Control of Biological Problems in Water Supplies
Presented by EDUCATION COMMITTEE OF AMERICAN WATER WORKS ASSOCIATION and
ENVIRONMENTAL PROTECTION AGENCY
Water Quality Office ¦ Water Hygiene Division
June 13,1971 • Denver. Colorado
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CONTROL OF BIOLOGICAL PROBLEMS
IN WATER SUPPLIES
Presented by
EDUCATION COMMITTEE OF AMERICAN WATER WORKS ASSOCIATION
AND
ENVIRONMENTAL PROTECTION AGENCY
WATER HYGIENE DIVISION - WQO
Denver, Colorado
June 13, 1971
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SPEAKERS
EDWIN E. GELDREICH
Principal Bacteriologist
Water Hygiene Division
WQO, EPA
5555 Ridge Avenue
Cincinnati, Ohio 45213
H. E. HUDSON, JR.
Partner, Hazen and Sawyer
360 Lexington Avenue
New York, New York 10017
ARLISS D. RAY
Associate Professor
of Civil Engineering
University of Missouri
Columbia, Missouri 65201
EDGAR A. JEFFREY
Training Officer
Water Hygiene Division
WQO, EPA
5555 Ridge Avenue
Cincinnati, Ohio 45213
KENNETH M. MACKENTHUN
Acting Director
Division of Technical Support
Water Quality Office, EPA
Washington, D. C. 20242
ROBERT M. SCOTT
Assistant Chief Public Health
Microbiologist
Division of Laboratories
Illinois Department of Public Health
134 North 9th Street
Springfield, Illinois 62701
HARRY W. TRACY
Manager, Purification Division
San Francisco Water Department
1000 El Camino Real
Millbrae, California 94030
HUGO T. VICTOREEN
Laboratory Director
Wilmington Water Department
16th and Market
Wilmington, Delaware 19899
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CONTROL OF BIOLOGICAL PROBLEMS
IN WATER SUPPLIES
Denver, Colorado
June 13, 1971
AGENDA
Moderator: E. A. Jeffrey
DAY AND TIME SUBJECT SPEAKER
Sunday, June 13
9:30 - 9:45
9:45 - 10:30
10:30 - 10:45
10:45 - 11:00
11:00 - 11:45
11:45 - 12:00
12:00 - 1:30
1:30 - 2:15
2:15 -
2:30 -
2:45 -
3:05 -
3:20 -
3:40 -
3:55 -
4:15 -
4:30 -
2:30
2:45
3:05
3:20
3:40
3:55
4:15
4:30
4:45
Opening Remarks
Cause and Extent of Biological
Problems in U. S. Waters
Discussion
Break
Cause and Control of Sponges in
Transmission Lines
Discussion
Lunch
Needs for Control of Bacterial
Populations in Potable Waters
Discussion
Break
Control of Iron and Other Nuisance
Organisms
Discussion
Control of Non-Indicator
Micro-Organisms
Discussion
San Francisco Experience with
Nuisance Organisms
Discussion
Summary
H. E. Hudson, Jr.
K. M. Mackenthun
D. L. King
A. D. Ray
J. L. Tuepker
E. E. Geldreich
R. Scott
H. T. Victoreen
H. W. Tracy
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CONTENTS
Lecture Outline Number
Cause and Extent of Biological Problems in 1
U. S. Waters
Cause and Control of Sponges in Transmission Lines 2
Needs for Control of Bacterial Populations in Potable 3
Water
Control of Iron and Other Nuisance Organisms 4
Control of Non-Indicator Micro-Organisms 5
San Francisco Experience with Nuisance Organisms 6
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INTRODUCTION
H. E. Hudson, Jr. Chairman
AWWA Education Committee
As one means of moving toward application of the "Water Quality
Goals", adopted in January 1968 by the American Water Works
Association as a part of a statement on water policy, the Associ-
ation has, for several years, enjoyed the collaboration of the
Bureau of Water Hygiene, United States Public Health Service in
the preparation and conduct of seminars and various aspects of
water quality problems, held in conjunction with the A WW A
national conferences.
These seminars related to water quality are held in parallel
with other seminars in fields such as labor negotiations, manage-
ment, safety, and emergency planning. Management and labor
negotiations seminars are being conducted today, in parallel to.
this one on biologic problems, m nearby rooms. Generally
the AWWA seminars are designed so that they can be repeated in
various parts of the country, under the sponsorship of AWWA
sections. In addition to this, the Association has been sponsoring
seminars on upgrading water treatment plant operation, and is
about to begin a new series of seminars at various parts of the
U.S. , on chlorination. Chlorination is of course very much
interwoven with the topics that will be discussed today at this
seminar, where you will be discussing and identifying the various
biologic problems in waters of North America ana in water
systems. These two topics, therefore, link up nicely.
The Association wants to express its appreciation to the Bureau
of Water Hygiene, which is now a part of the Office of Water
Quality in the Federal Environmental Protection Agency.
Members of the Association have long gained much from the work
of this Bureau, and AWWA has continued to give it strong support.
In particular, the Association wishes to express its thanks to
Ed Jeffrey for his work in preparing and conducting this seminar.
Ed and I first got acquainted over training activities in Rio de
Janeiro, and I'm glad to see that he is moving forward in this
field of work.
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CAUSE AND EXTENT OF BIOLOGICAL PROBLEMS
IN U. S. WATERS
Kenneth M. Mackenthun*
The subject that I have been assigned to
discuss is indeed broad. It encompasses
all of those living things that may interfere
in some way with man's optimum use of
water. Basically, it is safe to say that
biological nuisances are caused by man's
activities in the environment, either through
direct effect on water, atmospheric fallouts,
or run-off from the watersheds. Man's
"domesticated" uses of water demand a
certain level of quality. This level of
quality excepts biological nuisances of any
magnitude. On the other hand, man's
uses of water as a purveyor of wastes
either directly through a point source
effluent or indirectly from fertilized or
managed land drainage introduces constit-
uents into the receiving waters that tend
to produce biological nuisances through a
combination of selected organism toxicities
and biostimulation. These factors pave the
way for the production of an overabundance
of aquatic organisms or, more often, an
overabundance of a certain group of aquatic
organisms that has a detrimental and
decided impact. Basically those conditions
that create an environment that is more
favorable towards one group of organisms
than to another, and in turn may stimulate
the successfully resident group to over-
production, are the causes of biological
nuisances as we identify them.
uncovered reservoirs, but are common and
widespread in surface waters. Perhaps
none of the organisms found in surface
waters that may be used for domestic
purposes is directly injurious to health.
The chief complaints against them are
interferences with filtration or other water
treatment, and their effects on the palat-
ability and aesthetics of the water. Organ-
isms may accumulate in such numbers that
waters become unsightly and turbid and
many organisms impart disagreeable odors
and colors to the water.
To obtain information on the magnitude and
frequency of biological problems in water
supplies, a questionnaire was sent to over
1400 managers of those water systems
maintaining utility membership in the
American Water Works Association in
August 1969. In addition, the questionnaire
was mailed to the managers of water
supplies for the 100 largest cities and to the
50 State Sanitary Engineers. From this
questionnaire it was learned that organisms
had created problems for 25 percent of all
water treatment plant managers during the
past five years.
The most frequently reported problems
were associated with algal, iron bacteria,
and pond weed growths (Table 1).
The use for domestic water supply is one
of the more demanding of water quality
of the many that man makes of water.
Organisms that may cause, or have been
known to cause, problems in water supplies
include several species of algae, vascular
aquatic plants, protozoa, and diatoms that
produce tastes and odors and clog filters;
iron bacteria that produce tastes and odors
and clog pipes; copepods whose eggs pass
through filters; very small nematode worms,
sowbugs in the distribution system; midge
larvae or bloodworms; and snails and
mo llusks.
There may be present in surface waters
various types of organisms, both plants
and animals, which vary in complexity
and size. They are uncommon or absent
in ground water supplies unless stored in
'•"¦Acting Director, Division oi Tecnnical
Support, Water Quality Office, EPA
Table 1
Organisms Creating Problems in
Water Supplies
Number of Managers
Reporting Problem
Algae
164
Iron bacteria
54
Pond weeds
31
Midge larvae
17
Copepods
10
Asiatic clams
5
Nematodes
3
Manganese bacteria
3
Sulfur bacteria
3
Actinomycetes
2
Dead fish
2
Mosquito larvae
2
Fungi
2
Bryozoa
1
Water milfoil
1
Frogs
1
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Cause and Extent of Biological Problems in U. S. Waters
The types of water supply problem created by
the organisms were principally tastes and
odors, clogging of filters, organism in distri-
bution systems and the creation of the neces-
sity for increased reservoir maintenance
Table 2.
Table 2
Type of Water Supply Problem Created by
Organisms
dumber 01 Managers
Reporting Problem
Taste and odors
162
Clogging of filters
64
Organisms in distribution
50
system
Increased reservoir
45
maintenance
Reduced flows in distribution
6
system
Solids or color in finished
5
water
The place where a problem originated and
the type of organism involved were closely
related. The most common point of origin
for algal and pond weed problems were lakes,
reservoirs, and rivers. Iron bacteria were
associated with wells and distribution
systems. The origin of water supply problems
created by organisms is shown in Table 3.
Table 3
Origin of Water Supply Problem Created by
Organisms
Number of Reports
Indicating Origin
Supplying lake or reservoir 149
Supplying river 45
Supplying well 40
In distribution system 29
In clear well 6
Supply canal 4
Finish water storage 2
reservoir
Vegetational problems in water may derive
from the masses of floating, attached, and
rooted plants. These growths become
problems only when conditions of existence
promote excessive standing crops that inter-
fere with a water use.
Aquatic plants, like most other living matter,
are not all bad or all good. Their assets are
many and their liabilities are most often
associated with their degree of concentration
within the water media. A group of aquatic
weed control specialists may be inclined to
think only of the undesirable aspects because
of day-to-day association with problems
caused by an overproduction of the aquatic
crop.
Plants are a basic and extremely important
component of the aquatic ecosystem. As
the basis of the food chain they are the bread
of life for the aquatic'grazing animals. Such
animals range in complexity from the thousan
of different kinds of mostly microscopic zoo-
plankton to ducks and diving birds and fur-
bearing animals as well. Serving not only
as food in themselves, aquatic plants serve
also as the high rise apartment homes for a
myriad of different small animals that are
a vital link in the aquatic food web that cul-
minates in a fish population.
Aquatic plant apartment homes provide
protection from predators to their inhabitants
Studies have indicated that the plants with
the most finely divided leaves will harbor
the greatest abundance of animals. All
aquatic vegetation areas will support many
times the animal populations that may be
found in non-vegatational areas within the
same ecosystem. Plants are collectors
for fish and the fishing potential is increased
in areas adjacent to standing crops of
vascular plants. Many fish use these areas
as foraging grounds and dine contentedly
upon the apartment dwelling inhabitants of
the vascular plant community.
These same plants serve as the substrate
for the spawning of many organisms including
snails and fish such as the yellow perch.
Just forty years ago, Rudolfs and Huekelekia
noted the effects of sunlight and green
organisms on the reaeration of streams and
found that the dissolved oxygen in water
containing large quantities of algae could be
decreased from supersaturation to 17 percen
saturation by placing the water in darkness,
and could also be increased to 282 percent
saturation by subjecting it to diffused light.
If the darkness experiments had been
extended over a longer time period, these
early researchers would have no doubt found
the oxygen saturation decreasing into an
oxygen deficit. By adding oxygen during the
process of photosynthesis, plants decrease
the problems associated with decomposition
of organic materials that may be present.
Plants tend to purify their surrounding wate^
by other means. Certain algal and vascular"
plant populations have been found to possess
a bactericidal quality that reduces the
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Cause and Extent of Biological Problems in U. S. Waters
concentration of coliform bacteria and pre-
sumably pathogenic bacteria that would
other wise be associated with the specific
environment.
In photosynthesis, aquatic plants use carbon
dioxide and liberate dissolved and free
gaseous oxygen at times of supersaturation.
Since energy is required in the form of light,
photosynthesis is limited to the photic zone
where light is sufficient to facilitate this
process. During respiration and decom-
position, animals and plants consume dis-
solved oxygen and liberate carbon dioxide
at all depths where they occur. Because
excreted and secreted products and dead
animals and plants sink, most of the decom-
position takes place in the hypolimnion or
deeper waters. Thus, in stratified lakes;
there is a gradual decrease of dissolved
oxygen in this deep water zone. After the
dissolved oxygen is depleted, anaerobic
decomposition continues with evolution of
methane and hydrogen sulfide.
When aquatic plants are stimulated in some
manner to the production of a standing crop
so abundant that it interferes with a water
use, plant nuisances develop. It is at this
point where citizens, whose normal use of
the water has been restricted by an abundant
plant growth, demand remedial measures
and control of the nuisance. Plant nuisances
may curtail or eliminate bathing, boating,
water skiing and sometimes fishing; per-
petrate psychosomatic illness in man by
emitting vile stenches; impart tastes and
odors to water supplies, shorten filter runs
or otherwise hamper industrial and municipal
water treatment; impair areas of picturesque
beauty; reduce or restrict resort trade;
lower water front property values; interfere
with the manufacture of a product in industry
such as paper; on occasion become toxic to
certain warm blooded animals that ingest the
water; reduce the use potential of irrigation
waters through evapotranspiration; foul
irrigation siphon tubes and trash racks;
and cause skin rashes and hayfever-like
symptoms in man.
Dispersal of water plants is accomplished
by water transport, migratory birds, air
transport of algae, and by domestic and
other animals. Seeds may remain viable
after passing through the digestive tract
of animals, and seeds and other means of
propagation may be transported by animals
externally. Water plants usually produce
an abundance of seeds but propagation
through vegetative means is a most effective
methods of distribution. A small broken
portion of a healthy plant may soon re-
establish itself, when, in settling out of
the water, it roots again on a suitable substrate.
Most aquatic plants are perennials and are
well adapted to withstand heavy cropping by
animals.
Plant populations will develop in the aquatic
environment wherever conditions are suitable.
Plants have been found growing at a depth
of 500 feet in Lake Tahoe. Providing the
aquatic environment is nontoxic, light intensity
and nutrients are the principal controlling
factors for planktonic growths. Temperature
is an important factor with some of the
nuisance forms such as blue-green algae.
Light intensity, water temperature, wave
action, flow velocity, nutrient abundance,
water depth and type of substrate, all inter-
act to govern the establishment of weed
beds or weed sparsity. Both bottom sediments,
as well as the water contribute nutrients to the
plants. Sediments supply inorganic nutrients
through the plants relatively weak root system.
Many submersed plants, as well as algae,
continue active in winter, providing ice and
snow cover are not sufficiently opaque to
reduce light penetration so that growth is
impeded. Once established in an area, rooted
aquatic plants exhibit a high degree of persis-
tence and efficiency of propagation. Some
reproduce only by means of seeds formed in
insect pollinated flowers borne at or above
the water's surface. Others propagate by
buds, tubers, roots and node fragments in
addition to producing viable seeds. Factors
that limit growth include insufficient light,
insufficient nutrients, physical instability
because of water level fluctuation and current
and wave action, an unsuitable bottom stratum,
and competition by other plants and animals.
Considering that the physical properties of
the environment are favorable, the nutrients
become the prime stimulators and controllers
of aquatic plant production. The most
important required nutrients are carbon,
nitrogen, phosphorus, certain trace elements,
organic growth factors, and, in the case of
diatoms, silica.
Of the required nutrients, one will become
limiting for future growth if other physical
and chemical features of the aquatic environ-
ment are suitable. The term, "limiting, "
is one that has caused much misunderstanding
among many of those who attempt to evaluate
limiting factors. Something is always limit-
ing to the further growth of a biological
population. That which is limiting at one
level of production may not limit at another.
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Cause and Extent of Biological Problems in U. 5. Waters
The term, "limiting, " when associated with
eutrophi cation ana its control should refer
to that substance which when added will
stimulate a biological population to increase
and become restrictive, or more detrimental,
to a given water use.
Hutchinson in 1957 stated, "Of all elements
present in living organisms, phosphorus is
likely to be the most important ecologically,
because the ratio of phosphorus to other
elements in organisms tends to be greater
than the ratio in primary sources of the
biological elements. A deficiency of phos-
phorus is, therefore, more likely to limit
productivity." Research over the succeeding
3/ears has not produced evidence that would
discredit this basic observation. Evidence
indicates that: (1) High phosphorus concen-
trations are associated with accelerated
eutrophication of waters, when other growth
promoting factors are present; (2) aquatic
plant problems develop in reservoirs or
other standing waters at phosphorus values
lower than those critical in flowing streams;
(3) reservoirs and other standing waters
collect phosphates from influent streams and
store a portion of these within consolidated
sediments; and (4) phosphorus concentrations
critical to noxious plant growths vary, and
they produce such growths in one geographical
area at a given concentration but not in another.
Basic sources of nutrients to waterways are
(a) tributary streams carrying land runoff
and domestic and industrial wastes, (b) the
biological and chemical interchange between
bottom sediments and superimposed water,
and (c) precipitation from the atmosphere.
Tributary streams have been reported to
carry 21 pounds of phosphorus per square
mile of drainage area in sparsely settled
forested areas, 225 pounds of phosphorus
per square mile of drainage area in agri-
cultural areas, and more than 6, 000 pounds
in densely populated urban areas. The phos-
phorus contribution by domestic sewage is
about 3 pounds per capita per year. The
upper 1-inch stratum of bottom sediments
in Lake Sebasticook, Maine, contained a
calculated 50 pounds of phosphorus per acre.
The question is sometimes asked, how much
algae can be grown from a given amount
of phosphorus ? It has been found that the
maximum that could be grown in the laboratory
on sewage, an excellent growth media for
algae, was 1 to 2 g/1 (dry weight) and in the
field in sewage oxidation pounds the maximum
was 0.5 g/1. Thus, assuming optimal growth
conditions and maximum phosphate utilization,
the maximum algal crop that could be grown
from 1 pound of phosphorus would be 1, 0Q0
pounds of wet algae under laboratory
conditions or 250 pounds wet algae under
field conditions. Considering a cellular
phosphorus content of 0. 7 percent in algae,
1 pound of phosphorus could be distributed
among 1, 450 pounds of algae on a wet weight
basis. A considered judgment suggests that
to prevent biological nuisances, total phosphorus
should not exceed 100 p.g/1 P at any point with-
in the flowing stream, nor should 50 u.g/1 be
exceeded where waters enter a lake, reservoir,
or other standing water body. To enhance the
quality of this Nation's lakes, reservoirs
and estuaries, we must pursue a major effort
with diligence and speed to reduce to the
ultimate phosphates, and all other nutrients
where feasible, from all controllable sources.
With suitable environmental conditions, plants
will develop and avail themselves of the space
and available nutrients. With the application
of chemicals to a segment of the aquatic
environment, it is possible to change the
predominant growth from vascular plants
to planktonic algae or attached algae and visa
versa almost at will.
There are over 17, 000 species of algae and
fresh water forms are grouped into blue-green
algae, green algae, yellow-green algae,
golden-brown algae and diatoms, red algae,
euglenoids and dinoflagellates. Some of
these are capable of producing physiologically
active metabolites that may function as toxins,
growth inhibitors, or growth stimulators to
themselves or to associated algae. Some
algae are born to self-destruct. After a
growth period when extracellular products
have accumulated, it is thought that these
act as deterrents and that the plant, in a
sense, manufactures its own algicide. The
extracellular substances {possibly of a toxic
nature) have prevented the growth of certain
other species, and thereby the plant that
begins its development first in a body of
water quickly assumes a dominance, depend-
ing upon its inherent cell division rate. With
auto-destruction come a reduction in the
inhibitor, thus permitting another species
or group of species to develop.
Natural waters contain these active agents
that are secreted and excreted by fresh-water
algae. The toxicity of these agents to
other algae and bacteria and to fish varies
constantly and is not well understood in the
aquatic environment. It has been postulated
that algae secrete not just one substance but
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Cause and Extent of Biological Problems m U. 5. Waters
several, some antibiotic, other stimulating.
The amount secreted and the net result of
the secretions would be determined by the
prevalanc.e of one group of substances over
the other. Thus sequences of algal blooms
may be expected to occur under conditions
of a nutrient supply in excess of critical
values.
Chemical plant control measures to be
employed depend upon the type of nuisance
and local conditions. A good algicide or
herbicide must: (1) be reasonably safe to
use; (2) kill the specific nuisance plant or
plants; (3) be essentially nontoxic to fish,
fishfood organisms and terrestrial animals
that may use the water at the plant-killing
concentration; (4) not prove seriously
harmful to the ecology of the general aquatic
area; (5) be safe for water contact by humans
or animals, or be amenable to safeguards
during the unsafe period; and (6) be of
reasonable cost.
Ultimate control of nuisance aquatic organ-
isms can be accomplished only by drastic
alteration of the basic cause(s) of the problem.
The maintenance concept must be considered
by all users of streams, lakes, or reservoirs.
The water front is an aquatic extension of the
surrounding land. To achieve the most
lasting beauty, it must be maintained in a
fashion similar to that of the adjoining lawn
or the abuting parkway; otherwise nuisances
and unsightliness will prevail. Controls
developed to cure water ills are not singular
operations. The mechanisms triggering
nuisance development is usually such that it
will re-establish itself another year. Con-
tinuous surveillance and appropriate main-
tenance are necessary in water management
to ensure maximum multiple use.
Upon the introduction of waste nutritive
materials into watercourses, biological slimes
may develop to the extent that visible masses
appear. These are wooly coatings on sub-
merged objects or tufts and strands, some-
times 15 inches or more long, streaming in
the current from points of attachment. They
vary in color from milk}' white in fresh new
growths to dull grey-white, brown or rusty-
red, depending on age, nutrition, and type
and amount of solids they entrap from the
passing waters. Biological slimes bring
about an aesthetically unpleasant stream.
To the public they are an obvious sign of
stream pollution.
The secondary effects of biological slimes
m streams may be serious. The stream
slime community is composed of a variety
of microorganisms that are held together
as a mat, principally by Sphaerotilus.
Such interwoven mats entrap silt, sand,
fibers and other debris. The Sphaerotilus
masses offer shelter and support for other
organisms such as bacteria, protozoans,
nematodes, rotifers, and occasionally
midge larvae. During the process of decom-
position, or because of physical disturbances,
mats sometimes boil to the water's surface
in an unsightly, foul smelling eruption. These
boils may settle at or near the point of origin
or be carried downstream to areas where the
flow velocity permits settling. Here, sludge
banks are formed that give rise to anaerobic
conditions with subsequent, offensive
effects. These sludge banks may be formed
many miles downstream from initiating
pollution source, thus increasing the
stream reach of pollution.
A number of aquatic animals may create
biological problems of substantial magnitude.
Sponges in irrigation systems can cause
hydraulic problems when growing in
association with other aquatic animals and
plants. This undesirable effect is com-
pounded by the favorable substrate they
create for a wide variety of other aquatic
pest organisms.
Bryozoa growing on submerged aquatic
structures and in conduits have been known
to create serious hydraulic problems for
water distribution structures.
Midges or blind mosquitoes have created
severe nuisance problems around the shores
of a number of lakes. These have occurred
around lakes in New York City; Winterhaven,
Florida; Clear Lake, California; Lake
Winnebago, Wisconsin; and other areas.
Their abundance has been associated with
waters of high organic content. A study in
Clear Lake, California, indicated that in
44 square miles of lake the total seasonal
production was approximately 700 billion
gnats or 356 tons of organisms. One night's
emergence was estimated at 3j billion,
and bottom samples contained as many as
1,000 organisms per square foot.
Adult mayflies have caused damage in
certain local areas. The adults, which
emerge from the water more or less at the same
time, are fragile insects that die within
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Cause and Extent of Biological Problems in U. S. Waters
a few hours. When occurring in hordes,
their dead bodies may clog ventilator ducts
and sewers and may cause temporary traffic
difficulties. In 1940 they were piled four
feet deep on a highway bridge at Sterling,
Illinois. Crews of tow boats, which transport
freight on the Upper Mississippi River, find
mayflies to be a navigational hazard. Visi-
bility is greatly reduced by the mass of
insects in the searchlike beams. The
crushed insects render the deck, ladders, and
equipment of the boats slippery and dangerous.
Larvae mayflies are inhibited or killed when
organic pollution is sufficiently severe to
remove the dissolved oxygen from the waters
superimposed on the soft, loam lake and
stream beds.
Mosquitoes are implicated in the transmission
of parasitic and other diseases. Elephantiasis,
characterized by massive glandular swelling,
is a disease that occurs commonly among
the people of Puerto Rico and is transported
by many species of mosquitoes. The three
principal arthropod-borne encephalitis viruses
are transported also by mosquitoes. Mos-
quitoes, as a nuisance, attack man, domestic
animals, and foul and, when bites are inflicted
in large numbers, cause loss of weight and
health. It has been estimated that 500 mos-
quitoes will draw 1/20 of a pint of blood per
day from an exposed animal. They prefer
water with little wave action, an abundunt
cover of aquatic vegetation, an abundant food
in the form of humus or other organic matter
on the bottom, and surface floating particles
of microorganisms.
Leeches abound in warm protected shallow
water where there is little wave action and
where plants, stones, and debris offer
concealment. Many species of leeches are
encouraged by the presence of abundant
organic matter in their environment.
The Asiatic clam, Corbicula fluminea, has
become a pest in several areas and especially
has created nuisances in water distribution
systems in California. Accumulation of live
clams and clam shells caused serious
impairment of water delivery at turnout
valves, at ends of laterals and in irrigation
sprinkler systems. The clams contribute
to silt buildup in the canal by providing a
means to hold settled materials on the
bottom, and by removing suspended solids
from the water through filtration and deposi-
tion of the solids on the canal bed combined
with a proteinaceous slime that does not
readily go back into suspension. At one
station in the South Bay Aqueduct, California,
the density of the clam population range
between 380 and 1500 per square foot. The
highest density was over 5, 500 live clams
per square foot.
In general, standing water bodies give rise
to biological nuisances with much greater
frequency than do flowing waters. There
are over 1,500,000 lakes, ponds and
reservoirs in the United States. The
populace lives in contact with fresh surface
waters of which 90 percent by area are
lakes. The United States Great Lakes
have 33. 8 million acres, the Alaskan lakes
7. 4 million acres, and other United States
lakes 19. 5 million acres, making a total
of 60. 7 million acres of standing waters
within the United States. In the 48 contig-
uous States, the areas of natural and man-
made lakes are nearly equal, excluding the
Great Lakes. In these waters people swim,
water ski, boat, fish, and derive pleasures
from the aesthetic contribution to the
landscape.
The ''ounce of prevention" adage is most
applicable to lakes. Eutrophy creeps to a
crisis and as it does, values deteriorate.
When the crisis is reached, it requires
time to initiate a solution. Large sums
of money must be obtained in a short time.
Once a solution is found, it takes more time
to restore the lake. Eutrophy, correction,
and restoration may restrict lake uses
for years. Lake preservation is the
greater need. It is cheaper than restoration
and reduces the risk of eutrophy advancing
to a level where solutions are not applicable
and where the lake becomes moribund, green
and dies.
6
-------
CAUSE AND CONTROL OF SPONGES
IN TRANSMISSION LINES
Barrel L. King, Arliss D. Ray, J. L. Tuepker-
I INTRODUCTION
Biological growths or slimes frequently are
found within water transmission lines in
small numbers which cause little or no
alteration of water quality and have no
significant effect on the quantity of water
carried by the conduit. Occasionally, the
environment within such pipes is ideal for
selected aquatic organisms, and their groivth
is unrestrained. Massive growths within
raw-water transmission lines have a signi-
ficant potential for taste and odor production
in addition to decreasing the delivery capacity
by increasing resistance to flow and decreas-
ing the cross-sectional area of the conduit.
Any organism living attached within a raw-
water conduit lives there because the sum
(or product) of the various environmental
factors lies within its limit of tolerance.
Variation in one or more of these environ-
mental parameters exceeding the limit of
tolerance of an organism results in the
removal of that organism.
The inner surface of raw-water conduits is
an ideal site of attachment for many aquatic
organisms, including the fresh-water
sponges. The probability of development
of significant growths of sponges within
pipes is determined by both the biotic and
abiotic characteristics of the water.
H FRESH-WATER SPONGES
A sponge is an irregular mass of animal
tissue supported by numerous needle-like
silica spicules and permeated by an irregu-
larly arranged water exchange network.
Flagellated cells produce a water current
within the sponge mass and the sponge gains
both oxygen and food from this moving water.
Their food is comprised of small plant and
animal plankton suspended in the water
carried through the sponge mass.
*D. L. King, Associate Professor, University
of Missouri, Columbia, Missouri
A. D. Ray, Associate Professor, University
of Missouri, Columbia, Missouri
During the active growth phase sponges pro-
duce numerous thick-walled resting bodies
called gemmules. When the active growth
of the sponge dies, as it does when the water
cools in the autumn, the spicule-gemmule
assemblage remains attached to the substrate.
When water temperatures reach 50 -55 F in
the spring, the gemmules produce new active
sponge growth. Where there is little pre-
dation, such as within water conduits, this
spicule-gemmule mass may build up a hard
crusty material an inch or more in thickness.
IE REQUIREMENTS OF FRESH-WATER
SPONGES
A Relatively Clean Water Free of Pollution
and ExcessTurbidity.
1 The abrasive action of silt in the water
within the conduit damages the delicate
tissue of the sponge.
2 While pollution is generally harmful
to sponges, the effect of specific organic
and inorganic pollutants is unknown.
B Total Hardness of 75 mg/l or Less and a
Measurable Silicon Concentration.
1 The role of hardness and that of most
other mineral constituents is not well
understood.
2 Silicon is used in quantity to construct
the microscopia needle-like spicules
which serve as support for the sponge
tissue.
C Temperature in Excess of 50°F.
1 At lower temperatures the active, feeding
sponge growth does not develop.
2 Gemmules remain during periods of low
temperature and re-initiate active
sponge growth when conditions again
become suitable.
J. L. Tuepker, Administrative Assistant,
St. Louis County Water Company, St. Louis,
Missouri 1
-------
Causes and Control of Sponges in Transmission Lines
D A Moderately Productive Water.
1 Sponges feed by filtering the microscopic
plants and animals from the water.
2 In water with too great a productivity
bacteria may compete with the sponge
for attachment site.
IV IDENTIFICATION OF SPONGES
A Scrape organic accumulation from the
pipe and place the scrapings in a test
tube containing concentrated nitric acid.
B Place the test tube in boiling water. The
combination of heat and acid destroys the
organic fraction and leaves the distinguish-
ing silica spicules that can be seen with
any good microscope.
V SPONGES IN RAW WATER CONDUITS - A
CASE STUDY.
A The Problem
1 Prior to 1966 the St. Louis County
Water Company's south county water
treatment plant was not operated at
full capacity. When attempts were
made to increase delivery of processed
water toward full capacity, the intake
conduits delivered only about 75 percent
of design capacity.
2 Inspection of the inner surface of the
two 7, 500 ft. long, 30 in. diameter,
concrete-lined intake lines showed
them to be almost completely covered
with a hard, crusty growth almost one
inch thick,
3 The Hazen-Williams conveyance
coefficient, C, was calculated to be about
100 in 1960 and 95 in 1966. The normal
value for C in a concrete-lined pipe is
about 140. The relative constancy of
this coefficient over the six year period
from 1960-1966 indicates there is a
maximum thickness of sponge material
which can develop within these conduits.
While this maximum probably is deter-
mined by the increased shear within the
conduit, the amount of growth allowed
by such physical characteristics caused
a 25 to 30 percent reduction in the
delivery capacity of the pipes.
B Attempts to Control the Problem
1 Chlormation to chlorine residuals as
high as 10 mg/1 followed by back-
flushing at rates of 5 to 19 mgd did not
remove the growth.
2 Sponge growth was removed in IVIarch
1967 by a commercial pipe cleaning
firm at a cost of 30 cents/lineal ft. of
pipe. This restored the Hazen-
Williams coefficient, C, to 145.
3 By August 1967 this conveyance
coefficient decreased to about 110,
indicating significant re-growth of the
sponges.
4 In October 1967, attempts made to
clean these conduits by a less expensive
procedure using a plastic "pig'' resulted
in an increase of C to 125, indicating
that this procedure removed some but
not all of the attached sponge growth.
5 The rapid re growth of the sponge
material indicated that to maintain
good delivery characteristics within
the raw water conduits they would
have to be cleaned by the commercial
firm at least once and perhaps twice
each year.
C The Bioassay
1 Some of the attached material was
scraped from the pipe, examined
microscopically, and was shown to
consist of Trochospongilla leidvi.
This sponge is particularly obnoxious
in pipes because it produces numerous
basal gemmules which are tenaciously
attached to the pipe surface. It appeared
that the mechanical removal procedure
left a thin layer of the gemmules which
immediately initiated new growth when
the water temperature reached about
50°C.
2 Since the active living sponge tissue is
relatively delicate, it was reasoned that
chlormation at the point of intake would
control the sponge growth. The problem,
then, was to determine the minimum
chlorine concentration necessary to
control the sponges.
2
-------
Causes and Control of Sponges in Transmission Lines
3 Sheets of gemmules were used to
initiate active sponge growth in the
Laboratory where the resulting growth
wis subjected to various concentrations
of chlorine.
4 Microscopic examination of the sponge
growth after chlorination indicated that
5 mg/1 chlorine for an eight hour
period each day should allow control
of the sponge.
D Field Control
1 Chlorine at a concentration of 5 mg/1
is applied at the river intake with a
truck mounted 2,000 lb/day chlorinator
for an eight hour period each day during
that portion of the year when the water
temperature exceeds 50 F. Danger of
flooding of the intake structure during
periods of high water in the river
required that the chlorinators be port-
able. The chlorinators were mounted
on a used two-ton truck and connected
from the truck to the pipe lines by a
two inch pressure hose feeding a
chlorine solution through an insertion
tube into the pipe line. This makes the
chlorination installation completely
portable by disconnecting a boss coup-
ling attached to the two inch hose.
2 When the turbidity of the water exceeds
100 units, chlorination is not required
as the turbidity effectively "sand blasts"
the delicate living sponge tissue.
VI RELATIVE COSTS
A The cost of mechanical removal of the
sponge growth was 30 cents/lmeal foot
of pipe. With two 7500 ft. long conduits
to clean at least twice each year to
maintain some degree of control of the
sponge growth the cost of mechanical
removal would amount to about S9, 000
per year. This estimate includes
only the cost of the physical removal
and does not include the cost involved
in removing valves and metering
elements prior to the physical scraping
of the pipes; nor does it include the
cost associated with reduced capacity
of the plant due to growth and removal
of the sponges.
B The capital cost for the truck, chlori-
nators, and fittings amounted to 36, 425
and the additional labor cost is only
about 5 cents/mg. The cost of chlorine
used for that portion of the year when
the water temperature exceeds 50 F
and the turbidity is less than 100 units
is the only remaining cost and this
varies from year to year.
VIII EFFICIENCY
No problems with sponge growths within
these conduits have been encountered
since the chlorination practice was
initiated.
3
-------
THE NECESSITY FOR CONTROLLING BACTERIAL POPULATIONS
IN POTABLE WATER
Edwin E. Geldreich*
Bacteriological quality of potable water is
generally associated with attainment of the
desired goal for less than one total coliform
per 100 ml of water sample. Yet, there are
many other organisms common to the flora
of finished water whose numbers far exceed
the chance occurrence of one coliform in a
baseline specified at 100 ml. It is true that
there is no single medium, selected temp-
erature, or choice of incubation time that will
insure detection of all organisms present m
water. However, selected segments of this
population can be measured accurately and
their numbers related to water treatment
process efficiencies, to quality deterioration
in the reservoir or distribution lines, to
possible degradation in coliform detection
and to increased chance occurrence of
certain organisms that may become serious
"secondary pathogenic invaders" for de-
bilitated individuals in the community.
I MICROBIAL POPULATIONS IN FILTER
-BET35
The composition of the microbial flora in
potable water is a reflection of the organisms
introduced from the raw source water/*' ^
in the application of reconditioned stored
sand, 3) ancj from bird-droppings over
uncovered filter beds. Thus, it is not
unusal to recover a wide spectrum of organ-
isms from in-use filters, (Table ])including
anaerobic spore formers, indicator bacteria,
saprophytic mycobacteria, salmoneLlae, zoo-
glealstrains, bacteriophages, amoebas, roti-
fers, nematodes, algae, fungi, and actino-
mycetes. (•>> 7¦ 8, 9) This multitudinous con-
glomeration of microorganisms is to' be
expected since the purpose of the filtration
process is to remove more than 99 percent
of these organisms entrapped in coagulant
floes and in naturally occurring turbidity
particles. During the filtration process, not
only is there a substantial entrapment of
microorganisms but also a concurrent
increasing deposit of organic material on
the filter surface and to varying depths
within the compartment or filter bed. It is
"Chief Bacteriologist, Division of Water
Hygiene, EPA, Cincinnati, Ohio 45213
a result of these nutrient additions, plus the
presences of various physical factors and
biological forces, that shape the bacterial
composition and density in the filter bed.
II MONITORING FILTER BED EFFICIENCY
Rapid granular filtration effectiveness not
only relates to minimum filter depth, selected
particle size, and flow rate limits, but also
to careful maintenance of the filter bed.
Filters must be free of mud balls, uneven
depth cracks, and air-binding that blocks
uniform filtration. When these problems
occur, the filtration barrier begins to break
with the appearance of the microbial laden
chemical floe and the raw water sediments
in the filter effluent. In either event, a
protective shield of inorganic or organic
materials transports the adhering or
embedded organisms past the barriers of
treatment and disinfection then on to the
unsuspecting water consumer.
In an effort to anticipate this problem, so
that these filters may be taken out of service
for cleaning and necessary repairs, the
operator may, in part, look for evidence of
a significant bacterial increase over baseline
residual levels of organisms passing into
the filter effluent. Since it is impractical
to seek a measurement of all microorganisms
that might be present, this general bacterial
population data is derived from colony counts
made at 20°C for 48 hours on standard plate
count agar, with a suggested upper limit of
100 organisms per 1 ml of filter effluent. In
recent years, some water plant laboratories
have begun to develop this type of operational
data from pour plate cultures incubated at
35 C for 24-48 hours. The advantage of data
developed from 35 C incubation, lies in the
better correlation with a breakthrough of
substantial numbers of indicator bacteria and
possible pathogens. However, it is questionable
whether monitoring for bacterial breakthrough
alone will adequately correlate with entero-
virus or Endamoeba histolvtica occurrence
in the filter eiliuent.
1
-------
The Necessity for Controlling Bacterial Populations
TABLE 1
BACTERIAL POPULATIONS IN FILTER BED SAND*
Mean Density per gm Sand
Organisms
Surface Sand
Deep Sand
(25-150 mm)
Bacteria
Coliforms
Fecal coliforms
Bacterial plate count (37°C)
Aerobic spores
Anaerobic spores
Protozoa
Celiates and Flagellates
Amoebas
6, 300
75
770,000
430,000
9, 400
41,000
7, 100
110
51
350,000
350,000
5,100
740
1, 900
"Selected data from Tavlor.^
o
-------
The Necessity for Controlling Bacterial Populations
The key to effective bacterial and viral
reduction is in the skillful management of
the operational filters to prevent a floe
breakthrough. Pre-flocculation is essential
for optimum virus reduction. High turbidity
in raw -.vaters, like coagulants in action, does
entrap large numbers of organisms. These
turbid waters (Table 2) should receive suf-
ficient additions of coagulants to flocculate the
range of particle sizes that may be present
and could otherwise pass through the
treatment processes. As an example, only 1 to
22 percent of viruse was removed(lO) when
uncoagulated water containing PoLiovirus
type I was filtered through the recommended
filter media of proper particle size, (0. 78
mm) depth, (61 cm) and flow rate (2 gal I
min/sq.ft.). When Poliovirus type I was
inoculated into pre-flocculated water and
then filtered as previously defined, the
virus removal usually exceeded 98 percent
until the floe broke through the filter.
Apparently, a floe breakthrough of sufficient
magnitude to cause a turbidity of 0. 5 Jackson
units was usually accompanied by virus
penetration of the filter barrier.
Proper development of an adequate filter
media is essential for optimum removal of
amoeba cysts since normal disinfection
practices are inadequate for control of this
pathogen. Granular particle size should be
between 0. 5 and 0. 78 mm and of a minimum
depth of 61 cm (24 inches). Laboratory
experiments with Endamoeba histolytica
inoculated into pre-flocculated but unsettled
water indicated that a 99. 99 percent cyst
removal was possible if filtration rate did
not exceed 2 gallons per minute per square
foot. dD
Coliform bacteria have been observed to
consistently survive in water chlorinated to
provide 0.1 to 0. 5 mg/1 of free residual
chlorine after not less than 30 minutes'
contact simply because these bacteria were
imbedded in turbidity measured from 3. 8 to
84 Jackson units. d^O jn a study of three
waterworks treating surface waters by
chlorination only, coliform bacteria were
detected in the chlorinated samples at only
one of these waterworks. Inspection of
plant data indicated the Great Lakes source
water occasionally contained turbidities as
high as 100 Jackson units. (13) in another
problem, coliform bacteria were found in
one portion of the San Francisco water supply
that was chlorinated to provide substantial
chlorine residuals after hours of contact;
these bacteria survived in the bodies of
small Crustacea livingintnefinished waterd"*'
Enteric pathogens were found in an unfiltered
water supply in Calcutta, India, which was
reported to have been treated with chlorine
for a 10-minute contact time. In this case,
water turbidity was high and contact time
inadequate. Data on the distribution samples
indicated an absence of any residual chlorine
in 49. 8 percent of samples examined, (15)
suggesting a chlorine demand m the turbidity
may have consumed much of the applied
chlorine. These case histories illustrate
the necessity of producing a finished water
with a turbidity limit of 0. 2 Jackson units so
that disinfection will be most effective
against any residual pathogens that may be
present. Therefore, the answer to better
monitoring of filtration efficiency for both
viral and bacterial pathogen removal appears
to be in a continuous monitoring system for
turbidity levels in the filter effluent.
Although bacterial tests for filtration efficiency
are of limited value, special bacteriological
and chemical procedures for examination of
the filter bed may reveal this treatment pro-
cess to be the source of various taste and
odor organisms or their chemical by-products
that affect finished water quality. Future
developments for creating drinking water
from reclamation of wastewater will neces-
sitate critical bacterial monitoring for the
prodigious growth of some bacterial species,
namely, Pseudomonas that occur in or on
treatment processes involving reverse
osmosis, electro-dialysis membranes, sand
filtration, and carbon adsorption processes
embodied in tertiary treatment.
in QUALITY DETERIORATION IN
IMSTftlBU'riOa NETWORKS
The microbiological composition of potable
water transmitted through the distribution
network may not always be of the same
quality produced at the water plant. Finished
water with floe breakthrough or unfiltered
turbid waters receiving only disinfection treat-
ment prior to release to the finished water
reservoirs and distribution mains bring a
myriad of microoorganisms that can be
embedded in particles, or encysted or en-
trapped in Crustacea and nematodes oast the
barrier of aisiruection. d3, 14, 15,16f Open
reservoirs of finished water have been found
to be a source of hazardous pollution from
wild birds, particularly sea gulls, at locations
near coastal areas. (5.17) Biological growths
may frequently develop adjacent to shore lines
of open reservoirs, becoming the source of
taste and odor problems with concurrent
build-up of organic substances that accumulate
either in the reservoir sediment or circulate
to distribution lines. Development of sediment
3
-------
TABLE 2
SURVIVAL OF COXSACK1E VIRUS AND NATIVE BACTER1A IN OHIO RIVER
WATER AT 25°C TREATED BY COAGULATION AND SEDIMENTATION
Teat
Series
Initial Tur-
bidity (J. u. )
A12(S04)3
Dosage (nig/l)
Pinal Tur-
bidity (J. u. )
Coxaackie
virus
Percent Surviving
Coliform
Bacteria
General
Bacterial
Population11
1
00-100
15
5-10
4. 3
36. 2
24. 9
2
16-240
25
1-5
1.4
0. 2
0. 2
3
1G0-24O
25;25b
0. 1
0. 1
0. 01
0. 01
i Selected data from Chang et al. , Amer. Jour. Pub. Health 48, 159 (1058)
a - Standard Plate Count
b - Two-stage Coagulation and Sedimentation, Second stage Coagulant-Fe Cl.^
-------
The Necessity for Controllins Bacterial Populations
pockets and pipe sections encrusted with
chemical deposits form a protected habitat
for sheltering those organisms surviving
filtration and disinfection. Other sources
of a bacterial population injection into the
distribution lines may be more sudden,
coming from plumbing cross-connections,
loss of positive line pressure, plus line
breaks and their repairs. These abrupt
changes destroy the integrity of the finished
water and are frequently found to be
responsible for waterborne outbreaks.
When some microbial entity enters the
distribution network through storage
contamination, low pressure, and back
siphonage, the fate of these introduced
organisms will be tempered by various
environmental factors. These modifying
influences include exposure to any residual
levels of disinfectant, suitable source of
bacterial nutrients in pipe deposits, water
temperature, antagonistic action of other
organisms in the water flora, and sudden
pulsations in water pressure. Abrupt
changes in water pressure may slough-off
sheets of organisms from gelatinous film
deposits of deadend pipe sections
or send high density organism clouds
released from foci of growth in air
chambers to the main flow of distribution
water.19, 20, 21, 22, 23)
In terms of population levels developing
in the distribution network environment,
the microbiological quality can be influ-
enced by three possible conditions: (1) short-
term degradation associated with sudden
pulsations in water pressure; (2) persistent
low-density discharges of specific organisms
acclimated to the network environment; and
(3) high-level quality deterioration from sub-
stantial multiplication within some portions of
the system. Although high-level quality deteri-
oration may occur in a finished water receiving
ineffectual dismfection in the presence of
turbidity, (1°»24) regrowth is more fre-
quently a problem in waters that do not
receive the benefits of any disinfection.
Distribution water quality deterioration can
be temporarily reduced by line flushing and
application of high-dosage levels of disin-
fection. However, if the sources of these
microbial populations are not located and
appropriate action taken, deterioration of
the water quality will soon recur. Some of
the organisms are capable of long-term
persistence in protected areas of the dis-
tribution network where they can undergo
multiplication in the presence of trace concen-
trations of a source of carbon and nitrogen.
These nutrients may be introduced in the
source water receiving minimal treatment, '-0'
inle*sected with chemical treatment in the
processing of potable water or through
localized inputs of nutrients in cross-connection
contamination.
The microbial flora in potable water supplies
is highly variable in numbers and kinds of
organisms. Those bacterial groups most
frequently encountered in poor quality potable
waters include: Pseudomonas, Flavo-
bacterium, Achromooacter, Proteus,
Klebsiella, Bacillus, Serratia, Coryne-
bacterium, Spirillum, Clostridium ~4.rthro-
bacter, "nallionella,ind Leptothrixi0- > 28)
Substantial populations of some of these
organisms occurring in potable water supplies
may bring a new area of health risk to
hospitals, clinics nurseries, and rest
homes.(25, 30. 31, 32, 33) Although Pseudomonas
organisms are generally considered to be non-
pathogenic, they can become a serious
'secondary pathogenic invader" in post-
operative infections, burn cases, and intestinal-
urinary tract infections of very young infants
and the elderly population of a community.
These organisms can persist and grow in
water containing a minimal nutrient source of
nitrogen and carbon. If Pseudomonas becomes
established in localized sections oi the dis-
tribution lines it may persist for long periods
and shed irregular inputs into the consumers'
potable water supply.(34) f0 suppress their
development in protected areas, there must
be continual maintenance of a free chlorine
residual of 0. 3 to 0. 6 ppm in the distribution
system.
Flavobacterium strains can be prevalent in
drinking water and on water taps and drinking
fountain bubbler heads. A recent study of
stored emergency water supplies indicated
that 23 percent of the samples contained
Flavobacterium organisms with densities
ranging from 10 to 26, 000 per 1 ml.
Flavobacterium must be controlled in the
hospital environment because it can become
a primary pathogen in persons who have
undergone surgery.(35)
Klebsiella pneumoniae is another secondary
invader that produces human infection of the
respiratory system, genito-urinary system,
nose and throat, and occasionally this
organism has been reported a.s the cause of
meningitis and septicema.' Klebsiella
o
-------
The Necessity for Controlling Bacterial Populations
penumoniae, like Aerobacter aerogenes, (37)
can multiply in very minimal nutrients that
may be found in slime accumulations in
distribution pipes, water taps, air chambers,
and aerators.
IV COLIFORM SUPPRESSION
The inhibitory influence of various organisms
m the bacterial flora of water may be an
important factor that could negate detection
of the coliform group. (38, 39) strains of
Pseudomonas, Sarcina, Micrococcus.
Flavobacterium, Proteus, Bacillus, Actino-
mvcetes, and yeast have been shown to
suppress the detection of the coliform
indicator group.(40, 41, 42, 43) These
organisms can coexist in water, but when
introduced into lactose broth they multiply
at a rapid rate, intensifying the factor of
coliform inhibition.(44) Suspensions of
various antagonistic organisms in a density
range of 10, 000 to 20, 000 per 1 ml, added
to lactose tubes simultaneously with a sus-
pension of 10 E. coli per 1 ml, resulted in
reduction in coliform detection/41' This loss
of test sensitivity ranged from 28 to 97 per-
cent, depending on the combination of the
mixed strains.
Data from the National Community Water
Supply Study(45) on bacteriological quality
of distribution water from 969 public water
supplies were analyzed (Table 3) for bacterial
plate count relationship to detection of total
coliforms and fecal coliforms. It is interest-
ing to note that there was a significant
increase in total and fecal coliform detection
when the bacterial counts increased up to
1,000 per 1 ml. However, further increase
in the detection of either coliform parameter
did not occur when the bacterial count per
1 ml was beyond 1, 000 organisms. This
could indicate an after-growth of bacteria in
distribution system water or a breakpoint
where coliform detection was desensitized
by the occurrence of a large general bacterial
population that included organisms known to
suppress coliform recovery.
V CONTROL OF THE GENERAL BACTERIAL
POPULATION
Density limits for the general bacterial
population must be related, in part, to a
need to control undesirable water quality
deterioration and practical attainment for
water throughout the distribution system.
This necessity for monitoring the general
bacterial population is most essential in
those supplies that do not maintain any
chlorine residual in the distribution lines,
ana in special applications involving reuse
water and desalinization. This bacterio-
logical measurement would serve as a
quality control on water treatment processes,
and sanitation of distribution lines sections
and storage tanks that could be shedding
various quantities of organisms into the
system, thereby degrading the water quality.
Practical attainment of a low general
bacterial population can best be judged by a
study of data from the National Community
Water Supply Study. Data presented m
Table 4 correlated the general bacterial
population found in 969 public water supply
distribution systems, many of which contained
chlorine residual. Although the number of
samples on each distribution system in this
special study was small, it does reflect
bacterial quality conditions in numerous
large and small water systems examined
in each of the nine metropolitan areas.
These data indicate that the general bacterial
population in distribution lines can be con-
trolled to a value below 500 organisms per i
1 ml by maintaining a residual chlorine '
level in the system. Increasing the chlorine
residual above 0. 3 ppm to levels of 0. 6 and
1, 0 ppm did not further reduce the general
bacterial population by any appreciative
amount.
Any application of limit for the general
bacterial population in potable water will
require a definition of medium, incubation
temperature and incubation time so as to
standardize the population to be measured.
The 13th edition of Standard Methods for the
Examination of Water and Wastewater does
specify these requirements for a Standard
Plate Count (SPC) to be used in collection of
water quality control data. Since many
organisms present in potable waters are
attenuated, initial growth in plate count agar
frequently is slow, thus incubation t^me
should be extended to 48 hours at 35 C.
This time extention will permit a more
meaningful standard count of the viable
bacterial population. Samples must be
collected in bottles previously sterilized
within 30 days and adequately protected from
dust accumulation. Examination for a
Standard Plate Count should be initiated
within 8 hours of collection. This time may
be extended to periods up to 30 hours only
if these samples are transported in iced
containers.
6
-------
TABLE 3
HACTEftlAL PLATE COUNT vs. COLIFOUM DETECTION
IN DISTRIBUTION WATEK NETWORKS FOR 969 PUBLIC WAT 13R SUPPLIES
General nacterial Population*
Total Coliform
Fecal
Coliform
Density Range
per 1 ml
Number of
Samples
Occurrences
Percent
Occurrences
Percent
1 - 10
1401
64
4. 6
26
1.9
11 - 30
522
50
11. 3
25
4. B
31 - 100
500
120
20. 7
46
7. 9
101 - 300
378
89
23. 5
46
1 2. 2
301 - 500
157
48
30. 6
23
14. 7
501 - 1, 000
170
49
28. 8
22
12.9
1, 000
233
62
26. 6
19
0. 5
TOTAL
3441
41)1
207
^Standard Plate Count (40 hrs. incubation, 35°C)
-------
TABLE 4
Control of Bacterial Population in Potable Water
Distribution Systems by Chlorine Residual*
Acumulative
Percenta
ge - Chlorine Residual (m
g/1)
General Bacterial Pop-
ulation per 1 ml**
None
. 01-
. 10
. 11-
. 30
. 31-
. 60
. 61-
1.0
Over
1. 0
Total
Samples
<1 - 10
20. 6
44. 7
64. 0
72. 1
71.7
81. 3
1102
11 - 30
45. 5
63. 4
78. 6
81. 1
80. 0
89. 0
300
31 - 100
63.5
77. 0
87. 3
90. 9
89. 4
93. 6
286
101 - 300
75. 8
87. 9
96. 5
96. 2
93. 9
95. 9
194
301 - 000
81. 8
91. 3
98. 4
97. 0
97. 2
98. 2
82
501 - 1, 000
07. 7
95. 0
99. 7
97. 7
98. 9
99. 5
75
>1,000
100.0
100.0
100.0
100. 0
100. 0
100. 0
133
Total
Samples
860
322
317
265
180
219
2172
*Data from a survey of community water systems in 9 metropolitan areas.
**Standard Plate Count (4(1 hrs. incubation, 3f)°C)
-------
The Necessity for Controlling Bacterial Populations
With maintenance of a chlorine residual ana
turbidity of less than one Jackson unit, the
need for a bacteriological measurement of
the distribution system may become less
critical. For this reason, it is proposed
that such water supplies be monitored rou-
tinely, once each season or every three
months, for baseline data on the general
bacterial population and correlated with
chlorine residual and turbidity measurements
in the distribution lines. It is also recom-
mended that water plant personnel be alert
to unusual circumstances that may make it
desirable to monitor the general bacterial
population more often in a check of water
plant treatment efficiencies.
For these reasons, consideration should be
given toward the establishment of a general
bacterial population limit of 500 organisms
per 1 ml in distribution water. In theory,
limitation of the general bacterial population
to some practical low level would also
indirectly and proportionally limit any antago-
nistic organisms that could suppress coliform
detection and reduce the exposure and dosage
level for health effect organisms that might
be present.
VI SUMMARY
Although coliform detection is the primary
concern in potable water quality measurements,
attention must also be directed toward con-
trolling the general bacterial population. The
bacterial flora in finished water reflects the
characteristic organisms common to the filter
bed. Once organisms enter the distribution
system, they may be harbored in protective
slimes and sediments that develop in some
network pipes. Some of these organisms may
be a factor m suppression of coliform de-
tection and in creating health effects among
the very young, the debilitated, and the senior
citizens of a community. Data from a study of
969 public water distribution systems indicate
the critical level for suppression of coliform
detection occurred when the general bacterial
population exceeded 1,000 organisms per 1ml.
This bacterial population could be effectively
controlled to a value below 500 organisms per
1 ml by maintaining a residual chlorine level
in the system.
REFERENCES
1 Taylor, E. W. "The examination of Waters
and Water Supplies. " 7th edition, The
Blakiston Company (1958).
2 Victoreen, H. T. Panel Discussion on
Bacteriological Testing of Potable
Waters. Amer. Water Works Assoc.
Annual Conference, Washington, D. C.
June 21-26, 1970.
3 Taylor, E. W. 36th Report on the Results
of the Bacteriological, Chemical ana
Biological Examination of the London
Waters for the Years 1953-1954.
Metropolitan Water Board, London.
4 O'Connor, J. T. and Baliga, K. Y.
Control of Bacterial Growths in Rapid
Sand Filters. Jour. San. Eng. Div. ,
Proc. Amer. Soc. Civil Eng. 96:1377
(1970).
5 Taylor, E. W. 42nd Report on the Results
of the Bacteriological, Chemical and
Biological Examination of the London
Waters for the Years 1965-1966.
Metropolitan Water Board, London.
6 Willis, A. T. Anaerobic Bacilli in a
Treated Water SuDply. Jour. Appl.
Bact. 20:61 (1957).
7 Taylor, E. W, 43rd Report on the Results
of the Bacteriological, Chemical and
Biological Examination of the London
Waters for the Years 1967-1968.
Metropolitan Water Board, London.
8 Coin, L. , Menetrier, M. L., Labonde, J.,
and Hannoun, M. C. "Modern Micro-
biological and Virological Aspects of
Water Pollution. " Second International
Conference on Water Pollution Research,
1-18. Pergamon Press (1964).
9 Tennessee Department of Public Health.
"Epidemiologic Studies of Atypical and
Fast Bacilli in Tennessee. " Public
Health Service Grant No. CC00078,
133 pp. (1966).
10 Robeck, G. G. , Clarke, N. A. , and
Dostal, K. A. Effectiveness of Water
Treatment Processes in Virus Removal.
Jour. AWWA, 54:1275 (1962).
11 Bayliss, J. R., Gullans, O. , and Spector,
B. K. The Efficiency of Rapid Sand
Filters in Removing Cysts of the Amoebic
Dysentary Organisms from Water.
Public Health Reports, 51:1567 (1936).
9
-------
The Necessity for Controlling Bacterial Populations
12 Sanderson, W. W., and Kelly, S.
Discussion of "Human Enteric Viruses
in Water: Source, Survival, and
Removability, " International Confer-
ence on Water Pollution Research,
London, 2:523 (1962).
13 Walton, G. Effectiveness of Water Treat-
ment Processes as Measured by Coliform
Reduction. PHS Pub. 898 (1961).
14 Tracy, H. W. , Camarena, V. M. , and
Wing, F. Coliform Persistence in
Highly Chlorinated Water. Jour,
A WW A, 58:1151 (1966).
15 Sen, R. and Jacobs, B. Pathogenic
Intestinal Organisms in the Unfiltered
Water Supply of Calcutta and the Effect
of Chlormation. Ind. Jour. Med. Res. ,
57:1220 (1969).
16 Chang, S. L. , Berg, G., Clarke, N. A.
and Kabler, P. W. Survival and
Protection Against Chlorination of
Human Enteric Pathogens in Free-
living Nematodes Isolated from Water
Supplies. Amer. Jour. Trop. Med.
and Hyg., 9:136 (1960).
17 Alter, A. J. Appearance of Intestinal
Wastes in Surface Water Supplies at
Ketchikan, Alaska. Proc. of 5th
Alaska Science Conference, 81-84,
Anchorage - Sept. 7-10, 1954.
18 Committee on Water Supply, Bacterial
Aftergrowth in Water Distribution
Systems, Amer. Jour. Pub.
Health, 20:485 (1930).
19 Bayliss, J. R. Bacterial Aftergrowths
in Water Distribution Systems. Water
Works and Sewerage, 77:335 (1930).
20 Bayliss, J. R. Bacterial Aftergrowths
in Distribution Systems. Water Works
and Sewerage, 85:720 (1938).
21 Howard, N. J. Bacterial Depreciation of
Water Quality in Distribution Systems.
Jour. AWWA. , 32:1501 (1940).
22 Jewell, A. B. Bacterial After-Growths
in the Distribution System. Southwest
Water Works Journal, 23:13 (1942).
23 Hillmer, H. E. Data Concerning Study
of Water Quality and its Relationship
to New Water Distribution Systems in
Private Homes Which Have Not Been
Chlorinated. Presented to the City
Manager and Common Council of the
City of Fond au Lac, Wisconsin,
Oct. 13, 1970.
24 Nankivell, A. T. The Sand Filtration
and Purification of Chalk Waters. Jour.
Hyg. , 11:235 (1911).
25 Lantos, J. , Kiss, M. , Lanvi, B. , and
Volgyesi, J. Serological and Phage
Typing of Pseudomonas aeruginosa
Invading a Municipal Water suppiy.
Acta Microbiol. Acad. Sci. (Hungarv)
16:333 (1969).
26 Victoreen, H. T. Soil Bacteria and Color
Problem in Distribution Systems. Jour.
AWWA. 61:429 (1969).
27 Lueschow, L. A. and Mackenthun, K. M.
Detection and Enumeration of Iron
Bacteria in Municipal Water Supplies.
Jour. AWWA, 54:751 (1962).
28 Clark, F. M. , Scott, R. M. , and Bone, E.
Heterotrophic Iron-Precipitating
Bacteria. Jour. AWWA, 59:1036
(1967).
29 Culp, R. L. Disease Due to "Non-
pathogenic" Bacteria. Jour. AWWA,
61:157 (1969).
30 Roueche, B. Three Sick Babies. The
New Yorker, Oct. 5, 44:116 (1968).
31 Hunter, C. A. and Ensign, P. R. An
Epidemic of Diarrhea in a New-Born
Nursery Caused by P. aeruginosa.
Amer. Jour. Pub. Health, 37:1166
(1947).
32 Drake, C. H. and Hoff, J. C. Miscellaneous
Infection Section VI - Pseudomonas
aeruginosa Infections; 635-639. OTag-
nostic Procedures and Reagents,
A. H. Harris and M. B. Coleman,
editors, Amer. Pub. Health Assoc. ,
New York, 4th ed. (1963).
33 Maiztegui, J. I. et al. Gram-Negative
Rod Bacteremia with a Discussion of
Infections Caused by Herella Species.
Amer. Jour. Surgery, 1U7:701 (1964).
1 0
-------
The Necessity for Controlling Bacterial Populations
34 A Source of Pseudomonas Infection. New 40
England Jour. Mea. , 274:1430 (1966).
35 Herman, L. G. , et al. Detection and
Control of Hospital Sources of Flavo- 41
bacteria. Hospitals 39:72 (196FT
36 Leiguarda, R. H. and Polazzolo, A. Z.Q.D.
Bacteria of Genus Klebsiella in Water.
Rev. Obr. Sanit. Nac. (Argentina)
38:169 (1956) 42
37 Nunez, W. J. and Colmer, A. R.
Differentiation of Aerobacter-Klebsiella
Isolated from Surgarcane. Appl.
Microbiol., 16:1875 (1968). 43
38 Waksman, S. A. Antagonistic Relations of
Microorganisms. Bact. Reviews,
5-231 (1941).
44
39 Schiavone, E. L. and Passerini, L. M. D.
The Genus Pseudomonas aeruginosa in
the Judgement of the Potability of
Drinking Water. Sem. Med. , (1957). 45
Kliger, L. J. Non-lactose Fermenting
Bacteria from Polluted Wells and
Sub-Soil. Jour. Bact. , 4:35 (1919).
Hutchison, D. , Weaver, R. H. , and
Scherago, M. The Incidence and
Significance of Microorganisms Antago-
nistic to Escherichia coli in Water.
Jour. Bact. , 4b:^9 (1943).
Fisher, G. The Antagonistic Effect of
Aerobic Sporulating Bacteria on the
coli-aerogenes Grouo. Z. Immam
Forsch, 107:16 (195f).
Weaver, R. H. and Boiter, T. Antibiotic-
Producing Species ox Bacillus from Well
Water. Trans. Kentucky Acad. Aci. ,
13:183 (1951).
Reitter, R. and Seligmann, R. Pseudo-
monas aeruginosa in Drinking Water
Jour. Appl. Bact. , 20:145 (1947).
McCabe, L. J., Symons, J. M. , Lee, R. D.
and Robeck, G. G. Study of Community
Water Supply Systems. Jour. AWWA,
62:670 (1970).
11
-------
CONTROL OF IRON AND OTHER NUISANCE ORGANISMS
Robert M. Scott*
Iron bacteria as we shall use the term refers
to bacteria which use iron in their metabolic
functions as well as those whose metabolism
may directly or indirectly affect the state
of iron in water. This takes in a wide
variety of organisms, some of whose meta-
bolic activities have been studied, those
that are readily recognizable by morphology,
and those whose connections are more obscure.
The effects of bacterial deposition of iron in
water are quite apparent. They manifest
themselves by unpleasant taste and odors,
corrosion due to bacterial deposits, and
physical appearance, such as "dirty",
'rusty", or "red waters" causing staining
of laundry, plumbing, etc. Excessive
amounts of iron may cause tastes when used
for cooking and drinking.
For convenience in this discussion we may
break the iron bacteria down into three
separate catagories, as follows:
A True Iron Bacteria.
B Bacteria Whose Function Is Understood
and Whose Metabolism Is Known to Affect
Iron.
C Heterotrophic Iron-Precipitating Bacteria.
True Iron Bacteria
Bergey's Manual of Determinative
Bacteriology (1) classifies the iron bacteria
as follows:
Order II - Chlamydobacteriales
Family I — Chlamydobacteriaceae
Genus 1. Sphaerotilis
Genus 2. Leptothrix
Family in — Crenothrichaceae
Genus 1. Crenothrix
Order I — Pseudomonodales
Family V — Caulobacteriaceae
Genus 2. Gallionella
Family VI — Siderocapsaceae
Genus 1. Siderocapsa
Sphaerotilis (3 species) — These are filamentous
organisms which consist of a trichome
of short cells surrounded by a tubular
sheath. This organism is usually found
in polluted water and uses dissolved organic
material as a food source. In some instances
where the organic material is limited, iron
is deposited in the sheath. This last depends
upon environment more than any physiological
ability to utilize iron. Masses of Sphaero-
tilis sometimes have a brown ironlike ap-
pearance due to iron on the sheath, but the
individual cells are clear. These organisms
can be grown on pure culture agar media,
but bear little resemblance to natural appearing
specimens. Since they are so readily recog-
nizable from water samples by microscopic
examination there seems to be little value in
culturing them, other than for academic
research.
Leptothrix (12 species) — These organisms
have the general appearance of an empty
tubular sheath encrusted with iron or
manganese.
Crenothrix (1 specie) — These are filamentous
bacteria in which the trichome is usually
attached to substrate and surrounded with a
sheath which is usually encrusted with iron
or manganese. They may be found in all
types of water which contain iron salts.
They are generally credited with the ability
to live in an environment which has little
organic material, getting their carbon from
dissolved C02 and carbonates, and their
energy source from the oxidation of ferrous
iron to ferric hydroxide, which is deposited
in the sheath.
The classification of the filamentous iron
bacteria is somewhat confused since so many
of the species described have been observed
in natural conditions and which may have
different appearances due to the substrate
in which they have been growing. Crenothrix
and Leptothrix have not been grown in pure
culture and thus their physiology is in doubt.
Pringsheim(2) studying pure cultures of
Sphaerotilis was able to produce a variety
of forms by varying the amount of organic
material and iron present in the medium.
^Assistant Chief Public Health Microbiologist,
Department of Public Health, Division of
Laboratories
1
-------
Control of Iron and Other Nuisance Organisms
The available data at present indicates that
Crenothrix and Lepiothrix may be environ-
mental variants of Sphaerotilis.
Gallionella (5 species) — These bacteria
appear to have been proved to be true iron
bacteria in that they are autotrophs, deriving
their energy source from the oxidation of
ferrous salts to ferric salts, carbon from
carbon dioxide or carbonate and able to
synthesize its protein from ammonium
salts. These organisms are easily identi-
fied by the spiral twisting of the bands
which hold the cell, giving it a twisted
hair-pin appearance. Wolfe") has been able
to grow these organisms and study their
physiology. He found that calcium was an
essential element in the media. He used
ferrous sulfide as his iron source because
it provided a readily available source of
ferrous iron, and did not oxidize readily
in culture tubes. While he did not state
whether sulfide was necessary as a growth
factor, our experience in Illinois seems to
indicate that where we find sulfate-reducing
anaerobes present in deep water strata we
usually find Gallionella. This organism is
also easily recognized in nature and cultur-
ing is not necessary other than for research.
Siderocapsa (Genus I) - These cells are
spherical and usually are found in groups
of 6-8 embedded in a common capsule.
The capsules may be impregnated with
ferric hydrate or manganese. The mor-
phology can be demonstrated by dissolving
the iron with dilute HC1 and staining with
Gram's crystal violet. These bacteria
manifest themselves with profuse growths
of slime, discolorations of water, fouling of
mains and unpleasant odors.
Physiology of the Filamentous Iron Bacteria
—The physiology of Crenothrix, Leptothrix,
and Sphaerotilis is not too well understood
except that it is generally accepted that they
oxidize ferrous iron to ferric iron and the
resulting ferric hydrate is deposited in the
sheath of the trichome. They are widely
distributed in nature in waters where there
is considerable dissolved iron. They also
are able to oxidize manganese compounds.
They do not appear to be the direct cause
of corrosion, although they seem at times
to be associated with it. They move con-
siderable iron, and it has been estimated
that they can move about 500 times their
cell volume in iron. Most of the iron is
presumed to come from the water system
rather than the distribution pipes.
The iron bacteria are considered aerobic,
but this is debatable if aerobic means
utilization of dissolved oxygen, since in
many cases dissolved oxygen cannot be
demonstrated in the waters from which they
are found.
True autotrophic bacteria can grow and
develop in a medium containing no organic
material, their carbon rea.uirements being
met with dissolved carbon dioxide and
carbonates. It can be presumed that they
derive their energy from the oxidation of
ferrous iron to ferric hydrate by the following
reaction, described by Starkey(4):
4 Fe (HC03)2 t 2 H20 / 02 =
4 Fe (OH)3 / 8 C02
As said before, there seems to be evidence
that Sphaerotilis and Crenothrix may be the
same organism. The work of Pringsheim^-'
corroborates this. In actual practice they
are difficult to separate except that if
encrusted with iron in the sheath it is
Crenothrix and if not, Sphaerotilis. This
is a poor identification procedure, however.
This would then suggest that if an organism
grows in a medium containing organic matter,
the iron would not be used as an oxidizing
agent, whereas the same organism growing
in a medium containing no organic matter could
use ferrous iron, and carbonate for its
growth requirements.
The above described physiology is not com-
pletely accepted and the true mechanism is
not well understood, because of the difficulty
in growing these organisms in pure cultures.
These bacteria seem to be able to stand a
wide variety of temperature variations, from
6 degrees Centigrade to 30 degrees Centigrade;
light does not seem to affect them. The
amount of iron present does not seem to have
any bearing on growth since they occur in a
wide variety of iron concentrations. They
seem to thrive better in acidic waters. This
may be due to the fact that lower pH values
would tend to keep iron in the ferrous state.
They are reported to grow aerobically but this
does not explain their ability to grow in deep
wells where there is no measurable dissolved
oxygen. Baas Becking, Kaplan, and Moore'5)
in a comprehensive study found iron bacteria
existed in nature in a pH range from 2. 0-8. 9.
They also proved that they are "gradient1'
organisms, (&) that is, they develop where
there is a sharp increase in the electrode
potential of the environment.
9
-------
Control of Iron and Other Nuisance Organisms
Bacteria whose function is understood and
whose metaoolism is Known to aliect waler.
Sulfur bacteria — Sulfur Oxidizing bacteria
Order — Pseudomonodales
Family — Thiobacteriacea
Genus — Thiobacillus
There are nine species in this Genus, which
have the ability to oxidize H2S, thiosulfates,
and sulfur to HjSOg and H2SO4. They
tolerate acid conditions growing in pH as low
as 1.5 - 2.0. This low pH would cause cor-
rosion of pipes, and is probably more im-
portant in muds outside of the pipes than in
the pipes.
One Specie of the above group, called
Ferrooxidans, oxidize ferrous iron to
ferric iron autotrophic ally from acid mine
waters, with abjlity to grow in a pH of 2. 0
-3.0. Leathen*'\ working at the Mellon
Institute, isolated and studied these
organisms. This may explain the presence
of precipitated iron in strongly acid mine
waste.
Sulfur bacteria — Sulfate Reducing Bacteria
Order — Pseudomonodales
Family VII — Spirillaceae
Genus II — (3 species)
Desulfovibrio desulfuricans and two related
species have the ability to reduce sulfates to
sulfides. As a result ferrous iron is then
precipitated as black iron sulfide. These
organisms are important (and not uncommon)
in public water supplies because of the
offensive odor they produce and their effect
on the appearance of the water. Also sulfide
is a corrosive agent where the reaction of
the bacteria serves to activate the cathodic
hydrogen by serving as a donator.
Other bacteria not related to iron bacteria
that influence the state of iron in wateF!
Some bacteria may indirectly cause iron
to precipitate or be brought into solution
by altering the chemical composition or
the electro-chemical properties of a water.
These organisms are not related to the
iron bacteria but may produce changes, such
as:
1 Changing the oxidation-reduction
potential (UK)
Higher ORP tends to oxidize iron causing
precipitation, while a low ORP causes
iron to go into solution.
2 Changes in pH
Some bacteria produce ammonia from
decaying organic material. This would
tend to raise pH and thus precipitate
iron. Similarly an increase in COt
would cause iron to go into solution and
a decrease might cause precipitation.
Any environment in which acidity may
be increased would favor solution,
while decrease in acidity would tend to
precipitate iron.
3 Slime and encapsulated bacteria
Some bacteria growing under certain
conditions form large amounts of slirne
and others form capsules. It is entirely
possible that iron will absorb on the
capsule surface causing "zoogleal masses
of bacteria encrusted with iron. " Evi-
dence that this may happen is that the
brown iron rust impregnating the cells,
sometimes exhibits the prussian blue
reaction and sometimes not.
Heterotrophic iron-precipitating bacteria
This term is applied for convenience to any
bacteria which will effect a precipitation
of iron bound in a chelate or organic complex.
In studying zoogleal masses of bacteria en-
crusted with iron, and iron slimes where no
filamentous iron bacteria could be demon-
strated, Clark, Scott, and Bone isolated three
organisms—Serratia indica, Aerobacter
aerogenes, and Bacillus pumilus—that could
precipitate iron from a solution containing
ferric ammonium citrate. It was found that
the capsular coating was much enlarged and
the iron was fixed in the slime layer. It
was hypothesized that the bacteria utilized
the organic portion of the complex and the
iron remained fixed in the capsule and pre-
cipitated out of solution in the bacterial cell.
This could happen with most "humic" acids
formed by decomposition of vegetable matter.
Control: A sudden change of pH has been
found to be effective. If organisms are
adapted to a certain pH, a sudden change
will generally cause death to cells. When
the cells become adapted to this, a change
again will destroy more cells. Usually a
this can control the growth of a bloom. , ^
* . to ^
3
-------
Control of Iron and Other Nuisance Organisms
Flushing of hydrants will be helpful in pre-
venting accumulation of the slime or
filamentous forms. If this can be done by
surging, it is more effective.
Intermittant Chlorination: A method that has
been used with success is to heavily chlori-
nate (2-3 PPM) for about 24 hours and then
stop chlorination for 2-3 days, then heavily
chlorinate again with a resting period followed
by still a third chlorination. Surge flushing
should be carried out in the "resting stage'
as far as practical. The theory is that the
chlorination dose will destroy germinating
spores before they have a protective sheath
formed, then the sheaths will send forth
their spores and again be destroyed by
chlorination.
Removing Food Source: This of course is
one ol the primary steps. Waters not
exceeding 0. 3 mg/1 rarely have iron growths.
Conventional iron/removal methods will
accomplish this.
Wells: Deep wells containing iron growths
can often be controlled by heavy chlorination.
Sulfide bearing waters are usually accom-
panied by profuse growths of gallionella.
Copper has been used to control this, but
as far as my knowledge goes is not
recommended.
Conclusion: When iron slimes are noted it
is important to first identify the type of
organism before treatment is considered.
It can become a serious problem if it
develops since these organisms can impart
a very obnoxious taste and appearance in
the water.
REFERENCES
1 Bergeys Manual of Determinative
Bacteriology, 7th Edition.
2 Pringsheim, E. The Filamentous
Bacteria Sphaerotilis, Leptothris,
Clonothrix, and Their Relation to
Iron and Manganese. Phil. Tran.
Roval Sac. London Ser. B 2881453
1949.
Wolfe, R. S. Cultivations, Morphology,
and Classification of the Iron Bacteria.
JAWWA50 1241 (1958).
Starkey, Robert L. Transformation of
Iron by Bacteria in Water. JAWWA
Vol. 37 No. 10, Oct. 1945.
Baas Becking, L. G. M. , Kaplan, I. R.
and Moore, D. Limits of the Natural
Environment in Term of pH and
Oxidation Reduction Potentials. 'Journal
of Geology, Vol. 68, No. 3, May 1960.
Baas Becking, L. G. M. , Word, E. J. ,
and Kaplan, I. R. Iron Bacteria as
Gradient Organisms, (communication)
Leathen, W. W, , Mclntyre, L. D. and
Braly, S. A. A Medium for the Study
of the Bacterial Oxidation of Ferrous
Iron. Science 114:280 (1951).
Weart, J. G. and Margrave, G. E.
Oxidation-Reduction Potential Measure-
ments Applied to Iron Removal. Jour.
A WW A, 49:1223 (Sept.. 1957).
Scott, R. M. Studies on Rantoul, Illinois
Distribution Growths, unpublished,
1949.
Scott, R. M. Experiments on Iron
Precipitating Bacteria, unpublished,
1938-1939.
Electon Microscopy of Gallionella Ferrguinea.
Jour, of Bact. , Vol. 72, No. 2, pp 248-
252, 1956.
Standard Methods for the Examination of
Water and Waste Water, 11th Edition.
Clark, F. M., Scott, R. M., and Bone,
Ester. Heterotrophic Iron-Precipitating
Bacteria. Jour. AWWA, Vol. 59,
No. 8, August 1967.
3
4
5
6
7
8
9
10
11
12
13
4
-------
CONTROL OF NON-INDICATOR MICROORGANISMS
Hugo T. Victoreen*
I THE SIGNIFICANCE OF NON-INDICATOR
MICROORGANISMS
In recent years water utilities and public
health agencies have relied almost entirely
upon the frequency of coliforms as an indicator
of the bacterial quality of water. This prac-
tice assumes that bacteria capable of causing
disease will be under control when the number
of coliforms is under control.
Even if we make this highly questionable
assumption, most of us recognize the impor-
tance of another group usually described as
"nuisance organisms". A still larger group
has received virtually no attention because we
have been under the mistaken impression that
they are unrelated to water quality. These
"non-indicator" organisms may indeed
indicate trouble for a distribution system. At
the very least they indicate the decline of the
chlorine residual, and some of us find them
implicated in discolored water problems,"'
tubercle formation, (2) and odor problems, (3)
not to mention any direct relationship to public
health. (4, 5)
which any chlorine residual ceases to be
effective is frequently revealed by the
appearance of nitrfcte nitrogen, a product
of bacterial activity. Nitrite can not
coexist with free chlorine, but it can exist
in the presence of chloramines. Since
nitrite is apt to appear quite early in systems
carrying chloramines, the alleged persistence
of a combined residual is at least partly
illusory.
The reduction of chlorine in the distribution
system is caused not only by impurities
in the water, but by the interior surface of
the mains. This is most pronounced in
small mains where the ratio of surface to
volume is high. Cast iron itself is oxidi-
zable and the corrosion process liberates
soluble ferrous iron which exhibits a
chlorine demand as it is oxidized to in-
soluble ferric iron. The installation of
lined mains, the cleaning and relining of
old mains, and the production of a relatively
non-corrosive water will all assist in the
extension of a chlorine residual farther out
into the distribution system.
H CONTROL BY CHLORINATION
It must be kept in mind that free chlorine is
not just a disinfectant but an effective oxidizing
agent as well. Like all oxidizing agents it
decreases in concentration if it has the
opportunity to react with reducing substances.
If a free chlorine residual disappears in the
first several hours after entering the dis-
tribution system, it means that the water
contained, or was in contact with, enough
oxidizable material to destroy this residual.
This is not failure. This is the first step
toward success. We must go on to satisfy
the chlorine demand of the water and minimize
the chlorine demand of the main itself.
The addition of ammonia will convert a free
chlorine residual to a combined residual and
the chloramines thus formed will persist
for a longer time because they are weaker
oxidants. This might be justified if it were
not for the fact that ammonia is a bacterial
food and is always added in excess of the
theoretical ratio. The point in the system at
IH CONTROL BY NUTRIENT LIMITATION
All microbes on our planet must oxidize
reduced substances in order to obtain the
energy to drive their life processes. This
is even true of algae during the hours of
darkness. It is therefore useful to subject
the water in our purification plants to
extensive oxidation thus reducing the food
value of the material contained in this water.
This means that we will be practising free-
residual chlorination perhaps assisted by
potassium permanganate or chlorine dioxide.
At the Wilmington Water Department we
have found that prechlorination and perman-
ganate oxidation is most effective if we
can carry it out at a pH above neutral. In
doing this we seem to precipitate certain
amorphous organic materials which are
then incorporated in the floe. If we floccu-
late below neutral and then raise the pH
after filtration, we accumulate much more
organic debris in our mains and this
material is teeming with bacterial life.
* Laboratory Director, Wilmington Water
Department, Wilmington, Delaware
1
-------
Control of Non-Indicator Microorganisms
In order to satisfy the chlorine demand of
the raw water, a certain minimum retention
time is required. This is particularly true
in the winter months when temperatures are
low and reactions are slower. It is unfort-
unate that the engineers who have designed
our purification plants have not always
allowed for this.
Many soluble organic materials not removed
by oxidation and flocculation can be adsorbed
with activated carbon. Although carbon can
be used at various points in treatment, it is
most effective when used just ahead of the
filters. We stand the best chance of adsorb-
ing partly oxidized substances and substances
not amenable to oxidation if fresh carbon
surface is available at this point.
The production of a finished water approaching
the A WW A turbidity goal of 0.1 Jackson unit
also helps to maintain the finished water
chlorine residual. In summary, we can say
that all of those characteristics which we
associate with a high quality product are the
same characteristics which will minimize
chlorine loss and bacterial growth.
IV RECHLORINATION
In the real world where we all must labor,
there are old mains which have not been
relined, occasional lapses in treatment, and
plants operating beyond their capacity. It is
therefore necessary to rechlorinate at
suitable points in the distribution system.
Booster stations where water must be
repumped to higher pressure zones are
utilized for this purpose at three Wilmington
locations.
The most difficult situation confronting us is
the presence of an open finished water
reservoir in one of the Wilmington systems.
It is 21 feet deep with 260, 000 square feet
of surface area receiving pollen in the
spring, falling leaves in the autumn, trash
from children, particulate fall-out from the
urban atmosphere, and periodic visits by
sea gulls. Originally designed as a two
basin draw and fill reservoir, we have modi-
fied the piping so as to achieve a considerable
amount of circulation. We also rechlorinate
the water leaving the reservoir to an average
residual of 0. 8 ppm. This has improved the
water entering the distribution system from
the reservoir, but the changes we have made
at the plant pumping to this reservoir seem
to have been even more effective. These
changes are as follows:
A Free residual prechlorination frequently
aided by permaganate so as to produce
an in-plant residual which is 80 to 907:
free after 90 minutes contact.
B Flocculation at a pH of 7. 4 to 7. 8 and
maintaining this pH into the finished
water.
C Continuous use of activated carbon
ahead of the filters.
D Rechlorination after filtration to a free
residual of 1 ppm.
The phosphate content of the water provided ^ .
to this reservoir is held below 0. 04 ppm and . \ '
algae growth is stimulated when it rises . S
above this level. In spite of these efforts ^ \
it must be conceded that an open reservoir
is a liability when compared to covered
storage.
V DETECTION OF BACTERIAL GROWTH
The presence of bacteria is most conveniently
shown by an agar plate count and the tryptone
Glucose Yeast formula described in
Standard Methods^ is excellent for the
purpose. It is a broad spectrum medium
capable of growing a high proportion of the
miscellaneous bacteria present in tap water.
Comparative studies in our laboratory reveal
that an even higher proportion of the live
bacteria present in water can be recovered
using an agar less rich in total nutrient, but
containing a small amount of organic iron.* '
The 35°C 24-hour incubation described m
Standard Methods is not to be recommended,
for many bacteria simply will not grow to
colony size under these conditions. Incu-
bation should be at the temperature of a
warm room (24 to 29°C) for a minimum of
5 davs. ^
\
~ -r
If our agar plates are placed in an air tight j y £
container wherein a smokeless candle is ^
left burning, the candle will soon go out 1
and almost devoid of oxygen. Bacteria V~*
capable of growing to colony size under these
conditions can be found in water samples from
outlying sections of the distribution system
not carrying a chlorine residual.
For research purposes it is possible to
confirm plate count results using a Most
Probable Number method. The procedure
is the same as that used to enumerate
coliforms except that the broth employed
9
-------
Control of Non-Indicator Microorganisms
is a dilute Tryptone Glucose Yeast medium.
A positive tube is one which shows bacterial
turbidity after 5 days at 28°C. The tubes are
examined at the time the corresponding agar
colonies are counted and good agreement
usually results. This method is inexact but
very sensitive and is apt to detect anaerobic
as well as aerobic bacteria.
VI THE FEASIBILITY OF CONTROL
Wilmington has now extended a summertime
chlorine residual to an estimated 80% of its
service territory and the wintertime residual
extends slightly farther.
We have found that immediately beyond the
area protected by the chlorine lies a pioneer
zone characterized by the first appearance of
nitrite nitrogen and by a group of very hardy
bacteria. We feel that these bacteria are
attached to the main and dependent upon the
flow of the water for their nourishment.
Because their growth is nutrient limited, a
moderate increase in rate of flow actually
increases the growth rate.
Beyond this pioneer zone lies another which
we call the zone of accumulation. Here we
find the soft deposits which give rise to
customer complaints when disturbed. They
contain hydrated iron and manganese oxides,
organic matter which may in fact be dead
bacteria, and live bacteria of various types.
In addition to the control measures already
described, hydrant flushing should be con-
sidered. Flushing only in the zone of
accumulation gives temporary relief, but a
program which begins in the area showing
chlorine and works its way outward has a
more persistent effect since it may advance
the "chlorine front" into the pioneer zone.
It is now difficult to find any section of , x
Wilmington where the water will yield Q
bacterial plate counts of 5000 per ml. even
during the prime bacterial growth months
of May and June. Seven years ago when
our control program began, such counts
were common over wide areas and counts
of 50, 000 to 100, 000 per ml were not un-
known. We expect to continue the control
measures described above and we recommend
them to other utilities as being well worth
the effort.
REFERENCES
1 Victoreen, H. T. Soil Bacteria and Color
Problem in Distribution Svstems.
Jour. AWWA 61:9:429 Sept. 1969.
2 Maurelli, E. S. et al Loss in Capacity
of Water Mains. California Section
Committee ReDort. Jour. AWWA
54:10:1293, October 1962.
3 Silvey, J. K. E. Effect of Organisms
—Taste and Odors. Jour. AWWA
58:6:706 June 1966.
4 Culp, Russel L. Disease Due to "Non-
pathogenic Bacteria" Jour. AWWA
61:3:157 March 1969.
5 Hudson, Herbert E. High-Quality Water
Production and Viral Disease. Jour.
AWWA 54:10:1265.
6 Standard Method for the Examination
of Water and Waste Water. APHA,
AWWA and WPCF, New York (12 Ed.
1965)
7 Frerer et al, Canadian Journal of
Microbiology 9:420, 1963.
3
-------
SAN FRANCISCO EXPERIENCE WITH
NUISANCE ORGANISMS
Harry W. Tracy-
I INTRODUCTION
In order to have a clear picture of the problems
in the San Francisco Water Supply system by
nuisance organisms an understanding of the
basic system should be helpful.
The largest quantity of water is produced in
the Hetch Hetchy watershed, located in Yose-
rrnte National Park. This water is transmitted
some 150 miles by tunnel and pipe to the San
Francisco Bay Area, where the water not sold
enroute is discharged into Crystal Springs
Reservoir. From Crystal Springs Reservoir,
water either flows by gravity to the lower
elevations of the San Francisco Peninsula and
the City proper, or is pumped to nearby San
Andreas Reservoir. This latter reservoir
supplies the higher areas. The capacity of
Hetch Hetchy reservoir is 117 billion gallons,
Crystal Springs 22. 5 billion gallons and San
Andreas 6 billion gallons. All water flowing
from the local reservoirs is chlorinated and
fluoridated along with the customary copper
sulphate treatment for algae control of surface
waters.
Two other parts of the system should be
mentioned, although these waters have not
been involved particularly with nuisance
organisms. On the East Side of San Francisco
Bay the San Francisco Water Department
owns and operates two large reservoirs San
Antonio and Calaveras. The water from these
reservoirs passes through the 80 m. g. d. dual
media, Sunol Valley Water Filtration Plant
capable of complete treatment.
the San Francisco system and briefly relate
what was done about them, if anything.
The following described outbreaks of nuisance
organisms occurred during the period 1934 to the
present.
II AQUATIC ACTINOMYCETES
During Spring of 1956 there appeared in the
distribution system increasing incidents of
earthy taste and odor complaints, many by
our staff. Even the management was of the
opinion that the water was becoming poorer
and poorer; so a graduate student in micro-
biology was hired for the summer to investigate
the possibility of actinomycetes being the
source. Positive results were obtained from
many samples of water but a definite pattern
could not be established that would implicate
any part of the system.
Before finite data could be established the
need to raise our chlorine residuals to combat
persistant coliforms became our overwhelming
problem, and in the course of events chlorine
taste complaints took the place of earthy and
woody complaints. No doubt the chlorine
residuals of 1 mg/1, and better distribution
maintenance with regards to water quality-
were responsible for the control of these
organisms.
Those having complaints of this nature are
referred to the many articles in the Journal
by Professor. J. K. G. Silvey and his
co-workers.
Ill BRYOZOAN
All watersheds tributary to Crystal Springs
and San Andreas Reservoirs are owned by
the Water Department and only seven water-
shed keepers live on the 2 5,000 acres. The
other reservoirs are quite remote and human This outbreak occurred during the spring
activity is small compared to the area of 1956.
involved.
A routine complaint was turned over to the
The distribution and transmission system Department Laboratory for inspection. A
within San Francisco consists of eighteen customer has complained that little black
pressure zones; ten large covered reservoirs specks were caught in her nylons after
with capacities ranging from 2. 5 to 176. 7 washing. Inspection showed that they were
million gallons; 1, 163 miles of pipe and the coming from the tap and flushing of nearby
usual pumping stations, valves, etc. The fire hydrant produced clusters of the black
goal of this presentation is to describe the organisms up to the size of a quarter,
problems caused by nuisance organisms in
•'-Manager Purification Division, San
Francisco Water Department
1
-------
San Francisco Experience with Nuisance Onanisms
The black specks were identified as statoblasts
of Bryozoan, and Whipples description of a
Brooklyn Water Department outbreak in 1897
really shook the staff up. His description
states, "In a number of instances this material
stopped up the taps, and even large pipes were
choked". Great black masses of these
statoblasts could be imagined blocking our
pipes and valves.
The area for several blocks around was
flushed until no further statoblasts were
observed. In spite of a close inspection of
other parts of the system, including distribu-
tion and impounding reservoirs no additional
organisms were found. In fact these Bryozoan
have never been encountered again.
In the laboratory, attempts were made to
germinate the statoblasts to no avail and
it was assumed that the organisms were dead.
Several months later a test tube of statoblasts
in water which had laid on a tabie observed to
have formed colonies.
Although the San Francisco Water Supply has
experienced only the one outbreak, Bryozoan
are a common nuisance in water systems.
Prokopovich and Hebert'3) described a problem
in California's Delta-Mendota Canal and
Whipple also described outbreaks in Hartford,
Connecticut and Boston, Massachusetts.
IV CHLOROPHYTA: BULBOCHAETA AND
SPIROGRYRA
Although plankton net catches from the large
Hetch Hetchy reservoir contain high counts
of Crustacea the reservoir has never required
copper sulphate treatment.
During November 1964 a number of complaints
from the Department wholesale customers
were received, stating water meters were clogging
with a growth. At the time both Hetch Hetchy
and Calaveras Reservoirs were supplying the
Bay Division Lines and Department personnel
jumped to the conclusion that the growth
must be originating in Calaveras Reservoir
which was not filtered at this time and routinely
needs copper sulphate treatment. Accordingly
Calaveras was shut down and all customers
were supplied from Hetch Hetchy, but meters
continued to clog. Further investigations were
made and the trouble traced to the source
which was a short section of the Tuolumne
River where the Hetch Hetchy water ran in the
river a distance of twelve miles before entering
the tunnel and pipe system.
As this section of the system was in the
Yosemite National Park, permission to treat
parts of the river with copper sulphate was
requested of the Department of Interior, and
tests were run at the nearby Moccasin Fish
Hatchery. These tests showed treatment
could be effective without killing trout.
Prior to the conclusion of the above tests a
telegram was received from Washington
which read "Technicians this Service and
Bureau of Sport Fisheries and Wildlife
consider knowledge about use of the chemical
inadequate at this time to insure preservation
of ecological conditions based on potential
threat to the river's native aquatic organisms
and possible fish kill. We cannot grant
approval for this program. "
As the purpose of the treatment was to kill
some of the native aquatic organisms, the
project to copper sulphate the river was
abandoned without argument, but two small
regulating reservoirs were treated.
Luckily the organisms disappeared throughout
the system and have not reoccurred. Since
the above incident, this section of river has
been by-passed by a tunnel and the water
from Hetch Hetchy Reservoir enters the pipe
and tunnel system directly.
V HYDROIDES
Within the boundries of the City and County
of San Francisco lies the 2-j billion gallon
Lagana de la Merced or as the Water
Department calls it, Lake Merced.
Lake Merced, formerly connected with the
Pacific Ocean by a Channel which was closed
sometime between 1869 and 1894 is now a
freshwater lake and was used for domestic
water purposes from 1895 to 1932 at which
time it was placed on a standby basis. The
lake has characteristic freshwater fauna,
except for five species of definitely marine
affinities. The organism which could give
trouble in the system if this source were
ever used again is Cordylophora lacustris
(Allman) (Coelenterata Hyaroidea).
Hydroides were first noticed by the author
when the City's boating concessionaire
complained of growths covering the bottoms
of his row boats. This w-as solved by using
a non-fouling paint, but one cannot help
speculating on the problems which would be
encountered if an earthquake severed the
2
-------
San Francisco Experience with Nuisance Organisms
normal water supply lines to the city, in
which case Lake Merced would be the sole
source until repairs were made.
An experimental microstrainer was installed
to treat water from crystal Springs Reservoir
in conjunction with coliform investigations in
1963 and after a number of months of operation
hydroids established themselves in the tanks.
Although this organism did not cause opera-
tional difficulties, it was unsightly and the
tanks had to be manually cleaned. Strangely
these organisms have never become established
in the distribution system even though there
are distribution tanks and reservoirs in the
system with approximately the same light
intensity. It can only be assumed that the
chlorine residual is keeping the hydroids
from becoming a problem.
VI MYRIOPHYLLUM OR EURASIAN
WATER MILFOIL
Over a number of years this fern like plant
growth has been a problem in a 13.5 million
gallon open distribution reservoir within the
City of San Francisco. Every two to three
years the growth would be so abundant that
the weed would require harvesting by dragging
large rakes across the reservoir. Of course,
the solution was to clean, line and roof the
reservoir which was done in 1960.
Myriophyllum has now thoroughly established
itself in San Andreas Reservoir and because
of its size, 550 acres, it is impossible to
control it by dragging. The only effective
control has been to lower the reservoir and
let the banks dry out killing the surface
growth but, of course, not the roots and the
following year if the reservoir water level
remains relatively constant the cycle is
repeated.
With the construction of an 80 m. g. d. water
treatment plant employing coagulation, sedi-
mentation and dual media filtration, problems
are anticipated when the stems fragment
break off and either settle in the basins or
mat the filters thus shortening the filter runs.
This will at least transfer the problem from
the water consumer to ourselves. At the
present many consumers find the small fern
like leaves in their tap water and become
most unhappy when told they are not receiving
filtered water and there is nothing that can
be done to resolve the problem; although if
the problem seems severe and localized, the
mains in the area are flushed.
This summer some experiments are to be
performed utilizing a blanket of air bubbles
rising around the intake structure in an
attempt to keep the floating debris from
being sucked into the intake adits.
San Francisco is not alone with this problem
as the Watermilfoil has established itself in
reservoirs throughout the country. The
TWA project has fought the problem for
over ten years. If you are confronted with
this weed and your management asks where
it came from, Smith of the Vector Control
Branch TV A states, "We suspect that the
original TVA milfoil infestation in Peny
River embayment of Watts Bar Reservoir
started either intentionally or otherwise
from a misplaced fish bowl plant. From
such a modest beginning, it thrived but
remained unnoticed until its amazing spread
began to interfere with fishing and boating.
Within a very short time (probably 3 to 5 years)
it embraced about 1, 500 acres.
VII PLANKTON
This group of organisms give trouble in almost
any unfiltered surface supply by being observed
by the consumer as a white swimming
speck in a glass of water; by dying off in the
distribution piping and adding to the organic
load or, as has been found in San Francisco,
protecting coliform bacteria from the action
of chlorine. The author presented a paper
before this Association in 1966 detailing this
problem wherein Cyclops, Daphnia, etc. were
so protecting the coliform bacteria that 5 tubes
positive could be obtained from samples which
had chlorine contact times of 2 hours at a free
residual of 1. 4 mg/l. '6'
To the present time this problem continues
and laboratory tests confirm the original
hypothesis. Since the publication of the
original paper, discussions with the operators
of water systems similar to San Francisco's
also support this thinking.
VIII SLIME ORGANISMS
Hetch Hetchy water began to flow into the San
Francisco Water System in October 1934,
carrying a flow of 45 m. g. d. Part of this
flow, 16 m. g. d., was diverted into a 36-inch
wrought iron pipe that had been laid in 1887.
Three weeks after this diversion the carrying
capacity started to drop off at the rate of 0. 2
m. g. d. After another three week period the
3
-------
San Francisco Experience with Nuisance Organisms
flow decreased from 16. 2 to 13. 6 m. g. a. a
capacity loss of 15 per cent. Tests failed
to indicate any air pockets or other obstruc-
tions along the line and in March 1935 the line
was opened for inspection. A slimy, gelatinous
growth was found, light brown in color covering
the entire inner surface of the pipe from 1/8
to i inch thick.
Microscopic examination showed this growth
to contain the iron bacteria Crenothrix and
the sulphur bacteria, Beggiatoa.
Experiments were performed utilizing 4-inch
pipes, varying flows and chemical dosages.
Hydrated lime to raise the pH of the water to
10; and copper sulphate treatment did not
produce beneficial results. Treating the
water with l-§ lb of ammonia and 6 lb of
chlorine per m. g. was effective in controlling
the growth.
A large chlorination plant was then built and
growths have been controlled since by this
treatment with the only change being the use
of chlorine alone. 3" This use of'chlorine
alone has been effective probably due to the
higher flows of 100 - 275 m. g. d. now required
by consumption. Generally only a slight
taste or odor of chlorine is encountered.
At least once since the initial start of treatment
an outbreak of slime has occurred and this
was caused by lowering the chlorine dosage
below the required level for 100% control.
A visual inspection was made by shuting down
the tunnel system. This inspection showed
strings of mucoid slime hanging from the
tunnel ceiling. Heavy chlorination cleaned
the tunnel and maintenance of . 5 to . 75 mg/1
chlorine residual keeps the tunnel system
clean of these slime organisms.
IX FRESHWATER SPONGES
During July 1961 water being withdrawn
from San Andreas Reservoir through Outlet
No. 3 had a very objectionable odor. The
outlet system was drained and visual
inspection showed large freshwater sponges
growing on the walls of the 72-inch bitumastic
lined steel pipe upstream from the point of
chlorine injection. None of these growths
could be found downstream from the point
of chlorination but numerous growths could
be found all the way upstream to the adit
shut-off valve. No growths were ever found
on the intake screens.
Microscopic examination showed this growth
to be a freshwater sponge. Because growths
could not be found below the point of chlorine
injection the solution abviouslv was to move
the point of chlorine injection as far upstream
as possible, namely 1276 feet. Crews brushed
the walls of the pipes free of growths, the
line was flushed and placed back in service.
Funds were budgeted for a new chlorine
station and prior to its being placed into
service the pipeline was again inspected, and
found to have sponge growths, this required
another cleaning and the new chlorine station
was then placed in service. No problems have
been encountered since.
Strangely, there are two other outlets from
this same reservoir and neither of these two
have ever been found to have a sponge problem
although all points of chlorine injection
originally were about the same. Possible the
different velocities in the three lines could be
a factor, but one line has a greater velocity and
the other a lower velocity.
The author was interested to learn of a similar
outbreak which was observed during 1966 in
the St. Louis County Water District. Those
with a particular interest in freshwater sponges
and the resultant problems are particularly
referred to the article in the Journal by King,
Ray and Tuepker. This article goes into
scientific description of the freshwater sponge
Trochospongilla Leidyi and their methods of
control with chlorine, and there is no need to
repeat them here.
X SUMMARY
As San Francisco's experience with the
common blue-green and green algae is typical,
this paper has not included any discussion of
these algae. Although the local impounding
reservoirs do have growths which are easily
controlled through the normal bluestone
treatment, in a few instances the more
exotic organisms just vanished before control
methods were instituted.
Operators needing assistance in identification
or additional references are referred to the
work of Ingram and Bartsch^^O as excellent
source material.
Almost all the nuisance organisms encountered
in the San Francisco Water Supply System have
been susceptible to control by chlorination but
the resulting complaints of excessive chlorine
taste must be accepted. These complaints
become less numerous as water users become
accustomed to the chlorinous taste. The ultim;
goal, of course, is filtration and judicious use
of post chlorination.
4
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San Francisco Experience with Nuisance Organisms
REFERENCES
1 Silvey, J. K. G. and Roach, A. W.
Laboratory Culture and Odor Producing
Aquatic Actinomycetes. Journal AWWA,
51:20 (January 1959).
2 Microscopy of Drinking Water, by G. C.
Whipple. Revised by G. M. Fair and
M. C. Whipple (4th ed., 1927).
3 Prokopovich, N. P. and Herbert, D. J.
Sedimentation in the Delta Mendota
Canal. Journal AWWA, 57:375
(March 1965).
4 Miller, R. C. The Relict Fauna of Lake
Merced, San Francisco. Sears
Foundation: Journal of Marine Research.
17:375 (November 1958)
5 Smith, Gordon E. Eurasian Watermilfoid
(Myriophyllum spicatum) in the
Tennessee Valley. Paper presented at
the meeting of Southern Weed Con-
vention. Chattanooga, Tennessee.
(January 1962).
6 Tracy, H. W. et. al. Colifom Persistance
in Highly Chlorinated Waters. Journal
AWWA 58:1151 (September 1966).
7 Arnold, G. E. Crenothrix Chokes Conduits.
Engineering News - Record (May 23,
1936).
8 Arnold, G. E. Tesla Portal Chloramination
Station. Water Works and Sewage
(April 1938).
9 King, D. L. et. al. Freshwater Sponges
in Raw-water Transmission Lines.
Journal AWWA 61:473 (September 1969).
10 Ingram, W. M. and Bartsch, A. F.
Operator's Identification Guide to
Animals Associated with Potable Water
Supplies. Journal AWWA 52:1521
(December 1960).
o
-------
SUPPLEMENTARY MATERIAL
-------
MEANINGFUL APPLICATION OF BACTERIOLOGICAL DATA IN
POTABLE WATER SURVEILLANCE*
Edwin E. Geldreich**
The bacteriological quality of potable water
produced in water treatment and its subsequent
distribution is most readily monitored by the
search for coliform bacteria. Occurrence of
these organisms does signal some possible break
in the protective barrier to water-borne
pathogens. Analysis of data gathered in
our laboratory evaluation program indicates
(Table 1) that State Health Laboratories and
municipal water plant laboratories are
examining over 1. 6 million samples of
potable water each year from an estimated
20, 000 public water systems and 21 million
individual water supplies. This total number
for potable water examinations must be
closer to 3. 5 million since data from labora-
tory evaluation programs in the heavily
populated states of California, Hawaii,
Massachusetts, New York, Oregon and
Pennsylvania are currently not available
to the Federal laboratory evaluation service.
Sampling Frequency
These statistics are somewhat misleading in
terms of reflecting the degree of monitoring
of public water supplies since such numbers
do include substantial requests to State
Health Laboratories by citizens seeking
approval of their private well or cistern
water supply. Sampling frequency or public
water supplies has been based on a minimum
monthly number keyed to the population
served by a given water supply; thus requiring
fewer bacteriological samples from smaller
supplies.'1' Ironically, it is the water
systems serving populations of less than
50, 000 people that are more prone to show
unsatisfactory bacteriological results. Data
(Table 2) collected from the National Community
Water Supply Study(2' of 969 public water
supplies illustrates our particular concern
about bacteriological quality of potable
supplies serving populations of 10,000 or
less. For it was from 50 percent of these
smaller supplies that a history of unsatis-
factory bacteriological results could be
detected. Partly because of insufficient
personnel and program funds, the same study
revealed (Table 3) that 69 percent of the 969
water supplies received surveillance through
only half the minimum number of monthly
samples as recommended by the Public Health
Service, Drinking Water Standards.
TABLE 1
Magnitude of Bacteriological Examinations».
Laboratory
Number of
Laboratories
Total Examinations
Per Year
State
66
910,902
Municipal
240
678,805
Private
26
30,639
~No data currently available trom municipal and private laboratories
evaluated in California, Hawaii, Massachusetts, New York, Oregon,
Pennsylvania, and Wyoming.
* Presented at the American Water Works
Association Annual Meeting, June 21-25,
1970, at Washington, D.C.
**Chief Bacteriologist, Bureau of Water
Hygiene, Public Health Service, Cincinnati,
Ohio 45213.
-------
Meaningful Application of Bacteriological Data
TABLE 2
Analysis of 472 Public Water Supplies Having a History of
Unsatisfactory Bacteriological Results*
Population
Total Supplies
Studied
Percent Unsatisfactory Supplies
Based on Bacteriological Results
0 - 500
446
57
501 - 1, 000
101
56
1,001 - 5, 000
214
50
5,001 - 10, 000
75
42
10,001 - 25, 000
59
37
25, 001 - 50,000
36
38
over 50, 000
38
4
*Data trom National Community Water Supply Study,
Bureau oi Water Hygiene, May 1971).
TABLE 3
Monthly Sampling Frequency for Public Water Supplies*
Total Systems
Studied
Inadequate Sampling**
Population Served
Number ot
Systems
Percent
Occurrence
0 - 500
446
372
83. 4
501 - 1,000
101
71
70. 3
1,001 - 5, 000
214
108
50. 5
5,001 - 10,000
75
38
50. 7
10, 001 - 25, 000
59
34
57. 6
25,001 - 50,000
36
29
80. 6
50, 001 - 100, 000
16
8
50. 0
Greater than 100,000
22
10
45. 5
Total Systems
Percent deficient
969
670
69. 1
-Data i'rom National Community Water Supply Study, Bureau oi Water hygiene, May 1970.
-"Only 50% of USPHS Minimum monthly sample requirements.
2
-------
Meaningful Application of Bacteriological Data
Recently, this sampling frequency require-
ment has been challenged on the basis that
it has no scientific basis and is unrealistic.
If indeed trie existing PHS recommendations
are distorted, then the time has come for a
re-examination of these values based on a
study of state sanitary engineering records
across the country. Reconsideration of the
sampling requirements is on the agenda of
the intra-departmental Technical Task
Group on Revision of the PHS Drinking Water
Standards. Factors that may be considered
in any modification of the sampling require-
ments include: frequency of unsatisfactory
samples from supplies serving various
population levels, repeat sampling occurence
and the time interval for repeat sampling,
impact of peak water usage related to
seasonal shifts in population, adequacy of
plant capacity, proper sampling on the
distribution system, sample transit time
to the laboratory, quality of raw water
treated (some raw water sources consistently
contain more than 10, 000 total coliforms per
100 ml) and application of chlorination.
Sample points should be located throughout
the distribution system, including dead-end
mains, to establish adequate bacteriological
quality and to demonstrate that no contamina-
tion occurs through breaks in the distribution
system. Such careful monitoring of the water
quality in distribution lines is characteristic
of the large municipal plant operations.
Although a sufficient number of samples may
be collected on systems under 25, 000 pop-
ulation served, our study of sample records
indicates that 75 percent or more of these
required samples per month are taken from
the same locations: the municipal building,
the laboratory tap, the residence of some
city official, and a favorite restaurant or
tavern. Only an occasional attempt may be
made to obtain other samples that might
meaningfully measure water quality as
delivered through other portions of the distri-
bution lines.
Sample Transit Time
Every effort must be made to schedule ship-
ments of water samples to the State Laboratory
system to meet transportation scehdules.
Since week-end service of the U.S. Postal
Department is minimal and few State
Laboratories are open on Saturday morning,
sample collections should be avoided on
Thursday, Friday and any work day prior
to holidays unless specific arrangements are
made with the laboratory to process emer-
gency requests. Data presented in Table 4
illustrate the sample transportation problem
TABLE 4
Municipal Water Sample
Transit Time Observed
in Five Western States
Time (Days)
Percentage Occurrence
Idaho
Utah
Oklahoma
Colorado
Missouri
1
88. 6
88. 0
82. 8
68. 6
56. 6
2*
7. 2
6. 5
9. 2
18. 1
30. 1
3
3. 0
1.8
5. 2
11. 3
6. 5
4
0. 7
1. 1
1. 8
2. 0
6. 8
>4
0. 5
2. 6
1.0
NONE
NONE
Total Samples
Time Period
915 384
(Jan. -Oct. (6 days
1968) Sept. '69)
499
(11 days
Feb. '69)
204
(Jan. -Oct.
1968)
7, 185
(July, Oct.
1968)
* Maximum transit time limit for potable water samples.
3
-------
Meaningful Application of Bacteriological Data
as detected in the records from five western
states. To a large extent this problem is
related to the lack of coordination between
sample collection and timing of mailing to
meet the diminishing frequency of mail
movement. The problem can be further
aggravated in remote areas by the movement
of mail to regional centers outside a specific
state for sorting and consolidation prior to
reshipment back into the originating state
and on to the laboratory. Samples delivered
by car must be scheduled to reach the
laboratory promptly and not be postponed
to some more convenient time during the
next few days. Transporting samples for
several days in the high temperatures of a
car trunk or on the back seat of the auto-
mobile during the summer can drastically
alter the bacterial population.
These dramatic changes in the coliform
density as they relate to time-temperature
effects can be seen in data presented in
Table 5. In this study'^) three samples from
each source were examined in the winter and then
repeated the following summer. Five tube
MFN tests with three decimal dilutions were
used, employing lauryl sulfate trytose broth
in the presumptive tests and brilliant green
lactose broth in the confirmed procedure.
Each original sample was vigorously mixed
to insure even distribution of the contents.
Two portions were dispensed into sterile
containers and stored in the refrigerator at
5 C and at room temperature. The remainder
of the samples were used for coliform tests
initiated immediately to develop base-line
data. These results indicated that the validity
of data obtained in processing samples over
48 hours old is highly questionable since the
bacterial densities will not remain static.
Bacterial populations in samples containing
a source of protein nitrogen frequently will
increase in response to the nutrient source,
favorable water temperature and increased
transit time. In the absence of sufficient
nutrients but in contact with varying levels
of toxic metal ions and water temperature
above 15 C during transport, the bacterial
death rate will be accelerated. Just how
much it is accelerated is unpredictable.
Response to Unsatisfactory Samples
Once the bacteriological sample has been
collected in a sterile bottle containing a
dechlorinating agent and promptly transported
to the laboratory for processing, the prob-
ability of detecting any coliform population that
might be present has greatly increased. When
the laboratory detects an unsatisfactory
sample, one of several courses of action
will follow. In some State Health Depart-
ments, the laboratory promptly alerts the
engineering section to unsatisfactory
samples and local authorities are quickly
notified, with a high priority placed on
immediate repeat sampling. However, the
"hot-line" approach is not universal and
resampling is more likely to be delayed by
the slow processing of information either by
the laboratory or through the engineering
division. Frequently, the local sample
collector or water plant operator fails to
appreciate the urgency associated with
unsatisfactory results and one of several
actions may result. Repeat sampling may
be initiated on a daily basis but no action is
taken to find the source of contamination.
In essence, this approach would lead one to
conclude that the water plant operator and
his staff hope the problem will go away
with a prescription for sampling alone. In
many cases it will, of course, but in the
event of a hazardous source of contamination
the situation goes untended for days. In the
second instance, one extra sample is sub-
mitted for examination, but there is no
attempt to repeat the sampling on a daily
basis until 2 negative results are secured.
In the third case, the samples are not taken
from the original sample point where the
unsatisfactory result was originally reported.
However, the most common violation of the
sampling requirements is the lack of any
resampling until the next scheduled period
for routine sample collection. This interval
may represent a time delay of a week to a
month. As a result of these departures from
recommended procedures, any initial warning
of a possible system failure is not fully
investigated as a potential public health hazard.
At local water plant laboratories, some plant
records reveal bacteriological examinations
occur every few hours with little or no
measurement of finished water turbidity and
residual free chlorine in the distribution
system. These same records frequently are
pointed to with pride because there may never
have been a positive coliform result in the
finished water for periods of 5 to 20 years or
more. Such interpretation of data leads one
to suspect that these remarkable records relate
more to laboratory deviations that desensitized
coliform detection, rather than to an excellent
control on the treatment process. As an
illustration, one midwestern water plant
laboratory was observed by the state certi-
fying officer to be using nutrient broth in the
MPN procedure rather than lactose broth.
Since lactose sugar is not present in the
4
-------
TABLE 5
Time-Temperature Effects on Stored Split Water Samples
(Coliform analysis per 100 ml by MPN Method)
(2)
Sample
Date
Initial
Density
24
hrs
Storage at 5
48hrs.
°C
72
hrs.
Storage at Room Temperature*
24 hrs. 48 hrs. 72 hrs.
Private Well "A"
Mar.
18
33
49
33
13
49
33
4. 0
23
33
49
23
11
21
1 3
4. 5
25
23
33
7.
8
22
7.
8
13
7. 8
July
1
130
110
33
49
11
2.
0
6. 8
0
17
17
6.
8
46
23
2.
0
<2. 0
15
7. 8
4.
5
11
11
7.
8
<2.
0
<2. 0
Private Well "13"
Mar.
2
79
70
2.
0
2. 0
17
4.
0
4. 5
9
17
17
17
23
17
9.
3
4. 5
16
7. 8
17
6.
8
4. 5
13
13
<2. 0
June
3
79
33
31
33
17
130
22
10
33
23
13
49
11
17
23
24
490
350
490
490
>1.
600
3, 500
2, 200
Burnet Lake
Jan.
7
22
33
13
13
7.
8
23
9. 3
21
130
540
540
130
240
220
11
28
350
110
49
130
330
49
17
July
20
330
330
130
no
33
1 10
9, 200
27
490
170
330
220
110
79
23
Aug.
12
490
2,
400
1,
100
330
1.
400
490
1, 300
Public Water
Feb.
18
330
180
49
33
17
23
2. 0
Intake - Ohio
25
5, 400
1,
300
1,
700
490
210
79
130
River (Mile 463)
Mar.
4
3, 300
1,
300
1,
700
2,
800
1,
300
700
490
July
22
3, 100
790
1,
300
1,
300
1,
700
170
330
Aug.
3
1, 300
230
79
49
4.
5
4.
5
4. 5
Tit AAtli tnmi\at*ofnn<%
17
p..
230
22
¦ i _
2
2
3.
6
<2
<2
-------
Meaningful Application of Bacteriological Data
nutrient broth formulation, no gas production
was ever observed and any occurrence of
coliform bacteria went undetected.
Application of the Membrane Filter Test
Following acceptance of the membrane filter
procedure as a recommended test in the 11th
edition of Standard Methods, a significant
group of State Health Departments made the
change from the multiple tube procedure.
There reason for change was freqently related
to a desire for reduction of laboratory work
over Saturdays and Sundays. By using the
membrane filter procedure, the test was
completed within 22 hours as contrasted to a
minimum to maximum time of 48 to 96 hours
required by the multiple tube procedure. The
more recent increased acceptance of the
membrane filter procedure has been a re-
sponse to the increased necessity to monitor
streams for water quality criteria. To meet
this anticipated growing volume of water
examinations, additional laboratory directors
are planning to make the change in methodology.
They hope this decision will compensate for
the inability to increase either the laboratory
staff, space requirements or both. Currently,
the membrane filter procedure is being used
by 72 state and branch laboratories plus 125
municipal laboratories. These applications
range from use on those samples derived from
stream pollution investigations to all public
and private potable waters examined.
The indirect benefactor from this change has
been the sanitary engineer concerned with
potable water quality. By using the membrane
filter procedure, the technician is now
analyzing 100 ml of sample instead of the
usual 50 ml required in the 5 tube MPN test.
Thus the search for low levels of coliforms
in potable waters has been improved in two
directions i. e., examination of larger
sample portions and use of a more precise
testing method.
Laboratory Evaluation Program
The laboratory evaluation service^' ^ has
always been closely associated with the
application of established testing procedures
which would yield reliable, uniform, and
adequate results in all laboratories that
examine water samples. Such recognized
procedures as described in each edition of
Standard Methods for Water and Wastewater'6',
represent current techniques which have been
extensively studied and accepted by committee
action as being within the prescribed limits of
sensitivity, precision, and accuracy.
These techniques must always remain as
sensitive as the state-of-the-art permits.
This is of particular importance in the
constant watch for the low levels of coliform
bacteria that could signal the occurrence of
possible contamination by pathogenic micro-
organisms. Technicians must always attach
equal importance to every potable water
examination, regardless of the source or
the frequency of negative results. Monotony
of negative results does tend to breed
technical carelessness. Carelessness can
quickly lead to bad habits and deviations from ¦
standard procedures. Although occasional
deviations in techniques may in themselves
be insignificant, the cumulative effect of
several deviations will result in a decrease
test sensitivity to low density levels of
coliforms. Failure to detect coliforms when
they are present obviously poses a potential
health hazard to the consumers of such water.
Deviations in laboratory procedures will
continue as a result of many factors including:
attempted shortcuts, ignorance of technical
procedures, inexperience in new methods,
equipment failures, inadequate facilities,
technical carelessness, shifts of competent
personnel to other laboratory assignments
and lack of interest in this phase of public
health bacteriology. Thus, there exists a
continuing need for laboratory evaluation
services, both at the state and the municipal
levels, to keep the number of deviations to an
absolute minimum.
The optimum frequency of laboratory evalu-
ations at the state level appears to be every
three years. Our experience is that visits
at more frequent intervals yield little value
to either the staff or the program. Longer
intervals result in an increased number of
deviations observed. However, where there
are major difficulties or where there is a large
turnover of laboratory personnel, evaluations
must be performed more frequently, dependent
upon the individual situation. For these
reasons, the Surgeon General, in his memo-
randum of February 15, 1963 to all State
Health Officers^) stated that these laboratory
evaluations should be accomplished at least
every three years.
Status of Laboratory Procedures
Traditionally, the Public Health Service has
approved the state laboratories, which in turn
through qualified state laboratory survey
officers, certify the local laboratories within
each state. Study of these laboratory evaluation
6
-------
Meaningful Application of Bacteriological Data
reports made during 1960-1965 revealed^
significantly lower standards common to many
municipal laboratories. However, deviations
in procedures did increase among State
laboratory systems during 1966 to 1970
(Table 6) reflecting adoption of the membrane
filter test for potable water examinations.
Changes in procedures always require some
time period for technician acceptance,
methodology adjustments and acquisition of
necessary equipment items. A restudy of the
MF procedure as employed in water labora-
tories during the next five year period will
undoubtedly show a sharp reduction in these
deviation occurrences.
The multiple tube procedure has been the
traditional bacteriological test for water
quality for 70 years. Reports from state
evaluation officers indicate deviations in the
MPN test are occurring in 46. 9 percent of
the Municipal laboratories and at a rate
double that observed in the State systems.
The most serious errors include: not con-
firming cultures which produce small
quantities of gas, not confirming any pre-
sumptive positives during the first 24 hour
period, extended incubation times over
Saturday and Sunday, using insufficient or
excessive inoculum during presumptive
culture transfer to brilliant green lactose
broth, and failure to achieve colony isolation
when using the EMB confirmation procedure.
Major obstacles limiting improvements in
quality of laboratory procedures do relate
directly to limited financial budgets which
prevent the purchase or repair of essential
equipment and to low salary levels that do
not attract technicians that have the desired
academic background. In general, more
precise laboratory procedures, newer equip-
ment better laboratory housing, and more
experienced personnel are found in State
laboratories, although there are a few large
municipal laboratories with facilities and
staff resources of research capability. Many
of the deviations observed in Municipal
laboratories involve an urgent need for auto-
claves, incubators, pH meters, analytical
balances, and water stills. It was particularly
disturbing to note that 14 municipal labora-
tories did not have available the current
edition of Standard Methods for reference to
acceptable techniques. Inquiries directed to
some technicians revealed that their only
training in laboratory procedures was that
given by the plant operator or some previous
employee.
Every effort should be made by designated
state laboratory survey officers to up-grade
methods and equipment used in small water
plant laboratories. This could be accomp-
lished by recommending procedural improve-
ments that would lead to increased test
sensitivity; assisting with on-site training
when feasible; encouraging visits to the State
laboratory for an on-the-job training period
of several days; and establishing a direct
communication link between personnel of
these two levels of laboratory competencies.
Although the personnel of these small
laboratories may not have a background of
scientific training, per se, they are eager
to learn and to perform the bacteriological
control testing properly.
The State Health Departments are cognizant
of these problems. Some State Health
Departments have established a vigorous
program including an active laboratory
evaluation service, training school for local
laboratory personnel, and technical assistance
directed toward improving the quality and
reliability of the laboratory work performed.
These progressive leaders in public health
are to be commended for their early recog-
nition of the problem and resolution to act.
Unfortunately, a few State Health Departments
have developed little or no activity in this
program. Part of this inactivity is the result
of budgetary problems which negate the
necessary travel and salary funds needed to
operate a laboratory evaluation service. In
several states, no individual has yet been
designated or is actively participating as a
water laboratory survey officer. Finally,
our intended objective in this program is to
perform this service with the attitude of
stimulating and encouraging technicians to
improve the quality of work as a protection
against professional, legal, and public
criticism of data reliability. For most
laboratories, such an approach has resulted
in greater conformity and accuracy for micro-
biological methods used in our national
surveillance of public water supplies.
SUMMARY
A substantial amount of effort is made by
state and municipal laboratories to monitor
the bacteriological quality of both public and
private potable water supplies. However,
the adequacy of the sampling program on
those water supplies serving less than 50, 000
population is questioned since these supplies
frequently have the greater occurrence of
unsatisfactory samples and little attention to
prompt resampling.
-------
TA13LE 6
Status of MF and MPN Procedures Employed in Water Laboratories (19G6 - 1970)
Test Procedure
119 State and
Branch Labs.
287 Municipal Labs.
Labs.
Percent
Labs.
Percent
Labs.
Percent Labs.
Percent
Laboratories using MF test
72
60. 5
--
--
125
43.6
Deficiencies in MF procedures
--
--
46
63. 9
--
73
58. 4
] laboratories using MPN test
47
39. 5
--
--
1 62
56.4
--
Deficiencies in MPN procedures
--
--
11
23. 4
--
76
46. 9
Laboratories with deficiencies in
either test
--
--
57
47. 9
--
149
51. 1
-------
Meaningful Application of Bacteriological Data
Laboratory procedures for coliform detection
must be maintained at maximum sensitivity
levels and carefully follow recommended
Standard Methods for uniform results. Tech-
nicians in small quality control laboratories
must be up-graded in their technical knowledge
of bacteriology through various training
approaches to develop this resource. If
necessary, serious thought should be given
to consolidating small water plant laboratories
surrounding large metropolitan areas, to pool
manpower and equipment resources for an
improvement in the quality and quantity of
surveillance of potable water supplies.
REFERENCES
1 Public Health Service Drinking Water
Standards, Revised 1962. U. S.
Department of Health, Education, and
Welfare, Washington, D. C.
2 Bureau of Water Hygiene, "National
Community Water Supply Study"
May 1970.
3 Geldreich, E. E., Kabler, P. W.,
Jeter, H. L. and Clark, H. F. "A
Delayed Incubation Membrane Filter
Test for Coliform Bacteria in Water. "
Amer. Jour. Pub. Health, 45, 1462-
1474; 1955.
4 Black, L. A. "The Public Health Service
Program for Supervision of Laboratories
Analyzing Milk and Water Supplies used
on Interstate Carriers. " Conference of
State and Provincial Public Health
Laboratory Directors, New YorK
October 1944.
5 Geldreich, E. E. and Clark, H. F.
"Evaluation of Water Laboratories, "
Public Health Service Publication
Si^-EE-l, 1965.
6 Standard Methods for the Examination of
Water and Wastewater, 12th Ed.,
American Public Health Association.
New York, 1965.
7 Surgeon General's Memorandum to all
State Health Officers, "Public Health
Service Policy in the Application of
the 1962 Drinking Water Standards, "
February 15, 1963.
8 Geldreich, E. E. "Status of Bacterio-
logical Procedures Used by State and
Municipal Laboratories for Potable
Water Examination, " Health Laboratory
Science, 4 9-16; 1967.
ACKNOWLEDGMENTS
No study of this scope could be possible
without the assistance of many state sanitary
engineers, water plant managers, laboratory
directors, state survey officers, bacterio-
logists, sanitarians, and technicians who
share a sincere interest and concern about
potable water supplies and the meaningful
measurment of its bacteriological quality.
The willingness of this multi-discipline
group to openly discuss their problems,
make records available for analysis, and
conscientiously correct recognized deviations
in procedures is greatly appreciated.
9
-------
ENVIRONMENTAL PROTECTION AGENCY
Water Quality Office Indicating conformity with the 13th
Water Hygiene Division edition of Standard Methods for the
Examination of Water and Waste-
Bacteriological Survey for water (1971).
Water Laboratories
Survey By
X = Deviation
U
= Undetermined
O =
Not
Used
Laboratory
Location
Date
Sampling and Monitoring Response
1. Location and Frequency
Representative points on system
Frequency of sampling adequate
2. Collection Procedure
Faucets with aerators should not be used
Flush tap 1 min. prior to sampling
Pump well 1 min. to waste prior to sampling
River, stream, lake, or reservoir sampled at least
6 inches below surface and toward current
Minimum sample not less than 100 ml
Ample air space in bottle for mixing
Promptly identify sample legibly and indelibly
3. Sample Bottles
Wide mouth, glass or plastic bottles of capacity.
Sample bottles capable of sterilization and rinse . . . .
Closure:
a. Glass stoppered bottles protected with metal foil,
rubberized cloth or kraft type paper
b. Metal or plastic screw cap with leakproof liner . .
Sodium thiosulfate added for dechlorination
Concentration 100 mg/1 added before sterilization
Chelation agent for stream samples (optional)
Concentration 372 mg/1 added before sterilization
4. Transportation and Storage
Complete and accurate data accompanies sample . . . .
Transit time for potable water samples should not exceed
48 hrs, preferably within 30 hrs .
Transit time for source waters, reservoirs, and natural
bathing waters should not exceed 6 hrs
All samples examined within 2 hours of arrival
EPA-103 (Cin)
(Rev. 3-71)
-------
Laboratory
Location
Date
4. Transportation and Storage (Continued)
Sample refrigeration mandatory on stream samples,
optional on potable water samples
5. Record of Laboratory Examination
Results assembled and available for inspection
Number of Tests per year
MPN Test - Type of sample
Confirmed (+) (-) (Total)
Completed (+) (-) (Total)
MF Test - Type of sample
Direct Count (+) (-) (Total)
Verified Count (+) (-) (Total)
Data processed rapidly through laboratory and engineering sections . .
Unsatisfactory sample defined as 3 or more positive tubes per
MPN test or 5 or more colonies per 100 ml in MF test
High priority placed on alerting operator to unsatisfactory
potable water results
Prompt resampling for unsatisfactory samples
6. Laboratory Evaluation Service
State program to evaluate all laboratories which examine
potable water supplies
Frequency of surveys on a year basis
State survey officer (Name) . . . .
Status of laboratory evaluation service
Total labs known to examine water
approved laboratories
provisional laboratories
Laboratory Apparatus
7. Incubator
Manufacturer Model
Sufficient size for daily work load .
Maintain uniform temperature in all parts (± 0. 5°C)
Accurate thermometer with bulb immersed in liquid on
top and bottom shelves. . . . .
Daily record of temperature or use of recording thermometer
sensitive to 0. 5°C change
Incubator not subject to excessive room temperature variations
beyond a range of 50 - 80° F
EPA-103 (Cin)
(Rev. 3-71)
-------
Laboratory
Location
Date
8. Incubator Room (Optional) Manufacturer
Well insulated, equipped with properly distributed heating
and humidifying units for optimum environmental control. .
Shelf areas used for incubation must conform to 35° C ± 0. 5°
temperature requirement
Accurate thermometers with bulb immersed in liquid
Daily record of temperature at selected areas or use
recording thermometer sensitive to 0. 5°C changes ....
9. Water Bath
Manufacturer Model
Sufficient size for fecal coliform tests
Maintain uniform temperature 44. 5°C ± 0. 2°C
Accurate thermometer immersed in water bath
Daily record of temperature or use of recording
thermometer sensitive to 0.2°C changes
10. Hot Air Sterilizing Oven
Manufacturer Model
Size sufficient to prevent crowding of interior
Constructed to insure a stable sterilizing temperature ....
Equipped with accurate thermometer in range of 160-180°C
or with recording thermometer
11. Autoclave
Manufacture r Model
Size sufficient to prevent crowding of interior
Constructed to provide uniform temperature up to and
including 121° C
Equipped with accurate thermometer with bulb properly located
to register minimal temperature within chamber
Pressure gage and operational safety valve
Steam source from saturated steam line, or from gas or
electrically heated steam generator
Reach sterilization temperature in 30 min
Pressure cooker may be used only if provided with a pressure
gage and thermometer with bulb 1 in. above water level . .
12. Thermometers
Accuracy checked with thermometer certified by National
Bureau of Standards or one of equivalent accuracy
Liquid column free of discontinuous sections and graduation
marks legible
EPA-103 (Cin)
(Rev. 3-71)
3
-------
Laboratory
Location
Date
13. pH Meter
Manufacturer Model
Electronic pH meter accurate to 0. 1 pH units
14. Balance
Balance with 2 g sensitivity at 150 g load used for general
media preparations, Type
Analytical balance with 1 mg sensitivity at 10 g load used
for weighing quantities less than 2 g , Type .
Appropriate weights of good quality for each balance
15. Microscope and Lamp
Preferably binocular wide field, 10 to 15 diameters magnifi-
cation for MF colony counts, Type . . . .
Fluorescent light source for sheen discernment
16. Colony Count
Quebec colony counter, dark-field model preferred for
standard plate counts
17. Inoculating Equipment
Wire loop of 22 or 24 gauge chromel, nichrome, or platinum
iridium, sterilized by flame
Single-service transfer loops of aluminum or stainless steel, pre-
sterilized by dry heat or steam
Disposable single service hardwood applicators, pre-
sterilized by dry heat only
18. Membrane Filtration Units
Manufacturer Type
Leak proof during filtration
Metal plating not worn to expose base metal
19. Membrane filters
Manufacturer Type
Full bacterial retention, satisfactory filtration speed
Stable in use, glycerin free
Grid marked with non-toxic ink
Presterilized or autoclaved 121° C for 10 min. . . .
20. Absorbent Pads
Manufacturer Type
Filter paper free from growth inhibitory substances
Thickness uniform to permit 1.8 - 2.2 ml medium absorption
Presterilized or autoclaved with membrane filters
EPA-103 (Cin)
(Rev. 3-71)
4
-------
Laboratory
Location
Date
21. Forceps
Preferably round tip without corrugations
Forceps are alcohol flamed for use in MF procedure
Glassware, Metal Utensils and Plastic Items
2 2. Media Preparation Utensils
Borosilicate glass
Stainless steel
Utensils clean and free from foreign residues or
dried medium
23. Pipets
Brand Type
Calibration error not exceeding 2.5%
Tips unbroken, graduation distinctly marked
Deliver accurately and quickly
Mouth end plugged with cotton (optional)
24. Pipet Containers
Box, aluminum or stainless steel
Paper wrapping of good quality sulfite paper (optional)
25. Petri Dishes
Brand Type
Use 100 mm x 15 mm dishes for pour plates
Use 60 mm x 15 mm dishes for MF cultures
Clear, flat bottom, free from bubbles and scratches
Plastic dishes may be reused if sterilized in 70% ethanol for
30 min. or by ultraviolet radiation
2 6. Petri Dish Containers
Aluminum or stainless steel cans with covers, coarsely woven
wire baskets, char-resistant paper sacks or wrappings
27. Culture Tubes
Size sufficient for total volume of medium and sample portions . . . .
Borosilicate glass or other corrosive resistant glass
28. Dilution Bottles or Tubes
Borosilicate or other corrosive resistant glass
Screw cap with leak-proof liner free from toxic substances
on sterilization
Graduation level indelibly marked on side of bottle or tube
EPA-103 (Cin)
(Rev. 3-71)
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Materials and Media Preparation
29. Cleaning Glassware
Dishwasher Manufacturer Model
Thoroughly washed in detergent at 160° F, cycle time . .
Rinse in clean water at 180° Fj cycle time " • .
Final rinse in distilled water f cycle time
Detergent brand '
Washing procedure leaves no toxic residue
Glassware free from acidity or alkalinity
30. Sterilization of Materials
Dry heat sterilization (1 hr at 170°C)
Glassware not in metal containers
Dry heat sterilization <2 hrs at 170°C)
Glassware in metal containers
Glass sample bottles
Autoclaving at 121° C for 15 min
Plastic sample bottles
Dilution water blanks
31. Laboratory Water Quality
Still manufacturer Construction Material
Demineralizer with recharge frequency
Protected storage tank
Supply adequate for all laboratory needs
Free from traces of dissolved metals or chlorine ..........
Free from bactericidal compounds as measured
by bacteriological suitability test
Bacteriological quality of water measured once each year
by suitability test or sooner if necessary
32. Buffered Dilution Water
Stock phosphate buffer solution pH 7.2
Prepare fresh stock buffer when turbidity appears
Stock buffer autoclaved and stored at 5 - 10°C
1. 25 ml stock buffer per 1 liter distilled water
Dispense to give 99 ± 2 ml or 9 ± 0. 2 ml after autoclaving
33. pH Measurements
Calibrate pH meter against appropriate standard buffer prior to use . .
Standard buffer brand pH
Check the pH of each sterile medium batch or at least one batch
from each new medium lot number
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33. pH Measurements (Continued)
Maintain a pH record of each sterile medium batch,
the date and lot number
34. Sterilization of Media
Carbohydrate medium sterilized 121° C for 12 min
All other media autoclaved 121°C for 15 min
Tubes packed loosely in baskets for uniform heating and cooling.
Timing starts when autoclave reaches 121°C
Total exposure of carbohydrate media to heat not over 45 min. .
Media removed and cooled as soon as possible after sterilization
35. Storage
Dehydrated media bottles kept tightly closed and stored
at less than 30° C.
Dehydrated media not used if discolored or caked
Sterile culture media stored in clean area free from
contamination and excessive evaporation
Sterile batches used in less than 1 week
All media protected from sunlight
If media is stored at low temperatures, it must be incubated
overnight and any tubes with air bubbles discarded
Culture Media - Specifications
36. Lactose Broth
Manufacturer Lot No.
Single strength composition 13 g per liter distilled water .
Single strength pH 6. 9 ± 0. 1, double strength pH 6. 7 ± 0.1
Not less than 10 ml medium per tube
Composition of medium after 10 ml sample is added must
contain 0. 013 g per ml dry ingredients
37. Lauryl Tryptose Broth
Manufacturer Lot No.
Single strength composition 35. 6 g per liter distilled water
Single strength pH 6. 8 ± 0. 1, double strength pH 6. 7 ± 0. 1
Not less than 10 ml medium per tube
Composition of medium after 10 ml sample is added must
contain 0. 0356 g per ml of dry ingredients
38. Brilliant Green Lactose Bile Broth
Manufacturer Lot No.
2PA-103 (Cin)
(Rev. 3-71)
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38. Brilliant Green Lactose Bile Broth (Continued)
Correct composition, sterility and pH 7.2
Not less than 10 ml medium per tube
39. Eosin Methylene Blue Agar
Manufacturer Lot No.
Medium contains no sucrose, Cat. No.
Correct composition, sterility and pH 7.1
40. Plate Count Agar (Tryptose Glucose Yeast Agar)
Manufacturer Lot No.
Correct composition, sterility and pH 7.0 ± 0. 1
Free from precipitate
Sterile medium not remelted a second time after sterilization.
41. EC Medium
Manufacturer Lot No.
Correct composition, sterility and pH 6. 9
Not less than 10 ml medium per tube
42. M-Endo Medium
Manufacturer ; ¦ Lot No.
Correct composition and pH 7.1-7.3
Reconstituted in distilled water containing 2% ethanol
Heat to boiling point, promptly remove and cool
Store in dark at 2 - 10° C
Unused medium discarded after 96 hrs
43. M-FC Broth
Manufacturer Lot No.
Correct composition and pH 7. 4
Reconstituted in 100 ml distilled water containing 1 ml of
a 1% rosolic acid reagent
Stock solution of rosolic acid discarded after 2 weeks or
when red color changes to muddy brown
Heat to boiling point, promptly remove and cool
Store in dark at 2 - 10° C
Unused medium discarded after 96 hrs
44. Broth
Manufacturer Lot No.
Correct composition and pH
45. A2ar
Manufacturer Lot No.
EPA-103 (Cin)
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45. Agar (Continued)
Correct composition and pH
Multiple Tube Coliform Test
46. Presumptive Procedure
Lactose broth lauryl tryptose broth
Shake sample vigorously
Potable water: 5 standard portions, either 10 or 100 ml
Stream monitoring: multiple dilutions
Incubate tubes at 35° ± 0. 5°C for 24 ± 2 hr
Examine for gas any gas bubble positive
Return negative tubes to incubator
Examine for gas at 48 ± 3 hr from original incubation
47. Confirmed Test
Promptly submit all presumptive tubes showing gas production
before or at 24 hr and 48 hr periods to Confirmed Test
a. Brilliant green lactose broth
Gently shake presumptive tube or mix by rotating
Transfer one loopful of positive broth or one dip of applicator
from presumptive tube to brilliant green lactose broth
Incubate at 35° ± 0. 5°C and check at 24 hrs for gas production. . .
Reincubate negative tubes for additional 24 hrs
and check for gas production
Calculate MPN or report positive tube results
b. Endo or eosin methylene blue agar plates adequate streaking
to obtain discrete colonies separated by 0. 5 cm
Incubate at 35° ± 0. 5°C for 24 ± 2 hr
Typical nucleated colonies with or without sheen are coliforms . .
If atypical unnucleated pink colonies develop, result is
doubtful and completed test must be applied
If no colonies or only colorless colonies appear, the
confirmed test is negative.
48. Completed Test
Applied to all potable water samples or a proportion each three
months to establish the validity of the confirmed test in
determining their sanitary quality
Applied to positive confirmed tubes or to doubtful colonies
on differential medium
Streak positive confirmed tubes on Endo or EMB plates for
colony isolation
EPA-103 (Cin)
(Rev. 3-71)
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48. Completed Test (Continued)
Choice of selected isolated colony for verification should be one
typical or two atypical to lactose or lauryl tryptose broth and
to agar slant for Gram stain
Incubate at 35°C ± 0. 5°C for 24 hrs or 48 hrs
Gram negative rods without spores and gas in lactose tube
with 48 hrs in positive Completed Test
Membrane Filter Coliform Test
49. Application as Standard Test
Use as a standard test for determining potability of water after
demonstration by parallel testing that it yields information
equal to that from the multiple-tube fermentation procedure ....
50. MF Procedure
Filter funnel and receptacle sterile at start of series
Rapid funnel resterilization by UV, flowing steam or boiling water
acceptable
Membrane filter cultures and technician eyes should not be
subject to UV radiation leaks
Filtration volume not less than 50 ml for potable water; multiple
dilutions for stream pollution ___
Rinse funnel by flushing several 20 - 30 ml portions of sterile buffered
water through MF
Remove filter with sterile forceps
Roll filter over M-ENDO medium pad or agar so air bubbles
will not form
51. Incubation
In high humidity or in tight fitting culture dishes
At 35°C ± 0. 5°C for 22 - 24 hrs
52. Counting
All colonies with a metallic yellowish green surface sheen
If coliforms are found in potable samples, verify by transfers
to lactose broth, then to BGB broth for evidence of gas
production at 35°C within 48 hr limit
Calculate direct count in coliform density per 100 ml
53. Standard MF test with Enrichment
Incubate MF after filtration on pad saturated with lauryl tryptose
broth for 1 1/2 - 2 hr at 35° C ± 0. 5°C _
EPA-103 (Cin)
(Rev. 3-71) 10
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56. Delayed-Incubation Coliform Test (Continued)
Transfer MF cultures to standard M-Endo medium
at laboratory
Incubate at 35°C ± 0. 5°C for 20 - 22 hr
If at time of transfer, growth is visible, hold in refrigerator
till end of work day then incubate at 35° overnight
(16 - 18 hr period)
Count sheen colonies, verify if necessary, and calculate
direct count in coliform density per 100 ml
57. Additional Test Capabilities
Fecal streptococci Method
Pseudomonas aeruginosa Method
Staphylococcus Method
Salmonellae Method
Biochemical tests Purpose
Serological tests Purpose
Other Purpose
Laboratory Staff and Facilities
58. Personnel
Adequately trained or supervised for bacteriological
examination of water
Laboratory staff (Total) Prep room staff (Total)
59. Reference Material
Copy of the current edition of Standard Methods available
in the laboratory
State or federal manuals on bacteriological procedures for
water available for staff use
60. Physical Facilities
Bench-top area adequate for periods of peak work in
processing samples
Sufficient cabinet space for media and chemical storage
Office space and equipment available for processing water
examination reports and mailing sample bottles
Facilities clean, with adequate lighting, ventilation and
reasonably free from dust and drafts
61. Laboratory Safety
Proper receptacles for contaminated glassware and pipettes
EPA-103 (Cin)
(Rev. 3-71)
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53. Standard MF test with Enrichment (Continued)
Transfer MF culture to M-Endo medium for a final
20 - 22 hr incubation at 35°C ± 0. 5°C
Count sheen colonies, verify if necessary, and calculate
direct count in coliform density per 100 ml
Supplementary Bacteriological Methods
54. Standard Plate Count
Plate not more than 1 or less than 0.1 ml (sample or dilution)
Add 10 ml or more liquefied agar medium at a temperature
between 43 - 45° C
Melted medium stored for no more than 3 hr at 43 - 45° C
Liquid agar and sample portion thoroughly mixed by gently
rotating to spread mixture evenly
Count only plates with between 30 and 300 colonies, exception
being 1 ml sample with less than 30 colonies
Record only two significant figures and calculate as "standard
plate count at 35°C per 1 ml of sample"
35. Fecal Coliform Test
a. Multiple Tube Procedure
Applied as an EC broth confirmation of all positive
presumptive tubes
Place EC tubes in water bath within 30 min of transfers
Incubate at 44. 5° C ± 0. 2° C for 24 hrs
Gas production is positive test for fecal coliforms
Calculate MPN based on combination of positive EC tubes
b. Membrane Filter Procedure
Following filtration place MF over pad saturated with
M-FC broth.
Place MF cultures in water-proof plastic bag and submerge
in water bath within 30 min
Incubate at 44. 5°C ± 0. 2°C for 24 hrs
All blue colonies are fecal coliforms
Calculate direct count in density per 100 ml
56. Delayed-Incubation Coliform Test
After filtration, place MF over pad of M-Endo containing 3.2 ml
of a 12% sodium benzoate solution per 100 ml of medium
Addition of 50 mg cycloheximide per 100 ml of preservative
medium for fungus suppression is optional
Transport culture by mail service to laboratory within 72 hours . . . .
EPA-103 (Cin)
(Rev. 3-71)
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61. Laboratory Safety (Continued)
Adequately functioning autoclaves with periodic inspection
and maintenance
Accessible facilities for hand washing
Proper maintenance of electrical equipment to prevent fire
and electrical shock
Convenient gas and electric outlets
First aid supplies available and not out-dated
62. Remarks
EPA-103 (Cin)
(Rev. 3-71)
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