iing the culture at 100 C
at atmospheric pressure.
a Distillation of 10% of the
culture volume resulted
In 90% odor removal.
3 Odor was further coacentrated
by two methods
a Ether extraction of the
distilLing off of the ether
in vacuo.
(1) Resulted in yellowish
‘rown con entrate
having a threshold odor
of approximately 6 billion.
b Absorption on activated carbon
followed by elution of material
with chloroform
C Effect of Activated Carbon in Re-
moving the Earthy Odo:
1 The odor is practically elimi-
nated by 10 ppm carbon.
D Effect of Chlorine on Odor
1 Chlorine doea not eliminate
the odor but does not Intensify
the odor.
E Soil perfusion Tests
1 Conducted to determine the
extent to which actinomycetes
impart odors to a water environ-
1 Fogg, G.E., “The Metabolism of
Algae”, John Wiley and Sons, Inc.,
New Y k, N. Y., 1953.
2 Fox, Leo, ‘Mic oscoptc Organisms
in Drinking Water”, Taste and Odor
Journal, Vol. 19, No. 10, 1953.
3 Palmer, C. M., and Tarzwe]l , C. M.,
“Algae of Importance in Water
Supplies”, Public Works Magazine,
4 Whipple, G. C., Fair, G. M., ani
Whipole, M. C., “The Microscopy
of Drinking Water”, Fourth Edition,
John Wiley and Sons, Inc., New York,
N.Y., 1948.
This out1in was prepared by T. E. Maloney,
Former Res arch Biologist, Aquatic Bio1o
Activitie8, Research and Development,
Cin thnati Water Reearth Laboratory, FWPCA.

I INTRODUCTION b Chlorophyll. 1 mg per M 3 or less
The term oligotrophic was taken from the c Cells counts, less than 500 per ml
Greek words oligos - - small and trophein - -
to nourish, meaning poor In nutrients. 2 Zooplankton to phytoplankton volume
Lakes with low nutrient levels have low ratio, 19:1.
standing crops of plankton. The term is now
commonly applied to any water which has a B Quality
low productivity, regardless of the reason.
I European biologists have found
oligotrophic lakes to be dominated by
II PHYSICAL AND CHEMICAL CHARACTER- Chiorophyta (usually desmids),
ISTICS OF OLIGOTROPHIC LAKES* chrysophyta (such as Dinobryon , and
Diatomaceae (Cyclotella and Tabellaria).
A Very deep; high volume to surface ratio Eutrophic lakes are dominated by
Synedea, Fragilaria, Asterionella,
B Thermal stratification common, volume Melosira, blue-green algae, Ceratium,
of the hypolimnium large compared to the and Pediastrum. Nygaard devised
volume of the epilimnion several phytoplankton quotients based
on these relationships
C Maximum surface temperature rarely
greater than 15°C a Simple quotient
D Low concentrations of dissolved minerals Number of species of
and organic matter.
Chlorococcales if <1, oligotrophlc
1 Phosphorus, less than 1 microgram Desmidiaceae if > 1, eutrophic
per liter
b Compound index
2 N0 3 -Nitrogen, less than 200 micrograms
per liter Myxophyceae+Ch1orococca1e +CentraJ.es+Eu niace.ic
E Dissolved oxygen near saturation from
surface to bottom
if <1, oligotrophic
F Water very transparent, Secchi disk
readings of 20-40 meters are common if 1-2.5. mesotrophic
G Color dark blue, blue-green, or green if >2.5, eutrophic
c Diatom quotient
Centrales = if 0-0. 2, oligotrophic
A Quantity Pennales if 0.2-3.0, eutrophic
1 Standing crop very low
a Ash-free weight of plankton, less
than 0. 1 mg per liter (compared to
1 mg per liter or more in eutrophic
BI.ECO.mic.2. 10.66

Plankton in Oligotrophic Lakes
2 Several lists of trophic Indicators have
been publi8hed:
Two are listed here
Telling. Rawson 1
Swedish Lakes Canadian Lakes
Oltgotrophic Tabelleria flocculosa Oligotrophic Asterionella formosa
Dactylococcops is M elosira islandica 4
ellipsoldeus Tabellaria fenestrata
Tabellaria flocculosa
Mesotrophic Kirchneriella lunaris Dinobryon divergens
Tetraeadon Fragilarla capucina
Pediastrum spp. Stephanodiscus nlagarae
Fragilarta crotonenels Staurastrum spp.
Atthe zachariasil Melosira granulata
Mesotrophlc Fragtiaria crotonensis
Eutrophic Aphanizomenon 8 )• Ceratium hirundinella
A naba ena flos - aguae Pe din strum boryanuin
Anabaena circinalia
Pediastrum di plex
Pronounced M icrocystis aeruginosa naegelianum
Microcystis viridis Anabaena app.
Aphanizomenon floe - aquae
Microcystis aeruginosa
Eutrophic Microcystis flos-aquae

Plankton in Oligotrophic Lakes
Some discrepancies can be seen in the
ranking of species in the lists. These
may be the result of true differences In
the composition of the plankton, or may
be only apparent differences which
resulted from different sampling methods.
Many studies (e. g. those by Hilliard,
Olive, and Rawson) have been based on
netted samples, which may be highly
biased because they contain little of the
nannoplankton. Also, it is not uncommon
to characterize popu]ationa on the basis
of one or two samples collected during
the summer months.
3 The dominant plankton in four
oligotrophic North American lakes are
listed below. The Great Slave Lake
and Karluk Lake data are from netted
samples taken during the summer, and
monthly, re8pecttvely. The Lake
Superior and Lake Tahoe data are from
grab samples taken twice monthly, and
quarterly, respectively.
The dominant diatoms are generally
similar in the four lakes. Asterionella
formosa and Fragilaria crotonensis
are common to all. There are also
some obvious differences. Melos ira
islandica, the dominant diatom in the
Great Slave Lake and Lake Superior,
is absent from Lake Tahoe and Karluk
Lake. It was not found in Crater Lake
by Sovereign (1958), in the Mountain
lakes of Colorado by Olive (1955) or
Brin.ley (1950), and does not occur in
WPSS samples in streams west of the
Great Lakes. TabeUaria is also
absent from Lake Tahoe. It was
reported in Colorado lakes by Olive,
but was not abundant. Brinley makes
no reference to It, and Sovereign
indicated that it was rare In Crater
Lake samples. It i apparent that the
absence of these two diatoms from
Lake Tahoe is not related to the lake.
Except for the absence of Keratefla
cochlearis from Lake Tahoe, the
rotl.fer populations are very similar.
Data on other segments of the zoo-
plankton population are insufficient to
permit comparison.

Hash 5011,
Great Slase Lake
Lake Superior
Karluk Lake
Melosira islardica
Aslerlonella formosa
Dutobr on do .ergens
Cerattum hirunduiella
Pedlastrum bor anum
Tabellaria fenestrata
Cyclatella meneghuuana
Fragilarla crotonensis
Svnectra ulna
Eunotta Iunaris
Keratella cochlearls
KeUlcottia longtsptna
Diaptomus tenulcauthtua
Limnocalanus macru i-us
Senecefla calartosdes
Daphnla longispina
Bosmuta obtu Irogtrjs
Melosira islandica
TabeUaria fenestrata
Cyclotells kuteingiana
M elosira granulata
t! elosira amblgua
A aterlonella formosa
Synedra nana
Sc edesmus spp.
Arikiatrodesmus spp
Dictyosphaerlum app
A sterionella formosa
TabeUarta flocculosa
FragLlaria crotonensis
CycloteUa bodanica
Cymbefla turgida
Dictyosphaerlum app
Sphaerocystis app.
Staurastrum app
KerateUa cochlearis Not reported
KeUlcotua 1ongispsna
Fragllarta crotonensls
Svnedra nana
FragtIa rta construens
Fragilarta pinnata
Nitzschia actcularas
A sterloneila Formosa
Kelllcottln }onglsplna
Daphrna app.
Dmptomus t reflt
Epischura nevadensis
$. PSs.
Lake Tahoe
Ph) toplankton
Zoop lankton

Plankton In Oligotrophic Lakes
REFERENCES 9 Rawson, D.S., 1956. Algal indicators
of trophic lake types. Limnol.
1 Brinley, F.J. 1950. Plankton population Oceanogr. 1:18-25.
of certain lakes and streams in the
Rocky Mountain National Park, 10 Rodhe, W., 1948. Environmental
Colorado. Ohio 3. Sci. 50:243-250. requirements of fresh-water plankton
algae. Synib. Bot. Upsal. 10:1-149.
2 HiliLard, D. K. • 1959. Notes on the
phytoplankton of Karluk Lake, Kodiak 11 Ruttner, F. • 1953. Funthmentals of
Island, Alaska. Canadian Field- Limnology, 2nd ed., Univ. Toronto
Naturalist 43:135-143. Press, Toronto.
3 Jarnefelt, H., 1952. Plankton ala 12 Sovereign, H.E., 1958. The diatoms of
Indikator der Trophiegruppen der seen. Crater Lake, Oregon. Trans. Amer.
Ann. Acad. Sd. Fennicae A.IV:l-29. Microsc. Soc. 77:96-134.
4 Knudson, B.M., 1955. The distribution of 13 Teiling, E., 1955. Some rnesotrophic
Tabeiiaria in the English Lake District. phytoplankton indicators. Proc. lot.
Proc. mt. Assoc. Limnol. 12:216-218. Assoc. LImnol. 12:212-215.
5 Nygaard, G., 1949. Hydroblological studies 14 USPHS, 1962. National Water Quality
in some ponds and lakes It. The Network, Annual Compilation of Data,
quotient hypothesis and some new or PHS Pubi. No. 663.
little known phytoplankton organisms.
Kig. Danske Vidensk. Seisk. Biol. 15 Welch, P.S., 1952. Limnology, 2nd ed.,
Skrifter 7:1-293. McGraw Hill Book Co., New York.
8 Olive, J.R.. 1955. Some aspect8 of
plankton associations in the high
mountains lakes of Colorado. Proc.
lot. Assoc. Limnol. 12:425-435.
7 Rawson, D.S., 1953. The standing crop
of net plankton in lakes. J. Fish. Res.
Bd. Can. 10:224-237.
8 Rawson, D.S., 1956. The net plankton of This outline was prepared by C.!. Weber,
Great Slave Lake. 3. Fish. Res. Bd. Chief, Biological Methods Section,
Can. 13:53-127. Analytical Quality Control Laboratory,
NERC, EPA, Cincinnati, OH 45268.

The pollution of lakes inevitably results in a
number of undesirable changes in water
quality which are directly or Indirectly
related to changes in the aquatic community.
A Industrial Wastes may contain the following:
1 Sewage
2 Dissolved organics- -synthetics, food
processing wastes, etc.
3 Dissolved minerals- -salts, metals
(toxic and nontoxic), pigments, acids. etc.
4 Suspended solids--fibers, minerals,
degradable and non- degradable organics
5 Petroleum products- -oils, greases
6 Waste heat
B The Materials In Domestic Wastes which
affect Water Quality are:
1 PathogenIc fecal microorganisms
2 Dissolved nutrients: minerals, vitamins,
and other dissolved organic substances
3 Suspended solids (sludge)- -degradable
and non-degradable organic materials
C Pollution and Eutrophication
The discharge of domestic wastes often
renders the receiving water unsafe for
contact water sports and water supplies.
For example, some beaches on the eastern
seaboard and in metropolitan regions of
the Great Lakes are unfit for swimming
because of high coll.form counts. Other
effects of domestic pollution Include
changes In the abundance and composition
of populations of aquatic organisms.
1 As the nutrient level increases, so does
the rate of primary production.
2 Shore-line algae and rooted aquatics
become more abundant. For example,
problems have been experienced with
Cladophora and Dichotomosiphon along
the shores of Lakes Ontario, Erie,
and Michigan. These growths Interfere
with swimming, boating, and fishing.
and cause odors when the organisms
die and decay.
3 The standing crop of phytoplankton
increases, resulting in higher counts
and greater chlorophyll content.
Increases in phytoplarikton abundance
may result in taste and odor problems
in water supplies, filter clogging,
high turbidity, changes in water color,
and oxygen depletion In the hypolirnnion.
4 Populations of fish and larger swimming
invertebrates increase, based on the
increase in basic food production.
5 Changes in dominant species
a Diatom communities give way to
blue-greens. Toxic blue-greens may
pose a problem.
b Zooplankton changes include
replacement of Bosmina coregoni
by B. longirostris .
c Trout and whitefish are replaced by
perch, bass, and rough fish.
d Hypoliinnion becomes anaerobic in
summer, bottom sludge buildup
results in loss of fish food organisms,
accompanied by increase in density
of sludgeworms (oligochaeta).
The cultural eutrophication of a number of
lakes in Europe and America has been well
A Zurichsee, Switzerland
WP 1K ic. 4.70
32- 1

The Effects of Pollution on Lakes
1 1896 - sudden increase in Tabellaria B Haliwliersee, Switzerland
1 1897 - Oscillataria rubescens not
2 1898 - sudden appearance of Oscillatoria observed up to this time
rubescens which displaced Fragilaria
capucina 2 1898 - 0. rubescens bloomed,
decomposed, formed H 2 S, killing off
3 1905 - Melosira islandica var. helvetica trout and whitefish
C Lake Windermere, England (core study)
4 1907 - S phanodiscus hantzschii
appeared 1 Little change in diatoms from glacial
period until recent times
5 1911 - Bosmina longirostris replaced
B. coregoni 2 Then Asterionella appeared, followed
by Synedra
6 1920
1924 - 0. rubescens occurred in great 3 About 200 years ago, Asterionella
quantities again became abundant
7 1920 - milky-water phenomenon, 4 AsterioneJja abundance ascribed to
precipitation of CaCO 3 crystals ( 4 Oii) domestic wastes
due to pH increase resulting from
photosynthesis D Finnish Lakes
8 Trout and whitefish replaced by perch, Aphanizomenon. Coelosphaerium ,
bass, and rough fish Anabaena, Microcystis , are the most
common indication of eutrophy.
Zurichsee, Switzerland
Parameter Date Value
Chlorides 1888 1.3 mg/i
1916 4.9 mg/i
Dissolved organics 1888 9.0 mg/i
1914 20.0mg/i
Secchi Disk before 1910 16.8M 3.1M
1905- 1910 10.OM 2.1M
1914 - 1928 10.OM 1.4M
Dissolved oxygen, at 1910 - 1930 Minimum 100% saturation
100 M, mid-summer 1930 - 1942 9% saturation

The Effects of Pollution on Lakes
E Linsley Pond, Connecticut
1 Species making modern appearance
Include Astertonella formosa,
Cyclotella glomerata, Melosira
itailca, Fragilaria crotonensis,
Synecira ulna
2 Asterionella formosa and Melosira
Italics were considered by Patrick to
indicate high dissolved organics
3 Bosmina coregoni replaced by B.
F Lake Monona, Wisconsin
1 Began receiving treated sewage in 1920,
developed blue-green algal blooms.
G Lake Washington, Washington
1 1940 - Bosmina longirostris appeared
2 1955 - Oecillatoria rubescens seen for
the first time, and constituted 96% of
phytoplankton, July 1
H Lake Erie
1 Phytoplankton counts at Cleveland have
increased steadily from less than
500 cells/mi in the 1920’s to over
1500 cells/mi in the 1960’a
2 Abundance of burrowing mayflies
( Hexagenia spp.)Ln Western Lake Erie
decreased fro n 139/rn 2 in 1930, to
less than 1/rn in 1961.
Lake Michigan
1 Milky water observed in south end, and
in Limnetic region In mid-1950’s and
again in 1967.
2 During the period 1965-1967 the Chicago
water treatment plant has found it
necessary to increase the carbon dosage
from 23 lbs/mU gal to 43 lbs/mU gal,
and the chlorine dosage from 20 Ibs/mil
gal to 25 lbs/mu gal.
3 Phytoplankton counts in the south end
now exceed 10, 000/mi during the
spring bloom.
A Depth-surface area ratio: A large
hypolimnion will act as a reservoir to
keep nutrients from recirculating in the
trophogenic zone during the summer
stratification period. Raw8on found an
inverse relationship between the standing
crop of plankton, benthos, and fish, and
the mean depth.
B Climate: Low annual water temperatures
may restrict the response of the
phytoplankton to enrichment.
C Natural color or turbidity: Dystrophic
(brown-water) lakes may not develop
phytoplankton blooms because of the low
transparency of the water.
Except in cases where massive algal blooms
occur, the trophic status of lakes is often
difficult to determine. Core studies are
used to determine trends in diatom populations
which might indicate changes in nutrient
levels over an extended period of time.
The success of efforts to arrest the
eutrophication process, and where desirable,
reduce the trophic level of a lake, will
depend on a thorough knowledge of the
nutrient budget.
A Signincant quantities of nutrients may
enter a lake from one or more of the
following sources:
1 Rainfall
2 Ground water

The Effects of Pollution on Lakes
Oligotrophic Condition
1 Transparency > 10 meters
2 Phosphorus < 1 g/1
3 NO 3 - Nitrogen < 200 g/1
4 Minimum annual near 100% saturation
hypolimnetic oxygen concentration
5 Chlorophyll < i mg/rn 3
6 Ash-free weight of seston < 0. 1 mg/i
7 Phytoplankton count < 500/mi
8 Phytopiankton quotients
a number of species of Chiorococcales <1
number of species of Desmids
b Mlxophycease+Chlorococcales+centra le s+Euglenaceae <1
c Centrales 0 - 0. 2
9 Phytop]ankton species present (Bee outline on
plankton in oligotrophic lakes).
3 Watershed runoff c Many methods have been employed to
treat the symptoms, reduce the
4 Shoreline domestic and Industrial outfalls eutrophication rate, or completely
arrest and even reverse the eutrophication
5 Pleasure craft and commercial vessels process.
6 Waterfowl 1 Use of copper sulfate, sodium arsenite,
and organic algicides: It is not
7 Leaves, pollen, and other organic economically feasible to use algicides
debris from riparian vegetation in large lakes.
B The supply of nutrients from “natural” 2 Addition of carbon black to reduce
sources in some cases may be greater transparency. This is likewise
than that from cultural sources, and be frequently impractical.
sufficient to independently cause a rapia
rate of eutrophication regardless of the 3 Harvesting algae by foam fractionation
level of efficiency of treatment of domestic or chemical precipitation.
and industrial wastes.

The Effects of Pollution on Lakes
4 Reducing nutrient supply b ’ (a) removal
of N and P from effluents, (b) diversion
of effluents, and (c) dilution with
nutrient-poor water.
D Examples of lakes where control has been
attempted by reducing the nutrient supply,
1 Lake Washington, Seattle
The natural water supply for this lake
is nutrient poor
(Ca -8 mg/i. P <5Mg/I. TDS76 mg/I).
Since the turnover time of the water in
this lake is only three years, it was
expected that diversion of sewage
would result in a rapid improvement of
water quality. Diversion began in 1963,
and improvements were noticeable by
1965 - including an increase in
transparency, and a reduction in seston,
chlorophyll, and epilimnetic phosphorus.
Maximum phosphorus in
upper 10 meters
2 Green Lake, Washington
The lake has a long history of heavy
blooms of blue-green algae. Beginning
in 1959, low-nutrient city water was
added to the lake, reducing the con-
centration of phosphorus by 70% in the
inflowing water. By 1966, the lake had
been flushed three times. Evidence of
improvement l.a water quality was noted
in 1965. when Aphanizomenon was
replaced by Gleotrichia .
3 Lake Tahoe
This lake is still decidedly oligotrophic.
To maintain its high level of purity,
tertiary treatment incilities were
installed in the major sewage treat-
ment plant, and construction is now
underway to transport all domestic
wastes out of the lake basin.
1 Ayers, J.C. and Chandler, D.C . Eds.
Studies on the environment and
eutrophication of Lake Michigan.
Special Report No. 30. Great Lakes
Research Division, Institute of
Science and Technology. University
of Michigan, Ann Arbor. 1967
2 Brezonik, P.L., Morgan, W.H
Shannon. E.E., and Putnam, H.D.
Eutrophication factors in North
Central Florida Lakes. University
of Florida Water Res. Center
Pub. #5, 101 pp. 1969.
3 Carr, J.F , Hiltunen, J.K Changes
in the bottom fauna of Western Lake
Erie from 1930 to 1961 Limnol.
Oceanogr. 10(4):55l-569. 1965.
4 Frey, David G. Remains of animals
in Quatertiary lake and bog sediments
and their Interpretation.
Schweizerbartsche Ver)agsbuchhandlung.
Stuttgart. 1964.
5 Edmondson, W.T., and Anderson, G.C.
A rtificial eutrophication of Lake
Washington. Limnol. Oceanogr
l(1).47—53. 1956.
6 Fruh, E.G. The overall picture of
eutrophicatrnn. Paper presented
at the Texas Water and Sewage
Works Association’s Eutrophication
Seminar. College Station, Texas.
March 9, 1966.
7 Fruh, E.G , Stewuit, K.M., Lee, G.F.,
and Rohlich, G.A Measurements
of eutrophication and trends.
J. W. P.C. F. 38(8): 1237-1258 1966

The Effects of Pollution on Lakes
8 Hasler, A. D. Eutrophication of lakes
by domestic drainage. k co1ogy
28(4):383-395. 1947.
9 Hasler, A. D. Cultural Eutrophication
is Reversible. BIoscience 19(5):
425-443. 1969.
10 Herbat, Richard P. Ecological Factors
and the Distribution of Cladophora
glomerata in the Great Lakes.
Amer. Midi. Nat. 82(1):90-98. 1969.
11 National Academy of Sciences.
Eutrophication: Causes, Consequence a,
Correction. 661 pp. 1969.
(Nat. Acad. Sci. ,2101 Constitution
Avenue, Washington, DC 20418, 13.50).
12 Neel, Joe Kendall. Reservoir
Eutrophication and Dystrophication
following Impoundment. Reservoir
Fisheries Res. Symp. 322-332.
13 Oglesby, R. T. and Edmondson, W. T.
Control of Eutrophication.
J.W.P.C.F. 38(9):1452-1460. 1966.
14 Stewart, K.M. and Roh]ich, G.A.
Eutrophication - A Review.
Publication No. 34, State Water
Resources Control Board, The
Resources Agency, State of California.
This outline was prepared by C. 1. Weber,
Chief, Biological Methods Section,
Analytical Quality Control Laboratory,
NERC, EPA, Cincinnati, OH 45268.

A Qualitative - dealing with the taxonomic
composition of communittes
B Quantitative - dealing with the populat ion
density or rates of processes occurring
in the communities
Each kind of data has been useful in its own
A Certain species have been identified as:
1 Clean water (sensitive) or oligotrophic
2 Facultative, or tolerant
3 Preferring polluted regions
(see: Fjerdlnstad 1964, 1965; Gaufin
& Tarzwell 1956; Palmer 1963, 1969;
Rawson 1956, Teillng 1955)
B Using our knowledge about ecological
requirements the biologist may compare
the species present
1 At different stations in the same river
(Gaufin 1958) or lake (Holland 1968)
2 In different rivers or lakes (Robertson
and Powers 1987)
or changes in the species in a river or/lake
over a period of several years. (Carr
& Hiltunen 1965, Edmondson & Anderson
1956; Fruh, Stewart, Lee & Rohlich 1966,
Hasler 1947).
C Until comparatively recent times taxonomic
data were not subject to statistical treat-
Parameters of this type lnclude
A Counts - algae/mi; benthos/m
B Volume - mm 3 algae/liter
C Weight - dry wgt, ash-free wgt.
D Chemical content - chlorophyll,
carbohydrate; ATP; DNA; etc.
E Calories (or caloric equivalents)
F Processes - productivity; respiration
IV Historically, the chief use of statistics
in treating biological data has been In the
collection and analysis of samples for these
parameters. Recently, many methods have
been devised to convert taxonomic data into
numerical form to permit:
A Better communication between the
biologists and other scientific disciplines
B Statistical treatment of taxonomic data
C In the field of pollution biology these
methods Include:
1 Numerical ratings of organisms on the
basis of their pollution tolerance
(saprobic valency: Zelinka & Sladecek
(pollution index: Palmer 1969)
2 Use of quotients or ratios of species in
different taxonomic groups (Nygaard
BI. EN. 35. 12.70

Application of Biological Data
3 Simple indices of community diversity:
a Organisms are placed in taxonomic
groups which behave similarly under
the same ecological conditions. The
number of species in these groups
found at “healthy stations is com-
pared to that found at “experimental”
stations. (Patrick 1950)
b A truncated log normal curve is
plotted on the basis of the number
of individuals per diatom species.
(Patrick, Hohn, & Wallace 1954)
c Sequential comparison index.
(Cairns, Albough, Busey & Chanay
1988). In this technique, similar
organisms encountered sequentially
are grouped into “runs”.
d Ratio of carotenoids to chlorophyll
in phytoplankton populations:
0D 4301 0D 665 (Margalef 1968)
0D 4351 0D 670 (Tanaka, et al 1961)
e The number of diatom species present
at a station is considered Indicative
of water quality or pollution level.
(Williams 1964)
number of species (S )
number of individuals (N)
number of species (S)
g square root of number of individuals (.1 N)
S- 1
log N (Menhinlck 1984)
E n 1 (n - 1 ) (Simpson 1949)
N (N - 1)
where n 1 number of individuals
belonging to the i-th species,
N = total number of individuals
Information theory:
The basic equation used for
information theory applications was
developed by Margalef (1957).
1 NI
I — log
N 2NINI...N!
a b a
where I - information/lndjvjdua l;
N • N .. . N are the number of
inadivi uals i species a, b,
s, and N is their sum.
This equation has also been used
1) The fatty acid content of algae
(Mclntire, Tinsley, and Lowry
2) Algal productivity (Dickman 1968)
3) Benthic biomasa (Wi]hm 1968)
1 Cairns, J., Jr., Albough, D.W.,
Busey, F, and Chaney, M.D.
The sequential comparison index -
a simplified method for non-biologists
to estimate relative differences in
biological diversity in stream pollution
studies. J. Water Poll. Contr. Fed.
40(9):1607—1613. 1968.
2 Carr, J. F. and Hiltunen, J. K. Changes
in the bottom fauna of Western Lake
Erie from 1930 to 1961. Limnol.
Oceanogr. 10(4):551-569. 1965.
3 Dicknian, M. Some indices of diversity.
Ecology 49(6):1191-l193. 1968.
sd =
total organisms examined

Application of Bio1o tcal Data
4 Edmondson, W.T. and Anderspn, G.C.
Artificial Eutrophication of Lake
Washington. Limnol. Oceanogr.
1(1):47—53. 1956.
5 Fjerdlngstad, E. Pollution of Streams
estimated by benthal phytomicro-
organisms. I. A saprobic system
based on communities of organisms
and ecological factors. InternaUl
Rev. Gee. Hydrobiol. 49(1):63-13 1.1964.
6 Fjerdingstad, E. Taxonomy and saprobic
valency of benthic phytomicro-
organisms. Hydroblol. 50 (4) 475-6O4.
7 Fruh, E G.. Stewart, K.M., Lee, G.F.
and Rohlich, G.A. Measurements of
eutrophication and trends. J. Water
Poll. Contr. Fed. 38(8):1237—1258
8 Gaufin, A.R. Effects of Pollution on a
midwestern stream. Ohio J. Sd.
58(4):197—208. 1958.
9 Gaufin, A. R. and Tarawell, C. M. Aquatic
macroinvertebrate communities as
indicators of organic pollution In Lytle
Creek. Sew. md. Wastes. 28(7):908—
924. 1858.
10 Hasler, A. D. Eutrophication of lakes by
domestic drainage. Ecology 28(4):383-
395. 1947
11 HoUand , R.E. Correlation of Melosira
species with trophic conditions in Lake
Michigan. Limnol. Oceanogr.
13(3):555—557. 1968.
12 Margalef, R. Information theory In
ecology. Gen. Syst. 3 36-71. 1957.
13 Margalef, R. Perspectives In ecological
theory. Univ. Chicago Press. 1968.
14 Mclntire, C.D., Tinsley, 1.J. and
Lowry, R.R Fatty acids in lotic
periphyton: another measure of
community structure. J. Phycol.
5:26—32. 1969.
15 Menhinick, E. F. A comparison of some
species - Individuals diversity indices
applied to samples of field insects.
Ecology 45:859. 1964.
16 Nygaard, G. Hydrobiological studies in
some ponds and lakes. LI. The
quotient hypothesis and some new or
little-known phytoplankton organisms.
I g. Danske Vidensk. Selsk. Biol.
SkriIter 7:1-293. 1949.
17 Patten, B. C. Species diversity In net
plankton of Raritan Bay. .J. Mar.
Res. 20:57—75. 1962.
18 Palmer, C. M. The effect of pollution on
river algae. Ann. New York Acad.
Sci. 108:389-395. 1963.
19 Palmer, C. M. A composite ratmg of
algae tolerating organic pollution.
J Phycol. 5(1):78—82. 1969.
20 Patrick, R., Hohn, M. H. and Wallace,
J. H. A new method for determining
the pattern of the diatom flora. Not.
Natl. Acad. Sci., No. 259.
Philadelphia. 1954.
21 Rawson, D.S. Algal indicators of trophic
lake types. Limnol. Oceanogr.
1:18—25. 1956.
22 Robertson, S. and Powers, C. F
Comparison of the distribution of
organic matter in the five Great Lakes.
in: J.C. Ayers and D.C. Chandler,
eds. Studies on the environment and
eutrophication of Lake Michigan.
Spec. Rpt. No. 30, Great Lakes Res.
Div., Inst. Sd. & Techn., Univ.
Michigan, Ann Arbor. 1967.
23 Simpson, E. H. Measurement of diversity.
Nature (London) 163:688. 1949.
24 Tanaka, 0. H., Irie, S. Izuka, and Koga, F.
The fundamental Investigation on the
biological productivity in the Northwest
of Kyushu. I. The investigation of
plankton. Rec. Oceanogr. W. Japan,
Spec. Rpt. No. 5, 1-57. 1961.

Annileatfon of BioloEical Data
25 Teillng, E. Some mesotrophic phyto-
plankton indicators. Proc. Intern.
Assoc. L4mnol. 12:212-215. 1855.
26 WtIhm, J. L. Comparison of some
diversity indices applied to populations
of benthic macroinvertebrates In a
stream receiving organic wastes. J.
Water Poll. Contr. Fed. 39(1O):1673—1683.
28 WillIams, L. G. Possible relationships
between diatom numbers and water
quality Ecology 45(4):810-823. 1964.
29 Zellnka, M. and S]adecek, V. Hydro-
biology for water management.
State Pubi. Hou8e for Technical
Literature, Prague. 122 p. 1964.
27 Wt]hm, J. L.. U8e of biomass units in
Shannon’s formula. Ecology 49 153-156.
This outline was prepared by C.!. Weber,
Chief, Biological Methods Section, Analytical
Quality Control Laboratory, NERCI EPA,
Cincinnati, OH 45268.

I Sources of organic chemicals in water
are varied and of differing compiexity.(1)
A Natural pollutants , such as algae, actino-
mycetes. etc. contribute to organic
1 Tastes and odors associated with these
materials are probably not merely a
result of decomposition, but are closely
associated with materials produced
during the life cycle of the organisms
and plant8.
2 Discharge of nutrients in the form of
phosphorus and nitrogen compounds
from domestic or other wastes fre-
quently stimulate the production of
natural pollutants.
S Industrial wastes , due to the rate of
population Increase and industrial ex-
pansion, have made the problem of
effective water treatment an acute one in
many places.
1 The production of synthetic organic
chemical8 has risen steadily over the
past years, representing many new
and complex products - and of im-
portance to us - new and complex wastes.
2 The ideal method of handling lndustriai
waste is at Its source.
a However, what is often COflBidC red
good treatment, still results in
materials present in sufficient
quantities to affect the taste and odor
of water.
b Many problems are caused by slug
discharges, often accidental.
D Miscellaneous sources also contribute to
the problem.
1 Wastes from private and commercial
2 ChemIcals applied to the land may be
washed into streams.
3 Chemicals applied directly to water.
a Evaporation control
b Killing off rough fish
c Aquatic plant control
U Concentrations of organic chemicals
in water, even in comparatively minor
quantities may cause difficulties.
A Wastes may contain from a few mg/i to
several hundred mg/i of organic
B Surface waters may contain from a few
ig/1 of organics to several mg/i.
1 Some of the chemicals isolated from
water, along with the concentrations
which can be detected by odor, are: 2
Substance Detectable*, ig/l
C Domestic wastes in various stages of
5 Concentrations were determined by taking
the median of 4-12 observations.
Xyle flea
Refinery hydrocafbons
Petrochemical waste
Phenyl ether
Chlorinated phenohcB
50, 000
500 - 1,000
250 - 4, 000
300 - 1, 000
25 — 50
15 — 100
1 - 100
CH. OTS. 40a. 4.70

The Problem of Synthetic Organic Wastes
UI The damagin effects of organics in water
are becoming more apparent.
A Taste arid odor in water is usually the first
noticed effect from organics. This is a
serious public relations and economic
problem, it also may be a health problem.
B Organic contaminants may interfere with
coagulation, damage ion exchangers, and
create chlorine and carbc demand.
C In the stream they may have adverse
effects on aquatic forms that support higher
aquatic life, cause off-flavors in fish flesh,
or have direct toxic effects on fish.
IV The methods of study employed in the
collection and identification of organic
chemicals in water involve physical and
chemical methods and instrumental analysis.
A The comparatively small amounts of
organic materials may be concentrated
by adsorption on activated carbon.
1 This carbon is then extracted with
appropriate organic solvents, the
solvent extract is taken to dryness, the
weighed extract is subjected to solubility
group separation, and these individual
groups may then be analyzed by various
2 Employing the above method on Ohio
River water , the following results were
Chemical Group
% of
in p.g/l
Relative Odor
Water solubles
Ether and water
Neutral 14
Amine 4
Weak acid 8
Strong acid
5, 000+
B Chemical separation and analyses may be
ccompliahed by means of column chromato-
graphy, formation by derivatives, gas
chromatography, infrared and ultraviolet
spectroscopy, x-ray diffraction. etc.
1 Specific organic chemicals recovered
from river and drinking waters by
these methods include: synthetic
detergents (ABS), phenylether, phenol,
DDT, aidrin, o-nltrochlorobenzene,
-conedendrin, and xylene.
V Some of the types of problems that may
be attributed to organic wastes, and more
specifically to problems of taste and odor,
may be represented by the following examples:

The Problem of Synthetic Organic Waste8
A By applying the previously mentioned
carbon adsorption method, the odor po-
tential of organic pollutants and the
dilution necessary to reduce this odor
potential to a barely perceptible level has
been deterrnthed: 4
Industry Source
Conc. Required for
Detectable Odor i .’g/l
Dilution Factor
Sol. Org.
CHC1 3
Sol. Org.
Corn Refining
Meat Packing
Metal Fabrication
1, 000
1, 200
1, 400
3, 600
3, 600
1, 600
1, 000
1. 4
2. 8
2. i
4. 8
B Of all the organic pollutants that can affect
the taste and odor of drinking water,
phenol has been the most extensively
1 The potential sources of this chemical,
both natural arid synthetics have been
discussed. 5 )
2 The course of chlorination of phenol,
a common method of treatment in the
water plant, has been shown to proceed
by a process which starts with the pure
compound (in itself relatively tasteless)
and proceeds through strong-tasting
intermediates to tasteless end
products. (6)
C The effects of petrochemical wasteB 7
on water quality are becoming increasingly
important; It has been predicted that by
1970 the petrochemical production on a
tonnage basis may be equal to 41% of all
1 The three principal groups of petro-
chemicals are the paraffins, the
naphthenes, and the aromatics. From
these, over 200 basic products are
manufactured, having thousands of
subordinate uses.
2 Correspondingly, more than 100 identi-
fiable compounds have been found in
waste streams from petrochemical
1 Middleton, F. M. Taste and Odor Sources
and Methods of Measurement. Taste
and Odor Control Journal. 26:1. 1960.

The Problem of Synthetic Organic Wastes
2 Middleton, F. M., Rosen 1 A. A., and
Burttschell , R. H. Taste and Odor
Research Tools for Water Utilities.
Jour. A.W.W.A. 50:21. 1958.
3 Anonymous. Objectionable Organic Con-
taminants in Water. San.Eng.Center
Actlv. Rep. No. 25, 1855.
4 Sproul, 0. J., and Ryckman, D. W. The
Significance of Trace Organics in Water
Pollution. PCF 33:1188. 1861.
5 Hoak, R. D. The Causes of Tastes and
Odors In Drinking Water. Water and
Sewage Works. 104:243. 1957.
6 Burttscheli , B. H., Rosen, A. A.,
Middleton, F. M., and Ettinger, M. B.
Chlorine Derivatives of Phenol Caus-
ing Taste and Odor. Jour.A. W. W.A.
51:205. 1959.
7 Gloyna, E. F., and Malina, Jr. • J. F.
Petrochemical Wastes Effects on
Water. Indus. Water & Wastes, 7:
#5. 134. Sept. —Oct. 1962.
8 Baker, R. A. Problems of Tastes and
Odors. WPCF. 33:1099. 1961.
This outline was prepared by R. L. Booth,
Chief, Analyses Unit, Analytical Quality
Control Laboratory. NERC, OWP, EPA,
cincinnati, OH 45268.

- I Nil I i A N( I 01’ ‘II M I I ’ [ NG ‘A CTORS ‘10 P01 ‘U LA lION VARIATION
A All aquatic organism ’, do not react uniformly
to the various chemiLal, physical and
biologiLal features in their environment.
lhroiigh normal evolutionary processes
various organisms have become adapted
to certain combinations of environmental
conditions. The successful develo ment
and maintenance of a population or community
depend upon harmontous ecological balance
b tw en environmental conditions and
loleranc’e of the organisms to variations
In one or more 0! these Londitlons.
13 A factor whose presenLo 01 absence exerts
Nome r(stm aining Influence upon .i popii1 t ion
through Incompatibility with species
requirements or tolerance is said to be a
limiting luLtor . The principle ol limiting
factors is one of the major aspects of the
environmental control of aquatic organ1 ms
(Figure 1).
A Liebig’s Law of the Minimum enunciates
the first basic concept. In order for an
organism to inhabit a particular environ-
mt’nt, specified levels of the materials
necessary for growth and development
(nutrients, respiratory gases, etc. ) must
be present. If one of these materials is
absent from the environment or present
in minimal quantities, a given species
will only survive in limited numbers, if
at all (Figure 2)
This principle rests essentiaUy upon two basic
concepts. One of these relates organisms to
the environmental supply of materials essential
br their growth and development. The second
pertains to th tolerance which organisms
exhibit toward environmental conditions
I”igurc 1 ‘I h relationships ol limiting factors
to population gm owth and development
Figure 2. Relationships of environmental
factors and the abundance of organisms.
1 The subsidiary principle of factor
interaction states that high concentration
or availability of some substance, or
the action of some factor in the environ-
rnent, may modify utilization of the
minimum one. For exampleS
.i The uptake of phosphorus by the
algae Nitzchia closterlum is influenced
by the relative quantities of nitrate
and phosphate in the environment,
however, nitrate utilization appears
to be unaffected by the phosphate
(Reid, 1961).
b The assimilation of some algae is
closely related to temperature
The rate of oxygen utilization by fish
may he affected by many other sub-
stances or factors in the environment.
I ,
LWT yiO*u s
\ ttliTf bOpis
131. ECO.2fla.7.6’)

Signuicance of Limiting Factors’ to Population Variation
d Where strontium is abundant, mollusks
,rc able to substitute it, to a partial
extent, for calcium in their shells
(Oclum, 1959).
2 If a material is present in large amounts,
but only a small amount is available for
use by the organism, the amount available
and not the total amount present deter-
mines whether or not the particular
material is limiting (calcium in the form
of CaCO 3 ).
13 Shelford pointed Out in his Law of Tolerance
that there are maximum as well as minimum
values of most environmental factors which
an he tolerated. Absence or failure of an
organism can be controlled by the deficiency
or excess of any factor which may approach
the limits of tolerance for that organism
(Figure 3).
MtnIn ,un. IAmft of 1 5.n e of Optfmun Moolmum Ltmft of
rolursulon of F,,’toro Toler.ttøn
Atm, of
Ahun oc.
Oreitsit Abwt nc, Decru.in 1 Abient
_____J Ab JJ
Figure 4. Ftelationship of purely harmful
factors and the abundance of
3 Tolerance to environmental factors
varies widely among aquatic organisms.
a A species may exhibit a wide range
of tolerance toward one factor and a
narrow range toward another. Trout,
for instance, have a wide range of
tolerance for salinity and a narrow
range for temperature.
Figure 3. Shelford’s Law of Tolerance.
I Organisms have an ecological minimum
and maximum for each environmental
factor with a range in between called
the critical range which represents the
range of tolerance (Figure 2). The
actual range thru which an organism can
grow, develop and reproduce normally
Is usually much smaller than its total
range of tolerance.
2 Purely deleterious factors (heavy metals,
pesticides, etc.) have a maximum
tolerable value, but no optimum (Figure 4).
b All stages in the life history of an
organism do not necessarily have the
same ranges of tolerance. The
period of reproduction is a critical
time in the life cycle of most
c The range of tolerance toward one
factor may be modified by another
factor. The toxicity of most sub-
stances increases as the temperature
d The range of tolerance toward a given
factor may vary geographically within
the same species. Organisms that
adjust to local conditions are called
ecotypes .

Signhlicance of ‘Limiting Factors” _ pula t on Variation
e The range of tolerance toward a given
factor may vary seasonally. in general
organisms tend to be more sensitive
to environmental changes in summer
than in other seasons. This is
primarily due to the higher summer
4 A wide range of distribution of a species
is usually the result of a wide range of
tolerances. Organisms with a wide
range of tolerance for all factors are
likely to be the most widely distributed,
although their growth rate may vary
greatly. A one-year old carp, for
Instance, may vary in size from less
than an ounce to more than a pound
depending on the habitat.
5 To express the relative degree of
tolerance for a particular environmental
factor the prefix (wide) or steno
(narrow) is added to a term for that
feature (Figure 5).
Figure 5. Comparison of relative limits of
tolerance of stenothermal and
eurythermal organisms.
C The law of the minimum as it pertains to
factors affecting metabolism, and the law
of tolerance as it relates to density and
distribution, can be combined to form a
broad principle of limiting factors.
1 The abundance, distribution, activity
and growth of a population are deter-
mined by a combination of factors, any
one of which may through scarcity or
overabundance be limiting.
2 The artificial introduction of various
substances into the environment tends
to eliminate limiting minimums for
some species and create intolerable
maximums for others.
3 The biological productivity of any body
of water is the end result of interaction
of the organisms present with the
surrounding environment.
A The organism-environment relationship
Is apt to be 80 complex that not all factors
are of equal importance in a given situation;
some links of the chain guiding the organism
are weaker than others. Understanding
the broad principle of limiting factors and
the subsidiary principles involved make
the task of ferreting out the weak link in
a given situation much easier and possibly
less time consuming and expensive.
1 If an organism has a wide range of
tolerance for a factor which is
relatively constant in the environment
that factor is not likely to be limiting.
The factor cannot be completely
eUminated from consideration, however,
because of factor interaction.
2 If an organism is known to have narrow
limits of tolerance for a factor which is
also variable in the environment, that
factor merits careful study since it
might be limiting.

Significance of “Limiting Factors” to Population Variation
B Because of the complexity of the aquatic
environment, it is not always easy to
isolate the factor in the environment that
Is limiting a particular population.
Premature conclusions may result from
limited observations of a particular
situations. Many Important factors may
be overlooked unless a sufficiently long
period of time Is covered to permit the
factors to fluctuate within their ranges of
poBsible variation. Much time and money
may be wasted on control measures without
the real limiting factor ever being dis-
covered or the situation being Improved.
C Knowledge of the principle of limiting
factors may be used to limit the number
of parameters that need to be measured or
observed for a particular study. Not all
of the numerous physical, chemical and
biological parameters need to be measured
or observed for each study undertaken.
The aims of a pollution survey are not to
make and observe long lists of possible
limiting factors but to discover which
factors are significant, how they bring
about their effects, the source or sources
of the problem, and what control measures
should be taken.
D Specific factors in the aquatic environment
determine rather precisely what kinds of
organisms will be present in a particular
area. Therefore, organisms present or
absent can be used to indicate environ-
mental conditions. The diversity of
organisms provides a better indication of
environmental conditions than does any
single species. Strong physlo-chemical
limiting factors tend to reduce the diversity
within a community; more tolerant species
are then able to undergo population growth.
1 Odum, Eugene P. Fundamentals of
Ecology, W. B. Saunders Company,
Philadelphia. (1959)
2 Reid, George K. Ecology of Inland Waters
and Estuaries. Reinhold Publishing
Corporation, New York. (1961)
This outline was prepared by John E.
Matthews, Aquatic Biologist, Robert S. Kerr
Water Research Center, Ads. Oklahoma.

A Nutrients of importance include macro-
nutrients: those needed in large quantities,
and rnicronutrients: those needed in small
B These nutrients are important because
they promote biological reoponses which
may interfere with some desired u8e of
the water by man.
C Other factors (e. g. temperature, light)
affect the use of these nutrients and should
be considered in an evaluation of the effects
of nutrients upon the ecosystem.
U Algae, bacteria, fungi and aquatic plants
are the forms of life which nutrients affect
most directly.
A Algae are of Several Types
1 Phytoplankton are small algae suspended
in the water and form the basis of pro-
ductivity in the aquatic environment.
2 Benthic algae are those forms anchored
to substrates of rock and bottom
state tials,
3 Periphytic algal are those microscopic
forms attached to submersed substrates.
B Aquatic plants are of several types. In
general they may be referred to as rooted
or floating forms.
C Heterotrophic bacteria are fungi which
respond to organic nutrients Introduced
into water. Autotrophic bacteria may re-
spond and grow due to inorganic nutrient
A Liebtg’s “law” of the minimum the essen-
tial material avaUable in amounts most
closely approaching the critical minimum
needed will tend to be the limiting factor.
B Shellord’s “law” of tolerance survival
of an organism can be controlled by the
quantitative or qualitative deficiency or
excess with respect to any one of several
factors which may approach the limits of
tolerance for that organism.
C Q 10 “law” with a temperature increase
of 10 degrees centigrade metabolic pro-
cesses (rates) are approximately doubled.
IV The process of photosynthesis Is the fixa-
tion of the sun’s energy with the production
of organic matter by plants with chlorophyll.
A The general reaction is given below:
C0 2 +H 2 0 k CH 2 O+O 2
B Chlorophyll contains basically C, 0, H,
N and Mg, and in general makes up about
5% of the dry weight of algal cells.
A Oxygen production can be used as a
measure of photosynthesis because for
each mole of CO 2 reduced to organic
carbon one mole of free oxygen is liberated.
1 The value cf the molar 0 /C0 ratio
has been found experimen 2 tallyato vary
within wide limits.
B CO 2 Assimilation
1 The CO 2 taken up by algae does not all
originate from the dissolved gas. Some
algae can use bicarbonate directly as
a source of carbon.
W.RE.ntr.2e. 10.73

Nutrients: The Basis of Productivity
2 Hence measurement of CO 2 uptake from
water is a complicated problem which
must consider pH. HCO 3 , and CO 3
C Fixation of Carbon-14
1 ‘ h use of C 14 as a tracer of C’ 2 in
plant metabolism and productivity
estimation has been widely used since
the early nineteen fifties.
2 In this method a known amount of C 4
is added to the water and after a period
of time the proportion of C 14 in the
plant cells to C 14 added is found. The
amount of carbon assimilated Is then
estimated from the following equation.
3 Where K is a constant relating to the
slower uptake of C 14 .
4 The total carbon available is determined
D Uptake of Mineral Nutrients
1 The measurement of depletion of
nutrients in solution has been tried
but found unreliable.
E Chlorophyll
1 The quality of chlorophyll present has
been found to bear some relation to
productivity but not a reliable one.
V I Nutrients of significance in the growth and
production of algae and plants are discussed
A Carbon
1 Sources
a Gaseous CO 2
c C0
d Other carbon compounds
2 Effects of the removal of carbon upon
the water
a Lowered pH
b Deposition of CaCO 3
3 The quantity of carbon available is
great and it usually is not a limiting
B Nitrogen
1 Nitrogen can be taken up by most algae
as either ammonium salts or as nitrates.
Nitrites can also be used but a high con-
centration is usually inhibitory. Some
blue green algae can fix atmospheric
nitrogen. Certain algae varieties
require supplementary amines, growth
factors, etc.
2 The quantity of nitrogen in waters has
defmitely been shown to limit algal
C Phosphorus
1 Phosphate seems to be the only inorganic
source of this nutrient.
2 Limiting concentrations of P have been
found to range from .01 ppm at a mini-
mum and an inhibitory affect if P con-
centrations exceed 20 ppm.
3 Optimum concentrations have been found
to range from .018 ppm to 15 ppm.
4 Storage of inorganic phosphate by algae
has been demonstrated. The extent of
this storage may reach 80% of the total
phosphorus in algal cells.
D Silicon
1 Nutrient ratios in the algal cells of
some areas have been found to be Si 23:
N16 P1. It can be seen from this
ratio that silicon is an important ele-
ment in algal growth.
activity of
pt ytopla nkton
activity of
C1 4 0 added
total carbon
(K) assimilated
total carbon

Nutrients. The Basis of Productivity
2 Silicon is especially important in the
population growth of diatoms and may
be the limiting growth factor in these
E Inorganic micronutrients - Many elements
are needed in very small quantities by
algal cells. Some of these have a known
function in algal metabolism; others do
I Mg is a cation of major importance in
the chlorophyll molecule.
2 Co is known to be necessary for vitamin
B 12 .
3 Mn is necessary for 8everal enzyme
4 Mo, V, Zn, and Cu are necessary but
these functions are not as well known.
F Organic Micronutrients
1 Of 179 algal strains investigated about
40% required vitamin supplementation
for optimum growth. Principal growth
factors that were not synthesized in
sufficient quantity are given as follows
along with the percentage of the vitamin
deficient strains showing marked pro-
ductivity gain after supplementation:
a B 12 addition increased growth on
80% of the strains.
b Thiamin addition increased growth
on 53% of the strains.
c Biotin addition increased growth on
10% of the strains.
2 Algae can use and may require many
organic compounds depending upon
environmental conditions and the ability
of the organism to synthesize required
building blocks from mineral forms of
C.N. & P. This is an area for con-
linucd investig.ition with many unappre-
ciated or vaguely understood ecological
A Problems to man may result when the
total “primary production” by algae leads
to an increase in the total organic content
of the water that interferes with a desired
1 This may consist of a high algal popu-
lation that produces a water with high
turbidity, taste and odor, or other
undesirable effect. High respiratory
needs may lead to nocturnal oxygen
2 Certain algae may cause tastes and
odors, clog filters, or otherwise inter-
fere with potable water processing.
3 Death of large algal populations may
lead to tastes and/or odors through
bacterial decomposition. Oxygen
deficits may result at any time of day
in this process. Deposition of masses
of organic sediment or sludge may be
4 Other problems might be cited.
B The primary production of algae can also
serve as a supply of food to consumer
organisms (animals), resulting in increased
production at several (trophic) levels:
zo microbes, microinvertebrates,
macroinvertebrates, fishes.
1 Earlier notation cited the release of
oxygen during utilization of CO 2 during
algal photosynthesis. This encourages
fungal or bacterial breakdown of
2 Photosynthesis occurs in the presence
of adequate light and favorable condi-
tions. In darkness, the cells continue
to respire and may consume more
oxygen than they produced because
photosynthesis increases the organic

Nutrients The Basis of Productivity
3 Photo ynthenls tends to occur at the
sut fuct where light intensity is greatest.
Poor vertical mixing would result in
stratification of water supersaturated
with oxygen over oxygen deficient water
Depending upon conditions, a significant
fraction of the oxygen could be lost to
the atmosphere.
4 Increased productivity may result in
temporary reduction of the free dissolved
nutrient level in the water but harvesting
at some level is essential to prevent
later recycle.
A Once nutrients enter a body of water
they are cycled through a food chain.
B Factors affecting this food chain (e.g.
toxicity, removal) will affect the con-
centration and distribution of the nu-
This outline contains certain material
submitted by F. J. Ludzack and H. W.
4 Odum, H. T. Primary Production in
Flowing Waters. Limnology and
Oceanography. 1(2):102-117.
April 1956.
5 Ryther. John H. The Measurement of
Primary Production. Urnnology and
Oceanography. 1(2):72-84. April 1956.
6 Verduin, Jacob. Primary Production In
Lakes. Limnology and Oceanography.
l(2):85-91. April 1956.
7 Symposium: Factors That Regulate
the Wax and Wane of Algal Populations.
Inter. Assoc. of Theor. and Appi. Biol.
Communications No. 19. 1971.
8 Likens, G. E. Nutrients and Eutrophi-
cation. The limiting-nutrient contro-
versey. Am. Soc. Limnol. Ocean.
Spec. Symp. Vol. 1. 1972.
10 Fitzgerald, G. P. Nutrient Sources
For Algae and their Control. Water
Pollution Coat. Res. Ser. 16010 EHR
08/71. EPA. 77 p. 1971.
1 Golterman, H. L. and C]ymo, R. S.
Chemical Environment in the Aquatic
Habitat (Proc. of an IBP - symposium,
Amsterdam and Nieuwerslujs Oct. 1966).
322 pp. (N.y. Noord-HoUandsche
Tjltgevers Maatschapplj, Amsterdam.
2 Lewln, Ralph A. Physiology and
Biochemistry of Algae. Academic
Press. 1962
3 Odum, Eugene P. Fundamentals of
Ecology. W.B. Saunders Co. 1959.
This outline was prepared by Michael E.
Bender, Biologist, Formerly with Training
Activities, Ohio Basin Region, SEC.
9 Bartoch, A.F.
EPA - R3 - 72 -
Role of Phosphorus In
Ecol. Res. Ser.
001. 45 p. 1972.

This topic covers a wide spectrum of items
often depending upon the individual discussing
the sub)ect and the particular situation or
objectives that he is trying to “prove”.
Since the writer Is not a biologist, these
viewpoints are “from the outside-looking in ”.
Any impression of bias is Intentional.
A Some Definitions are in Order to Clarify
1 EutrophIcation - a process or action of
becoming eutrophic, an enrichment.
To me 1 this Is a dynamic progression
characterized by nutrient enrichment.
Like many definitions, this one is not
precise, stages of eutrophication are
classified as aug- , meso-, and eutrophic
depending upon Increasing degree. Just
how a given body of water may be
classified is open to question. It
depends upon whether you look at quiet
or turbulent water, top or bottom
sample 8, season of the year, whether
it is a first impression or seasoned
judgement. It also depends upon the
water use in which you are interested,
such as for fishing or waste discharge.
The transitional stages are the major
problems - it is loud and clear to a
trout fisherman encountering carp and
2 Culture
Fostering of plant or animal growth.
cultivation of living material and
products of such cultivation, both fit.
Some degree of control Is implied but,
the control may have limitations as
well as advantages. Human cultural
development has fostered human num-
bers successfully, but, has promoted
rapid degradation of his natural environ-
3 Nutrients
A component or element essential to
sustain life or living organisms. This
includes many different materials,
some in gross quantities - others in
minor quantities. Deficiency of any
one essential item make living
impossible. Nutrients needed in large
quantities include carbon, hydrogen,
oxygen, nitrogen, phosphorus, sulfur
and silica. N and P frequently are
loosely considered as “the “nutrients
because of certain solubility, con-
version and “known” behavior
4 Algae
A group of nonvascular plants, capable
of growth on mineralized nutrients with
the aid of chlorophyll and light energy -
known as producer organisms, since
the food chain is based directly or
indirectly upon the organic material
produced by algae.
B Now that we have “backed into” the title
words via definitions, some of the
ramifications of eutrophication, nutrient
enrichment, and cultural behavior are
A All nutrients are interchangeable in form,
solubility, availability, etc. There are
no “end” products. We can isolate, cover,
convert to gas liquid or solid, oxidize,
reduce, complex, dilute, etc. - some
tfme, some place, that nutrient may
recycle as part of cultural behavior.
1 Water contact is a major factor in
recycle dynamics just as water
represents two—thirds or more of cell
SI. ECO. hum. 3.5 71

jj .tc wid Cullui al Liitrophicatlon
ui’. ss and app.ars to be the medium in
wiiwh living foritis started. Waste
dispo’,ul intertelationships (Figure 1)
stiggi .ts physical interielationships of
soil, air and water. The wet apex of
tins triangle is the basis for Life. It’s
difficult to isolate water from the soil
tn atmosphere - water contact means
,i thition of available nutrients.
/\ ‘OSAL
ATER $ ii
2 Figure 2 takes us into the biosphere (1)
via the soluble element cycle. This
refcr .s mainly to phosphorus interchange
Phosphorus of geological origin may be
solubilized In water, used by plants or
animals and returned to water. Natural
inovt ment is toward the ocean. Less
phosphorus returns by water transport.
Phosphorus does not vaporize, hence,
atmospheric transport occurs mainly
as windblown dust. Man and geological
upheaval, partially reverse the flow of
phopshorus toward the ocean sink.
3 The nitrogen cycle starts with ele-
mental nitrogen in the atmosphere.
It can be converted to combined form
by electrical discharge, certain
bacteria and algae, some plants and
by industrial fixation. Nitrogen gas
thus may go directly Into plant form
or be fixed before entry. Denitrification
occurs mainly via saprophytes.
(Figure 3) Industrial fixation is a
relatively new contribution to
[ I i •’
— ii liUflh it
._ — _______— liTt i l li
III upullic
,i, i i u
I I niuululit

( 1 . 1 1 t h
H I ll
Phi, i i luthil uy
11 11 11
tillilTiT! llIl

Algae and Cultural Eutrophication
4 Carbon Convei sion (Figure 4) show
most of the carbon in the ioi m of
geological carbonate (1) but bicarbonate
and CO 2 readl]y are converted to plant
cell mass and into other life forms.
Note the relatively small fraction of
carbon in living mass. -
Nutrient cycles could go on, but, life
depends upon a mixture of essential
nutrients under favorable conditions.
Too much of any significant item in the
wrong place may be consldeied as
pollution. Since toxicity is related to
chemical concentration, time of exposure
and organism sensitivity, too much
becomes toxic. If it happens to be too
much growth, its a result of eutrophication.
‘How much’ is generally more important
than the ‘what’? Both natural and manmade
processes lead to biological conversions,
to pollution, to eutrophication and to
toxicity. Man is the only animal that can
concentrate, speed up, invent, or otherwise
alter these convu’rsions to make a r’ollossal
roe ‘ , S.
1 Life forms have been formulated In
terms of e ehienthl or nutrient com-
ponents many times. The simplest is
C 5 H 8 0 N. A more complex formula
is ’ C 10 H 76 0 80 N 20 Ca 6 C1 7 I’ 2 CuF 2
SiMgMn 2 K 2 NaS 21 Zn. This includes
16 elements. More than 30 have been
implicated as essential and they still
would not “live”, unless they were
correct] y assembled. As a nutrient
Mnemonic I -I. COPKINS - - Mg(r)-
CaFe-MoB does fa,irly well. It also
indicates Iod ine-I, Iron-Fe,
Molybdenum-Mo, and Boron-B that
were not included earlier.
2 The Law of Distribution states that
“Any given habitat tends to favor all
suitable species - any given species
tends to be present in all suitable
habitats. ‘ Selection tends to favor the
most suitable species at a given place
and time.
3 Laebigs Law of the Minimum, states
that “The essential material available
in amounts most closely approaching
the critical minimum will tend to be
the limiting growth factor.”
4 Shelfords law recognizes that there
will be some low concentration of any
nutrient that will not support growth.
Some higher concentration will stimulate
growth. Each nutrient will have some
still higher concentration that will be
bacteriostatic or toxic. This has been
discussed earlier but was considered in
a different manner.
The biological “balance’ appears to be a very
transitory condition in cultural behavior.
Man favors production. A steady state
“balance” does not persist very long unless
energy of the system is too low to permit
significant growth. A progression of species
where each predominent form thrives for a
time, then is displaced by another temporarily
favored group is usual. ‘Yearly events in the
lawn start with chickweed, then dandelion,
plantain, crab grass, rag weed, etc , in
successive predominence. Occasionally,
more desirable grasses may appear on the
lawn. Grass is a selected unstable “culture”
B Nutrient - Growth Relationships

A Igae and Cultural Eutrophication
A Figure 3 shows a biological progression (2)
following introduction of wastewater in an
unnamed stream. Sewage or slime bacteria
proliferate rapidly at first followed by
dilates, rotifers, etc.
5. S.ct.,l. th,iv. sad finally b.csm. pt.y .1 Ks ciliate., which in •wni sea Is.d is, the roth. ,, sad crustacoani.
H Figure 4 shows another progression of
bottom dwelling larva. Here the sequence
of organi8ms changes after wastewater
introduction from aquatic Insects to sludge
worms, midges, sow bugs and then to
re-establishment of insects.
Figure 6. n. p .l.tl.. .i F1.r. 7 is c .mpu.d of a oath.. .1 issal..
I . , individusl • .ci , soak .swltiplying and dying sff a, sins. c..dith.ns swy.
N. PER m l.
(N0 PtR ML)
• ;.. .,. . 0
Iii ‘ I
I ••
2 1
24 12 0
3 -
12 24 36 41 SO 72 84 96 108
14 1 I I i 11 II it I I S I I I

_Algae and Cultural Eutrophication
U Another progression after waste
introduction changes the blota from
an algal culture to sewage moulds with
later return to algal predominence,
Figures 5, 6, and 7 are shown separately
only because one visual would be unreadable
with all possible progressions on it. There
are progressions for fungi, protiata, Insect
larvae, worms, fish, algae, etc. Each
species will perform as it may perform.
It it cannot compete successfully, it will
be replaced by those that can compete
under prevailing conditions at the time.
Conditions shift rapidly with rapid growth.
IV The interactions of bacteria or fungi and
algae (Figure 8) are particularly
significant to eutrophication.
A The bacteria or the saprophytic group
among them tend to work on preformed
organic materials - pre-existing organics
from dead or less favored organisms.
Algal cells produ’e the organics from
light energy chlorophyll and mineralized
nutrients. This is a happy combination
both: The algae release the oxygen
for use by tht bacteda while the bacteria
release the CO 2 needed by the algae.
Since the algae also acquire CO 2 from
the atmosphere, from wastewater and
from geological sources, it always ends
up with more enrichment of nutrients In
the water - more enrichment means more
growth and growing organisms eventually
clump and deposit. The nature of growth
shifts from free growth to rooted forms,
starting In the shallows. Another
progression occurs (Figures 9 and 10).
It is this relationship that favors profuse
nuisance growth of algae below significant
waste discharges. There is a tremendous
pool of carbon dioxide available in
geological formations and in the air.
Transfer to the water Is significant and
encourages algal productivity and eventual
eutrophication of any body of water, but,
this does not occur as rapidly as when the
water body is super saturated with CO 2
from bacterial decay of wastewater
discharges or benthic deposits from them.
• zi.gure ‘s .,,*, . 1.. .. .. dl.ch.,g.. N t. ..uUd. .N.I. .. l*vm 1 ,wNt.
This.. ,. .ss.deNd wINt sI..d, dSg.NIIM s .w. I. N t. Urn, curve. Tb. sludge Is
dr..mp.-.d gi.d..l$p : as c.adIp4..s c i . ., up, .1g.. gals a f..th.Id .itd .t.ftIply.

Algae and Cultural Eutrophication
r o o o@ u

A1 ae and Cultural Eutrophication
LgiiO AL ZO /\ D
LI Nitrogen and phosphorus are essential
for growth. They also are prominently
considered in eutrophication control,
Algal cell mass Is about 50% carbon,
l % nitrogen and approximately 1%
phosphorus not considering luxury uptake
in excess of immediate use. Phosphorus
is considered as the most controllable
limiting nutrient. It’s control Is corn-
plicated by the feedback of P from benthic
sediments and surface wash. Phosphorus
removal means solids removal. Good
clarthcatlon is es8entlal to obtain good
removal of P. This also means improved
removal of other nutrients- a major
ddvantago of the 1’ removal route. I3oth
N & P arc easily converted from one form
to another, most forms are water soluble.
Cnntrol of eutrophication is not entirely
possible. Lakes must eventually fill with
benthic sediments, surface wash and
vegetation. Natural processes eventually
Lause filling. Increased nutrient discharges
from added activities grossly increase filling
A We produce more nutrients per capita per
day In the United States than In other
nations and much more today than 100
years ago. More people in population
centers accentuate the problem.
B Technology is available to remove most
of the nutrients from the water carriage
1 This technology will not be used unless
water is recognized to be in short
2 It will not be used unless we place a
realistic commodity value on the water
and are willing to pay for cleanup for
reuse purposes.
C Removal must be followed by isolation of
acceptable gases to the atmosphere
acceptable solids into the soil for i euse
or storage. Water contact cannot be
prevented, hut it must be limited or the
enrichment of the water body is hastened.

Algae and Cultural Eutrophication
I IEFERENCES _______________________________
This outline was prepared by F. J. Ludzack,
1 A collection of articles on the Biosphere Chemist, National Training Center,
Sd. Am. 223 (No. 3). pp. 44-208. Water Programs Operations, EPA, Cincinnati,
September 1970. OH 45268.
2 Bartch, A.E. and Ingrain, W.M.
Stream life and the Pollution Environ-
ment. Public Works 90:(No. 7)104-
110. July 1959.

A Plankton growths are as natural to aquatic
areas as green plants are to land areas
and respond to the same stimuli.
B Mart i8 currently harnessing plankton forms
to accomplish useful work.
1 For generation of oxygen
a Stabilization of waste waters in
oxidation ponds
b Oxygen recovery from CO 2 in space
2 For augmentation of food supply
a Fish ponds
b Nitrogen fixation in rice growing
c Harvesting of algae for direct use
as food
C A growing knowledge of the nutrient re-
quirements of plankton organisms will
lead to a more enlightened approach to
ways and means of controlling their growth
when desirable.
A Chemical
1 Inorganic
a Copper sulfate is used most exten-
sively. It is most effective in pre-
ventive rather than curative treat-
ment. It has long lasting effects in
soft waters but is short-lived in hard
waters due to precipitation of the
Cu as a basic carbonate The pre-
cipitated material accumulates m
bottom muds and is toxic to certain
benthal forms, some of which serve
as important fish food.
Dosages are normally based on the
alkalinity of the water. When alka-
linity is < 40 mg/I. the recommended
dosage is 0. 3 mg/l of CuSO 4 5H 2 0
in total volume of water. When
alkalinity is > 40 mg/l, recommended
dosage is 2. 0 mg/l in 8urface foot
of water.
b Chlorine is preferable to copper
sulfate in the control of certain
forms of algae. However, it is
difficult to apply in most instances
and is very short-lived due to photo
catalytic decomposition of HC1O —
HC1 + 0
2 Organic - Numerous organic compounds
have been evaluated, especially in re-
lation to control of blue-green algae.
“Phygo&’, 2, 3 -dichloronaphthoquinone,
has been field tested but is too specific
in its action for general application.
A Theory - Ecological control is based upon
the principle of preventing or restricting
growth by limiting one or more of the
essential requirements. This is an ap-
plication of Liebig’s Law of the Minimum.
The logical avenues of control are as
1 Elimination of light
2 Limiting nutrient materials
B Light - Many cities have solved the prob-
lem of plankton growths by the use of
covered reservoirs, underground and
elevated. Concurrently, they have solved
contamination problems created by birds
and atmospheric fallout In open reservcxxrs,
131. MIC. con, lOb. 4.70

Control of Plankton in Surface Waters
some success has been obtained by
limiting light through the use of a film of
activa led carbon
C Nutrients - Since phytoplankton (algae)
serve as the base of the food chain, know-
ledge concerning their nutrient require-
ments is required for ecological control,
when limitation of light is impractical.
The nutrient requirements of phytoplankton
are as follows ’
I Nature of - The major nutrients are
a Carbon dioxide
b Nitrogen - ammonia and nitrates
(also N2)
c Phosphorus - phosphates.
Minor nutrients are
d Sulfur - sulfates
e Potassium
f Trace inorganics - magnesium, iroa,
g Trace organics-vitamins, amino
2 Sources of - See Fig 1
a Atmosphere
b Groundwater - springs
c Storm water or surface runoff
d Waste waters - domestic sewage and
industrial wastes.
3 Significance of each major nutrient
a Carbon dioxide - Sec Fig 2
Usually present in great abundance.
Rapidly replenished from at mosphere
and bacterial Uccuinpusition of organ-
ic matter No reasonabLe possibility
of human control Nature, however,
does provide some control through
elevated pH levels if carbon dioxide
becomes depleted rapidly.
b Nitrogen - Like land plants, certain
algal forms prefer nitrogen in the
form of NH 3 (NH 4 t ) and others prefer
it in the form of N0 3 , Both forms
often become depleted during the
growing season and reach manmum
concentrations during the winter
season. A level of 0. 30 mg/I of
inorganic nitrogen at the time of the
spring turnover is considered to be
the maximum permissible level
All natural surface waters are
saturated with nitrogen gas This
serves as a source of nitrogen for
bacteria and algae capable of fixing
c Phosphorus - A key element in all
plant and animal nutrition. The
critical level is considered to be
0 01 mg/i at the time of the spring
turnover. Phosphorus is needed
to sustain nitrogen fixing forms,
D Practice Of
1 Exclusion of hght - Practice we u
established l it distribution system
reservoirs but impractical on large
storage reservoirs,
2 Nutrient limitation
a Control of surface run-off quality
1) Agricultural
2) Other
b Diversion of sewage plant effluents
1) Madison , Wisconsin
2) Detroit Lakes, Minnesota
3) Pending - State College, Pa.
c Tertiary treatment of sewage
I) Nitrogen removal - Because of
the several forms is very difficult.


CO 2
H 2 0
CO 2
+ CO
I :
+H 2 0

Control of Plankton in Surface Waters
Also, may be unsuccessful in
control unless phosphorus Is con-
trolled 1 too, because of nitrogen
fixing forms.
2) Phosphorus removal - Phosphorus
can be effectively removed by
coagulation methods employing
lime, alum or ferric salts. It
Is expensive and no one has
proven its value beyond laboratory
d By Biological Engineering
Laboratory studies have shown that
effluents essentially free of plant
fertilizing elements can be produced
by biological treatment of wastes
with proper ratios of C to N and P.
E Practical Aspects
1 Diversion
2 Nutrient control
Muilican, Hugh F. (Cornell Univ.)
Management of Aquatic Vascular Plants
andAlgae. pp. 464-482. (inEutro-
phication: Causes, Consequences, and
Correction. Nat. Acad. Sci.) 1969.
This outline was prepared by C. N. Sawyer,
Director of Research, Metcalf & Edcbr
Engineers, Boston, Massachusetts.
3 ExperIences
a Madison
b Detroit Lakes
c State College
d Lake Winniequam, N. H.

A Information on the number, kinds, and
effects of interference organisms in a
particular water supply is essential for
determining adequate control measures.
B Collection of the biological data should be
on a regular routine basis.
C Interpretation of data requires information
on relationship of number and kinds of
organisms to the effects produced.
D It is generally more satisfactory to an-
ticipate and prevent problems due to these
organisms than it is to cope with them later.
A Use of algicides
1 Application of an algicide is to prevent
or destroy exces8ive growths of algae
which occur as blooms, mats or a high
concentration of plankton.
2 Algicide may be applied to control even
low concentrations of certain algae such
as Synura.
3 Copper sulfate is the only algicide in
common use at present.
a Application may be by dusting.
spraying or dissolving from a porous
container over all or part of the water
surface, or by continuous feeding
of the algicide at the intake of the
reservoir or pre-treatment basin.
b Effective dosage depends upon the
Alkalinity and pH and temperature
of the water and the amount and
kinds of algae to be controlled.
Bartch states that the following
arbitrary dosages have been round
to be generally effective and safeS
M.O. alkalinity > 50 p.p. in
2 p. p. m. in the surface foot of
water only (5. 4 pounds per acre).
M.O. alkalinity <50 p.p,m. 0.3
p. p. m. in total volume of water
(0. 9 pound per acre foot).
c Application of copper sulfate should
be limited to the minimum effective
dosage because of its corrosive
properties, and its toxicity to fish and
other aquatic animals.
4 Other algicides
a Promising types include inorganic
salts, organic salts, rosin amines,
antibiotics, quinones, substituted
hydrocarbons, quaternary ammonium
compounds, amide derivatives and
phenols. Cuprichloramine which is
a combination of copper, chlorine and
ammonia, and also chlorine dioxide
have shown promise as general aigi-
b For domestic water supplies they will
have to be not only economically fea-
sible but nontoxic to animal life and
to green plants other than algae.
c Due to higher costs they will prob-
ably be used only when adequate plank-
ton and algal records are kept, which
would permit early localized treat-
d Algicides selectively toxic to the
particular algae of greatest signifi-
cance would be useful.
5 Mechanical removal or spreading out
to permit rapid drying may be the sim-
plest way of handling massive growths
which are detached and washed ashore.
6 Turbidity due to sLit keeps down the
plankton population. In shallow reservoirs,
fish which stir up the bottom mud will
aid in keeping turbidity due to silt high.
7 Provisions for keeping the amounts of
nutrients to a minircium may be em-
phasized more in the future.
8 For new reservoirs, clearing the site
BI.MIC.con.6b.4. 70

Control of Interference Organisms in Water Supplies
of vegetation and organic debris before
filling will reduce the algal nutrients.
Steep rather than gentle elopes will
reduce the areas which allow marginal
growths to occur.
A Coagulation and sedimentation
1 When well regulated they often will re-
move 90 per cent or more of the plank-
2 With low plankton counts, a coagulant
aid may be required.
3 Frequent removal of sludge from the
basins, especially during the warm
seasons may help to reduce tastes and
odors originating from decomposing
organic sediment.
B Sand filtration
I Both slow and rapid sand filters tend to
reduce the plankton count of the effluent by
90 per cent or more, when well regu-
2 For rapid filters, accumulated plankton
can be removed or reduced by surface
scraping and by back washing.
C Micro-straining
1 This involves the passing of the water
through a finely woven fabric of stain-
less steel. AU but the smaller plankton
organisms tend to be removed from the
water. It is being used in some treat-
ment plants in England and elsewhere.
D Activated carbon
1 The slightly soluble, organic, taste
and odor compounds tend to be readily
adsorbed by the activated carbon. It
is probably most often applied prior to
coagulation, but may be used prior to
filtration or in the raw water.
E Chlorination
1 Treatment with chlorine is practiced
primarily to destroy pathogenic organ-
isms. The dosages commonly used are
toxic also to many algae and to some of
the other groups of aquatic organisms.
However, dead as well as living organ-
isma are often capable of causing tastes
and odors and of clogging filters.
F The depth and position of the intake for
entrance of raw water into the treatment
plant may determine the kinds and amount
of plankton which will be drawn into the
plant. Plankton algae generally are more
concentrated near the surface of the water
in lakes and reservoirs.
A Maintenance of a chlorine residual con-
trols the chlorine sensitive organisms.
B Other pesticides such as cuprich.loramine
have been used in attempts to control the
resistant organisms such as worms,
nematodes and copepod eggs.
C Flushing of infested portions of the system.
especially dead ends may be practiced.
D Covering of treated water reservoirs to
prevent the entrance of light wifi stop the
growth of algae.
E Organisms associated with pipe corrosion
are probably the most active when the water
itself is corrosive.
F Mechanical cleaning of the distribution
system may be an effective but expensive
method of reducing infestations of attached
A Adequate control is dependent upon ade-
quate procedures for detecting and record-
ing of organisms.
B Control may involve the following:
1 Use of an algicide or pesticide.

Control of Interference Organisms In Water Supplies
2 Mechanical cleaning of distribution d By reducing the amount of fertilizing
lines, settling basins, and filters, nutrients entering the reservoir
screens, intake channels and reservoir
margins. e By encouraging a balanced develop-
ment of the aquatic organisms
3 Modification of coagulation, filtration,
chemical treatment or location of raw
water Intake. REFERENCE
4 Use of adsorbent, such as activated Mackenthun, Kenneth M. The Practice of
carbon, for taste and odor substances. Water Poliution Biology. FWPCA.
U. S. Dept. of Interior, Washington, DC.
S Modification of reservoir to reduce the 1969.
opportunities for massive growths.
a By covering treated water reservoirs
b By IncreaBing the depth of the water This outline was prepared by C. M. Palmer,
Former Aquatic Biologist, Biological Treat-
c By eliminating shallow marginal meat Research Activities, Cincinnati Water
areas Research Laboratory, FWPCA, SEC.

A Administrative Proceedings
1 Rule making
a Setting up of regulations having
general application, e.g. • stream
classifications and implementation
plan target dates
b Factors of safety and absolute
prohibitions may be appropriate
2 AdjudicatIons
a Determinations by agency having
expertise with respect to particular
discharge or discharger. e.g.,
approval of plans and ape s and
time schedule of a particular
B Court Actions
1 Civil in behalf of state or federal
a Actions to compel action or sus-
pension of action - nuisance, health
hazard, etc. , - -including court
action following federal conference
- -hearing procedure
b Violations of Water Quality Standards
c Violations of Effluent Standards or
discharge permits
d Tort or contract actions relating to
design and/or operation of treatment
2 Criminal (dependent on content of
applicable statutes)
a Discharge of specific materials
b Discharges from specific industries
c Littering
d Discharges harmful to fish and/or
e Discharges harmful to specific
types of receiving waters
f Discharges of poisons
NOTE- -In some of these situations
doing the act may constitute
the violation; in otherH
proof of intent or knowledge
of effects may also have to
be proved.
3 Private actions for damages or
to compel action
a Alleged harm to plaintiff, e.g.,
poUution of stream IdUing animals
C Procedural Matters
1 See Attached sheet “Administrative
and Court Proceedings” on Burden
of proof, fact finding, and methods of
presentation of evidence.
D Classes of Evidence - General Rules
1 Facts - direct
a The material was floating from
the outfall.
2 Derived values - expert testimony
- test results and/or opinion as to
a The D. 0. was zero; the waterway
was polluted; the plant can be built
in 6 months.
3 Hearsay
a Joe told me
W,Q.lc. la. 3.74

Case Preparation and Courtroom Procedure
4 Relevancy
5 Admissibility vs. weight
a Even i i admissible, the weight to be
given is up to fact finder-- credi-
IE Admissibility of Results of Sampling
and Testing (Numbers)
1 Sampling
a Chain of custody
b Tags. etc.
c Containers
d Place and tune
e Retention of samples (Proving that
the sample represents what Is at
issue in the action (relevancy),that
there has been no opportunity for
tampering; and availability of
portions for analysis by other aide
(non-transitory criteria) ).
2 Analysis
a Who performed (Can identity of
each participant be shown?)
b Admission through supervisor -
c Scientific acceptance of method.
Is there a particular method required
to be used by the agency?
d Propriety of conduct
e Retention of bench cards and other
indicia of results. (Your attorney
can make arrangements to substitute
copies for originals).
3 Tests
a Comparison with actual conditions
b Mathematical models - how can a
computer be cross-examined?
F Admissibility of Expert Opinion on
Causes and Effects
1 Who has special knowledge - and of
what particular areas?
2 Indicators
3 Significance of numerical determin-
ations or observations
4 Consistency with own prior publications
and testimony
5 Have underlying facts been or need to
be proved- -first hand information of
this and/or comparable situations.
6 Use of treatises
G Conduct on the Witness Stand
1 General
a On direct - know what counsel will
ask and let him know generally
what you will answer, but don’t
make it sound rehearsed.
b Use layman’s language to extent
c Listen to question and answer it
to best of your ability.
d Speak so that court reporter, judge,
Jury, and counsel can hear you.
e Speak in language that will be
understood; don’t talk down.
f Answer only what you are asked
- - don’t volunteer; however, answer
with precision.
g There is nothing wrong with asking
to have a question repeated or
h There is nothing wrong with saying
that you consulted with your
attorney before you testified, but
beware of the question “Did Mr. C
tell you what to say?”

Case Preparation and Courtroom Procedure
I There is nothing wrong with thinking
out your answer before responding.
j You are not expected to know all
the answers- -if you do not know,
admit it.
k Don’t attempt to answer questions
outside your area of personal
knowledge (hearsay) or beyond your
expertise. (Your may be an expert
on conducting laboratory tests, but
not on epidimeological inferences
from results).
1 Don’t try to answer before the judge
rules on objection.
m Show that you are an impartial
dispenser of Information and/or
opinion, not a protagonist.
n Don’t be afraid to admit what may
appear to be damaging.
2 If you are testifying as an expert:
a Establish qualifications - - give
information relevant to your area
of expertise - - educational (in-
cluding this course?), work,
publications, number of times you
have testified previously.
a Review and refamiliarize self with
materials before you discuss with
your attorney.
b Be in a position to present all facts
known to you simply and concisely:
Who, What, When, Where, and
Why, How.
c Don’t overlook facts and/or test
results because you don’t think
they’re important. Let attorney
decide what he needs.
d Use of standard report forms
e Ability to recommend additional
witnesses with needed specialized
f Ability to aid In cross-examination
of other side’s experts and reconcile
opinions and/or results
g Be candid - sometimes better not
to start a lawsuit or accept a
settlement than lose in the end.
H Non-Verbal Presentation of Evidence
1 Exhibits - including photographs
2 Summaries
b Differentiate between physical facts
(measurements and observations)
and opinion ( derived values) .
c Be prepared to discuss theory (in-
cluding assumptions) instruments
used, techniques(including choice
of a particular technique), physical
limita ons and errors, inter-
d If experiments were conducted,
be able to justify both as to theory
and relevancy to this litigation.
e If you’re being paid to testify,
admit it.
3 ScientifIc personnel as advisers to
3 Buslnes and/or government records
a Prepared contemporaneously and
in usual course of activities
4 Pre-prepared direct examination
a Usually limited to actions before
ICC, FPC, and other federal
I Criminal Procedure
1 Privilege Against Self Incrimination
(available only to persons)
a Warning and suspects
b Effect of duty to report spills

Case Preparation and Courtroom Procedure
c Effect of duty to obtain license or This .itline was prepared b r David I.
permit and/or furniah operating iedroff , Enforcement Anaiyst, Office of
reports Enforcement and General Counsel,
Cincinnati Field Investigations Center,
d Immunity from prosecution 5555 Ridge Avenue, Cincinnati, OH 45268.
2 Double Jeopardy
3 Unreasonable search and seizure Deacripto s : Courtroom Procedure,
Law Enforcement, Legal Aspects, Sampling,
a Available to persona and Water Analysis. Water Pollution
corporations Control, Water Quality Standards
4 Procedures and need for arreBt and
search warrants - -possible cause
Administrative & Court Proceedings,
and cerpts from Revised Draft of
Proposed Rules of Evidence for the
United States Courts can be found on the
following pages.

Court or Agency Fact Finder Burden of Proof Comments
State Pollution Control Agency As per statute - Hearing may be conducted by hearing
Agency usually weight of examiner, agency member, or full
evidence, agency. Appeal may be on facts and
Rule making-adjudi- law or law alone, depending on statute. 0
Federal Water PoLlution
Control Act
Conference Head of agency Reports acceptable.
Hearing Hearing Board Specific testimony. o
Court Judge Uses prior material, and may take
additional testimony.
Civil Case - -
- for money only Judge or jury Weight of evidence
- In lunction
preliminary or Judge Must show immediate Must also show likelihood of success at
temporary harm or danger, final hearing - bond required for non-
government plaintiff.
permanent Judge Usually clear and convincing. “Balance Equities”
- administrative Judge - whether Sometimes have complete new trial.
appeal “arbitrary and
capricious” or sub-
stantial evidence.
C ” Criminal case Jury unless waived. Beyond reasonable Proof of intent may be required.
includes penalties doubt.

Case Preparation and Courtroom Procedure
Excerpts from Revised Draft of Proposed
Rule 102.
These rules shall be conBtrued to secure fairness In administration, elimination
of unjustifiable expense and delay, and promotion of growth and development of
the law of evidence to the end that the truth may be ascertained and proceedings
justly determined.
Rule 101.
(a) Questions of Adnttssthility Generally. Preliminary questions concerning the
qualification of a person to be a witness, the existence of a privilege, or the
admissibility of evidence shall be determined by the j idge, subject to the pro-
visions of subdivision (b). In mald.ng hia determination he is not bound by the
rules of evidence except those with respect to privileges.
(b) Relevancy Conditioned on Fact. When the relevancy of evidence depends upon
the fu].filhxnent of a condition of fact, the judge shall admit it upon, or subject to,
the introduction of evidence sufficient to support a finding of the fuiflhlnient of the
Rule 815.
At the request of a party the judge shall order witnesses excluded so that they
cannot hear the testimony of other witnesses, and he may make the order of his
own motion. This rule does not authorize exclusion of (1) a party who Is a natural
person, or (2) an officer or employee of a party which Is not a natural person
designated as Its representative by its attorney, or (3) a person whose presence
is shown by a party to be essential to the presentation of his cause.
Rule 611.
(a) Control by Judge. The Judge may exercise reasonable control over the mode
and order of interrogating witnesses and presenting evidence so as to (1) make the
interrogation and presentation effective for the ascertainment of the truth, (2) avoId
needless consumption of time, and (3) protect witnesses from harassment or undue
(b) Scope of Cross-Examination. A witness may be cross-examined on any matter
relevant to any ls9ue In the case, including credibility. In the interests of justice.
the judge may limit cross-examination with respect to matters not testified to on
direct examination.

Case Preparation and Courtroom Procedure
Rule 613.
(a) Examining Witness Concerning Prior Statement. In examining a witness
concerning a prior statement made by him, whether written or not 1 the state-
ment need not be shown or its contents disclosed to him at that time, but on
request the same shall be shown or disclosed to opposing coun e1.
Rule 201.
(b) Kinds of Facts. A judicially noticed fact must be one not sublect to reasonable
dispute In that it Is either (I) generally known within the territorial jurisdiction of
the trial court or (2) capable of accurate and ready determination by resort to
sources whose accuracy cannot reasonably be questioned.
(g) Instructing Jury. The judge shall instruct the jury to accept as established
any facts judicially noticed.
Rule 401.
“Relevant evidence” means evidence having any tendency to make the existence
of any fact that is of consequence to the determination of the action more probable
or lees probable than it would be without the evidence.
Rule 402.
All relevant evidence is admissible, except as otherwise provided by these rules,
by other rules adopted by the Supreme Court, by Act of Congress. or by the
Constitution of the United States. Evidence which is not relevant is not admissible.
Rule 601.
Every person is competent to be a witness except as otherwise provided in these

Case Preparation and Courtroom Procedure
Rule 602.
A witness may not testify to a matter unless evidence is introduced sufficient
to support a finding that he has personal knowledge of the matter. Evidence to
prove personal knowledge may, but need not, consist of the testimony of the
witness himself. This rule is subject to the provisions of Rule 703, relatIng to
opinion testimony by expert witnesses.
Rule 702.
If scientific, technical, or other specialized knowledge will assist the trier of
fact to understand the evidence or to determine a fact in issue, a witness qualified
as an expert by knowledge, skill, “xperience, training, or education, may testify
thereto in the form of an opinion or otherwise.
Rule 703.
The facts or data In the particular case upon which an expert bases an opinion or
inference may be those perceived by or made known to him at or before the hearing.
If of a type reasonably relied upon by experts in the particular field in forming
opinions or inferences upon the subject, the facts or data need not be admissible
in evidence.
Rule 705.
The expert may testify in terms of opinion or inference and give his reasons
therefore without prior disclosure of the underlying facts or data, unless the
judge requires otherwise. The expert may in any event be required to disclose
the underlying facts or data on cross-examination.
Rule 706.
(a) Appointment. The judge may on his own motion or on the motion of any
party enter an order to show cause why expert witnesses should not be appointed,
and may request the parties to submit nominations. The judge may appoint any
expert witnesses agreed upon by the parties, and may appoint witnesses of bin
own selection. An expert witness shall not be appointed by the judge unless he
consents to act. A witness so appointed shall be informed of his duti.es by the
judge in writing, a copy of which haU be filed with the clerk, or at a conference
In which the parties shall have Opportunity to participate. A witness so appointed
8hail advise the parties of his findings, if any, his deposition may be taken by any
party, and he may be called to testify by the judge or any party. I-Ic shall be subject
to cross-examination by each party, Including a party calling him as a witness.

Case Preparation and Courtroom Procedure
Rule 801.
The following definitions apply under this Article
(a) Statement. A “statement Is (1) an oral or written assertion or
(2) nonverbal conduct of a person, if it is intended by him as an asserticn.
(b) Declarant. A “deelarant” is a person who makes a statement.
(c) Hearsay. “Hearsay” is a statement, other than one made by the declarant
while testifying at the trial or hearing, offered in evidence to prove the truth
of the matter asserted.
Rule 802.
Hearsay is not admissible except as provided by these rules or by other rules
adopted by the Supreme Court or by Act of Congress.
Rule 803.
The following are not excluded by the hearsay rule, even though the declarant is
available as a withess:
(5) Recorded Recollection. A memorandum or record concerning a matter about
which a witness once had knowledge but now has insuffici nt recollection to enable
him to testify fully and accurately, shown to have been made when the matter was
fresh in his memory and to reflect that knowledge correctly. If admitted, the
memorandum or record may be read into evidence but may not itself be received
as an exhibit unless offered by an adverse party.
(6) Records of Regularly Conducted Activity. A memorandum, report, record,
or data compilation, in any form, of acts, events 4 conditions, opinions, or
diagnoses, made at or near the time by, or from information transmitted by.
a person with knowledge, all in the course of a regularly conducted activity, as
shown by the testimony of the custodian or other qualified witness, unless the
sources of informatioi or other circumstances indicate lack of trustworthiness.
(18) Learned Treatises. To the extent called to the attention of an expert withess
upon cross-examination or relied upon by him in direct examination, statements
contained in published treatises, periodicals, or pamphlets on a subject of
history, medicine, or other science or art, established as a reliable authority
by the testimony or admission of the witness or by other expert testimony or by
judicial notice. If admitted, the statements may be read into evidence but may
not be received as exhibits.

Case Preparation and Couitroom Procedure
Rule 901.
(a) General Provision. The requirement of authentication or identification as a
condition precedent to admissibility is satisfied by evidence sufficient to SUpport
a finding that the matter In question is what its proponent claims.
(b) Illustrations. By way of illustration only, and not by way of limitation, the
following are examples of authentication or identification conforming with the
requirements of this rule:
(1) Testimony of Witness with Knowledge . Testimony that a matter is what it is
claimed to be.
(3) Comparison by Trier or Expert Witness . Comparison by the trier of fact or
by expert witnesses with specimens which have been authenticated.
(9) Process or System . Evidence describing a rocess or system used to produce
a result and showIng that the process or system produces accurate result.
Rule 406.
(a) Admissibility. Evidence of the habit of a person or of the routine practice of an
organization, whether corroborated or not and regardless of the presence of eye-
witnesses, 18 relevant to prove that the conduct of the person or organization on a
particular occasion was in conformity with the habit or routine practice.
(b) Method of Proof. Habit or routine practice may be proved by testimony In the
form of an opinion or by specific instances of conduct sufficient In number to warrant
a finding that the habit existed or that the practice was routine.
Rule 612
If a witness uses a writing to refresh hie memory, either before or while testifying,
an adverse party is entitled to have it produced at the hearing, to inspect it. to
cross-examine the witness thereon, and to introduce in evidence those portions whith
relate to the testimony of the witness.
Rule 1006.
The contents of voluminous writings, recordings, or photographs which cannot
conveniently be examined In court may be presented In the form of a chart, summary,
or calculation. The originals, or duplicates, shall be made available for examination
or copying, or both, by other parties at a reasonable time and place. The judge may
order that they be produced in court.

The following key Is intended to provide
an introduction to some of the more
common freshwater animals Technical
language Is kept to a mlnlmwn
in using this key. start with the first
couplet (la. ib), and select the alternative
that seems most reasonable. If you
selected HiaIl you have identified the
animal as a member of the groups Phythm
PROTOZOA If you selected “Ib’, proceed
to the couplet indicated. Continue thiq
process until the selected statement is
terminated with the name of a group
If you wish more information about the
group, consult references (See reference
l1 t )
B !. AQ.21b. 5.71

i’ y to Selec’tcd Gioiips of Freshwater Ammals
l.i ‘l lie hoily of the organis n comprising
a single microscopic independent
cell, or many similar and indepen-
dently functioning cells associated
in a colony with little orno differ-
enre between the cells i e with-
out fern ing tissues, or body com-
prised of masses of rnultinucleate
protoplasm Mostly microscopic,
single celled animals.
it) The body of the organism com-
prised of many cells of different
kinds, i.e., forming tissues.
May be microscopic’ or macro-
2a Body or colony usually forming
irregular masses or layers some-
times cylindrical, goblet 8haped,
vase shaped, or tree like. Size
range from barely visible to
2h Body or colony shows some type 4
of definite symmetry.
‘ Ia Colony surface rough or bristly
in appearance under microscope
or hand lens. Grey, green, or
brown. Sponges.
Phylum PORIFERA (Fig. 1)
‘lb Colony surface relatively smooth.
General texture of mass gelatinous,
transparent. Clumps of minute
individual organisms variously
distributed. Moss animals,
Phylum BRYOZOA (Fig. 2)
4a Microscopic. Action of two
ciliated (fringed) lobes at an-
terior (front) end in life often
gi er appearance of wheels.
Body often segmented, accordian-
like. Free swimming or attached.
Rotifers or wheel animalcules,
(liotifera) (Fig. 3)
4h Larger, worinlike, or having 5
strong skeleton or shell.
5a Skeleton or shell present. Skel- 15
eton may be external or internal.
5b Body soft aridfor wormlike.
Skin may range from soft to
6a Three or more pairs of well 19
formed jointed legs present.
Phylum ARTHROPODA (Fig. 4)
6b Legs or appendages, if present, 7
limited to pairs of bumps or hooks.
Lobes or tenacles. if present,
soft and fleshy, not jointed.
7a Body strongly depressed or 8
flattened in cross section.
3 7b Body oval, round, or shaped like 10
an inverted “U” in cross section.
8a Parasitic inside bodies of higher
animals. Extremely long and flat,
divided into sections like a Roman
girdle. Life histor) may involve
an intermediate host. Tape worms.
Class CESTODA (Fig. 5)
8b Body a single unit. Mouth and 9
digestive system present, but no
9a External or internal parasite of
higher animals. Sucking discs
present for attachment. Life his-
tory may involve two or more in-
termediate hosts or stages. Flukes.
9b Free living. Entire body covered
with locomotive cilia. Eye areas
in head often appear ‘crossed’.
Free living flatworms.
Class TURBELLARIA (Fig. 6)
lOa Long, slender, with snake-like
motion in life. Covered with glis-
tening cuticle. Para8itlc or free-
living. Microscopic to six feet in
length. Round worms.
(Fig. 7)
lOb Divided into sections or segments 11
#1 1 —

Key to Selected Groups of Freshwater Animil’ .,
10,’ tJnc( Inentcd head blunt one 18
i two retractile tentacles.
Flat pointed, tail.
liii Head a more or less well-formed,
hard, capsule with jaws, eyes,
and antennae.
(Figs. RA, 8C)
lib Head structure soft, except 12
jaws (if present). Fig. 8E.)
12a }k ’acl conical or rounded, lateral
appendages not conspicuous or
12h Ilpad somewhat broad and blunt.
Retractile jaw’; usually present.
Soft fleshy lobes or tentacles,
often somewhat flattened, may be
present in the head region. Tad
usually narrow. Lateral lobes
or fleshy appendages on each
segment unless there is a large
sucker disc at rear end.
Phylum ANNELJDA (Fig. 9)
13a Minute dark colored retractile
jaws present, body tapering
somewhat at both ends, pairs or
rings of bumps or “legs” often
present, even near tail.
13b No jaws, sides of body generally
parallel e’ccept at ends. ThLcken-
ed area or ring usually present
if not ill the way back on body.
Clumps of minute bristles on most
segments Earthworms, sludge-
l4a Scgmentb with bristles and/or fleshy
lobes or other extensions. Tuhc ’
builders, borers, or burrowers.
Often reddish or greenish in
color, Brackish or fresh water.
Nereid worms.
14b Sucker disc at each end, the large
one posterior. External blood-
sucking parasites on higher anirnal .
often found unattached to hoqi.
Class HIRUDINEA (Fig 9B)
15a Skeleton internal, of true bone. 40
15b Body covered with an external 16
skeleton or shell.
(Figs. 10, 13, 17, 18, 24.
25, 28)
13 16a External skeleton jointed, shell 19
covers legs and other appendages,
often leathery in nature.
16b External shell entire, not jointed. 17
unless composed of two clam-
like halves.
(Figs. 10, 11, 12)
17a Half inch or less in length. Two
leathery, clam-like shells. Soft
parts inside include delicate
jointed appendages. Phyllopodc
or branchiopods.
Class CRUSTACEA, Subcbssei,
and OSTRACODA (Fig. 11)
17b Soft parts covered with thin
skin, mucous produced, no jointea I c
14 iSa Shell single, may be a spiral cone.
Class GASTROPODA (Fig 13)
18b Shell double, two halves, hinged
at one point. Mussels, clams.
Class BIVALVIA (Fig. 10)
19a Three pairs of regular walking
legs, or their rudiments. Wings
present in all adults and rudiments
in some larvae,
Class INSECTA (Figs. 22, 24D
25, 26, 28, 29)
19b More than three pairs of legs 20
apparently present.
20a Body elongated, head broad anu flat
(Fig. 8)

Ke) to Selected Gr p of Freshwater Animals
with strong jaw8. Appendages follow-
ing first three paiis of legs are round-
dcii tapering filaments. Up to 3
inches long. Dobson fly and fish fly
Class INSECTA Order
20b Four or more pairs of legs. 21
2 1d Four pairs of legs. Body rounded,
bulbous, head minute. Often
brown or red. Water mites.
Phylum ARTHROPODA, Cla8s
(Fig. 15)
21h Five or more pairs of walking
or swimming legs; gills, two
pairs of antennae. Crustaceans.
22a Ten or more pairs of flattened,
leaflike swimming and respiratory
appendages. Many species swim
constantly in life, some swim
upside down. Fairy shrimps,
phyllopods. or branchipods.
(Fig. 16)
22b Less than ten pairs of swimming 23
or respiratory appendages.
23a Body and legs inclosed in bi-
valved (2 halves) shell which may
or may not completely hide them.
2 lb Body and legs not enclosed in
bivalve shell. May be large or
(FIgs. 17, 18, 19)
24a One pair of branched antennae
enlarged for locomotion, extend
outside of shell (carapace).
Single eye usually visible.
“Water fleas”
Subclass CLADOCERA (Fig. 12)
24h Locomotion accomplished by 25
body legs, not by antennae.
25a Appendages leaflike, flattened,
more than ten pairs.
(See 22 a)
25b Ammal less than 3 mm, in length.
Appendages more or less slender
and jointed, often used for walking.
Shells opaque. Ostracods.
(Fig. 11) Subclass OSTRACODA
26a Body a series of six or more 27
similar segments, differing
mainly in size.
26b Front part of body enlarged into
a somewhat separate body unit
(cephalothorax) often covered
with a single piece of shell (cara-
pace). Back part (abdomen) may be
relatively small, even folded
underneath front part. (Fig. 19b)
27a Body compressed laterally i.e.,
organism is tall and thin. Scuds.
Subclass AMPHIPODA (Fig. 17)
27b Body compressed dorsoventrally,
i.e., organism low and broad.
Flat gills contained in chamber
beneath tail. Sowbugs.
Subclass ISOPODA (Fig. 18)
28a Abdomen extending straight out
behind, ending in two small pro-
24 jections. One or two large masses of
eggs are often attached to female.
Locomotion by means of two enlarged,
uribranched antennae, the only large
26 appendages on the body. Copepods.
Subclass COPEPODA (Fig. 19)
28b Abdomen extending out behind ending
in an expanded “flipper ’ or swim-
ming paddle. Crayfish or craw fish.
Eyes on movable stalks. Size range
usually from one to six inches.
29a Two pairs of functional wings,
one pair may be more or less har-
dened as protection for the other
pair. Adult insects which normally
live on or En the water. (rigs. 25, 28)

Key to Selected Groups of Freshwater Animals
29b No functional wings, though
p.ids in which wings are develop-
inj tony h visible. Some may
r ‘o’mhle adult insects very
closely, others may differ ex-
treincly from adults.
30a External pads or cases in which 35
wings develop clearly vieible.(Figs.
2426, 27)
30b More or less wormlike, or at 31
least no external evidence of
wing development.
31a No jointed legs present. Other
structures such as hooks, sucker
dii cs, breathing tubes may be
present. Larvae of flies,
midges. etc.
31b Three pairs of jointed thoracic 32
legs, head capsule well formed.
32a Minute (2-4mm) living on the
water 8urface film. Tail a
strong organ that can be hooked
into a “catch” beneath the
thorax, When released animal
jumps into the air. No wings
arc ever grown. Adult spring-
Order COLLEMBOLA (Fig. 20)
32b Larger (usually over 5 mm) 33
wormlike, living beneath the
33a Live in cases or webs in water.
Cases or webs have a silk
foundation to which tiny sticks,
stones, and/or bits of debris
are attached. Abdominal segments
often with minute gill filaments.
Generally cylindric In shape.
(‘addisfly larvae.
Order TBJCHOPTERA (Fig. 21)
33b Free living, build no cases. 34
34a Somewhat flattened in cross
‘ction and massive in appear-
.lfl( e, Each abdominal segment
with rather stout, tapering, lateral
filaments about as long as body
is wide. Aiderflies, fishflies, and
Order MEGALOPTERA (Fig. 22, 14)
34b Generally rounded In cross section,
Lateral filaments if present tend
to be long and thin. A few forms
extremely flattened, like a suction
cup. Beetle larvae.
Order COLEOPTERA (Fig. 23)
35a Two or three filaments or other 37
structures extending out from
end of abdomen.
35b Abdomen ending abruptly, unless 36
terminal segment itself is extended
as single structure.(Figs. 24A, 24C)
36a Mouth parts adopted for chewing.
Front of face covered by extensible
folded mouthparts often called a
“mask”. Head broad, eyes widely
spaced. Nymphs of dragonflies
or darning needles.
Order ODONATA (Figs.24A, 24C, 24E)
36b Mouthparts for piercing and sucking.
Legs often adapted for water lo-
comotion, Body forms various.
Water hugs, water scorpions, water
boatmen, hackswimmers, electric
light bugs, water striders, water
measurers, etc.
Order HEMIPTERA (Fig. 2
37a Tail extensions (caudal filaments)
two. Stonefly larvae.
Order PLECOPTERA (Fig. 26)
37b Tail extensions three, at times
greatly reduced in sue.
38a Tail extensions long and slender.
Rows of hairs may give extensions
a feather-like appearance.
Mayfly larvae.
(Fig. 27)
38b Tail extensions flat, elongated
plates. Head broad with widely
spaced eyes, abdomen relatively
long and slender. Damseifly
Order ODONATA (Fig. 24D1
Order DIPTERA (Fig. 8)

l’.ei to Selpeted Groups of Freshwater Animals
‘a l.xt ’riia1 wings or wing co ers
form a hard protective dome
over the Inner wings folded
beneath, and over the abdomen
I teetles.
(Fig 28)
39b flxternal wings leathery at base,
Mernbranareoua at tip. Wings
sometimes very short Mouth-
parts for p1 rcing and sucking
l3odv form various. True bugs
Order HEMIPTERA (Fig. 25)
40a Appendage present in pairs
(fins. legs, wings)
40h No paired appendages. Mouth
a round suction disc
41a Body long and slender Several
holes along side of head
lib Rndy plump, oval Tail extendin
out abruptly. Larvae of frogs an
toads. Legs appear one at a time
during metamorphosis to adult
form. Tadpoles.
42a Paired appendages are legs
42b Paired appendages are fins,
gills covered by a flap
(operculum) True fishes.
43a Digits with claws, nails, .or hoofs
43b Skin naked \in claws or digits
Frogs, toads, and salamanders
42 44a Warm blooded
41 44b Cold b1i oded. Body covered
with horny scales or plates
451 Body covered with feathers
Class AyES
45b Body covered with hair

Key to Selected Gro ps of Freshwatcr Animals
1EFERENCES - Invertebrates
I Eddy, Samuel and Hodson, A.C.
Taxonornic Keys to the Common
Animals of the North Central States.
Burgess Pub. Company. Minneapolis.
162 p. 1961.
2 Edmondson, W. T. (ed.) and Ward and
\Vhipple’a Freshwater Biology. John
Uley & Sons, New York. pp. 1-1248.
3 Jahn, T. L. and Jahn, F. F. How to Know
the Protozoa. Wm. C. Brown Compai ,
Dubuque, Iowa. pp. 1-234. 1949.
4 Kiots, Elsie B. The New Field Book of
Freshwater Life. G.P. Putnam’s Sons.
398 pp. 1966.
5 Kudo, R. Protozoology. Otarles C.
Thomas, Publisher, Springfield, Illinois.
pp. 1-778. 1950.
6 Palmer, E. Lawrence. Fieldbook of
Natural History. Whittlesey House,
McGraw-Hill Book Company, Inc.
New York. 1949.
7 Pennak, R.W. Freshwater Invertebrates
of the United States. The Ronald Press
Company, New York. pp. 1-769. 1953.
8 Pimentel, Richard A. Invertebrate
Identification Manual. Reinhold
Publishing Corp. 151 pp. 1967.
9 Pratt, U. ‘.. A Manual of the Common
Invertebrate Animals Exclusive of
Insects. The Blaikston Company,
Philadelphia, Pa. pp. 1-854. 1951.
1 American Fisheries Society. A List of
Common and Scientific Nami-’s of Fishcs
from the United States and Canada.
Special Publication No. 2, Am. Fish
Soc. Executive Secretary AFS.
Washington Bid. Suite 1040, 15th &
New York Avenue, N. V I. Washington,
DC 20005. (Price $4.00 paper,
$7.00 cloth). 1970.
2 Bailey. Reeve M. A Revised List of
the Fishes of Iowa with Keys for
identification, IN Iowa Fish and
Fishing. State of Iowa, Super, of
Printing. 1956. (Excellent color
3 Eddy, Samuel. How to Know the
Freshwater Fishes. ‘Nm. C. Brown
Company. Dubuque, Iowa. 1957.
4 Hubbs, C.L. and Lagler, K.F. Fishes
of the Great Lakes Region. Bull.
Cranbrook Inst. Science, Bloomfield
Hills, MichIgan. 1949.
5 Lagler, K,F. Freshwater Fishery
Biology. Wm. C. Brown Company,
Dubuque, Iowa. 1952.
6 Trautman, M. B. The Fishes of Ohio.
Ohio State University Press, Columbus.
1957. (An outstanding example of a
State study).
Descriptors: Aquatic Life, Systematics.

Key to Selected Group. of Freshwater Animals
3W RoWer, KrstsUa
Up to • 3 mm,
IC. Retlf.r Philodiss
Upto .4 mm.
4C. Joimed leg
5. Tapeworm beads
TunIa . Upto
25ydm. long
7. Nematodes. Free Uvthg
forms commoni) ’ up to
1 nun., occasionafly
i At \
‘ \
1. 5pongLlla spiculs.
Upto .2 mm. long.
3*. Retlfer, arthra
Upto .3 mm.
2B. Bryotosi mass. Up to
several feet diam.
2A. Brycios PlumMsll* . isdividuala ep
to 3 mm. hdertwtasd ma.e . maybe
very satonelve.
4*. Joi ed leg
Caddiaf ly
Jo et.d log
SB. Turb.U ri.. f
Up to 1.6 cm.
aria M..ost a
.1 IV £ m.

K.y to Selected Groups of Freshwater Animals
SB. Diptera, Mosquito
pupa, Upto5mm.
SA. Dipitra. Mosquito larvae
Up to 15 mm. long.
8C. Diptera,
Up to 2 cm.
12B. Branchiopod,
Bosmina. Up
to 2mm.
chironomid BE.
Diptera. crane fly
pupa. Upto2.Sczn.
BA. Annelid.
• 1 gj td
worm. up to
1$ meter
SD. Dipters. Rattailed maggot
Up to 25mm. without tub..
- 1 )
bA. PeI.cyopod. Alasmidonta
Side view, up to 18 cm. long.
SB AnneUd. l..ch up to 20 cm.
108. Alasmidonta , end view.
12A. BranchiopOd,
Daphnia . Up
to 4mm.
hA. Ostracod, Cypericu .
Side view, up to 7 mm.
111. Cypericua , end view.
41 9

Key to Selected Group f Freshwater Anim 1s
13. Gastropod, Caapelom*
Up to 3 Inches.
14. segaloptera, Siali .
Alderfly larvae
Up to 25 am.
16. Fairy Shrimp, Eubranchipuu
Up to $ cm.
clopoid copepod
Up to 25 am.
15. Water mite,
up to 3 mm.
18. leopod, Asellus
Up to 25 am.
20. Collembola, Podura 1OA.
Up to 2 am. Tong
Up to 3 mm.

hey to Selected Groups 01 Freshwater Anirnai,’,
‘ (\
t J
2 1C. I
21. Trichoptera, larval cases,
mostly 1-2 cm.
2 • Odonata, tail
of daaeelfly
(side view)
(248, D)
241). Odonata, damseifly
nymph (top view)
22. Megaloptera ,alderfly
Up to 2 cm.
Odonata, front view
of dragonfly nymph
showing “mask”
partially extended
(24A, E, C)
24C. Odonata, tail of
dragonfly nymph
(top view)
23A. Beetle larvae,
Usually about 2
23B. Beetle larv ,
cm. Usually *boUt
J. cm.
24A. Odonata, dragonfly
nymph up to 3 or
4 cm
41 ’11

Key to Selected Groups of Freshwater Animals
M —
tip to 3cm .
ISA. Dipt.ra. Crsi
fly. Up to *1 cm.
25B. Hemiptera,
Water Scorpion
About 4 cm.
25A. H.mlptirs.
Water Boatman
About 1 cm.
tJpto 5cm.
3M. Co1sopt.r ,,
Water Ioavsse r
bs.tle, Up to 4 cm.
31B. Colsoptera
Dytiseid beetle
Usuallyupto4 cm.
39B. Dlpt.rs, Mosquito
UptoIO mm.

I Plant atube.thread. strand, ribbon, or membrane, frequently visible to the unaided eye 2
1’ Plants of microscopic cells which are isolated or in irregular, spherical, or microscopic
clueter5, cells not grouped into threads . . . 123
2 (I) Plant a tube, strand, ribbon, thread, or mernhrsne composed nf cells . 3
Plant a branching tube with continuous protoplasm, not divided into cclls . 120
3 (2) Plant a tube, strand, ribbon, thread, or a mat of threads . . ... . 4
3’ . Plant a membrane of cells one cell thick (and 2 or more cells wide) 116
4 (3) Cells in isolated or clustered threads or ribbons which are only one cell thick or wide 5
44 Celia in a tube, strand, or thread aLL (or a part) of which is more than one cell thick or
wide . ... 108
5 (41 Heterocysts present . 6
5’ Heterocysts absent , , . 23
6 (5) Thread. gradually narrowed to a point at one end . . 7
6’ Threads same width throughout. . . . . 12
7 (61 Threads as radii, In a gelatinous bead or mass . . . 8
7’ Threads not in a gelatinous bead or mass . ... 11
8 (7) Spore (akinete) present, adjacent to the terminal heterocyst ( Gloeotrichia ) 9
8’ No spore (akinste) present ( Rivularia ) .. . ... - . . .. 10
9 (8) GelatInous colony a smooth bead. . ... . .. . Cioeotrichia echinulata
9’ Gelatinous colony irregular .. . , Oloeotrlchia natans
10 (8’) Cslls near the narrow end as long as wide . Rivularia
10’ Cells near the narrow end twice as long as wide .. .. Rivularia haematites
11 (7’) Cell, adjacent to heterocyst wider than heterocyst . . . Calothrlx braunii
Il ’ Cells adjacent to heterocyst narrower than hetsrocyst . . Calothrix p4rietina
12 (6’) Branching present . , , 13
12’ BranchIng absent . .. . 14
13 (12) Branches in pairs . , Scytonema tolypothricotdee
13’ Branches arising singly . , . . . .. . . Tolypothrix tenuie
14 (12’) Heterocyst terminal only ( Cyclindrospermum) . . 15
14’ Hstrocyste intercalary within the filament) . 16
15 (14) Heterocyst round . . . . . Cy1indrosi rmum musci cola
15’ Heterocyst elongate . , . ... Cylindrospermum stagnale
16 (14’) Thread. encased in a gelatinous bead or mess . . . .. . 17
16’ Threads not encased In a definite gelatinous mass . . is
I i (16) Heterocysts and vegetative cells rounded Nostoc pruniforme
I ?’ Heterocysts and vegetative cells oblong . . . . . Nostoc csrneum
18 (16’) Heterocyste and vegetative cells shorter than the thread width Nodularia gpumIgena
18’ Heterocysts and vegetative cells not shorter than the thread width, . . . . . .19
19 (18’) Heterocyets rounded ( Anabaena ) 20
19’ Heterocysts clindric. . .. . Aphanleomenon flos-aquae
20 (19) Cells elongate, depressed in the rniodle. heterocyste rare. Anabaena conetricta
20’ Cells rounded, heterocyets common . ... . 21
21 (20’) Heterocyste with lateral extensions. . . Anabaena planctonica
21’ Ileterocyste without lateral extensions .22
B1.MIC ,c la ,8b ,8 ,5 9 42—1

22 (21’)
23 (S’)
24 (23)
25 (23)
26 (25’)
27 (26’)
28 (27)
29 (28)
30 (29’)
32 (28’)
33 (32)
34 (32’)
35 (27’)
36 (35)
37 (36’)
38 (37)
39 (35’)
40 (39’)
42 (41)
43 (40’)
44 (43)
Microcoleus subtorulosue
• . Lyngbya ocracea
Lyngbya lagerheimii
Lyngbya digueti
Lyngbya ver.icolor
Phormidium uncinstum
Phormidjum autumnale
Phormidium inundatum
• . . Phormjd [ um retail
• O ,cillatorja ornata
• . 38
O.cillatoria limosa
Oscjljatorja curviceps
Oscillatoria princep.
Oacjll&torja rube,cer
Osciliatorla putrida
• . . . 42
Oscillatoria lauterbornit
Oscillatoria chlorina
• . 48
Oscillatoria pseudogeminata
rhreads 4-8ii wide . Anabaena fioe-
Thread. 8- )4, wld. ’ • . Anabaena circina3is
Branching absent . . 24
Branching (including “false” branching) present . . 84
Cell pigments distributed throughout the protoplasm
Cell pigments limited to plaetid.
Thread, short and formed as an even spiral • ..
Threads very long and not forming an even spiral
Several parallel thread. of cells in one common aheath ____________ ____________
One thread per sheath if present
Sheath or gelatinous matrix present . . • . . . . .28
No sheath nor gelatinous matrix apparent ( Oscillatoria ) . . . 35
Sheath dIstinct, no gelatinou, matrix between threads ( Lyngbya ) . 29
Sheath indistinct or absent, threads Interwoven with gelatinous matrix between ( Phormidium )
. 32
Cells rounded ,
Cells short cyltndric
Threads in part forming spirals
l’hrends straight or bent but nut in spirals
Maximum cell length 3 5g , sheath thin
Maximum cell length 6 5. . sheath thick
Ends of some threads with $ rounded swollen “cap” cell
Ends of all threads without a “cap” cell .
End of thread (with “cap”) abruptly bent
End of thread (with “cap”) .traight .. .
Thread. 3-5, in width .
Thread. 5-12, in width
Cell. very short, generally less than 1/3 the thread diameter
Cells generally 1/2 as long to longer than the thread diameter
Cross walls constricted . .
Cross wall, not constricted . .. .
End . of thread, if mature, curved
Ends of thread straight .
Thread. 10-14, thick , .
lhreads 16-60, thick
Threads appearing red to purplish
Threads yellow-green to b)ue-green
Thread, yellow-green • ,
Threads blue-green . . . • .
Cells 4-7 times a. long as the •t read diameter
Cell, less than 4 times as long a, the thread diameter
rrominent granules (“pseudovacuc le.”) in center of each cell
No prominent granules In center of cells
Cells 1/2-2 times as long a. the thread diameter
Cell. 2-3 time. as long as the thread diameter
Cell walls between ccli, thick and transparent
Cell walls thin appearing a. a dark line

45 (44’) End, of thread straight . . . Oscillatoria agardhii
45’ Ends of mature thread. curved . . . . . . . .46
46 (45’) Prominent granule. present eepeclsliy at both ende of each cell . . Oacillatoria tenuia
46’ Cell, without prominent granule. .. . . 47
47 (46’) Cross wall. conatricted . . . . .. Oscillatorla chalybea
47’ C rose wall, not conetrictad Oscillatoria formoaa
48 (43’) End of thread long tapering . . . Oscillatoria eplendida
48 Fed of thread not tapering . . . . . .. . . . . Oscillatoria amphibia
49 (24’) Cell, separate from one another and enclosed in a tube ( Cyrnbella ) . 251’
49’ Cell, attached to one another as a thread or ribbon 50
50 (49’) Ccli. separating readily Into disco or short cylinder.. their circular face showing radial
marking. . . . . 233
50’ Cells either not separating readily, or if eo. no circular end wall with radial marking. 51
51(50’) Cells in a ribbon, attached elde by side or by their corners . 52
SI’ Cell. In a thread, attached end to end . . . 56
52 (51) Numerou, regularly spaced marking. in the cell wall . 53
52’ Numerous markings in the cell wall absent ( Scenedesmus ) . . . 128
53 (52) Wall marking, of two types, one coarse, one fine .. . . 185
53’ Wall marking. all fine ( Fragilaria ) . . 54
54 (53’) Cell, attached at middle portion only . Fragilaria crotonensls
54’ Cells attached along entire length .. .,,, , .55
55 (54’) Cell length 25-l00 t Fragilaria capucina
55’ Cell length ‘7-ZSsi ... . . .. Fragilarla conetruens
56 (51’) Plastid in the form of a sj ral band ( Spirogyra ) . . . . .. . . 57
56’ Plastid not a spiral band .‘ . 61
57 (56) One plastid per cell . . 58
5?’ Two or more plsstids per cell 60
58 (57) Threads i 8 -Zbp wide Spirogyra communie
58’ Threads Z8-50 wide 59
59 (58’) Threads 28 - 4 0p wide . , .. . Spirogyra variana
59’ Thread. 40-SOp wide . . . . . . Spirogyra porticalis
60 (57’) Thread. 30-45 . wide. 3.4 plastid. per ,etl . . . Spirogyra fluviatilie
60’ Threads 50-80, wide. 5-8 pla.tid. per cell . . Spirogyra majuscula
61(56’) Plaetid. two per cell , . . . . . . . . 62
61’ Pla.tids either one or more than two per cell . 66
62 (61) Cells with knob . or granules on the wall . . 63
62’ Cell, with a smooth outer wall , . . , 64
63(62) Each cell with two central knob, on the wall Deemtdium grevillit
63’ Each cell with a ring of granule. near one end Hyalotheca mucoaa
64(62’l Cells denne green, each pla,tid reaching to the wall ygnema sterile
64’ Cells light green. plastids not completely filling the cell 65
65 (64’) Width of thread U’ 32p. maxlrnun, cell length 6Op Zygnema inaigne
65’ WIdth of thread .30 -36, ,, maximum “all length IZOp . Zygnerna pectinatum
66 (61’) Plastid a wide ribbon. psesing through the cell axle ( Mougeotia ) . 67
66’ Plastid or plastids close to the cell wail (parietal) 69
C’ t’ ’’’
2003W 3hSk7o .e (
C r I k. Orc ’qc 97 D

1 ,7 ((.1,) Thread, with occasional ‘knee-joint” bend. .. . . . Mougeotia genuflexa
1,7’ lhrende straight . .
( H (67) Thread. l’)-Z 4 wide. pyrenoids 4-16 per cell. . . Mougeotia sphaerocarpa
68’ Thread. 2O -34 , wide. pyrenoid. 4-10 per cell . Mougeotia acalaris
69 (66’) Occasional cell. with one to .everal transverse wall lines near one end ( Oedogonium ) 70
69’ Occasional terminal transverse wall line. not present. .. . .. 73
70 (69) Thread diameter less than 24g 71
70’ Thread diameter 25j,i or more . 72
71(70) I’hread diameter 9 -l4 (* . . . Oedogonium .uecicum
7) Thread diameter l4-23g . Oedogonium bo.cii
72 (70) Dwarf mile plant. attached to normal thread, when reproducing Oedogonium idioandro .porum
72’ No dwarf male plant, produced . .. Oedogonium gre.nde
73 (69’) Cells with one plastid which has a smooth surface , 74
73’ Cells with several plastids or with one nodular piastid . . . . . . 78
74 (73) C cli . with rounded ends . . Stichococcus bacillari .
74’ Cells with flat end. (Ulothrtx) 75
75 (74’) Thread. lOg or lee, in diameter . . . 76
75’ ‘1 breads more than lOp in diameter . . . 77
76 (75) Thread. 5- 6 p in diameter . . . . . Ulothrix variabjije
76’ Thread. h.lOp in diameter . tJiothri.x tenerrima
77 (75’) rhread. ll-l?p in diameter . Ulothrtx aeguali .
77’ Threads 20-60 in diameter , , ‘ . ‘ . Ulothrix Zonata
78 (73’) Iodine test for starch positive; one nodular plastid per cell . .79
78’ Iodine test for starch negative, .everal plastids per cell . . . . 80
79 (78) Thread when broken, forming “H” ehape segments Microapora amoena
79’ Thread when fragrr(ente4 separating irregularly or between cells ( Rhizocionium) . 100
HO (78’) Side walls of cell. straight. net bulging A pa t?rn of fine lines or dote present in the wall
but often indistinct ( Melosire ) 81
HO’ Side wall, of cells slightly bulging Pattern of wall markings not present ( Tribonema) , 83
HI (80) Spine-like tceth at margin of end walls .. ‘ . . 82
81’ No spine-like teeth present , . . Melosira variant
82 (81) Wall with (the granule., arranged obliquely .. . . . Melosirs crenulata
H. ’ Wall with coarse granules, arranged parallel to side. . Meloeira ranulata
83 (80’) Plastid. 2-4 per cell . . . . . Tribonema minue
Hi’ Plastide more than 4 per cell . , . . ‘ .. Tribonema oombyclnum
84 (23’) Plastids present, branching “true” . 85
144’ Plastide absent, branching “false” Plectonema tomaqiniana
145 (84) Branches reconnected, forrnIn , net . . Hydrodictyon reticulaturn
85’ Branches not forming a distinct net , . . . . . . 86
86 (85’) Fach cell in a conical sheath open at the broad end tDinobryon ) .. . 87
M I” N , conical sheath sroun ,I each cell . 90
8 ? (86) l3ranchee diverging, often almost at a right angle . Dinobryon divergena
87’ ranche. compac.. often almost parallel . ‘ 88
88 (87’) Narrow end ol sheath sharp pointed . , , , 8q
8$’ Narrow end of i heath blunt pointed . , , Dinobryon .ertuiaria

89 (88) Narrow end drawn out into a stalk . .. . . . Dinobryon atipitaturn
$9’ Narrow end diverging at the base . .. . . . . . - Dinobryon sociale
90 (86’) Short branchee on the main thread in whorls of 4 or more (Nitella) . . . 91
90’ Branching commonly single or in paire . . .92
91 (90) Short branches on the main thread rebranehed once . Ntte llaflexi lis
91’ Short branches on the main thread rebranched two to four times Nitella gracilis
92 190’) Terminal cell each with a colorless spine having an abruptly swollen base ( Bulbochafle l 93
92’ No terminal spines with abruptly swollen bases . . 94
93 Vegetative cells Z0-48p long . .. Bulbochaete rnirabilis
93’ Vegetative cc Its 4$-€Sp tong .... . . . . Bulbochaete insign)j
94 (92’) Cell, red, brown, or violet . . Audouinella violacea
94’ Cells green . . . . 95
95 (94) Threads enclosed in a gelatinous head or mass . . . . 96
95’ Threads not surrounded by a gelatinous mass . . . . 99
96 (95) Abrupt change in width from main thread to branches ( Draparnaldia ) 97
91 , ’ Gradual change in width from main thread to branches ( Chaetophora ) 9 1 1
97 (96) 3 ranches (from the main thread) with a central, main axis . . Draparnaldia plumosa
97’ Rrsnchrs diverging and with no central main axis Draparnaldia glomerats
yR (96’) End cells long-pointed, with colorless tips . Chaetophora attenuata
98’ End cells abruptly pointed, mostly without long colorless tips Chaatopltora elegans
99 (95’) Light and dense dark cells intermingled In the thread . . .. Pithophora oedogogonia
99’ Most of the calls essentially alike in density . . . . . . . . 100
100 (99’) Branches few in number, and short, colorless Rhiaoclonium hierogiyphicum
100’ Branches numerous and green . .. ... 101
1011100’) ‘Terminal attenuation gradual, involving two or more cells ( Stigeoclonium ) .. 102
101’ Terminal attenuation absent or abrupt. involving only one cell ( Cladoohora ) 104
102 (10)) Branches frequently In pairs 103
102 ’ Branches mostly single Stigeoclonium stagnatile
103 (102) Cells in main thread 1-2 times as long as wide Stigeoclonium lubricum
103 ’ Cells in main thread 2-3 times as long as wide Stigeocloniom tenue
104 (101’) Branching often appearing forked, or in threes .....,...... Cladophora aegagropila
104’ Branches distinctly lateral 105
lO S 1104’) Branches forming acute angle with main thread, thus forming clusters.Cladophora glomerats
105’ Branches Forming wide angles with the main thread . . . ,. 106
106 (105’) Threads crooked and bent Ciadophorafracta
l O b ’ lhreads straIght . . . . . . . 107
107 (106’) Branches few, seldom rebranching . Cladophorn insignis
107’ Branches numerous, often rebranching Cladophora crispata
108 (4’) Plant or tubc with a tight surface layer of cells and with regularly spaced awellings (nodes)
. . Lemanen annulata
108’ Plant not a tube that has both a tight layer of surface cells and nodes - . 109
109 (lO S’ ) Cells spherical end loosely arranged In a gelatinous matrix Tetraspora geiatinosa
109’ Cells not as loosely arranged spheres. . , 110
110 (109’) Plants branch .. . . I D
1 10’ Plants not branched . . .. . . . . Schizomeris leibleinil
I II (110) Clustered branching . . . . . . . . . . - 112
Ill’ Branches single , , . 115

112 (III) Threads embedded in gelatinous matrix ( Batrachosperm ) . 113
HZ’ No gelantlnou. matrix (Char.) . . 114
113 (lIZ) Nodal masses of branches touching one another Batrachospermum vagum
113’ Nodal masses of branches separated by a narrow space . Batrachospermum moniliforme
114 (112’) Short branches with 2 naked cell, at the tip Citarn globularje
114’ Short branches with 3-4 naked cells at the tip . Chara vulgaris
115 (Ill’) Heterocy.te present. plastids absent . Stigonema minutum
115’ Heterocyets absent. pla.tid . present Compsopogon coeruleus
116 (3’) Red eye spot and two flagella present for each cell 125
116 No eye spots nor flagella present . . . 117
II? (116’) Round to oval cells, held together by a flat gelatinous matrix ( A menellurn ) .. . 131
117’ Cell, not round and not enclosed in a gelatinou, matrix . . 118
118 (117’) Cell, regularly arranged to an unattached disc. Number of cells 2, 4. 8, 16, 32. 64. or
128 . . . . . . 133’
118’ Cells numerous, membrane attached on one surface 119
119 (118’) Long hairs extending from upper surface of cells . . . Chaetopeltis megalocystis
119’ No hairs extending from cell surfaces Hildenbrandia rivularle
120 (2’) Constriction at the base of every branch Dichotomosiphon tuberosus
120’ No constrictions present in the tube ( Vaucheria) . . , . .. . . . . 121
121 (120’) Egg sac attached directly, without a stalk, to the main vegetative tube Yaucheria sessilis
121’ Egg sac attached to an abrupt, short, side branch 122
122 (121’) One egg sac per branch . Vaucheria terrestris
122’ Two or more egg sacs per branch Vaucheria geminata
123 (1’) Cells in colonies generally of a definite form or arrangement . 124
123’ Cells isolated, in pairs or in loose, irregular aggregate. . 173
124 (123) Cells with many transverse rows of markings on the wall .. . . . 185
124’ Cell. without transverse rows of marking. 125
125 (124’) Cells arranged as a layer one cell thick 126
125’ Cell cluster more than one cell thick and not a flat plate . . . .. . . 137
126 (1251 Red eye spot and two flagella present for each cell Gonium pectorale
126’ No red eye spots nor flagella present . 127
127 (1Z( , ’) Cells elongate, united side by side in 1 or 2 rows ( Scenedesmus ) 128
127’ Cells about as long a. wide .. . . . . . . . . 131
128 (127) Middle cells without spines but with pointed ends Scenedesmus dimorphus
128’ Middle cells with rounded ends . .. . .129
129 (128”) Terminal cells with spines .,, , . , 130
129’ Terminal cells without spines . Scenedesmu. bijuRa
130 (129) Terminal cells with two spines each . , . . .. Scenedosmus guadrtcauda
130’ Terminal cells ith three re ” more spines each Scenedesmue abundans
131 (117) Cells In regular rows, immersed in colorless matrix ( Agmenellum Quadrjduplicatum ) 132
131’ Cells not immersed In colorless matrix . . . . , . . . 133
II? (Ill) Cell diameter I 3 to L Agmenellum guadriduplicatum , tenuissima type
13t’ Cell diampter 3 -Sp . Agmenellum guadriduplicatum. glauca type
133 1131’) Coils without spines, projections, or Incision.. .. . . .. . Crucigenia guadrata
133’ Cell, with spine., projections, or incision. . . . . 134

134 (133’) Call. round.d . . Micractintum pusillum
134’ Cell, angular (PedIsetru, , ,) . , 135
135 (134) Nu,narou. spaces between elIs . . . Pediastrum duplex
1 1 5 ’ Cells fitted tightly together . 136
l3( (135’) Cell incisions deep and narrow. , . . Pediastrum tetres
136’ Cell Incisions shallow and wide Pediastrum boryanum
137 (125’) Celle sharp-pointed at both ends, often arcuate 138
137’ Cells not sharp-pointed at both ends; not arcuate. . . 141
138 (137) Cells embedded in a gelatinou. matrix . Ktrchneriella lunaria
138’ Cells not embedded in a gelatinous matrix ... 139
139 (138’) Cells all arcuate, arranged back to back . Selenastrum g aci1e
139 Cells straight or bent in various ways, loosely arranged or twisted together
( Ankistrode.mus ) 140
140 (139’) Cells bent . . . . . . Ankistrodesmus falcatue
140’ Cells straight Ankistrodeemus falcatus var acicularts
141 (137’) Flagclla present, eye spots often present 142
141’ No flagella nor eye spots present . . 152
142 (141) Each ‘cl i in a conical eheath open at the wide end ( Dinobryon ) . 86
142’ IndIvidual cell, not In conical sheaths . , , 143
141 (142’) Fach cell with l-Z long .iralght rods extending , . . Chryeoephaerella longispina
143’ No long straight rods extending from the call. 144
144 (143’) Cc 11cc touching one another Ins dense colony 145
144’ Cell, embedded separately In a colorle, naatr 149
145 (144) Cell, arranged radially, facing outward .. 146
145’ Cells all facing in one direction 147
146 (145) Plastid. brown, eye spot absent . . . Synura uvelia
l4f ’ Plastid. green, eye spot present in each cell . . . . . Pandorina morum
147 (145’) Eaich cell with 4 flagella . Spondylomorum guaternarium
147’ 1a, h cell with 2 flagella ( Pyrobotrys ) . . . 148
148 1147’) Eye spo t In the wider (anterior) end of the cell Pyrobotry8 etellata
148’ Eye spot In the narrower (posterior) end of the cell . Pyrobotrys precut ,
149 (144’) Plastid. brown . Uroglenopsus americana
149’ Pla.tlds green 150
150 (149’) Cells 16. 32, or 64 per colony . Eudorina elegans
I SO’ Cells more than tOO per colony . . . 151
151 (1501 Colony spherical each cell with an eye spot. . Volvox aurcus
151’ Colony tubular or irregular no eye spots ( Tetraspora ) ioq
152 (141’) Flongate cells, attached together at one end, arranged radially ( Actinastrum ) 153
152’ Cells not elongate, often spherical.. . . 154
153 (152) Cells cylindric . . . . . . . Actinastrum gracullumum
153’ Cells listInctly bulging . Acttnastrum hantzschui
154 (1521 PlasI ide present . . ‘ 355
154’ Plaetids absent, pigment throughout each protoplast 168
155 (154) Colonies, including the outer matrix, orange to red-brown Botryococcus braunui
It S’ Matrix If any, not bright colored, cell pla.tids green 156

156 (155) Colonies round to oval . . .. . . . . . . . . . 160
56’ ColonIes not round, oftrn Irregular In form . . . 157
l’;7 (156’) StraIght (flat) wall, between adjacent celia ( Phytoconis ) 278
IS ?’ Wall. b t wean neighboring cell. rounded . . . 158
58 (157’) Cells arranged a. a surface layer in a large gelatinou, tube ( Tetra.pora) . .. 109
158 Colony not a tube, cell, in irregular pattern .. . 159
159 (158) Large cell, more than twice the diameter of the small cell. ( Chlorococcum ) . . . 280’
159’ Large cell, not more than twice the diameter of the email cell. ( PalmelIa ) 281
160 (156) Cell, touching one another, tightly grouped. . ... . . Coeia.truin microporum
160’ Cells loosely grouped . . . . 161
161 (160’) Colorless threads extend from center of colony to cells. . . 162
161’ No colorless threads attached to cell, in colony . . . 164
162 (161) Cell, rounded or straight, oval ( D1ctyosphaer um ) . . . ‘ . 163
162’ Cell, elongate, some cello curved Dimorphococcus lunatus
163 (1621 Cell. rounded. . Dictyo.phaerium pulchellum
l6 ’ Cells straight, oval Dictyosphaerium ehrenbergianum
164 (161’) Cell, rounded 165
164’ Cells oval . Oocysti, borgei
169 (164) One plastid per cell . . 166
165’ Two to four plastids per cell . . . Cloeococcu, achroeteri
161 (165) Outer matrix divided into layers ( Cloeocyetla ) . . . 167
16(.’ Outer matrix homogeneous Sphaerocy tjs achroeteri
167 (166) Colonies angular , . . . Gloeocy,tis planctonica
167’ Colonies rounded Gloeocy.tj. giga .
168 (154’) Cell, equidistant from center of colony ( Gompho.phaeria ) 169
168’ Cells irregularly distributed in the colony . .. . . 172
169 (168) Cell, with pseudovacuoles . .. . ... , Gompho.paeria wichurar
169’ Cells without pseudovacuoles . . . 170
170 (169’) Cells -4 in diameter ( Comphosphaeria lacustris ) . . . 171
170’ Cells ovate . . . . . . Compboephaeria aponina
171 (170) Cells .phorical . . . .. Oomphosphaeria lacuetri,, kuetaingianum type
Ill’ Cells 4-15 in diameter , . . . Gomphosphaerta lacustri,, collin.li type
172 (168’) Cells ovid, divl .Ion plane perpendicular to long axis ( Coccochlorip ) . . . . . 286
172’ Cells rounded, or division plane perpendIcular to short axis ( Aztacystis ) 286’
173 (123’) Calls with an abrupt median transver.e groove or incision . , 174
173’ Cells wIthout an abrupt tranever.e median groove or incision . 184
174 (173) Cell, brown, flagella present (armored flagellates) , . 175
174 C dl. green, no flagella (deimids) . . .. 178
175 (174) Cell with 3 or more long horns Ceratium hirundinella
175’ Cell without more than 2 horn, 176
176 (175’) Cell wall of very thin smooth plates Clenodinium palustre
176’ Cell wall of very thick rough plates ( Peridinium ) 177
177 (176’) Ends of cell pointed . . . Peridinjum wiscon,inense
177’ End, of cell rounded . . . . Peridinfum cinctum
178 (174’) Margin of cell with sharp pointed . deeply cut lobes or long .pikeo . .. , 179
178’ Lob.., if prelent, with rounded end, . . .. . 182

119 (178) Median incision narrow, linear . , MJcras erjas truncita
179’ Median Incision wide. “V’ or ‘U’ shaped ( Staurastrum ) 180
180 (179) Margin of cell with long spikes.. Staurastrurn paradoxum
180’ Ms rgtn of cell without long spikes 181
181 (180”) Ends of lobes with short spines Staurastrum polymorphum
181’ Ends of lobes without spine. Staurastrum punctulttum
182 (178’) Length of cell about double the width Euastrum oblongum
182’ Length of cell one to one ..nd one.half times the width ( Cosrnarium ) 183
183 (182’) Median incision narrow linear Cosmariurn botrytis
183’ Median tnciston wide, “U” shaped Co.marium pprtianurn
184 (173’) Cells triangular Tetraedron muticum
184’ Cell, not triangular 185
185 (124) Cells with one end distinctly different from the other 186
Cell, with beth ends essentially alike 225
186 (185) Numerou. transverse (not spiral) regularly spaced wall markings present (diatoms) . 187
186’ No transverse regularly spaced wall marking. 193
187 (186) Cell, curved (bent) In girdle view Rholcosphenia curvata
187’ Cells not curved In girdle view 188
188 (187’) Cell, with both fine and coarse transverse linea Merjdton circulare
188’ Cells with transverse line, all alike in thickness 189
189 (188’) Cells essentially linear to rectangular, one terminal swelling larger than the other
( Asterloneila ) 190
189’ Cells wedgs-.haped; margins sometimes wavy ( Goinphonema) . . .. 191
190 (189) Larger terminal swelling 1-1/2 to 2 tIme. wider than the other Asterionella formosa
190’ Larger terminal swelling less than 1 -1/2 time, wider than the other. . Aa erionella g c llima
191 (189’) Narrow end enlarged in valve view Gomphonema geminatum
19!’ Narrow end not enlarged in valve view 192
192 (191’) Tip of broad end about as wide as tip of narrow end in valve view . Gomphonema parvulum
192’ Tip of hroad end much wider than tip of narrow end in valve view Comphonema olivaceurn
193 (186’) Spine present at each end of cell Schrooderia setigera
193’ No spine on both ends of cell 194
194 (193’) Pigments in one or more plastids 195
194’ No plastid. pigments throughout the protoplast Entophysalis lemaniae
195 (194) Cells in a conical sheath ( Dinobryon ) 86
195’ Cells not In a conical sheath 196
196 (195’) Cell covered with scales sod long spines M*ilo,nena. caudata
196’ CelIa not covered with scale, and long spines 197
197 (196’) Protoplasts separated by a space from a rigid sheath (lorica) . 198
197’ No loose sheath around the cells 202
198 (197) Cells Compressed (flattened) .. Phacotus lenticularis
198’ Cells not compressed 199
199 (198’) Lorica opaque, yellow to reddish or brown Trachelomonag crebea
199’ Lorica transparent, colorless to brownish ( Chrysococcus) . . . 200
200 (199’) Outer membrane (lorica) oval Chrysococcu, ovalis
200’ Outer membrane (lorica) rounded 201

101 (200’) Lorica thickened around opening . . . . . . Chrysococcue rufeecena
20)’ L.orica not thickened around opening .... . Chrysococcu. major
202 1)97’) Front end flattened diagonally . . . 203
202’ Front i ’nd not flattened diagonally . 206
203 (202) Plnetid. bright blue-green ( Chroomonse ) 204
203’ Plastids brown, red, olive-green. or yellowish 205
204 (203) Cell pointed at one end Chroomona. nordetetii
204 Cell not pointed It one end Chroomonas setoniensia
205 (203) Gullet present, furrow absent Cr’yptomona. eroas
205’ Furrow present, gullet absent Rhodomonas lacustris
206 (202’) Plastids yellow-brown Chrotnulina ro.snoffj
206’ Plastid. not yellow-brown, generally green 207
207 (206’) One plastid per cell 208
207’ Two to aeveral plastid. per cell . . 211
208 (207) Cells tapering at each end . Chiorogoniwn euchiorum
208’ Cells rounded to oval 209
209 (208’) Two flagella per cell ( Chismydomonas ) . .210
209 Four flagella per cell Caterta multifilia
210 (209) Pyrenoid angular: eye spot in front third of cell Chlamydoinonas reinhardi
210’ Pyrenoid circular, eye spot in middle third of cell Chlamydomonas globosa
211 (207’) Two pla.tids per cell .
211’ Several plastids per cell 212
212 (211’) Cell compressed (flattened) ( caal 213
212’ Cell not compressed 214
213 (212) Posterior spine short. bcnt Phacu. pleuronectea
213’ Posterior spine long, straight Phacue longicauda
214 (212) Cell margin rigid . 215
214’ Cell margin flexible ( Eunlena ) . 217
215(214) Cell margin with spiral ridges Phacus pyrum
2 )5’ Cell margin wIthout ridge., but may have spiral lines ( Lepocinclis) . . . . 216
216 (215’) Posterior end with an abrupt, spine-like tip . . Lepocinclieovtun
216’ Posterior end rounded Lepocindli . texta
217 (2)4’) Green plastids hidden by a red pigment in the cell Euglens eangulnea
217’ No red pigment except for the eye spot . . . . . 218
218 (2)7’) Pla.tid. at least 1/4 the length of the cell . . .219
218’ Plastide discoid or at least shorter than 1/4 the length of the cell.. . . 220
219 (2)8) Plastids two per cell . . Euglena
219’ PlistSdn several per cell, often extending radiately from the center Euglena viridis
220 (2)8’) Posterior end extendLng a. an abrupt colorless spine . . . 221
220’ Posterior end rounded or at least with no colorles, spine. .. . 222
221 (220) Spiral morkinga very prominent and granular Euglens apirogyra
22)’ Spiral markings (airly prominent, not granular Euglena oxyuria
. 4Z (220’) Small, length 35-Sip . . . Euglena gracilis
/2 1’ Medium to large, length 69,. or more . . 223
123 (222’) Medium in sue, length 69-2001. . . . . 224
/ 2 1’ Large in alze, length Z50-Z90i . . . Euglena ehrenbergii

//4 (223) lastidi, with irregular edge, flagellum 2 times as long as cell. . Euglena polymorpha
224 Plastids with smooth edge, flagellum about 1/2 the length of the cell . . Euglena deses
2Z’ (lH’’ ( Cr 1 1, distinctly bent (arcuate), with a spine or narrowing to a point at both ends 226
zzc’ Cells not arcuate ‘ 230
?26 ( 22 g .) Vacuole with particles showing Brownian movement at each end of cell Cells not in
clu.ters ( Closterium ) . . . .. . . . 227
226’ No terminal vacuole. Cells may be in cluster, or colonies . 228
227 (226) Cell wide, width 30-7O . . . Closterium moniliferum
227’ Cell long and narrow width up to 5 p Clo.terium aciculare
228 (226’) Cell with a narrow abrupt spine at each blunt end . Ophiocytium capitatum
228 No blunt ended cell, with abrupt terminal spines . . . . 229
229 (228’) Sharp pointed ends as separate colorless spines . . 193
229’ Sharp pointed ends as part of the green protoptast 137
230 (225) One long spine at each end of cell . . . . . . . . 231
230’ No long terminal spines . . . . . . . . 232
231 (230) Cell gradually narrowed to the spine . , 13
231’ Cell abruptly narrowed to the spine . . . . . Rhizosolenia gracili .
232 A regular pattern of fine lines or dots in the wall (diatoms) . . 233
232’ No regular pattern of fine lines or dot. in the wall... . . . 276
233 (50. Cell, circular in one (valve) view, short rectangular or square in other (girdle) view 234
233’ Cells not circular in one view 240
234 (233) Valve surface with an Inner and outer (marginal) pattern of striae ( Cyclotella) . . . 235
234’ Valve surface with one continuou, pattern of .trtae ( Stephanodiecue ) . . . 238
235 (234) Cell, small, 4-lOg in diameter. . . . . . . . Cycletella glornerata
235’ Cells medium to large. 10-80 in diameter . , . . . . . . 236
236 (235’) Outer hall of valve with two types of lines, one long, one short . , 237
236’ Outer half of valve with radial lines all alike . Cyclotella meneghtniana
237 (236) Outer valve tone constituting more than lIZ the diameter . Cyclotella bodanica
237’ Outer valve Lone constituting more than 1/2 the diameter. Cyclotella compta
238 (234’) C cl i 4- 2 5p in diameter . 239
238’ Cell 2 S- 65 g in diameter. Stephanodiscu, niagaree
239 (238) Cell with two transverse band,, In girdle view Stephanodiscus binderanus
239’ Cell without two transverse bands, in girdle view . Stephanodiscue hantc,chii
240 (233’) Cell, flat, oval ( Cocconeis ) . . ‘ 241
240’ Cell. neither flat nor oval , , 242
241 (240) Wall markings (.triae) (8.20 in lO , . .. . Cocconeis pediculue
241’ Wail markings (striCcl 23-25 In I0i . . Coccorteis placentula
242 (240’) Cell sigmoid in one view. . ‘ 243
242’ Cell not sigmoid in either round or point ended (valve) or square ended (girdle) surface
view 244
243 (242) Cell .igrnoid in valve surface view . . Gyrosigma attenuatum
43’ Cell slgmoid in square ended (girdle) surface view Nitzechia acicularis
244 (242’) Ccli longitudinally uneymniet rical in at leaet one view 245
244’ Cell longitudinally Ayninietrii .il 254
24c (244) Cell well will, both fine and coarse transverse line, (striae and costae) 246
245’ Cell well with fine transverse lines (striac) only 247

/4/, (245) Valve Ia e about as widc at , , , ,d ,Ile as git die face . . . Epithernia turgida
‘4/i ’ Valve face if?. or c ,. as whir at middle as girdle face Rhopalodia
247 245) inc of pores dod raphe located at edge of valve lace . . . . . . 248
/4 ! ISaphe out at ,‘xtren ,e edge of valvi face 250
4411 (247) l(aphe of r .u h valve atjncent to the tame girdle outface . Henteichla amphioxys
24R’ Raph. ( each valve adjacent to different girdle surfaces ( Nitasehia ) . 249
24’) (248) Cell 20-65p long N&tzschia
249’ Cell 70- 18G 0 long Niti.chia linearia
150 (247) Cell longitudinally unsymmetrical in valve view . . 251
250 Cell longitudinally unsymmetrIcal in gIrdle view . . Achnanthes microcephala
251 (250) l5aphc bent toward one side at the middle . .. . Amphora ovalie
251’ Raphe a smooth curve throughout ( Cymbella ) . . . . . . . 252
252 (251’) Cell only slightly unsymmetricaL Cymbella ce,ati
Cell distinctly unsymmetrical . 253
253 (252) Striation. distinctly cross lined, width l0-30 s . . Cymbella prostrata
253’ StriatIon. indistinctly cross line . t , width S-lZ a . . Cymbella ventricosa
154 (244’) Longitudinal line (rapho) and prominent marginal markings near both edges of valve 255
?54 ’ No marginal longitudinal line (raphel nor keel, raphe or pseudoraphe median 257
Z ’ c (254) Margin of girdle face wav . . Cymatopleura solea
255’ Margin of girille face straight ( Surirella ) . . . . . 256
256 (4S5 ( Cell width S.23g . . . . . . . . . . . . Surirella Ovata
256’ Cell width 40-601 . . Surirella splendida
457 (254) Gridle face genrTally In view and with two or more prominent longitudinal linee In valve
view, swollen central oval portion bounded by a line ( Tabeliaria ) . 258
257’ Girdle lace with less than two prominent longitudinal lines. In valve view, whole central
portion not bounded by a line , . . . .. 259
258 (2”7) Girdle face lee, than /4 as wide aa long . . . . Tabellaria fenestrata
258’ GIrdle face m.,re than 1/2 as wide as long . . Tabellaria flocculosa
259 (257’) Valve (are with both coarse and fine transverse lines Diatoma vulgare
259’ Valve face with transverse lines, if vIsIble alike in thickness . 260
260 1259’) Valvi face naviculoid true raphe present . . 261
2h0’ Valve face linear to linear-lanceolate. true raphe absent . 270
261 (260) Valve lace with wide transverse line. (co,tae( ( Pinnularia ) 262
Valve face with thin transverse lines (striar) , 263
i ( ,4 (21,1) C.ell 5 - 6 ii broad . . . . . . . Pinnularia subcapitata
Cell 34-50 i broad . . . Pinnularia nobilis
4 / 121,1’) Transverse lines (strlar) ab ,cnt a ross transverse axis of valve face
Staurpneis phoenicenteron
I ransverse lines (ilriae) present acros, transverse axis of valve face. 264
264 (2611 fl&phe strictly median ( Navicula ) 265
4 / , 5’ Raph located nlightly I.. one side 252
.‘65 (/64) Fnda of valve face abruptly narrowed to a beak . . Navicula exigun var
‘(.5 Ends of valve face gratially oar rowed . 266
,/ 6 (/1.5’) Moat of striation, air,, tly tranavi rec . . . Navicula gracili ,
266’ Most of striation. ratliai (oblique) . . 267

267 (266) Striac distinctly cum 1 ,o,cd of dot (pu ctae) Navicula lonceolata
267 Striac essentially a. continuous lines . . 268
268 (267) Central clear area on valve face rectangular Navlcula graciloicles
268 Cent rd clear area on valve face oval 269
269 (268’) C.ll length 29 -40p. ends slightly capitate. Navicula cryptocephala
Ciii length 3O l20li, ends not capitate . Naviculo radlosa
270 (260’) Knob at one end larger than at tb othcr ( Astertonella ) 1 ) 19
270 Terminal knobs if present equal in st e ( Synedra ) 271
271 (270’) Clear space (pseudonodule) in central area Synedra pulchella
271’ No p.eudonoduie in central area. 272
272 (271) Sides parallel In valve view, each end with an enlarged nodule Synedra capitata
27.: SIdes converging to the ends in valvi view 271
273 (272) V4IVC linear ti lanceolate-linear, R-ll striac per IO )J. Synedra ulna
273 Valve narrowly linear-lanceolate, (2-lB otrtae per lOp .274
?74 Valve 5-61J wide . Synedra acue
274’ Valve 2.4k Wi(le . . . . . . 275
275 (274) Cells up ti , (iS time, as long as wide. entral area absent to small oval
Synedra acus var radian .
275 ’ Cells 90-14(1 times as long as wide, central area rectangular
Synedra acuavar augustissima
276 (232’) Green to brown pigment in one or more plastid 277
276’ No plastid., blue end green pigments throughout protopleot 284
277 (276) Cello long and narrow or flat 233
277’ Celle rounded 278
278 (277) Straight, flat wall between adjacent cells in colonies . - Phytoconis botryoides
278 Rounded wall between adjacent cells in colonies 279
279 (278’) Cell eIther with 2 opposite wall knob, or colony of 2-4 cells surrounded by distinct mem-
brane or bo’h . 164
279’ Cdl without 2 wall knobs, colony not of 2-4 cello surrounded by rltotince membrane 280
280 (279’) Cells essentIally similar in sice within the colony 28)
280’ Cello of very dIfferent sires within the colony Chlorococcum ‘ umicola
2)11 (159’) Cello , ‘,nhe ,Ided in art extensive gelatinous matrix Palm.’lla mucooa
2141’ Cells tth lIttle or no gelatinous matrix around them Chlorella ) 2 ) 12
282 (281’) C. 110 rounded 281
282’ Cells rIlIpsoid 1 to ,,vold Chiorella ellipsoidea
283 (282) Cell 5 -lOp in diameter. pyrenoid indistinct Chlorella vulgaris
483’ Ci II 3 -5 in diameter, pyrcnoid distini t Chlorella pyrenoidosa
2)14 (276’) Cell a spiral rod . . 285
284’ ( elI not a opiral rod . 286
?85 (25) Thread aeptate (with crosewallo) Arthrospira Jennerl
2115’ Thread non-aeptate (without eroaswallo) Spirulina nordotedtit
286 (172) Cello dividing in a plane at right . .ngles to the long axis , Cocc’ochiorjs stagnina
286’ (172’) Cello op. ri al or dividing in a plane parallel to the long axis ( Anacystis ) 287
2117 (2111) Cell containing pseudovacuoles Anacystis cyanca
287’ Cell not containing pseudovacuoles 288

88 (287’) Cell 2-6k in diameter, heath often colored montana
288 Cr11 6 -SOp in diameter. sheath colorless . .. . . . 289
289 (288’) C cl i 6 -IZ a in diameter. cell. in colonieo Cr. mostly spherical . . . Anacystis thermalis
289’ Cell lZ-5O In diameter, cells in colonies ar, often svgular.. . , . Anacy.ti. dimidiata

The following work is more easily defined in terms of what it is not,
than what it is: it is not a ukeyhl in the usual biological sense of the
word, nor is it a glossary of a dictionary. It is rather a device for
determining what general kind of organism or group is designated by
some unrecognized name, be it common or scientific.
if one has access to one or more of the references cited, he can find
the same name, and learn much more about It; but not everyone has
all of these books, and the Information is often couched in highly
technical terms.
The nonbiologist would be well advised to read Part I before attemp-
ting to use the Finder. The experienced biologist on the other hand
may proceed directly to the index and quickly be referred to the larger
group to which his unknown organism belongs.
No professional systematist will find himself completely at home. In
an effort to present a relatively simple concept of relationships
couched in standard terms for all groups or organisms, some violence
was done to certain highly sophisticated systems of classification. It
is hoped, however, that the layman will find accuracy sufficient for his
needs, and the specialist will be referred to technical literature where
he can satisfy his needs for greater detail.
While every effort has been made to ensure accuracy, it is inevitable
that errors have crept in. Please call them to our attention.
Grateful appreciation is extended to Michael E. Bender and Charles L.
Brown, Jr., both former Biologists with the Water Pollution Training
A ctlvitles, for their valuable contributions and encouragement.
H.W. Jackson
Chief Biologist
National Training Center

Part I. The System of Classification
A Every type of living creature
has a favorite place to live.
There are few major groups that
are either exclusively terres-
trial or aquatic. The following
remarks will therefore apply in
large measure to both, but pri-
mary attention will be directed
to aquatic types.
B One of the first questions usu-
ally posed about an organism is:
“What is it?”, usually meaning
“What is it’s name?”. The nam-
ing or classification of bio-
logical organisms is a science
in itself (taxonomy). Some of
the principles involved need to
be understood by anyone working
with organisms however.
1 Names are the “key number”,
“code designation”, or “file
references” which we must
have to find Information
about an unknown organism.
2 Why are they so long and why
must they be in Latin and
Greek? File references in
large systems have to be long
in order to designate the
many divisions and subdivi-
sions. There are over a
million and a half items (or
species) included in the
system of biological nomen-
clature (very few libraries
have a million books).
3 The system of biological no-
menclature is regulated by
international congresses.
a It is based on a system of
groups and super groups,
of which the foundation
(which actually exists in
nature) is the species.
Everything else has been
devised by man and is sub-
ject to change and revision
as man’s knowledge and
understanding increase.
b The basic categories em-
ployed are as follows:
(1) Similar
grouped ______
(2) Similar genera are
grouped into families
(3) Similar familes are
grouped into orders
(4) Similar orders are
grouped into classes
(5) Similar
grouped _____
(6) Similar phyla are
grouped Into kingdoms
4 The scientific name of an or-
ganism Is its genus name plus
its species name. This is ana-
logous to our system of sur-
names (family names) and given
names (Christian names).
a The generic (genus) name
is always capitalized and
the species name written
with a small letter. They
should also be underlined
or printed in italics when
used in a technical sense.
For example:
Homo sapiens - modern man
Homo neanderthalis -
neanderthal man
Esox niger — Chain pickerel
Esox lucius - northern pike
Esox masquinongy -
b Common names do not exist
for most of the smaller and
less familiar organisms.
For example, if we wish to
refer to members of the
species are
into genera
classes are
into phyla
BI. AQ. 24. 5.71

: ..DEVELOPMENT)f MuLrjcL [ ,.uL4R.oRcOENoC Ift ORG NISMS. -
IA 97
( si’)!’i; 1 .f-I -

Classification - Finder
genus Anabaena (an alga),
we muet simply use the
generic name, and:
Anabaena planctonica,
Anabsena constricta, and
Anabaena flos—aguae
are three distinct species
which have different signi—
ficances to water treatment
plant operations.
5 A complete list of the various
categories to which an organism
belongs is known as its “classi-
fication”. For example, the
classification of a type of
frog spittle, a common fila—
mentous alga, and a crayfish
or crawdad are shown side by
side below. Their scientific
names are Spirogyra crassa and
Cambarus sciotensis .
a Examples of the classifica-
tion of an animal and a
b These seven basic levels of
organization are often not
enough for the complete de-
signation of one species
among thousands; however,
and so additional echelons
of tenii’i are provided by
grouping the various cate-
gories into “Super. ..“
groups and subdividing them
Into “sub.. .“ groups as:
Superorder, Order, Suborder,
etc. Still other category
names such ns “tribe”, “di-
vision”, “va iety” , “race”,
“section”, etc. are used on
c Additional accuracy is gained
by citing the name of the
authority who first described
a species (and the date) m-
mediately following the spe-
cies name. Authors are also
often cited for genera or
other groups.
d A more complete classification
of the above crayfish is as
Kingdom Animalia
Phylum Arthropoda
Class Crustacea
Subclass Malacobtraca
Order Decapoda
Section Nephropsidea
Family Astacidae
Subfamily Cambarinae
Genus Cambarus
Species sciotensis Rhoades
e It should be emphasized that
since all categories above
the species level are essen-
tially human concepts, there
is often divergence of opin-
ion in regard to how certain
organisms should be grouped.
Changes result as knowledge
f The most appropriate or cor-
rect name for a given species
is also sometimes disputed,
and so species names too are
changed. The species itself,
as an entity in nature, how-
ever, is relatively timeless
and so does not change to
man’s eye.
A Living organisms (as contrasted to
fossil types) have long been group-
ed into two kingdoms: Plant King-
doms and Animal Kingdoms. Modern
developments however have made this
(Frog Spittle) (Crayfish)
A rthropoda

Classification - Finder
Simple pattern technically unten-
able. It has become evident that
there are as great and fundamental
differences between certain other
groups and these (two), as there
are between the traditional “plant”
and “animal”. The accompanying
chart consequently Shows the
Fungi as a third kingdom.
B The three groups are essentially
defined as follows on the basis
of their nutritional mechanisms:
1 Plantae: Photosynthetic;
synthesizing their own organic
substance from inorganic min-
erals. EcologicaLly known as
2 Animalia: ingest and digest
solid particles of organic food
material. Ecologically known
3 Fungi: extracellular digestion
(enzymes secreted externally).
Food material then taken in
through cell membrane where it
is metabolized and reduced to
the mineral condition. Ecolo-
gically known as REDU RS .
C Each of these groups includes
simple, single celled representa-
tives, persisting at lower levels
on the evolutionary stems of the
higher organisms.
1 These groups span the gaps be-
tween the higher kingdoms with
a multitude of transitional
forms. They are collectively
called PROTISTA .
2 Within the protista, two prin-
ciple sub-groups can be defined
on the basis of relative com-
plexity of structure:
a The bacteria and blue algae,
lacking a nuclear membrane,
may be considered as the
lower protista or MONERA .
b The single celled algae and
protozoa having a nuclear
membrane, are best referred
to simply as the higher

Classification - Finder
Part II. Biological Classification
What 18 it?
“Algae” defined
CLASS Myxophyceae
Order Chroococcale
Order Hormogonales
Suborder Hetercystineae
CLASS Chlorophyceae
Order Volvocales
Order Ulotrichales
Order Cbaetophorales
Order Chlorococcales
Order Siphonales
Order Zygnematales
Order Tetrasporalee
Order Ulvalee
Order Schizogoniales
Order Oedogonialea
Order Cladophorales
CLASS Charophyceae
Order Charales
green algae or yellow—brown
CLASS Xanthophyceae
Order Rhizochloridales
Order Heterocapsales
Order Heterococcales
CLASS Chrysopbyceae —
yellow-green algae
Order Chrysomondalas
Order Rhizochrysidales
Order Chrysosphaerales
Order Chrysocapsales
Order Chrysotrichales
CLASS Bacillariophyceae -
Order Pennales - pennate
Order Centrales — centric
di atoms
noid algae
brown algae
CLASS Desmokontae
Order Desmononadales
CLASS Dinophyceae —
Order Gymnodiniales
Order Peridiniales
Order Dinocapsales
Order Chioromonadales
CLASS Cryptopbyceae
CLASS Rhodophyceae
Order Rangiales
Order Nemallonales
Order Gelidiales
131. AQ. 24 5.71

Classification — Finder
a r
Order Cryptonemiales
Order Gigartinales
Order Rhodymenjales
Order Ceraniales
PHYLUM PHAEOPH’YTA - brown algae
CLASS Phaeophyceae
Order Ectocarpales
Order Sphacelarjales
Order Tilopterjda].ee
Order Chordjales
Order smarestjalas
Order Punctarialea
Order Dictyosiphonales
Order Laminarjales
Order Fucalee
Order Dictyotales
CLASS Hepaticae — 1iver orts
CLASS Musci - mosses
Emergent vegetation
Rooted plants — floating leaves
Submerged vegetation
Free floating plants
CLASS Mastigophora
Subclass Phytomastigina
Subclass zoomastigiria
CLASS Sarcodina - amoebojd
Order Amoebina
Order Foraminifera
Order Radiolarja
Order Heliozoa
Order Mycetozoa (Myxomycetes)
CLASS Ciliophora - ciliates
Order Holotricha
Order Spirotricha
Order Peritricha
Order Chonotrjcha
CLASS Suctorja — suctorja
CLASS Sporozoa
CLASS Calciepongea
CLASS Hyalospongea
CLASS Demospongea
CLASS Hydrozoa - hydroide
CLASS Scyphozoa — Jellyfish
CLASS Actinozoa (Anthozoa) —
CLASS Turbellarja — turbella— 109
CLASS Trematoda - fluke
CLASS Cestoidea — tapeworme
G PHYLUM NEMATODA - threadworme,1l3
4 ‘ —6

Classification — Finder
Horsehair worms
headed worms
J PHYLUM RaFIFERA - rotifer, 118
wheel animalculee
CLASS Polychaeta — polychaet 122
CLASS Oligochaeta — briltie 123
legged animals
CLASS Crustacea — crustaceans 129
Subclass Branchiopoda 130
Order Anostraca — fairy 131
Order Notostraca — tadpole 132
Order Conchostraca - clam 133
Order Cladocera — water fleasl34
Subclass Ostracoda — seed 135
shrimps, ostracodes
Subclass Copepoda - copepods 136
Subclass Branchiura — fish 137
Subclass Cirripedia — 138
Subclass Malacostraca
Order Leptostraca
Order Hoplocardia
(Stomatopoda) — mantis
Order Syncarida
Order Peracarida
Suborder Mysidacea
Suborder Cumacea
Suborder Tanaidacea
Suborder leopoda — sowbugs
Suborder Amphipoda — scuds 148
Order Eucarida 149
Suborder Euphaueiacea — 150
kr ill
Suborder Decapoda — shrimp,l51
lobster, crab
Macrurous group (4 tribes) 152
shrimps, prawns, lobsters,
c ny fish
Brachyurous group 153
(2 trIbes) — crabs and hermit
CLASS Insecta — the insects 154
AIINELIDA - segmented
Hirudinea — leeohe
Sipunculoidea - peanut
Orders represented by imma-
ture stages only.
Order Plecoptera — stone— 156
Order Ephemeroptera - 157
may files
Order Odonata - dragon and 158
damself lies
Order Megaloptera — alder- 159
flies, dobsonfiles, fisbflies
Order Neuroptera — spongilla—l60

Classification — Finder
Order Trichoptera — caddjs— 161
Order Lepidoptera — aquatic 162
Order Diptera - two winged 163
Orders including aquatic 164
Order Coleoptera - beetles 165
Order Hemiptera — true bugs 166
CLASS Arachnoidea — spiders, 167
Scorpions, mites
Order Xiphosoura - horse— 168
shoe or king crabs
Order Hydracarina — aquatic 189
Order Pantopoda (Pycnogonida)_ 170
Order Tardigrada 171
CLASS Amphineura - chitons 173
CLASS Gasteropoda — snails 174
Order Prosobranchiata 175
Order Opisthobranchiata ],76
Order Pulmonata — air breath— 177
log snails
CLASS Scaphopoda - tusk 178
she l ls
CLASS Bivalvia 179
CLASS Cephalopoda - squid, 180
Octipua, nautilus
H PHYLUM SRYOZOA (Ectoprocta) — 181
Moss animals
she 1 is
CLASS Asteroidea — starfishes
CLASS Ophiuroidea — brittle
CLASS Echinoidea - sea urchins
CLASS Holothuroidea — sea
CLASS Crinoidea — sea lilies
Subphylum Henichordata —
Acorn worms
Subphylum Urochordata —
tunicates, sea squirts
Subphylum Cephalochordata -
Subphylum Vertebrata
( Craniata) — vertebrates
CLASS Agnatha — jawlesa 196
Order Myxinifornes — 197
hagf ishes
Order Petromyzontjformes — 198
CLASS Chrondrichthys — 199
cartilage fishes
Order Squaliformes — sharks 200
Order Rajiformes — skates, 201
Order Chimaerifo es — 202
CLASS Osteichthys (Pisces) — 203
bony fishes
Order Acipenseriformes — 204
Order Polyodontidae — 205
paddle fishes

Classification — Finder
Order Semionoteformes - ears 206
Order Amliformes - bowfisa 207
Order Clupeiformee — soft 208
rayed fishes
Family Clupeidae — herrings 209
Family Salmonidae — trouts, 210
Family Esocidae — pikes, 211
Order MyctopbifOrmes — 212
lizard fishes
Order Cypriniformea — 213
Family Cyprinidae - minnowS, 214
Family Catoetomidae - euckers2l5
Family Ictaluridae — fresh— 216
water catfishes
Order Anguiliiformes — eel— 217
like fishes
Order Notacanthiformeb - 218
spiny eels
Order Beloniformes — needle— 219
fishes, flying fishes
Order Cyprinodontiformes - 220
killifishes, livebearers
Order Gadiformes — coda and 221
hake 8
Order Gasterosteiformea — 222
Order Lampridiformea — Opahe, 223
ribbon fishes
Order Beryciformes — beard— 224
Order Percopsiformes — trout 225
and pirate perches
Order Zeiformes - dory 228
Order Perciformes — spiny— 227
rayed fishes
Family Serranidae — sea 228
Family Centrarchidae - 229
sunfishes, freshwater
Family Percidae — perch 230
Family Sciaenidae — drum 231
Family Cottidse — sculpins 232
Family Magilidae — mullets 233
Order Pleuronectiformes — 234
Order Echeneiformes — remoras235
Order Gobiesociformes — 236
Order Tetraodontiform — 237
Order Batrachoidiformes — 238
Order Lophiiformes — 239
goose fishes
LA8S Amphibia — frogs, toade,240
CLASS R.eptilia — turtles, 241
snakes, lizards
CLASS Ayes - birds 242
CLASS Mammalia — whales, 243
seals, walrusses
A Bacteria 251
Eubacteria 252
Actinomycetes 253
Myxobacteria 254
Spirochaetes 255
Other bacterial types 256
“Phycomycete” group

ClasRification - Finder
CLASS Chytridionycetes 262
CLASS Oornycetes 263
CLASS Zygomyce tee 264
CLASS Ascoinycetes 265
CLASS Basidlomycetes 266
CLASS Fungi Imperfectj 267