2t,

ELEMENTS

OF

WATER BACTERIOLOGY

WITH SPECIAL REFERENCE TO

SANITARY WATER ANALYSIS.

BY

SAMUEL GATE PRESCOTT,

Assistant Professor of Industrial Biology,
AND

CHARLES-EDWARD AMORY WINSLOW,

Instructor in Sanitary Bacteriology,

IN THB

Massachusetts Institute of Technology.

NEW YORK:
JOHN WILEY & SONS.
London: CHAPMAN & HALL, Limi'j
1904.

Copyright, 1904,

BY

S. C. PRESCOTT

AND

C.-E. A. WINSLOW.

I^OBKRT I^mrMMOND. rmNTEK. NEW VOKK.

DEDICATED
TO

212I(lU'am Ci)oiiip0on ^elfgtotclt

BY TWO OF HIS PUPII.S,
i A TOKEN OF GRATEFUL AFFECTION.

PREFACE.

The general awakening of the community to the
importance of the arts of sanitation — accelerated by the
rapid growth of cities and the new problems of urban
life— demands new and accurate methods for the study
of the microbic world. Bacteriology has long since
ceased to be a subject of uiterest and importance to
the medical profession merely, but has become intimately
connected with the work of the chemist, the biologist,
and the engineer. To the sanitary engineer and the
public hygienist a knowledge of bacteriology is indis-
pensable.

In the swift development of this science during the
last ten years perhaps no branch of bacteriology has
made more notable progress than that which relates to
the sanitaiy examination of water. After a brief period
of extravagant anticipation, and an equally unreason-
able era of neglect and suspicion, the methods of the
practical water bacteriologist have gradually made their
way, unta it is recognized that, on account of their deli-
cacy, their directness, and their certainty, these methods
now furnish the final criterion of the sanitary condition
of a potable water.

VI PREFACE.

A knowledge of the new science early became so
indispensable for the sanitary expert that a special
course in the Bacteriology of Water and Sewage has for
some years been given to students of biology and sani-
tary engineering in the Biological Department of the
Massachusetts Institute of Technology. For workers in
this course the present volume has been especially
prepared, and it is fitting, we think, that such a manual
should proceed from an institution whose faculty, gradu-
ates, and students have had a large share in shaping the
science and art of which it treats. We shall be grati-
fied, however, if its field of usefulness extends to those
following similar courses in other institutions, or occupied
professionally in sanitary work.

The treatment of the subject in the many treatises on
General Bacteriology and Medical Bacteriology is neither
special enough nor full enough for modern needs. The
classic work of Grace and Percy Frankland is now ten
years old; and even Horrocks' valuable "Bacteriological
Examination of Water" requires to be supplemented by
an account of the developments in quantitative analysis
which have taken place on this side of the Atlantic.

It is for us a matter of pride that Water Bacteriology
owes much of its value, both in exactness of method
and in common-sense interpretation, to American sani-
tarians. The English have contributed researches of the
greatest importance on the significance of certain intesti-
nal bacteria-, but with this exception the best work
on the bacteriology of water has, in our opmion, been
done in this country. Smith, Sedgwick, Fuller. Wlnpple,
Jordan, and their pupils and associates (not to mention

PREFACE.

vii

Others) have been pioneers in the development of this
new field in sanitary science. To gather the results of
their work together .'n such form as to give a correct
idea of the best American practice is the purpose of
this little book; and this we have tried to do, with such
completeness as shaU render the volume of value to the
expert and at the same time with such freedom from
undue technicality as to make it readable for the layman.
It should be distinctly understood that students using
it are supposed to have had beforehand a thorough
course in general bacteriology, and to be equipped foi
advanced work in special lines.

The Biological Laboratories,
Mass. Institute of Technology,
Boston, March lo, 1904.

TABLE OF CONTENTS.

CHAPTER I.

The Bacteria in Natural Waters

CHAPTER n.

The Quantitative Bacteriological Examination of Water 19

CHAPTER in.

The Interpretation of the Quantitative Bacteriological
Analysis

35

CHAPTER IV.

Determination of the Number of Organisms Developing
AT the Body Temperature

43

CHAPTER V.

The Isolation of Specific Pathogenes from Water 49

CHAPTER VI.

Methods for the Isolation of the Colon Bacillus 58

CHAPTER VII.

Significance of the Presence of B. Coli in Water 74

CHAPTER vni.
Presumptive Tests for B Coli

• • 94

X

TABLE OF CONTENTS.

CHAPTER IX.

PAGE

Other Intestinal Bacteria 99

CHAPTER X.

The Significance and Applicability of the Bacteriolog-

iCAL Examination 109

Appendix 119

References 129

Index 143

ELEMENTS OF WATER BACTERIOLOGY

CHAPTER I.

THE BACTERIA IN NATURAL WATERS.

Bacteria are the most numerous and the most widely
distributed of living things. They are present not merely
at the surface of the earth or in the bodies of water which
partially cover it, as is the case with most other living
things, but in the soil itself, and in the air above, and in
the waters under the earth.

Probably no organisms are more sensitive to external
conditions, and none respond more quickly to slight
changes in their environment. Temperature, moisture,
and oxygen are of importance in controlling their distri-
bution; but the most significant factor is generally the
amount of food supply. Bacteria and decomposing or-
ganic matter are always associated, and for this reason
a brief consideration of the general relation of bacteria
to their sources of food supply and to other forms of
organic life must precede the study of their distribution
in any special medium.

3

4

ELEMENTS OF WATER BACTERIOLOGY.

First, then, the bacteria, though possessing greater con-
structive power than any animal organism, lack the power
of green plants to build up their own food from purely
inorganic materials, and must live upon the products of
the growth of higher forms. A few species which have
become adapted to a parasitic or semi-parasitic mode
of life occur on the surface of the normal plant or animal
body or penetrate the deeper layers of diseased tissues,
feeding upon the fluids of the body or on the extraneous
material collected upon its surface. Even these bacteria,
however, may generally be cultivated imder saprophytic
conditions, and the vast majority of other forms live as
true saprophytes on dead organic matter wherever it
may occur in nature,, and particularly in that diffuse layer
of decomposuig plant and animal material which we call
the humus, or siirface layer of the soil. Wherever there
is life, waste matter is constantly being produced, and
this finds its way to the earth or to some body of water.
The excretions of animals, the dead tissues and broken-
down cells of both animals and plants, as well as the
wastes of domestic and industrial life, all eventually find
their way to the soil. In a majority of cases these sub-
stances are not of such chemical composition that they
can be utilized at once by green plants as food, but it is
first necessary that they go through a fermentation or trans-
formation in which their chemical composition becomes
greatly changed; and it is as the agents of this trans-
formation that bacteria assume their greatest importance
in the world of life.

THE BACTERIA IN NATURAL WATERS.

5

We may take a comparatively simple excretory prod-
uct, urea, as an example. Through the activity of an
enzyme produced by certain bacteria this compound
unites with two moleciiles of water and is converted into
ammonium carbonate,

C0< ■ + 2H,0 = (NHj2C03.

While green plants can derive their necessary nitrogen
in part, at least, from ammonium compounds it is a well-
established fact that this element may be obtained much
more readily from nitrates, and it is, therefore, essential
as a further step that some means be employed to oxidize
the nitrogen. This process of oxidation is known as
nitrification, and takes place in a succession of steps,
the organic nitrogen being first converted to the form of
ammonium salts, and these in turn to nitrites and nitrates,
the oxygen used commg from the air. Several groups
of organisms are instrumental in bringmg about this con-
version. It is generally assumed that one group attacks
the ammonium compounds and changes them to nitrites
while another group completes the oxidation to nitrates'
In the latter form nitrogen is readily taken up by green
plants to be built up into the more complex albuminoid
substances (organic nitrogen) through the constructive
power of chlorophyll.

This never-ending cycle is illustrated in the accom-
panying figure, devised by Sedgwick (Sedgwick, 1880)
to lUustrate the transformations of organic nitrogen L

6

ELEMENTS OF WATER BACTERIOLOGY.

nature, the increasing size and closeness of the spiral on
the left-hand side indicating the progressive complexity
of organic matter as built up by the chlorophyll bodies
of green plants in the sunlight, and the other half of the
figure the reverse process carried out largely by the bac-
teria. In nature there are many short circuits, as, for
instance, when dead organic matter is used as food for
animals and built up into the living state again without
ever being nitrified and acted upon by green plants; but
the complete cycle of organic nitrogen is as indicated on
the diagram.

We have dwelt thus at length upon the general relation
between bacteria and organic decomposition because in
this relation will be found the master key to the distri-
bution of bacteria in water as well as in other natural
habitats. It is true that certain peculiar forms may at
times multiply in fairly pure waters; but in general large
numbers of bacteria are found only in connection with
the organic matter upon which they feed. Such organic
matter is particularly abundant in the surface layer of the
soil. Here the processes of nitrification proceed most
rapidly. Here the bacteria are most abundant; and in
other media their numbers vary according to the extent
of contact with the living earth. Natural waters particu-
larly group themselves from a bacteriological standpoint
in three well-marked classes, according to their relation
to the rich layers of bacterial growth upon the surface of
the globe. There are first the atmospheric waters which
have never been subject to contact with the earth; second,

THE BACTESIA IN NATURAL IVATERS.

the surface-waters immediately exposed to such contami-
nation in streams and ponds; third, the ground- waters
from which previous pollution has been more or less
removed by filtration through the deeper layers of the
soil.

Even rain and snow, the sources of our potable waters
are by no means free from germs, but contain them in
numbers varying according to the amount of dust present

NITROGEN A3
S ORCANIC NITROGEN
/(ALBUMINOID AMMONIA)

Fig. I.

in the air at the time of the precipitation. After a long-
continued storm the atmosphere is washed nearly free
of bacteria, so that a considerable series of sterile plates may
often be obtained by plating i-c.c. samples. These re-
sults are in harmony with the observations of Tissandier
(reported by Duclaux, 1897), who found that the dust in
the air amounted to 23 mg. per cubic meter in Paris

8 ELEMENTS OF WATER BACTERIOLOGY.

and 4 mg. in the open country. After a rainfall these
figures were reduced to 6 mg. and .25 mg., respectively.

With regard to what may be considered normal values
for rain we have no very satisfactory figures. Those
obtained by Miquel (Miquel, 1886) during the period
1 883-1 886, showing that rain contains on the average 4.3
bacteria per c.c. in the country (Montsouris) and 19 per
c.c. in Paris, are probably lower than would be yielded by
the present methods of examination. Snow shows rather
higher numbers than rain. Janowski (Janowski, 1888)
found in freshly fallen snow from 34 to 463 bacteria per
c.c. of snow-water, and his results indicate that the num-
ber is independent of the temperature at the time of
snowfall.

As soon as the rain-drop touches the surface of the
earth its real bacterial contamination begins. Rivulets
from ploughed land or roadways may often contain several
hundred thousand bacteria to the cubic centimeter; and
furthermore the amounts of organic and mineral mat-
ters which serve as food materials, and thus become a
factor in later multiplication of organisms, are greatly
increased.

In the larger streams several conditions combine to
make the bacterial numbers lower. Ground-water con-
taining little microbic life enters as a diluting factor from
below. The larger particles of organic matter are re-
moved from the flowing water by sedimentation; many
earth bacteria, for which water is an unfavorable medium,
gradually perish; and in general a new condition of equi-

THE BACTERIA IN NATURAL WATERS.

9

librium tends to be established. A good river- water under
favorable conditions should thus contain only a few hun-
dred bacteria. Heavy rains which introduce wash from
the surrounding watershed may, however, at any time
upset this condition of equilibrium and surface-waters
are apt to show sudden fluctuations in their bacterial con-
tent. Particularly in the spring and fall high numbers
manifest themselves, and seasonal variations arise, such
as are well shown in the appended table.

NUMBER OF BACTERIA PER CUBIC CENTIMETER IN CERTAIN
SURFACE-WATERS.

Water.

Yr.

Observer.

Jan.

Feb.

March.

April.

May.

June

Boston tap!

water 1

Merrimac River .
Thames River. . .

River Ourcq. . -j

1892-

1895

1901

1888

1887-

1890

j- Whipple*

Clark t
Franklandt

j- Miquel §

135
5,600
92,000

143,370

21 1

2,SOO

40,000

63,720

102

8,900
66,000

47,780

1 ,400
13,000

22,660

53
1,400
1 ,900

29,340

86

3000
3500

7340

Water.

Yr.

Observer.

July.

Aug.

Sept.

Oct.

Nov.

Dec.

Boston tapS

water I

Merrimac River.
Thames River. . .

River Ourcq. . -j

1 8g 2-

189s
1 90 1
1888
1887-
1890

j- Whipple*

Clarkt
Franklandl

|- Miquel §

73
1 760
1070

7730

81

1580
3000

8520

86

1760
1740

8070

55
1,900
1,130
1 2,560

56

1,300
1 1 ,700

135,700

52

5, 100
10,600

153.200

* Whipple, 1896.
X Frankland, 1894.

t Massachusetts State Board of Health, 1902.
§ Miquel, 1891.

Two factors influence this seasonal distribution of bac-
teria. First, during the summer months the water flow-
ing in open rivers is largely derived from springs and
subterranean sources, while during the autumn and
spring months, there is a much greater proportion of
"run-off" water contaminated by contact with the sur-

10 ELEMENTS OF WATER BACTERIOLOGY.

face of the earth. In the second place, during these
rainy seasons the amount of dissolved organic matter is
far greater than in summer, thus making the food-supply
of the bacteria more abundant.

In standing waters all the tendencies which make for
the reduction of bacteria are intensified, and ponds and
lakes often give numbers under a hundred. The student
will find numerous analyses of natural waters in Frank-
land's classic work (Frankland, 1894). He notes, for
example, that the Lake of Lucerne contained 8 to 51 bac-
teria per c.c. Loch Katrine 74, and the Loch of Lintral-
then an average of 170. The water of Lake Champlain
examined by one of us (S. C. P.) in 1896 contained on
an average 82 bacteria per c.c. at a point more than two
miles out from the city of Burlington. Certain surface
water-supplies near Boston studied by Nibecker and one
of us (Winslow and Nibecker, 1903) gave the following
results :

Number of

Average

City.

Number

Samples.

of Bacteria

per cc.

Wakefield

7

59

Lynn

6

16

Plymouth

6

35

Cambridge

5

94

Salem

5

232

Medford

5

524

Taunton

4

13

3

141

Russell found similar small numbers in sea-water at
Naples (Russell, 1891) and Wood's Hole (Russell, 1892),

THE BACTERIA IN NATURAL WATERS. ii

and in salt as in fresh water the amount of bacterial life
decreases in general as one passes downward from the
surface and outward from the shore.

The principal factors in the destruction of the bacteria
in water during storage appear to be sedimentation, the
activity of other micro-organisms, light, temperature, and
food-supply, and perhaps more obscure conditions such
as osmotic pressure.

The subsidence of bacteria either by virtue of their own
specific gravity or as the result of becoming attached to
particles of suspended matter is unquestionably partly,
if not largely, responsible for changes in the number of
bacteria in the upper layers of water at rest or in very
sluggish streams. The results of numerous inves-
tigations by different workers seem to indicate that
sedimentation takes place slowly, and that the differ-
ence in numbers between the top layer and the bottom
layer of water in tall jars in laboratory experiments of
only a few days' duration is very sHght or quite within
the Hmits of experimental error (Tiemann and Gartner,
1889). Different species may, of course, be differently
affected, (Scheurlen, 1891). It must be remembered,
that in natural streams bacteria are to a great extent
attached to larger sohd particles upon which the action
of gravity is much more important. Jordan (Jordan
1900) IS firmly of the opinion that in the lower part'
of the Illinois River, where there is a fall of but 30
feet m 225 miles, the influences summed up by the term
sedimentation are sufficiently powerful to obviate the

12

ELEMENTS OF WATER BACTERIOLOGY.

necessity for summoning another cause "to explain the
diminution in numbers of bacteria," and he further adds:
" It is noteworthy that all the instances recorded in the
literature where a marked bacterial purification has been
observed are precisely those where the conditions have
been most favorable for sedimentation."

Little is known as to the share of other organisms in
hastening the decrease of bacteria in stored water. Doubt-
less predatory Protozoa play some part here, and cer-
tain bacteriologists have believed that the toxic waste
products of some species of bacteria materially check
the development of other forms. Horrocks (Horrocks,
1901), Garre (Garre, 1887), Zagari (Zagari, 1887),
and Freudenreich (Freuderireich, 1888) have shown
that an "antagonism" exists when bacteria are grown
in artificial culture media such that the substratum
which has supported the growth of one form may be
rendered antiseptic to another. It is difiicult, however,
to beheve that any poisons are produced of such enor-
mous power as to cause this effect in a stream or a
lake, and there is no evidence in support of such a
view.

Temperature has a direct relation to bacterial life, and
the number of parasitic bacteria at least may be quickly
lessened by the action of cold. Sedgwick and one of us
(Sedgwick and Winslow, 1902) have shown that of
typhoid bacilli in ice or cool water over 40 per cent will
perish in three hours and 98 per cent and upwards in two

I

weeks.

THE BACTERIA IN NATURAL WATERS.

13

Many investigations conducted since the pioneer re-
searches of Downes and Blunt (Downes and Blunt, 1877)
have confirmed the results reported by them, viz., direct
sunhght is fatal to most bacteria in the vegetative state
and even to spores if the exposure be sufficiently long,
while diffused hght is harmful in a lesser degree. Opin-
ions vary as to the degree to which light is active in
destroying the bacteria in natural water. Buchner
(Buchner, 1893) found by experiment that the bacteri-
cidal power of light extends to a depth of about three
meters before it becomes imperceptible. On the other
hand, Procaccini (Procaccini, 1893) found that when sun-
light was passed vertically through 60 cm. of drain-water
the lower layers contained nearly as many bacteria after
three hours' treatment as before the exposure. The
middle and upper portions showed a great falling off in
numbers, however.

But few studies have been made of the effect of light on
bacteria in flowing water. Jordan (Jordan, 1900) has
investigated several Illinois streams and arrived at the
conclusion that in moderately turbid water, at least, the
sun':, rays are virtually without action. On the other
hand, Rapp has observed a considerable reduction of
the bacteria in the Isar at PuUach after the period of
diurnal insolation, as shown by the table on the following
page.

Although it is hard to estimate the exact importance
of each factor, the general phenomena of the self-purifi-
cation of streams are easy to comprehend. A smaU

14 ELEMENTS OF WATER BACTERIOLOGY.

Examinations or the Isar at Pullach (Rapp, 1903).

(a) carried out SEPTEMBER 26, 1 898, NO RAIN HAVING FALLEN FOR

THREE WEEKS.

Temperature

Time of the
Experiment.

Bacteria
per cc.

of the Water.

of the Air.

13.0° c.

8.8° C.

7.30 P.M.

146

12.1° C.

7.0° C.

9.30 P.M.

270

10.5° c.

6.2° C.

5.00 A.M.

370

10.2° C.

8.2° C.

8.00 A.M.

320

(b) CARRIED OUT NOVEMBER 28, 1 898, NO RAIN HAVING FALLEN FOR

SOME TIME.

5-5° C.

3.0° c.

6.00 P.M.

266

5-5° C.

2.5° c.

8.00 P.M.

402

5-5° C.

2.0° c.

10.00 P.M.

482

5.0° C.

2.0° C.

3.00 A.M.

532

4.5° c.

2.5° c.

7.30 A.M.

400

brook immediately after the entrance of polluting mate-
rial from the surface of the ground contains a large num-
ber of bacteria from a diversity of sources. Gradually
those organisms adapted to life in the earth or in the
bodies of plants and animals die out, and the forms
for which water furnishes ideal conditions sundve
and multiply. It is no single agent which brings this
about, but that complex of little-understood conditions
which we call the environment. If any one thing is of
prime importance it is probably the food-supply, for only
certain bacteria are able to multiply in the presence of the
small amount of organic matter present in ordinary po-
table waters. As Jordan (Jordan, 1900) has said: "In
the causes connected with the insufficiency or unsuitabihty

THE BACTERIA IN NATURAL WATERS.

15

of the food-supply is to be found, I believe, the main rea-
son for the bacterial self-purification of streams."

In general we have seen then that surface-waters tend
continually to decrease in bacterial content after their
first period of contact with the humus layer of the soil.
In that other portion of the meteoric water which pene-
trates below the surface of the earth to join the reservoir
of ground-water, later to reappear as the flow of springs
and wells, this diminution is still more marked since
the filtering action of the earth removes not only most
of the bacteria but much of their food material as well.
Indeed many observers formerly believed that all ground-
waters were nearly free from bacteria, because often no
colonies appeared on plates counted after the ordinary
short periods of time. If, however, a longer period of
incubation be adopted considerable numbers may be
obtained.

For convenience we may divide ground-waters into two
groups, namely: first, springs and shallow open wells, and
second, "tubular" (driven) or deep wells. This division is
a convenient one because ordinary springs and wells form
a group by themselves in respect to the possibility of
aerial and surface contamination, their water often being
fairly rich in bacterial life. Egger (Wolffhugel, 1886) ex-
amined 60 wells in Mainz and found that 17 of them
contained over 200 bacteria to the cubic centimeter.
Maschek (Maschek, 1887) found 36 wells out of 48 exam-
ined in Leitmeritz which had a bacterial content of over
500 per c.c. Fischer (Horrocks, 1901) reported 120 wells

i6

ELEMENTS OF WATER BACTERIOLOGY.

in Kiel which gave over 500 bacteria per c.c. and only 51
v^^ith less than that number.

Several unpolluted springs and open wells were ex-
amined by Sedgwick and one of us (Sedgwick and Pres-
cott, 1895) with the following result:

Spring No. I. 252, 255, 258 bacteria per c.c.

" " 2. 163, 149, 134

" 3. 92, 98, 105

" " 4. 95, loi, 106 "

" " 5. 193, 213, 218 "

" "6. 216, 208, 201 "

No.

OPEN WELLS.

509, 525 bacteria per C.C.

248, 190 " " "

602, 560 " " "

335. 332 " "

2084, 2063, 2287 bacteria per c.c.

8905, 8905, 8640 " " "

702, 910, 871 " " "

720, 712, 763 " " "

It should be noted that the above results were obtained
by incubating the plates for considerable periods of time.
In the ordinary standard 48-hour period but very few
bacteria develop from normal ground-waters. Thus in
an examination of spring-waters made by the Massa-
chusetts State Board of Health in 1900 (Massachusetts
State Board of Health, 1901), of 37 springs which were
practically unpolluted and had less than 10 parts per
100,000 excess of chlorine over the normal, 54 samples
were examined and gave an average of 41 bacteria per
C.C. Only 6 samples showed figures over 50. On the
other hand, the analysis of the bottled samples of the
same waters as sold after exposure to contamination in

THE BACTERIA IN NATURAL WATERS. 17

bottling and the multiplication of the water bacteria
gave numbers rising to 243,000, only 14 out of 50 samples
being under 1000.

It now remains to consider the other great division of
ground-waters, namely, deep, "driven," or "tubular"
wells, which, if carefully constructed, should ordinarily be
perfectly free from all surface-water contammation. The
numbers of bacteria in such sources has been reported
by but few investigators. A series of wells in and near
Boston was found to give the following figures (Sedgwick
and Prescott, 1895).

r^^"-^ Depth, Feet. Bacteria per ,
269, 254

2.

100

^ 454 206, 214

t '54 150, 135

228

7-

10.
II.
12.

■ ■ , 192, in?
Second sample ^^^^

139, 140

Second sample 4^8, 416

377 48, 54

158, 149
'^7 1240, 1376

^3° 440, 480

525

60, 57
750 38

Second sample.

^3 180

14

Agam It should be noted that the period of incubation
for these samples was at least five days. Fifteen driven
wells m the neighborhood of Boston examined in igo,
showed at the end of 48 hours an average of only r 8
colonies per c.c. ^

xS ELEMENTS OF WATER BACTERIOLOGY.

It is plain that water absolutely free from bacteria is
not ordinarily obtained from any source and that even
deep wells contain quite appreciable numbers. The
peculiar character of the organisms present in the latter
case is manifested in many cases by the slow develop-
ment at room temperature (no growth until the third
day in some cases), the entire absence of liquefying
colonies, and the abundance of chromogenic species.

CHAPTER II.

THE QUANTITATIVE BACTERIOLOGICAL EXAMINATION

OF WATER.

That the customary methods for determining the num-
ber of bacteria do not reveal the total bacterial content,
but only a very smaU fraction of it, becomes apparent
when we consider the large number of organisms, nitrify-
ing bacteria, cellulose-fermenting bacteria, strict ans-
robes, etc., which refuse to grow, or grow only very slowly
in ordinary culture media, and which, therefore, escape
our notice. On the one hand certain obligate parasites
cannot thrive in the absence of the rich fluids of the ani-
mal body; on the other hand the prototrophic bacteria
adapted to the task of wrenching energy from nitrates
and ammonium compounds are unable to develop
in the presence of so much organic matter. This
IS made clear by the use of special media hke the
Nahrstoff Heyden agar (Hesse and Niedner, .808)
which are particularly adapted to the needs ;f these
a er organisms; or by direct microscopic exami^

n^enrtionlVftlT^^^^ '^'^ f^'^ ^°"-cl the .eco.n-

^- -f. ti. A., as given in the Appendix.

19

20 ELEMENTS OF WATER BACTERIOLOGY.

1902) showed that the numbers obtained by the ordi-
nary procedure were only from 5 to 50 per cent of
those obtained by the use of Heyden's Nahrstoff agar.
For practical sanitary purposes, however, our methods are
fairly satisfactory. Within limits, it is of no great impor-
tance that one method allows the growth of more bacteria
than another. When we are using the quantitative analy-
sis as a measure of sewage pollution only two things are
essential. First, media should be of standard composi-
tion, so that results obtained at different times and by
different observers may be comparable. In this respect
the work of G. W. Fuller, G. C. Whipple, and other
members of the Committee on Standard Methods of the
American Pubhc Health Association has placed the art of
quantitative water analysis in a state, veiy satisfactory, by
contrast with the chaos which prevails in England and
Germany. Secondly, it is desirable that the section of
the total bacterial flora which we obtain should be thor-
oughly representative of that portion of it in which we
are most interested— the group of the quickly growing,
rich-food-loving sewage forms. In this respect our meat-
gelatin-peptone appears to be unrivalled. The follow-
ing table from Gage and Phelps's valuable paper shows
clearly that the standard media bring out the difference
between pure and polluted waters much more clearly than
does the Nahrstoff medium. To emphasize this difference
with constancy is all that we require of a method for prac-
tical work.

QUANTITATIVE BACTERIOLOGICAL EXAMINATION. 21

Table showing Percentages of Bacteria Developing on Regu-
lar Agar and on Nahrstoff Agar for Different Classes of
Waters. (Gage and Phelps, 1902.)

regular agar.

Class of Water.

Days' Count.

2

3

4

s

6

7

8

Ground-water . . .

0

6

6

Filtered water

6

5
7

6

6

6

Merrimac River

6

7

7

7

7

7

Filtered sewage

14
34

7
17
44

7
18

8

19
46

8

19
46

9

9

40

19

19

46

46

NAHRSTOFF AGAR.

Ground-water. . ,
Filtered water. . .
Merrimac River.
Filtered sewage..
Sewage

6

43

78

37

69

80

29

78

93

26

65

93

39

75

95

92

97
95
100

93
98

97

97
100

100
100
99
99
100

100
100
100
100

TOO

The procedure for the quantitative determination of
bacteria in water consists, in brief, in mixing a deiinite
amount of a suitably collected specimen of the water with
a sterile sohdifiable culture medium and allowin- it to
develop for a sufficiently long time to permit reproduction
of the bacteria and the formation of visible colonies which
may be counted. The process is divided naturally into
four stages-samphng, plating, incubating, and counting
—which will now be discussed.

Saupling.--KXX samples of water for bacteriological
exammation should be collected in clean, sterile boTtles
-th wide mouths and glass stoppers, preferably of the flat

22 ELEMENTS OF WATER BACTERIOLOGY.

mushroom type. It is desirable that these bottles should
have a capacity of at least loo c.c.

They should be cleaned thoroughly before using, by
treatment with sulphuric acid and potassium bichromate
or with alkaline permanganate of potash followed by
sulphuric acid, dried by draining, and steriHzed by dry
heat at i6o° C. for at least one hour, or by steam at 115°-
120° for fifteen minutes. If not to be used immediately
the neck and stopper should be protected against dust
or other contamination by wrapping with lead-foil. For
transportation the bottle should be enclosed in a suitable
case or box.

The greatest care must be taken that the fingers do not
touch the inside of the neck of the bottle or the cone of the
stopper, as the water thereby would become seriously
contaminated and rendered unfit for examination. It is
well known that bacteria are found abundantly upon
the skin, and Winslow (Winslow, 1903) has shown that
even B. coli is present upon the hands in a considerable
number of cases.

In order to obtain a fair sample, great precautions must
be taken, and these will vary with the different classes of
waters to be examined and with local conditions. If a
sample is to be taken from a tap, the water should be
allowed to flow at least five minutes (if from a tap in regu-
lar use) or for a longer period in case the water has been
standing in the house service system, since in the small
pipes, changes in bacterial content are liable to occur.

QUANTITATIVE BACTERIOLOGICAL EXAMINATION. 23

certain species dying, and the sample, therefore, not being
a fair average of the water in the mains.

If a sample is to be taken from a pump similar pre-
cautions are necessary. The pump should be in con-
tinuous operation for several minutes at least, and prefer-
ably for half an hour before the sample is taken, in order
to avoid excessively high numbers due to the growth of
bacteria within the well and pump, the bacterial condition
of the water as it passes through the ground being what
we wish to determine. Thus Herasus (Herajus, 1886) in
a well-water which had been but little used during the
preceding thirty-six hours found 5000 organisms per cc;
when the well was emptied by continuous pumping a
second sample, after an interval of half an hour, gave only
35- Maschek (Tiemann and Gartner, 1889) obtained
similar results shown in the following table:

EFFECT OF PUMPmc ON THE BACTERIAL CONTENT OF WELL-WATER.

WeU-water after continuous pumping for fifteen minutes. . . ^rS

" " << << ,

many hours 140

^^t^^ '. 68

after contmuous pumping for fifteen minutes ... c,S

" << << ,

many hours i^n

later

, 73

After a proper interval of pumping the sample of a well-
water may be collected from the pet-cock of the pump or
from a near-by tap. With a hand-pump, as in sampling
domestic shallow wells, the water is, of course, pumped
directly into the sample bottle. The difficulties in
secunng an average sample from this latter source are

24 ELEMENTS OE WATER BACTERIOLOGY.

often great, since if the flooring about the pump is not
tight, as is usually the case, continued pumping may wash
in an unusual amount of surface pollution.

In sampling surface-waters, the greatest precautions
must be observed to prevent contamination from the
fingers. In still waters the fairest sample is one taken
from several inches down, as the surface itself is likely to
have niunerous dust particles floating upon it. The
method most frequently recommended is to plunge the
bottle beneath the surface to a depth of a foot or so, then
remove the stopper and allow the bottle to fill.

Another method which is comparatively free from
objection and which has been employed by the writers is
to remove the stopper first and then, holding the bottle by
the base, plunge it mouth downward into the water, turn-
ing it at the desired depth so as to replace the enclosed
air by the water. Whenever any current exists the
mouth of the bottle should be directed against it in
order to carry away any bacteria from the fingers. If
there is no current, a similar effect can be produced by
turning the bottle under water and giving it a quick for-
ward motion. In rapidly flowing streams it is only
necessary to hold the bottle at the surface with the mouth
pointed up-stream.

For taking samples of water at greater depths, a num-
ber of devices have been employed, aU of which are fairly
satisfactory. The essentials are, first, a weight to carry
the bottle down to the desired depth, and, second, a device

QUANTITATIVE BACTERIOLOGICAL EXAMINATION. 25

for removing the stopper when that depth is reached. The
student will find one good form of apparatus described
in Abbott's " Principles of Bacteriology" (Abbott, 1899);
an admirable one was devised by HiU and EUms (Hill
and EUms, 1898). Miquel and Cambier (Miquel and
Cambier, 1902) and other authors recommend the use of
a sealed glass bulb with a capillary tube which can be
broken off at any desired moment.

As soon as a sample of water is collected its conditions
of equilibrium are upset and a change in the bacterial
content begins. Even in the purest spring-waters which
contain but few bacteria when collected, and in which the
amount of organic matter is infinitesimal, enormous num-
bers will be found after storage under laboratory con-
ditions for a few days or even a few hours. In some cases
the rise in numbers is gradual, in others very rapid. The
Frariklands (Frankland, 1894) record the case of a deep-
well water in which the -bacteria increased from 7 to
495,000 in three days. Miquel (Miquel, 1891), from his
researches, arrived at the conclusion that in surface-waters
the rise is less rapid than in waters from deep wells or
springs, and that in the latter case the decrease, after
reaching a maximum, is likewise rapid and steady. Just
how far protection from Hght, increase in temperature,
and a destruction of higher micro-organisms is responsible
for the increase, and to what extent an exhaustion of food-
supply or the formation of toxic waste products causes
the succeeding decrease, we are not aware; but the facts
are well estabhshed.

20 ELEMENTS OF WATER BACTERIOLOGY.

Whipple has exhaustively studied the details of this
multiplication of bacteria in stored waters and has shown
in the table given below that there is first a slight reduc-
tion in the number present, lasting perhaps for six hours,
followed by the great increase noted by earHer observers.
It is probable that there is a constant increase of the
typical water bacilli, overbalanced at first by a reduction
in other forms, for which this is an unsuitable environment.

BACTERIAL CHANGES IN WATER DURING STORAGE.

(Whipple, 1901.)

Sample.

Initial
Temper-
ature.

Temp,
of Incu-
bation
of

Sample.

Number of Bacteria per c c.

Initial.

After
3 Hours.

After
6 Hours.

After
24 Hours.

After
48 Hours.

C.

C.

A

7.6°

17.0°

260

215

230

900

27,000

B

7.6°

17.0°

260

245

25s

720

10 850

C

7.6°

12.5°

260

270

231

600

2,790

D

7.6°

12.5°

260

270

245

710

I ,800

E

7.6°

2.4°

260

243

210

675

1,980

■ F

7.6°

2.4°

260

■235

270

560

1,980

G

11.0°

12.8°

77

55

58

lOI

10 250

H

11.0°

12.8°

77

S3

74

87

2175

I

11.0°

23.6°

77

51

52

11,000

41.400

J

6.7°

20.0°

430

375

245

385 000*

K

6.7°

20.0°

430

345

405

750,000*

L

23.2°

23.0°

51°

340

230

8,000

20,000

M

23.2°

2.5°

525

300

220

380

2.200

0.0005 psr cent peptone added to the water.

Wolffhugel and Riedel (Wolffhugel and Riedel, 1886)
noted the dependence of this multiplication on the amount
of the air-supply, vessels closed with rubber stoppers show-
ing lower numbers than those plugged with cotton. Simi-
larly, Whipple found that the multiplication of bacteria

QUANTITATIVE BACTERIOLOGICAL EXAMINATION. 27

was much greater when the bottles we're only half full
than when they were filled completely; and also, as shown
in the following very striking table, that the size of the
bottle markedly influenced the growth.

EFFECT OF SIZE OF VESSEL UPON THE MULTIPLICATION OF WATER
BACTERLA DURING STORAGE.
(Whipple, 1901.)

Sample

A
B
C
D
E

F
G
H
I
J

Bottle.

I -gallon
2Tquart
I -quart

1 - pint

2 - ounce

1 - gallon

2 - quart
I -quart

1 - pint

2 - ounce

Temp, of
Incuba-
tion.

Number of Bacteria per c.c.

C.
13°
13°
13°
13°
13°

24°
24°
24°*
24°
24°

Ini-
tial.*

After
3 hrs.

After
6 hrs.

77

63

6S

77

59

63

77

63

63

77

57

6r

77

55

58

77

81

97

77

92

59

77

84

77

77

46

77

5=;

52

After
1 2 hrs

47
60

47

36

47

275
62
46

100

145

After
24 hrs.

42

45
46

38

lOI

290
180
340
2,950
11,000

After
48 hrs.

175
690

325
630
10,250

300
250
900
7,020
41,400

* Average of five plates.

These results and those of other observers make it
obvious that samples must be examined shortly after
collection and that they must be kept cool during their
storage. If fairlypure waters are placedupon ice and kept
between o and ro», they wiU show no material increase
m twelve hours. With polluted water, however, another
danger .s here tntroduced. Samples of such wa er when
packed .n ,ce show a marked decrease due to the larg:
number of sens.ttve mtestinal bacteria present. Jordan
(Jordan, r^) fou^d that three samples of river-water

28 ELEMENTS OF WATER BACTERIOLOGY.

packed in ice for forty-eight hours fell off from 535,000 to
54,500; from 412,000 to 50,500, and from 329,000 to
73,000, respectively. It is, therefore, necessary to adhere
strictly to the recommendations of the A. P. H. A. Com-
mittee that the interval between sampling and examina-
tion should not exceed twelve hours in the case of rela-
tively pure waters, six hours in the case of relatively
impure waters, and one hour in the case of sewage.

Plating. — The bottle containing the sample of water is
first shaken at least twenty-five times in order to get an
equal distribution of the bacteria. If the number of
bacteria present is probably not greater than 200, i c.c. is
then withdrawn with a sterile i c.c. pipette and deUvered
into a sterile Petri dish of 10 cm. diameter. To this is
added 5 c.c. of standard 10 per cent gelatin at a tempera-
ture of about 30° C. or standard agar (7 c.c.) at 4o°-42° C
Should the number of bacteria per c.c. probably exceed
200, dilution is necessary. This is best accomplished by
adding i c.c. of the water in question to 9, 99, or 999, etc.,
c.c. of sterile tap water according to the amount of dilution
required. The diluted sample is then shaken thoroughly
and I c.c. taken for enumeration. In order to determine
the number of bacteria originally present it is only neces-
sary to multiply by the factor 10, 100, or 1000, etc.

When a sample of water from an unknown source is to
be examined it is generally desirable to make a series of
plates at each of the above dilutions, selecting those which
give nearest to 200 colonies on the plates after incubation
as the ones on which to rely for the count. A much

QUANTITATIVE BACTERIOLOGICAL EXAMINATION. 29

smaller number will not give average figures, and if more
than 200 colonies are present on a plate many bacteria
will be checked by the waste products of those which first
develop and the count obtained will be too low. After
the addition of the diluted sample and the nutrient medium
their thorough mixture in an even layer on the bottom
of the plate is obtained by careful tipping and rota-
tion.

It was formerly customary to mix the water with the
gelatin in the tube before pouring into the plate, but this
method is objectionable because there is always a small
residue of medium remaining in the tube which will
retain varying numbers of bacteria and thus interfere
with the accuracy of the count. Before pouring the
medium into the plate the mouth of the tube should
be flamed to remove any possibility of contamina-
tion.

The exact composition of the medium used is, of course,
of prime importance in controlling the number of bac-
teria which will develop. The reaction of the medium
was found as early as 1891 to be important, for Reinsch
(Reinsch, 1891) showed in that year that the addition
of one one-hundredth of a gram of sodium carbonate to
the Hter increased sixfold the number of bacteria develop-
ing. Fuller (Fuller, 1895) and Sedgwick and one of
ourselves (Sedgwick and Prescott, 1895), working inde-
pendently, estabhshed the fact that an optimum reac-
tion existed for most water bacteria and that a deviation
either way decreased the number of colonies developing.

20 ELEMENTS OF WATER BACTERIOLOGY.

The following table from Gage and Phelps shows con-
clusively the effect of the general composition of the
nutrient medium.

TABLE SHOWING PERCENTAGES OF BACTERIA DEVELOPING ON MEDIA OF
DIFFERENT COMPOSITIONS.
(Gage and Phelps, 1902.)

Days' Count.

meaium.

2

3

4

s

6

7

8

9

19

60

78

85

95

99

99

100

Nahrstoff pepton agar

10

22

26

28

30

30

3°

30

II

16

22

23

24

24

24

24

Meat agar

8

13

16

17

17

17

17

17

Plain agar

8

10

13

14

14

14

14

14

7

0

1 1

II

II

II

II

II

Nahrstoff glycerin agar

6

10

II

II

II

II

II

II

Nahrstoff meat agar

7

7

8

8

10

10

10

10

Meat gelatin

12

19

24

26

26

26

26

26

Pepton gelatin

.7

12

18

20

20

20

20

20

8

10

II

12

13

13

13

13

I

6

12

13

13

13

13

13

5

6

9

II

13

13

13

13

Finally, Whipple (Whipple, 1902) has shown that not
only the particular kind of gelatin used but its exact
physical condition as afifected by sterilization and other
previous treatments will materially affect the results
obtained. It is evident, therefore, that only the strictest
adherence to some standard method can ensure com-
parable results; the ordinary nutrient gelatin should
then in all practical sanitary work be made up in exact
accordance with the direction of the A. P. H. A. Commit-
tee as given in the Appendix.

QUANTITATIVE BACTERIOLOGICAL EXAMINATION. 3I

Incubation. — " Incubation should take place in a dark,
well-ventilated chamber where the temperature is kept,
substantially constant at 20° and where the atmosphere
is practically saturated with moisture." — ^A. P. H. A.
Report. It has, been shown by Whipple (Whipple, 1899)
and others that the number of bacteria developing in
plate cultures is to a certain extent dependent upon the
presence of abundant oxygen and moisture. Thus
reckoning the number of bacteria developing in a moist
chamber at 100 the percentage counts obtained in an
ordinary incubator were as follows: 75 when the relative
humidity of the incubator was 60 per cent of saturation;
82 when it was 75 per cent; 98 when it was 95 per cent.'
This source of error may be avoided by the use of ven-
tilated dishes and by the presence of a pan of water in
the incubating chamber.

According to American and German practice, plates
made for sanitary water analysis are counted at the end of
forty-eight hours. French bacteriologists still recom-
mend longer periods, and the following table from Miquel
and Cambier (Miquel and Cambier, 1902) shows that
many bacteria fail to appear in our ordinary analysis.
It is, however, as we have before pointed out, certain
peculiar water forms which develop so slowly, sewage
bactena almost without exception being rapid growers
The longer period of incubation is, therefore, not only
inconvenient but undesirable, since it obscures the differ-
eace between good and bad waters.

32

ELEMENTS OF WATER BACTERIOLOGY.

EFFECT OF THE LENGTH OF INCUBATION OF WATER BACTERIA IN GEL-
ATIN UPON THE NUMBER OF COLONIES DEVELOPING.
(Miquel and Cambier, 1902.)

Colonies Developed.
20

136

254

387

53°

637

725

780

821

859

892

921

951

976

1000

Length of Incubation.

1 day

2 days

3
4

5
6

7

8

9
10
II
12

13
14
15

Counting. — The number of bacteria is determined by
counting the colonies developed upon the plate, either
with the naked eye or preferably, with a low-power lens.
This is actually done by placing the plate upon a glass
plate ruled in centimeter squares and over a black tile;
or the tile itself may be ruled. The colonies then appear
as whitish round or oval specks in and upon the medium.
As has already been said, it is desirable that this number
should not exceed 200, for when the number is very high
the colonies grow only to a small size and make counting
laborious and inaccurate, and many do not develop at all.
On the other hand, too few bacteria upon the plate prob-
ably give inaccurate resuks also. The greatest accuracy
is probably obtained with numbers ranging from 50 to
200.

QUANTITATIVE BACTERIOLOGICAL EXAMINATION. 33

When it is possible to do so, all the colonies on the
plate should be counted. When they exceed 400 or 500
it is often easier and fully as accurate to count a frac-
tional part of the plate and estimate the total number
therefrom. This should not be done, however, except
in case of necessity.

It is customary in determining numbers to make plates
in duphcate, thereby affording a check upon one's own
work and enhancing the value of the results through
the greater accuracy obtained. Owing to the lack of
precision in the method, the hmit of experimental error is
a wide one. It should be possible,, however,, for careful
manipulators to obtain results within 10 per cent of each
other, and a closer agreement than this is hardly to be
expected. It has been suggested by the committee of
the American Public Heakh Association to adopt the fol-
lowing mode of expressing results.

NUMBERS OF BACTERIA FROM

1-50 shall be recorded to the nearest unit

51-100
101-250
251-500
501-1,000
1,001-10,000
10,001-50,000
5o,.oo 1-100,000
100,001-500,000
500,001-1,000,000
1,000,001-5,000,000

S
10

25
50
100
500
1,000
10,000
50,000
100,000

The determination of numbers of bacteria in water in
the field has frequently been attempted. Since the labora-

34 ELEMENTS OP WATER BACTERIOLOGY.

tory method of "plating out" is not appUcable for field
work the Esmarch tube process has often been employed.
This consists in introducing into a tube of melted gelatin
or agar i c.c. of the water and then rotating the tube until
the medium has sohdified in a thin layer on the inner wall
of the tube. In the writers' opinion, this method is open
to several objections and does not give as trustworthy re-
sults as the laboratory method, even if the samples have
to be several hours in transit between the place of col-
lection and the laboratory.

CHAPTER III.

THE INTERPRETATION OF THE QUANTITATIVE BAC-
TERIOLOGICAL ANALYSIS.

The information furnished by quantitative bacteri-
ology as to the antecedents of a water is in the nature of
circumstantial evidence and requires judicial interpre-
tation. No absolute standards of purity can be estab-
lished which shall rigidly separate the good from the bad.
In this respect the terms "test" and "analysis" so univer-
sally used are in a sense inappropriate. Some scientific
problems are so simple that they can be definitely settled
by a test. The tensile strength of a given steel bar, for
example, is a property which can be absolutely deter-
mined. In sanitary water analysis, however, the factors
involved are so complex and the evidence necessarily so
indirect that the process of reasoning much more re-
sembles a doctor's diagnosis than an engineering test.

The older experimenters attempted to establish arbi-
trary standards, by which the sanitary quaHty of a water
could be fixed automaticaUy by the numbers of germs
alone. Thus Miquel (Miquel, 1891) furnished a table
according to which water with less than 10 bacteria per c.c.
was "excessively pure," with 10 to 100 bacteria, "very

35

36 ELEMENTS OF WATER BACTERIOLOGY.

pure," with loo to looo bacteria, "pure," with looo to
10,000 bacteria, "mediocre," with 10,000 to 100,000 bac-
teria, "impure," and with over 100,000 bacteria, "very
impure." Few sanitarians would care to dispute the
appropriateness of the titles applied to waters of the last
two classes; but many bacteriologists have placed the
standard of "purity" much lower. The limits set by
various German observers range, for example, from 50 to
300. Even Dr. Sternberg (Sternberg, 1892), in a much
more conservative fashion, has stated that a water con-
taining less than 100 bacteria is presumably from a deep
source and uncontaminated by surface drainage; that
one with 500 bacteria is open to suspicion ; and that one
with over 1000 bacteria is presumably contaminated by
sewage or surface drainage. This is probably as satisfac-
tory an arbitrary standard as could be devised, but any
such standard must be appHed with great caution. The
source of the sample is of vital importance in the inter-
pretation of analyses; a bacterial count which would con-
demn a spring might be quite normal for a river; only
figures in excess of those common to unpolluted waters
of the same character giye the indication of danger. Fur-
thermore, the bacteriological tests are far more deHcate
than any others at our command, very minute additions
of food material causing an immense multipHcation of
the microscopic flora. This deHcacy necessarily requires,
both in the process of analysis and the interpretation of
results, a high degree of caution. As pointed out in the
previous chapter, the touch of a finger or a particle of

QUANTITATIVE BACTERIOLOGICAL ANALYSIS. 37

dust may wholly destroy the accuracy of an examination.
Even the slight disturbance of conditions incident upon
the storage of a sample after it has been taken may in a
few hours wholly alter the relations of the contained
microbic Ufe. It is necessary, then, in the first place to
exercise the greatest care in allowing for possible error in
the collection and the handling of bacteriological samples;
and in the second place, only well-marked differences in
numbers should be considered as possibly significant.

In the early days of the science, discussion ran high as
to the interpretation of bacteriological analysis; and par-
ticularly as to the relation of bacterial numbers to the
organic matter present in a water. Different observers
obtained inconsistent results, and Bolton (Bolton, 1886)
concluded that there was no relation whatever between
the chemical composition of a water and its bacterial con-
tent. Tiemann and Gartner (Tiemann and Gartner,
1889) furnished the key to the difficulty in their state-
ment that there are two great classes of bacteria, the
great majority of species normally occurring in the earth
or in decomposing organic matter which require abun-
dance of nutriment, and certain pecuHar water bacteria
which can multiply in the presence of such minute traces
of ammonia as are present in ordinary distilled water.
Bacteria of the second class under abnormal conditions,
as in bottled samples, or, at times, in a well or the basin of
a spring, may occur in great numbers where there is but
little organic matter. In normal surface-waters, how-
ever, such growths have not been recorded as far as wc

38 ELEMENTS OF WATER BACTERIOLOGY.

are aware. Here the numbers of bacteria present will
depend, other things being equal, upon the extent to
which he water has been contaminated with decomposing
organic matter, either by pollution with sewage o: by
contact with the surface of the ground. The bacterial con-
tent will therefore vary as the extent and character of the
contamination varies It measures not merely organic
matter but organic matter in a state of active decay, and
thus, like the ammonias and other features of the sanitary
chemical analysis, clearly indicate, fresh organic pollution
with the added advantage that the presence of the stable
nitrogenous compounds often present in peaty waters
introduces no error in the bacteriological analysis.

In judging of a surface-water the student will be aided
by reference to the figures given for certain normal sources
in Chapter I; the Boston tap water with 50 to 200 bac-
teria per c.c. (Whipple, 1896) and the water of Lake
Zurich with an average of 71 in summer and 184 in winter
(Cramer, 1885) may be taken as typical of good potable
waters; and numbers much higher than these are open
to suspicion, since aU contamination whether contributed
by sewage or by washings from the surface of the ground
is a possible source of danger. The excess of bacteria
in surface-waters during the spring and winter months is
by no means an exception to the general rule that high
numbers are significant, since the peril from supplies of
this character is clearly shown by the spring epidemics of
typhoid fever which at the times of melting snow ^aslt
communities making use of unprotected surface-waters.

QUANTITATIVE BACTERIOLOGICAL ANALYSIS. 39

Streams receiving direct sewage pollution exhibit a simi-
lar excess of bacteria at aU times, numbers rising to an
extraordinary height near the point of entrance and fall-
ing off below as the stream suffers dilution and the sewage
organisms perish. Miquel (Miquel, 1886) records 300
bacteria per c.c. in the water of the Seine at Choisy, above
Paris, 1200 at Bercy in the vicinity of the city, and 200,000
at St. Denis after the entrance of the drainage of the city.
Prausnitz (Prausnitz, 1890) found 531 bacteria per c.c.
in the Isar above Munich, 227,369 near the entrance of
the principal sewer, 91 11 a': a place 13 kilometers below
the city, and 2378 at Freising, 20 kilometers further
down. Jordan (Jordan, 1900), in his study of the fate of
the sewage of Chicago, found 1,245,000 bacteria per c.c.
in the drainage canal at Bridgeport, 650,000 twenty-nine
miles below at Lockport, and numbers steadily decreasing
below to 3660 at Avery ville, 159 miles below the point of
original pollution. Below Averyville the sewage of Peoria
enters and the numbers rise to 758,000 at Wesley City,
decreasing to 4800 in 123 miles flow to KampsviUe.

In ground-waters we have seen that bacteria may be
present in considerable numbers, but that they wiU
generaUy be organisms of a peculiar character, incapable
of development on the ordinary nutrient media in the
standard time. Thus in forty-eight hours we often obtain
counts measured only in units or tens. When higher
numbers are present, the general character of the colonies
must be taken into account, since besides the slowly-
growing foims certain other water bacteria which

40

ELEMENTS OF WATER BACTERIOLOGY.

require a comparatively small amount of nutriment may
multiply at times in a deep well or the basin of a
spring. In such a case, however, the appearance of the
plates would at once reveal the peculiar conditions, for the
colonies would all be of one kind and that distinct from
any of the sewage species. Thus Dunham (Dunham, 1889)
reports that the mixed water from a series of driven wells
gave 2 bacteria per c.c, while another well, situated just
like the others, contained 5000, all belonging to a single
species common in the air. Plates from polluted water
contain, on the other hand, a wide variety of species.

The process of slow sand filtration for the purification
of unprotected surface-water is essentially similar to the
action which takes place in nature when rain soaks through
the ground to appear in wells and springs; and it is in the
examination of the efBuent from such municipal plants
that the quantitative bacteriological analysis finds, per-
haps, its most important apphcation. The chemical
changes which occur in the passage of water through
sand at a rate of 1,000,000 or 2,000,000 gallons per acre
per day are so slight as to be negligible. The bacteria
present should, however, suffer a reduction of 98 or 99 per
cent, and their numbers are, therefore, used as the standard
for measuring the efiiciency of such filtration plants. At
Lawrence, in 1901, Clark found an average of 3017 bac-
teria per cc. in the raw water of the Merrimac River, while
the number present in the filtered water was only 26
(Massachusetts State Board of Health, 1902). Mechan-
ical filtration gives similar results. Fuller at Cincinnati

QUANTITATIVE BACTERIOLOGICAL ANALYSIS. 41

(Fuller, 1899) records 27,200 organisms per c.c. in the
water of the Ohio River between September 21, 1898, and .
January 25, 1899, while the average content of the effluent
from the Jewell filter was 400. In well-managed purifi-
cation plants the bacteria in the effluent are determined,
daily, and any deviation from the normal value at once
reveals disturbing factors which may impair the efficiency
of the process. In Prussia official regulations demand
such systematic examinations and prescribe 50 as the
maximum number of bacteria allowable in the filtered
water. In the same way the condition of an unpurified
surface supply may be determined by daily bacteriological
analyses and warnings of danger issued to the public, as
has been done at Chicago and other cities. In general,
any such regular determination of variations from a nor-
mal standard furnish ideal conditions for the bacteriolog-
ical methods; and the detection by Shuttleworth (Shuttle-
worth, 1895) of a break in a conduit under Lake Ontario
by a rise in the bacteria of the Toronto water-supply may
be cited as a classic example of its appHcation.

Often, however, the expert is called to pass upon the char-
acter of a water of which no series of analyses is available
and whose surroundings it may be impossible for him to in-
spect. It has been said that single bacteriological analyses
of this kind are valueless ; but this we believe cannot always
be maintained. Knowing the normal bacterial range
for a given class of waters, even an isolated analysis may
show such an excess as to have great significance, as a
few practical examples may make clear (Winslow, 1901).

42 ELEMENTS OF WATER BACTERIOLOGY .

In the spring of 1900 the city of Hartford, Conn., was
using a double supply from the Connecticut River and
from a series of impounding reservoirs among the hills.
A single series of plates showed from 4000 to 7000 bac-
teria per c.c. in the water of the river, while the reservoir
water contained 300 to 900. The abandonment of the
river supply followed, and at once the excessive amount
of typhoid fever in the city was curtailed.

In the fall of 1900, Newport, R. I., experienced an out-
break of typhoid fever, and when suspicion was thrown
upon the public water-supply, chemical analysis of the
latter was not wholly reassuring; but there were only
334 bacteria per c.c. in the water from the taps, while a
well in the infected district gave 6100. It was no surprise
to find, on a further study of the epidemic, that the well
was largely at fault and the pubhc supply not at all.

In the case of ground- water the evidence is usually even
more distinct. At Framingham, Mass., in 1903, high
chlorin content in the pubhc supply, drawn from a filter
gallery beside a lake, had led to pubhc anxiety. Five
samples from different parts of the system showed aver-
ages of I, 2, 2, 2, and 4 bacteria per c.c; and taking this
in conjunction with the other features of the bacterio-
logical analysis, it was possible to report that any pollution
introduced upon the gathering ground had at the time of
analysis been entirely removed. In such a case the bac-
teriological methods give a certainty attainable by no
other sanitary process.

CHAPTER IV.

DETERMINATION OF THE NUMBER OF ORGANISMS
DEVELOPING AT THE BODY TEMPERATURE.

The count of colonies upon the gelatin plate measures
as we have pointed out, the number of those micro-organ-
isms associated with the decomposition of organic matter
wherever it may occur. In this great class, however,
there are a few species, like B. subtiHs and B. ramosus,
which will grow under a great variety of conditions and
which are Hkely to be present with more or less constancy
in water, and others which through a semi-parasitic
mode of Hfe have become specially adapted to the pecu-
liar conditions characteristic of the animal body. The
latter in particular possess the property of developing
most actively at the temperature of the human organism,
37° C, which ahogether checks the growth of the majority
of normal earth and water forms. The determination of
the number of organisms growing at the body temperature
may throw light, then, on the presence of direct sewage
pollution, since the bacteria from the alimentary canal
flourish under such conditions, while most of those derived
from other sources do not. The count at 37° helps to
distinguish contamination by wash of the soil of a virgin
woodland from poUution by excreta, since in the latter

43

44 ELEMENTS OF WATER BACTERIOLOGY.

case the proportion of blood-temperature organisms is
much smaller than in the latter. Furthermore, this
method is free from much of the error introduced by the
multiplication of bacteria after the collection of a sample,
since most of the forms which grow in water during
storage cannot endure the higher temperature and conse-
quently do not develop upon incubation.

The body-temperature count must, of course, be made
upon agar plates, otherwise the technique is the same
which has already been described for the routine quan-
titative bacteriological analysis. The period of incuba-
tion ordinarily adopted by the writers is twenty-four
hours, as httle development occurs after that timg. Diffi-
culty is sometimes caused by the spreading of colonies
of certain organisms over the surface of the plate in the
water of condensation which gathers; this may be avoided
by inverting the plates after the agar is once well set.

Additional evidence as to the character of a water
sample may be obtained with little extra labor by adding
a sugar and some sterile litmus to the agar medium and
observing the fermenting powers of the organisms present,
as first suggested by Wurtz (Wurtz, 1892) for the separa-
tion of B. coli from B. typhi. It happens that the most
abundant intestinal organisms belonging to the groups
of the colon baciUi and the streptococci decompose dex-
trose and lactose with the formation of a large excess of
acid, while many other organisms, even if they grow
abundantly at the body temperature, are not favored by
the presence of the sugar and litmus. The decomposi-

DETERMINATION OF ORGANISMS.

45

tion of the latter sugar is almost entirely wanting among
the commoner saprophytic bacteria, and therefore lac-
tose is most commonly used in making sugar agar, 2 per
cent being added to the medium just before the final filtra-
tion (between steps 15 and 16 in the standard process
of media making given on p. 120). In pouring the plate
a cubic centimeter of sterile litmus solution should be
added; and in counting, the colonies of the acid-forming
organisms will be clearly picked out by the reddening of
the adjacent agar. Only those which show this clearly
should be considered as significant, since certain bacteria
of the hay-bacillus group produce weak acid and faint
coloring of the litmus.

When polluted waters are examined in this manner the
number of organisms developing on the lactose-agar plate
will be very high, almost equalling in some cases the total
count obtained on gelatin. Chick (Chick, 1901), using
a lactose-agar medium vsdth the addition of one-thousandth
part of phenol, found of colon bacilli alone 6100 per c.c.
in the Manchester ship canal, 55-190 in the polluted
River Severn, and numbers up to 65,000 per gram in road-
side mud. In an examination of water from the Charles
River above Boston, total 37° counts ranging from 9800 to
16,900 have been found. Two twenty-four-hour exam-
inations of Boston sewage made at the Sanitary Research
Laboratory of the Institute of Technology during the
summer of 1903 gave an average of 3,660,000 bacteria
per c.c. on gelatin at 20° and 2,310,000 per c.c. on lactose
agar at 37° with 550,000 acid-formers.

46 ELEMENTS OF WATER BACTERIOLOGY .

In unpolluted waters not only the absolute number of
organisms developing at the body temperature, but its
ratio to the gelatin count, is very different. Rideal (Rideal,
1902) states that the proportion between the two counts
in the case of a London water in a year's examination
was on the average one to twelve. Mathews (Mathews,
1893) in 1893, gave the following figures, the contrast
between the ponds and streams which were presumably
exposed to pollution on the one hand, and the wells,
springs, and taps on the other, being marked.

Sources of Water.

Wells, springs
Reservoirs —

Ponds

Taps

Streams

Average Number of
Colonies per c.c.

Gelatin, 20°.

1664

153
296
242
273

Wurtz Agar,
37-5°.

28
43
95
24

lOI

In 1903 Nibecker and one of ourselves (Winslow and
Nibecker, 1903) made an examination of 259 samples
of water from presumably unpolluted sources in Eastern
Massachusetts, including pubhc supplies, brooks, springs,
ponds, driven wells, and pools in the fields and woods,
with a view to testing the value of the body-temperature
examination. In many cases the samples showed high
gelatin counts, since some of the waters were exposed to
surface wash from vacant land, but the average number
of organisms developing on lactose agar at 37° was less

DETERMINATION OF ORGANISMS. 47

RELATION OF 20° AND 37° COUNTS IN SAI.IPLES OF WATER FROM APPAR-
ENTLY UNPOLLUTED SOURCES.

(Winslow and Nibecker, 1903.)

Gelatin
Plates,
20°.

Litmus-
lactose-
agar
Plates, 37°.

Dextrose Broth
Tubes.

Source of Samples.

r of Samples

e Number
lonies.

e Number
ilonies.

Showing
Colonies.

r of Tubes.

r of Tubes
Gas.

r of Tubes
Gas i-o.

r of Tubes
Gas 2-1.

'id

So

XI

¥

%

a
3

2

<

<

S

5

94

II

0

IS

0

0

0

n Vfififilrl inrl ^+r\n n a m Cll Tin 1 V
VV clKcilclU. cili(-l o HJllClldlil oUjjpi^

7

59

6

0

21

0

0

0

6

16

3

0

18

0

0

0

I

18

2

3

0

0

0

T*IvTnoiitn Qiinnlv ifani

6

3S

2

0

18

0

0

0

T^pa nnnv ciinnlv ( fnn i

3

141

21

0

9

2

2

0

Tlprlnfim oiir^r^Mr il"Qr>i

6

3.717

I

0

18

0

0

0

^^f* w 111 1 T*vr>oT"t Cll r^nl V i ta n 1

6

36

9

0

18

0

0

0

Salem supply (tap)

5

232

14

0

IS

0

0

0

4

13

2

0

12

0

0

0

Sharon (well) (tap)

3

738

46
8

2

9

3

0

3

0

I e

0

0

2

4,700

0

0

6

0

0

0

Westerly, R. I., supply (tap). . .

1

12

0

3

0

0

0

6i

223

7

0

18^

1 1

I X

Driven wells

IS

18

I

0

45

0

0

32

294

2

0

95

13

13

IS

167

9

0

45

I

I

T\yr„i*-,j

I

Pools in fields

22

36s

4

31

0
0

3
66

0

2

0
2

Pools in woods

Roadside pools

22
10

181
811

3
4

0
0

65
30

0
2

0
2

Stream, Blue Hill Reservation. .

I

D

0

0

Flow from rocks. . . .

Ponds fed by springs

2

6

47
188

0
2

0
0

3
6

18

0
0

0
0
0

Drainage from manured pasture.
Swamps

Rain-water after twelve hours'

1

1.23s

27

0

3

0

0

3

269

6

0

9

5

5

0

heavy fall..

Shallow well in Lynn woods. ...

7
I

IS

2
I

0
0

21
3

0
0

0
0

38

3

259

4

775

41

48 ELEMENTS OF WATER BACTERIOLOGY.

than 8 per c.c. The highest individual counts obtained,
as will be seen by reference to the table on the preceding
page, were 95 in a meadow pool, 83 in a brook, and 74
in a barnyard well, the latter probably actually polluted.
Only two samples in the whole series, one from the well
above mentioned, gave any red colonies on the agar
plates.

Thus it is clear that organisms growing at the body tem-
perature and those fermenting lactose are not numerous
in normal waters, the total count rarely exceeding 50,
with acid producers generally entirely absent. On the
other hand, the numbers on the Htmus-lactose-agar plate
will be likely to run into hundreds with a good proportion
of red colonies when polluted waters are examined. The
method is, therefore, one of the most useful at the dis-
posal of the bacteriologist. It yields results within
twenty-four hours, and the conclusions to be dravra from
it are definite and clear.

CHAPTER V.

THE ISOLATION OF SPECIFIC PATHOGENES FROM

WATER.

The discovery of the organisms which specifically
cause the infectious diseases naturally led to the hope
that their isolation from polluted water might become
the most convincing proof of its sanitary quality. The
typhoid bacillus and the spirillum of Asiatic cholera were
in this connection of paramount importance, and to the
search for them many investigators devoted themselves.

In the earlier examinations of water for the typhoid
bacillus an attempt was made to use media which espe-
cially favored the growth of the microbe sought for, or to
begin with some process of "enrichment" in which the
sample was incubated under conditions which would
favor the growth of the pathogenic organisms while
checking the development of the common water bacteria.
It was apparent that the body temperature and the pres-
ence of a slight excess of free acid furnished such condi-
tions, and most of the methods suggested rest upon
these principles. The most general perhaps is that of
Parietti (Parietti, 1890), which consists in the addition
of the water to a series of broth tubes containing increas-

49

so ELEMENTS OF WATER BACTERIOLOGY.

ing amounts of a solution of 4 per cent hydrochloric acid
and 5 per cent phenol. From tubes in which growth
occurs after twenty-four hours at 37°, the organisms
present may be isolated in pure cultures by some plating
method and identified by subcultures.

The great difficulty with the enrichment processes is
that the conditions which favor the multiplication of the
typhoid bacillus are suited in an even higher degree to
B. coli and other intestinal organisms. Being present in
almost all cases in much higher numbers than B. typhi,
these germs develop most abundantly, and effectually
mask any disease germs originally present. In order to
obviate this difficuky, Hankin (Hankin, 1899), after
adding successively increasing portions of Parietti solu-
tion to tubes inoculated with the water to be tested,
selected the second highest tube of the series in which
growth occurs for the inoculation of a new set, finally
plating as above. He beheved that the chance for over-
growth in this method is somewhat decreased; but in
the hands of other investigators it has not met with marked
success. Klein (Thomson, 1894), in his investigations,
made use of the Berkefeld fiher to concentrate the or-
ganisms in the sample. Some recent observers have
abandoned the enrichment process altogether and recom-
mend direct plating upon phenolated gelatin or on the
Eisner (Eisner, 1896) medium made by adding 10 per
cent of gelatin and i per cent of potassium iodide to an
infusion of potato whose reaction has been adjusted to
20 on Fuller's scale. In all cases, however, the chance

ISOLATION OF SPECIFIC PATHOGENES. $1

of success is small; as is well shown by the experiments
of Laws and Andrewes (Laws and Andrewes, 1894), who
entirely failed to isolate the typhoid bacillus from the
sewage of London and found only two colonies of the
organism on a long series of plates made from the sewage
of a hospital containing forty typhoid patients. So Wathe-
let (Wathelet, 1895) found that of 600 colonies isolated
from typhoid stools and having the appearance charac-
teristic of B. coli and B. typhi only 10 belonged to the
latter species.

At the other end of the process the identification of the
pure cultures isolated is again subject to considerable
uncertainty. The typhoid bacillus belongs to a large
group which contains numerous varieties differing from
each other by minute degrees. The inability to repro-
duce the disease by inoculation in available test animals
omng to their natural immunity is a serious drawback;
and the specific biochemical characters of the organism
are, as it happens, mostly negative ones, as shown by com-
parison with B. coh, to which it is supposed to be allied.

COMPARISON OF THE CHARACTERS OF B. COLI AND B. TYPHI.
(Horrocks, 1901.)
C. coli. B. typhi.

(i) Surface Colonies, Gelatin (i) Much thinner than those
Plates.— Thicker, and grow more of B. coli, and grow more slowly,
rapidly than those of B. typhi. After forty-eight hours' incuba-
After forty-eight hours' incubation tion at 22° C. they are hardly
at 22° C. they are usually large visible to the naked eye.
and characteristic.

52

ELEMENTS OF WATER BACTERIOLOGY.

C. coli.

(2) Gelatin-stab. — Quick growth
on the surface and along the line
of inoculation.

(3) Gelatin-slope. — Thick broad
grayish-white growth with a cre-
nated margin.

(4) Witte's Peptone and Salt
Solution. — Indol produced.

(5) Milk. — Coagulated.

(6) Litmus-whey, one week at
37° C. Acid produced, usually
requiring from 20 to 40 per cent of
N

— alkali to neutralize it.
10

(7) Neutral -red Glucose-agar. —
Marked green fluorescence.

(8) Glucose-gelatin and Lac-
tose-gelatin Shake Cultures, and
Glucoseragar-stab. — Marked gas
formation.

(9) Gelatin, 25 per cent, incu-
bated at 37° C— Thick film
appears on the surface.

(10) Potato. — As a rule, a thick
yellowish-brown growth.

(11) Proskauer and Capaldi's
Media. No. I, after twenty hours
growth, medium acid. No. II,
Growth, medium neutral or faintly
alkaline.

(12) Nitrate-broth. — Nitrate re-
duced to nitrite.

(13) Microscopical Appear-
ances.— A small bacillus often Uke
a coccus, not motile as a rule.

(14) Flagella. — Usually i to 3,
short and brittle; sometimes 8 to
12, long and wavy.

B. typhi.

(2) Slow growth on the surface
like the colonies; along the line of
inoculation the growth is much
thinner, and often ends below in a
few white points consisting of dis-
crete colonies.

(3) Thin narrow grayish-white
growth, crenated margin not
marked as in B. coli.

(4) No formation of indol.

(5) Unchanged after a month.

(6) Very small amount of acid
produced, requiring not more than

N

6 per cent of — alkali to neutralize
10

it.

(7) No change.

(8) No gas formation.

(9) No film appears on the sur-
face, but a general growth takes
place throughout the tube.

(10) Thin transparent growth
hardly visible to the naked eye.

(11) No. I, no growth or change
in the reaction of the medium.
No. II, Growth, medium acid.

(12) Reduction of nitrate not so
marked.

(13) Usually longer than B.
coli; highly motile, with a quick
serpent-like movement.

(14) Usually 8 to 12, long and
wavy.

ISOLATION OF SPECIFIC PATHOGEN ES. S3

(15) Agglutination. — As a rule, (15) Marked agglutination with
no agglutination with a dilute anti- dilute anti-typhoid serum,
typhoid serum.

Of the many obsen'ers who have reported the isola-
tion of the typhoid bacillus from water all but the most
recent are quite discredited, on account of the insufficiency
of their confirmatory tests, and even the latest results
should be received with caution. Since the introduction
of the Widal (Widal, 1896) reaction, founded on the
fact that typhoid bacilli examined under the microscope
in the diluted blood-serum of a typhoid patient lose their
motility and "agglutinate" or clump together, an import-
ant aid has been furnished in the diagnosis. Yet serum
tests are notably erratic, and insufficient to identify an
organism without an exhaustive study of biochemical
reactions, especially as many organisms are agglutinated
by typhoid serum in a more or less dilute solution. . The
discovery of the Bacillus dysentcriag of Shiga,* which
closely resembles the typhoid bacillus, has made the
identification of the latter more dubious than ever.

It seems probable that in some recent cases the typhoid
bacillus has indeed actually been isolated from polluted
water, as by Kubler and Neufeld (Kubler and Neufeld,
1899), who examined a farmhouse well at Neumark in
1899, and Fischer and Flatau (Fischer and Flatau, 1901),
who discovered an organism responding to a most ex-
haustive series of tests for the typhoid bacillus in a well at

For an account of the Biology of B. dysenteric the student
referred to an article by Dombrowsky, 1903.

54 ELEMENTS OF WATER BACTERIOLOGY.

Rellingen in 190 1. In these cases the water was directly
plated upon Eisner's medium or phenolated gelatin with
no preliminary process of enrichment.

The search for the typhoid bacillus is usually suggested
when an outbreak of the disease has cast strong suspicion
upon some definite source of water-supply. By the time
an epidemic manifests itself, however, the period of the
original infection is long past, and the chances are good
that any of the specific bacilli once present will have dis-
appeared. While elaborate experiments have shown
that B. typhi may persist in sterilized water for upwards
of two months and in unsterilized water from three days
to several weeks, the number of the organisms present is
always very rapidly reduced (Frankland, 1894). Epi-
demiological evidence confirms the results of Laws and
Andrewes which teach that the number of typhoid bacilU
even in polluted water probably is never very great, while
the fate of Lowell and Lawrence in 1890-91 seems strongly
to demonstrate that even a small number of virulent
organisms can bring about an almost wholesale infection.
Indeed if the virulent organism were as abundant as some
recent results would indicate (Remlinger and Schneider,
1897), the human race would long since have been exter-
minated. On the whole it seems that since a positive
result is always open to serious doubt, and a negative
result signifies nothing, the search for the typhoid bacillus
itself, however desirable theoretically, cannot be regarded
at present as generally profitable.

The isolation of the cholera bacillus from water can

ISOLATION OF SPECIFIC PATHOGENES. 55

probably be accomplished with somewhat less difficulty
than is encountered in the case of B. typhi. Schottelius
(SchottcHus, 1885) was the first to point out the necessity
for growing this organism in an alkaline medium, and
Loeffler (Loeffler, 1893) found that its isolation from
water could be successfully accomplished by adding 10 c.c.
of alkaline peptone broth to 200 c.c. of the infected water
and incubating for twenty-four hours at 37°, when the
organism could be found at the surface of the medium.

Somewhat earlier than this Dunham (Dunham, 1887)
had made a special study of the chemical reactions of the
cholera bacillus and found that the organism would grow
abundantly in a solution containing i per cent peptone
and .5 per cent salt (Dunliam's solution), producing the
"cholera-red or nitroso-indol reaction." This medium
was brought into practical use by Dunbar (Dunbar, 1892),
who succeeded in isolating the organisms from the water
of the Elbe in 1892, during the cholera epidemic at Ham-
burg.

Koch (Koch, 1893) prescribed the following method
for the isolation of the organism from water:

To 100 C.C. of the water to be examined is added 1
per cent peptone and i per cent salt. The mixture is
then incubated at 37° After intervals of ten, fifteen and
twenty hours the solution is examined microscopically
for comma-shaped organisms, and agar plate cultures are
made which are likewise incubated at ^57° Tf
colomes showmg the characteristic appearance of the
cholera bacillus are found, these are examined micro-

S6 ELEMENTS OF WATER BACTERIOLOGY.

scopically, and if comma-shaped organisms are present,
inoculations are made into fresh tubes to be further tested
by means of the indol reaction and by inoculation into
animals. The existence of other spirilla of some patho-
genic power renders necessary the greatest care and
caution in claiming positive isolations. That no great
improvement on Koch's method has been made during
the last ten years seems apparent from the statements of
KoUe and Gotschlich (KoUe and GotschHch, 1903), who
employed "the peptone method with subsequent agar
cultivation" in the isolation of the organisms from faeces
of cholera patients during the epidemic in Egypt in 1902,
and who have made notable epidemiological and clinical
researches upon this disease.

Other pathogenic organisms have been isolated from
waters, according to the accounts of numerous investi-
gators, but from the sanitary point of view the typhoid
and cholera bacilli are of most importance since these are
manifestly the germs of disease most likely to be dissemi-
nated through this medium. For the detection of B.
anthracis and other spore-forming pathogenic bacteria
which may at times gain access to water from stockyards,
slaughter-houses, etc., the method suggested by Frank-
land (Frankland, 1894) may be adopted. The water
to be examined is heated to 90° for two minutes and then
plated, the characteristic colonies of the anthrax organism
being much more easily discerned after the destruction of
the numerous non-sporing water bacteria. Again, water
is sometimes the means of distributing the germs of

ISOLATION OF SPECIFIC PATHOGEN ES. 57

dysentery and diarrhoea, as sho-wn by the decrease of
these diseases in BurHngton, Vt. (Sedgwick, 1902), and
other communities where pure water-supplies have been
substituted for polluted ones. It is possible that the
examination of water for the B. dysenteries may in the
future help to throw important light on the sanitary con-
dition of a water.

CHAPTER VI.

METHODS FOR THE ISOLATION OF THE
COLON BACILLUS.

The Bacillus coli was first isolated by Escherich
(Escherich, 1884) from the fasces of a cholera patient.
It was subsequently found to be a normal inhabitant of
the intestinal tract of man and many other animals and
to occur regularly in their excretions, and on this account
it became of the highest interest and importance to
sanitarians, since its presence in water-supplies was
regarded as direct evidence of sewage pollution.

This organism may be described as a short, usually
motile rod, with diameter generally less than one micron
and exhibiting no spore formation. It forms thin irregu-
lar translucent films upon the surface of gelatin, called
"grape-leaf colonies" by the Germans, produces no
liquefaction, and gives a wire-nail-like growth in stick
cultures. It forms a white translucent layer of character-
istic appearance upon agar, produces a more or less
abundant, moist, yellovidsh growth on potato, and tur-
bidity and some sediment in broth; it ferments dextrose
and lactose with the formation of gas of which the ratio is

H 2 . .

^^=— , accordmg to most mvestigators; one vanety

' 58

ISOLATION OF THE COLON BACILLUS. 59

ferments saccharose with a gas ratio approaching — ,

and another does not. As a rule, a strong acid reaction
is developed in all sugar-containing media. The or-
ganism reduces nitrates to nitrites and sometimes to
ammonia. It coagulates casein in Htmus milk, and
reduces the litmus with subsequent slow return of the
color (red), and forms indol in peptone solution. Many
cukures of this organism are fatal to guinea-pigs when
• the latter are inoculated subcutaneously with i c.c. of a
twenty-four-hour bouillon culture, and most cultures
produce death when this amount is inoculated intraperi-
toneally. Akhough not a spore-forming bacillus, and in
general not possessing great resistance against anti-
septic substances, B. coli seems to be less susceptible to
phenol than are many other forms, especially certain
water-bacteria.

The Wurtz litmus-lactose-agar plate (Wurtz, 1892), as
noted in Chapter IV, furnishes one ready method for the
isolation of B. coU from water, and it was used by Sedg-
wick and Mathews for the purpose as early as 1893
(Mathews, 1893). The process is based upon the fact
already alluded to, that B. coH readily ferments lactose
with the formation of acid. If, therefore, plates are made
with agar containing both lactose and litmus, the colon
colonies develop as red spots in a blue field. Since or-
ganisms other than B. coli may also develop red colonies,
it becomes necessary further to examine the red colonies
in order to prove that they are made up of colon bacilli.

6o ELEMENTS OF WATER BACTERIOLOGY.

This is clone by fishing from isolated suspicious-looking
colonies, replating and inoculating into the usual media
for diagnostic work.

For success in examining polluted waters by this method
it is necessary to get a sufficient dilution so that colonies
may be well isolated, and to this end it is advisable that a
number of different dilutions be employed, a series of
plates being prepared from each. Under any conditions
the detection of the colon bacillus is seriously hampered
by the development of other forms. Certain observers
have therefore added phenol to the agar medium, com-
bining the effect of high temperature and an antiseptic
to check the growth of water-bacteria. Copeland for
this purpose added to his tubes .2 c.c. of a 2% solution
of phenol (Copeland, 1901). Chick (Chick, 1900)
found that 1.33 parts of phenol in 1000 materially de-
creased the number of colon baciUi which would develop,
while I part gave very satisfactory results, the plates
showing pure cultures of B. coli.

The test for the colon bacillus may, however, be made
still more dehcate by a prehminary enrichment of the
sample by growth in a liquid medium for twenty-four
hours at 37°, thus greatly increasing the ■ proportion of
these organisms present before plating. As suggested
in the classic researches of Theobald Smith (Smith, 1892),
this method may be made approximately quantitative by
the inoculation of a series of tubes with measured por-
tions of the water. If, for example, of ten tubes inocu-
lated each with of a cubic centimeter, four show

ISOLATION OF THE COLON BACILLUS. 6i

the B. coli, we may assume that some 40 of these organ-
isms were present to the cubic centimeter. Irons (Irons,
1901), in a comparative study of various methods for the
isolation of B. coh, showed that the prehminary enrich-
ment frequently gave positive results when the results
of the direct use of the agar plate were negative and
concluded that " where the waters to be examined are
much polluted, or where the amount of B. coH is small
and the colony count large, the lactose plate for plating
water direct is inferior to the dextrose fermentation-
tube." Gage obtained similar results (Gage, 1902).
The method of direct plating in phenol-agar is quicker
and there is a certain danger that colon baciUi originally
present may be overgrown and Idlled out in the enrich-
ment process. If the incubation is not too prolonged,
however, this need not occur.

The medium ordinarily used for the prehminary enrich-
ment is ordinary broth to which 2.5 per cent of dextrose
has been added, and the reaction brought to the neutral
point. Into each of a number of fermentation-tubes of this
medium a measured quantity of the water to be examined
is inoculated, and the culture is incubated for twenty-four
hours at 37.5° C. At the end of this time the tubes are
examined for gas formation. If gas is found, a small
amount of the culture should be added, after suitable
dilution, to htmus lactose agar and plated.

With polluted waters it wall be found advantageous to
plate out on the first appearance of gas (4-8 hours). It
has been shown by one of us (Prescott, 1.902^) that a very

02 ELEMENTS OF WATER BACTERIOLOGY.

rapid development of B. coli takes place in the first few
hours after dextrose solutions are inoculated with intes-
tinal material, and a nearly pure gro^\th of colon baciUi
often results, while other bacteria multiply more slowly.
With highly polluted waters gas formation wiU probably
begin within twelve hours, but with fewer colon bacilli
present the duration must be increased. If the period
of incubation be too long continued, trouble in the subse-
quent steps of the isolation may be encountered because
of the overgrowth of B. coli by the sewage streptococci,
which are almost invariably present, and which by their
greater acid-producing powers may check the growth of
the colon bacilli.

As has already been stated, phenol has less inhibitory
action upon B. coh than upon normal water-bacteria,
hence a broth containing this substance may be emplo3^ed
for preHminary enrichment; and this medium has been
used in place of dextrose broth for many of the studies
made in connection with the Chicago drainage canal
(Reynolds, 1902). Phenol broth consists of ordinary
broth to which o.i per cent phenol is added, and the
method of procedure is to add i c.c. of the water to 10 c.c.
of the sterihzed phenol broth and incubate at body
temperature for twenty-four hours. Litmus-lactose-agar
plates are then made and the examination of the red
colonies carried on as described for the dextrose-broth
method. The dextrose broth furnishes, however, a much
more delicate test than the carljol broth when the num-

ISOLATION OF THE COLON BACILLUS.

63

ber of colon bacilli present is small, as is clearly shown by
the following table from Irons.

PROPORTION OF POSITIVE RESULTS IN TESTS OF POLLUTED AND
UNPOLLUTED WATERS BY DEXTROSE FERMENTATION-TUBE AND
CARBOL-BROTH METHODS.

(Irons, 1901.)

Dextrose
Fermentation-
tube.

Carbol-broth
Method.

Polluted waters

+ - 7

33 31 5

+ - 7
38 30 I

Relatively unpolluted waters

56 38 25

37 61 21

Furthermore, the dextrose fermentation-tube possesses
a second advantage in the fact that a decisive negative
test may be obtained within twenty-four hours of the
original planting of the sample. If no gas is formed in
the tube, we commonly assume that B. coli is absent and
carry the examination no farther.

When it is desired to examine samples larger than i c.c.
for B. coh it becomes necessary to modify the enrichment
process by adding the nutrient material to the water
instead of the reverse. For this purpose phenol dex-
trose broth (consisting of broth with 10 per cent dex-
trose, 5 per cent peptone, and .25 per cent phenol) may
be added to the sample of water to be enriched as sug-
gested by Gage (Gage, 1901). Generally 10 c.c. of the
broth is added to 100 c.c. of the water. The sample is
then incubated at 37° for twenty-four hours, and if at the
end of that time growth has taken place, a cubic centi-

64 ELEMENTS OF WATER BACTERIOLOGY.

meter is inoculated into a dextrose tube. If this tube
shows gas formation after twenty-four hours at 37°, a lit-
mus-lactose-agar plate is made and the other diagnostic
tests applied.

Our experience has shown that it is not specially advan-
tageous to apply the colon test in such large samples,
since the significance of B. coli when present in numbers
less than i per c.c. is extremely doubtful. On the other
hand the danger of overgrowth is greatly increased by
this process and negative results may often be obtained
when the organisms are really present. Ilunnewell and
one of ourselves (Winslow and Hunnewell, 1902'^) found
that of 48 samples of certain polluted river waters 18 gave
B. coli when i c.c. was inoculated directly into dextrose
broth, while in only 4 cases was a positive result obtained
after preliminary treatment of 100 c.c. in carbol broth.
In 153 samples from presumably unpolluted water B. coli
was found 5 times in i c.c. and 11 times by the examination
of the larger sample. The authors, therefore, concluded
as follows:

"It appears evident that the use of large samples in
applying the colon test to the sanitary analysis of drink-
ing-water is not advantageous. In comparing the results
of the tests in i c.c. and in 100 c.c, it will be noted that the
proportion of lactose fermenting organisms and of colon
bacilU in the unpolluted waters was more than doubled
in the latter; thus waters of good quality are more likely
to be condemned by the use of large samples. On the
other hand, in the polluted waters a considerable propor-

ISOLATION OF THE COLON BACILLUS. 65

tion of the colon bacilli originally present were lost during
the incubation of the large samples, so that waters of bad
quality actually appeared to better advantage by the use
of 100 c.c. with preliminary incubation in phenol broth."

Similarly Whipple (Whipple, 1903) notes that 2.9 per
cent of some samples of water examined by him gave
positive tests with .1 c.c. but not with i c.c, while 4.3 per
cent gave positive tests with .i c.c. or i c.c. and negative
tests vnih. 10 c.c. Again, in another series of samples
examined, of those which gave positive samples in smaller
portions 5.3 per cent were negative in 10 c,c., 4.7 per cent
in ICQ c.c, and 7.7 per cent in 500 c.c

In all practical processes of examining water for B. coli
one essential step is the isolation of pure cultures upon
the litmus-lactose-agar plate, whether the plate be inocu-
lated from the water direct or from a preliminary enrich-
ment culture. In the first case a measured quantity of
water must be added. In the second case, since the
enrichment-tube was inoculated with a known amount of
water all further work is purely qualitative and it is only
necessary to obtain such a number of colonies upon the
lactose plate that the isolation of a pure culture shall be
easy. In practice the following procedure has been
found generally successful. After the dextrose-tubes have
been incubated for twelve to twenty- four hours at 37°,
from those which show gas, one loopful is carried over
to a tube containing 10 c.c of sterile water, and of this
water one loopful is taken for the inoculation of the plate.
Ordinarily this will give colonies which are sufficiently

ELEMENTS OF WATER BACTERIOLOGY.

well separated, but a second plate, inoculated from the
dilution water with a straight needle instead of a loop,
furnishes a desirable safeguard.

The litmus-lactose-agar plates made in this manner
should be incubated for from twelve to twenty-four hours at
the body temperature (37°), at the end of which time if B.
coli is present red colonies upon a blue field will be visible.
If a pure culture of B. coH is obtained, the litmus-lactose-
agar plate may become blue again after forty-eight hours
owing to the formation of amines and ammonia by the
action of the bacteria upon the nitrogenous matter present.
If the dilution is too low, the resulting colonies will be
small and imperfectly developed, making it difficult to be
sure of pure cultures for the subsequent tests. A great
number of colonies will also prevent the change of reac-
tion from acid back to alkaline. Since many bacteria
ferment lactose with the formation of acid, it is erroneous
to regard all colonies as those of B. coli; in all cases
several colonies from each plate should be isolated upon
agar streaks and further studied in subculture.

In the selection of those red colonies which are to be
fished from the litmus-lactose-agar plate the appearance
of the growths must be closely noted. A colony of irregu-
lar contour, surrounded by a very faint area of reddening,
will probably belong to some member of the B. mycoides
group (Winslow and Nibecker, 1903); small, compact,
bright-red colonies are characteristic of the streptococci,
and Gage and Phelps (Gage and Phelps, 1903) have
pointed out that of these there are two types, one of a

ISOLATION OF THE COLON BACILLUS. 67

brick-red color, and of such consistency as to be readily
picked up by the needle-point and the other smaller and
of an intense vermiHon color. The colonies of the colon
bacillus are usually well formed, pulvinate on the sur-
face and fusiform when growing deeper down.

The agar streak made from the litmus-lactose-agar plate
shows after twenty-four hours certain marked character-
istics. The most distinct types are two, the abundant,
first translucent, later whitish and cheesy growth, cover-
ing nearly the whole surface of the agar, characteristic of
B. coli and its allies, and a very faint growth, either con-
fined strictly to the streak or made up of faint isolated
colonies, dotted here and there over the surface. The
latter cultures are typical of the sewage streptococci, and
a microscopic examination will generally settle their status
at once. Of the more luxuriant growths, some of which
are stringy to the needle many will generally prove to be
atypical, and if any of the weakly fermenting forms (B.
mycoides) are present, a dull wrinkled growth will be
produced.

Having submitted the sample of suspected water to a
preliminary enrichment process, and having isolated pure
cultures of suspicious organisms from the Utmus-lactose-
agar plate, the third step is the examination of the specific -
reactions of the organisms thus obtained. Just what
characters to use in defining the "colon bacillus" is a
matter of prime importance. The whole question of
species among the bacteria is an extremely complex one,
since around each definite species are grouped forms

ELEMENTS OF WATER BACTERIOLOGY.

differing from the type in one or two of its character-
istics.

As Whipple says (Whipple, 1903), "The type form of
Bacillus coli is one which can be defined within reason-
ably narrow limits, but when the organism has been away
from its natural habitat for varying periods of time, and
has existed under abnormal conditions, its ability to react
normally to the usual tests appears to be greatly impaired.
Its power to reduce nitrates may be lost, or on the other
hand may be increased; its power to produce indol may
be lost, or on the other hand, it may be increased; its
power to coagulate milk, even, is sometimes reduced,
although seldom entirely lost; its power to ferment car-
bohydrates may be altered so that the amount of gas
obtained in a fermentation-tube, as well as its ratio of
H to CO2, is quite abnormal. But in spite of all these
facts, the bacillus tested may have been originally a true
Bacillus coli."

The more of such atypical forms which are included
the greater will be the number of positive isolations. At
present our definitions must be more or less arbitrary;
each observer will consider as true colon baciUi those
which fulfil his particular set of tests, and will class as
pseudo-colon organisms those which do not. If we find,
having established such an arbitrary standard, that the
colon bacillus, as determined by it, is found in waters
known to be polluted, and not, as a rule, in those Imown
to be free from pollution, the sanitarian can afford to
ignore the theoretical question of specific values and

ISOLATION OF THE COLON BACILLUS. 69

make confident use of the practical test. It is, of course,
higlily desirable that some standard set of reactions
should be commonly adopted by sanitary bacteriologists;
and it is to be hoped that some such uniform scheme may
soon be drawn up by the Society of American Bacteriolo-
gists or by the American Public Health Association. At
present the plan developed by the Massachusetts State
Board of Health (Massachusetts State Board of Health,
1899) is most widely prevalent in this countr}^ It in-
volves the use of six simple, definite, positive tests — the
growth in gelatin, lactose-agar, dextrose broth, milk,
nitrate solution, and peptone solution.

For recording the results of the various tests applied,
the appended blank form has been in use at the Massa-
chusetts Institute of Technology. On the upper part of
the sheet are noted the results of the gelatin count and
the Htmus-agar count at 37°. In the second case the
number of acid-formers is placed in brackets after the
total numbers. The lower Dart is used for the B. coH
isolation.

MASSACHUSETTS INSTITUTE OF TECHNOLOGY.

Gelatin-plate cultures. No. colonies Agar-plate cultures at

Bacteriological Examination of Water.

Sample No.
Examined by-
Place of collection

Date of collection
Hour of collection
Remarks

quantitative analysis.

48 hours

per CO.

No. colonies per c.c.

Average
Remarks

24 hours
Average
Remarks

70

ELEMENTS OF WATER BACTERIOLOGY.

QUALITATIVE ANALYSIS.

Preliminary fermentation-tube

Litmus-lactose-agar plates

Agar streak culture

Fermentation-tube

Nitrate test

Milk test

Indol reaction

Gelatin stab culture

Remarks

Opposite the words "Preliminary fermentation-tube"
the results of the first enrichment test in the five or ten
tubes inoculated are recorded by plus or minus signs.
Under the columns headed by plus signs the presence or
absence of typical red colonies on the litmus-lactose-agar
plate is similarly indicated. The third line serves for
the results of the microscopic and macroscopic appear-
ance of the agar streak. If this gives the proper type of
growth and rod-shaped organisms are found, a dex-
trose fermentation-tube, a nitrate tube, a milk tube, a
peptone tube, and a gelatin stab are inoculated from it.

After twenty-four hours incubation at 37°, the first
three of these subcultures are examined and the results
recorded in the proper columns by plus and minus signs.
The amount of gas in the closed arm of the dextrose tube
may be conveniently measured by the Frost gasometer
(Frost, 1901). Then a few centimeters of strong sodium
or potassium hydrate are added and mixed with the broth
by cautiously tipping the tube and a second measurement
determines the amount of gas absorbed (assumed to be
CO2). The gas should first fill from a third to two-

ISOLATION OF THE COLON BACILLUS. 71

thirds of the closed ann and about one-third of this should
be absorbed. The nitrate tube is tested for nitrites by
adding a drop of the following solutions in succession:

A . Sulphanillc acid .5 gram

Acetic acid (25% sol.) 150.0 c.c.

B. Naphthylamine chloride .1 gram

Distilled water 20.0 c.c.

Acetic acid (25% sol.) 150.0 c.c.

A red or violet coloration indicates the presence of
nitrites. The milk tube is merely heated to boiling over
the free flame; if coagulation occurs the test is considered
positive.

After seventy-two hours incubation at 37° the peptone
solution is examined for indol by adding i c.c. of a .02 per
cent solution of sodium or potassium nitrite and i c.c. of a
I to I solution of sulphuric acid. Both the tube and
the reagents should be cooled on ice before mixing, and
the tube should be left in a cool place for an hour after-
ward to allow time for the characteristic rose-red color
of nitroso-indol to develop. The gelatin tube is kept at
20° for seven days according to the procedure adopted
at the Massachusetts Institute of Technology. It seems
undesirable in practice to prolong the test much beyond
this point, although some slowly liquefying organisms
are doubtless included, which would be thrown out by a
longer incubation. The extent of this source of error
as well as the relative importance of the various other
diagnostic tests is weU shown in the following table of

72

ELEMENTS OF WATER BACTERIOLOGY.

o

+->

c

6

•dno

•SjC'EQ oi

•[opuj aonpojj;

•aqntv-uoii'B^uauua^

•uinog 3onpoj£

•SBQ aonpojj

OCO On 00

IT) LO LO

O O CO t^co 00 w

O CN cs o

O O vovo ^ O u^

O O fD tovO 00 O

CO W M M N to

O OO On O OnOO O

W H CS

O »0 On OnOO XTi
H CM

On On LO

O w H O O

+

O 'tJ- w ro

vO 00 GO 00 i>-0

+

ro M to li-) OOO
t-,. r-* LDOO

+

M 'O O !>. N
00 r^MD O ^

I VO O 00 H I^CO

T 00 O^OO O^O 00

O CO ro O ^ O

+

H tooo NO
00 00 t^NO i/-)NO

-uoo

' NO NO O O On
' CO CO Tl- M CO »o
I N M 01 U-)

ci
ho

O l-l H M CO O

•pat3IuuAi-iina

•a^Bitj JBSy zijnjw 3ut
-SSBJ suox^Binauuaj AjBunii;iaJd jo ^uaQ J3J

3

o
CO

O CO ^00 N "d-co

O O ON CO O NO

vo CO --d- o
■ 00 00 00 o

CO in O 0) CO On
t^OO NO H to
W ^ H CO

u

I "

o

^N4_j

fi< W CO C/3 M

ISOLATION OF THE COLON BACILLUS. 73

the results obtained at Lawrence during a period of
eighteen months.

It should be noted that considerable differences often
appear in these biochemical reactions between tubes of
the same batch of a medium, inoculated with approxi-
mately the same amount of the same culture. This has
been shown very markedly for the amount of the gas and
the proportion of carbon dioxide in the dextrose tube by
Fuller and Johnson (Fuller and Johnson, 1899), Penning-
ton and Kiisel (Pennington and Kiisel, 1900), Gage
(Gage, 1902), and one of ourselves (Winslow, 1903).
Variations in the nitrate reduction are often even more
marked, one tube, perhaps, showing a strong reaction
and another none. In important cases, therefore, it is
desirable to inoculate the subcultures in dupHcate.

CHAPTER VII.

SIGNIFICANCE OF THE PRESENCE OF B. COLI IN WATER.

Ten years ago the B. coli of Escherich occupied a posi-
tion of very great prominence in the eyes of sanitarians.
If it was not considered to be in itself a dangerously patho-
genic germ, it was at least regarded as a suspiciously close
relation of the typhoid organism. At this time, therefore,
the alleged presence of either of these forms was quite
sufficient to condemn a water-supply.

Investigation soon showed, however, that the Bacillus
coli was by no means confined to the human intestine.
Dyar and Keith (Dyar and Keith, 1893) found it to be the
prevailing intestinal form in the cat, dog, hog, and cow.
In the goat and rabbit they reported that no single organ-
ism was constantly present; and in the case of the horse,
the place of the colon bacillus was taken by a new form,
described by the authors under the name of B. equi intes-
tinalis. About the same time, Fremhn (FremHn, 1893)
found colon baciUi in the feces of dogs, mice, and rabbits,
but not in those of rats, guinea-pigs, and pigeons. Smith
(Smith, 1895) recorded the presence of the same or-
ganism, in almost pure cultures, in the intestines of dogs,

74

TEE SIGNIFICANCE OF B. CO LI IN WATER. 75

cats, swine, and cattle ; and he also found it in the organs
of fowls and turkeys after death. Brotzu (Brotzu, 1895)
reported B. coli and aUied forms as very abundant in the
intestine of the dog; and BeHtzer (Belitzer, 1899) still
more recently isolated typical colon bacilli from the
intestinal contents of horses, cattle, swine, and goats.
Russell and Bassett (Russell and Bassett, 1899) stated that
studies made by E. B. Hoag in Professor Russell's labora-
tory indicated the presence of baciUi of the colon group,
fermenting dextrose with a gas formula of f , in the feces
of " a considerable number of different species of mam-
malia, as well as that of birds, fish, etc.," while similar
organisms with the inverted gas formula ^ were con-
sidered by the same authors to be characteristic of decom-
posing organic matter of vegetable origin, free from sus-
picion of fecal contamination. Moore and Wright
(Moore and Wright, 1900) recorded the finding of the
colon baciUus in the horse, dog, cow, sheep, and hen;
and in a later report (Moore and Wright, 1902) they noted
its occurrence in swine and in some, but not all, the speci-
mens of rabbits examined. In frogs it was not found.
Amyot (Amyot, 1902) failed to find B. coH in the intes-
tines of 23 fish representing 14 species. The possibihty
that colon bacilli may be introduced into unpolluted
waters through the agency of fish was considered again
by Johnson, in a paper read at the Washington meeting
of the American Public Health Association and not yet
pubhshed. Colon bacilli were found present in the
intestinal tract of fish taken from polluted waters, and

76 ELEMENTS OF WATER BACTERIOLOGY.

the conclusion was drawn that the migration of fish from
a contaminated stream or lake to an unpolluted one may
explain the occasional finding of B. coH in small samples,
or the more regular detection of it in large volumes of the
water.

Many bacteriologists have gone much further and
afiirmed that the colon bacillus was not a form character-
istic of the intestine at all, but a saprophyte having a wide
distribution in nature. The first of this school, perhaps,
was Kruse (Kruse, 1894), who in 1894 protested against the
arbitrary conclusions drawn from the colon test as then ap-
plied. He pointed out that the characters usually observed
marked, not a single species, but a large group of organisms.
As ordinarily defined, he added, "the Bacterium coli is in
no way characteristic of the feces of men or animals.
Such bacteria occur everywhere, in air, in earth, and in
the water, from the most diJTerent sources." Even if the
relations to milk and sugar media be considered, "micro-
organisms with these characteristics are also widespread."
Dr. Kruse gave no experimental data on which his opinion
was based. In the same year Beckmann (Beckmann,
1894) isolated a bacillus which he identified by pretty
thorough tests as B. coli from the city water of Strass-
burg, a ground- water which he believed could by no pos-
sibility be subject to fecal contamination. Large quan-
tities of water were used for the isolation.

Refik (Refik, 1896) recorded the constant presence of
colon baciUi in water of all sorts, public supplies, wells,
cisterns, and springs in the neighborhood of Constant!-

THE SIGNIFICANCE OF B. COLI IN WATER. 77

nople, but the only characters which these "colon bacilli"
exhibited in common were the "classical growth" upon
potato, the possession of less than 8 cilia, and the power
of active development on certain media upon which the
typhoid bacillus did not grow. A more careful and sig-
nificant piece of work on the same line was published by
Poujol in the succeeding year. 'This author reported
(Poujol, 1897) the isolation of B. coli from 22 out of 34
waters studied by him in relation to their use as public
suppHes. The waters were from various sources — springs,
wells, and rivers — but all were of fair quality and many
quite free from any possibihty of contamination. Samples
of 100 c.c. were used for analysis; in the only case in which
a smaller amount was also tested, broth inoculated with
10 drops of the water and placed at 45° C. remained
sterile. The author concluded that "fecal contamination
can only exceptionally be invoked to explain the presence
of B. coli in water. As the bacteria of the subterranean
water are contributed to it from the surface of the earth
by the water which filters downward, I am rather inclined
to believe in a general diffusion of B. coli either on the
surface of the earth, where it might be deposited with
the dust of the air, or in the superficial layers of the earth,
which may form one of its normal habitats." Therefore,
the author considered that caution should be exercised
in condemning a water on account of the presence of B.
coli, except, as he added, "for those cases where it exists
in considerable quantity."
Certain ItaHan observers appear to have come to even

78 ELEMENTS OF WATER BACTERIOLOGY.

less conservative conclusions. Abba (Abba, 1895) found
colon bacilli constantly present in certain unpolluted
waters near Turin. Moroni (Moroni, 1898; Moroni,

1899) reported the examination of numerous deep and
shallow wells and unpolluted springs about Parma, as
well as of the public water-supply of the city, for the colon
bacillus and concluded that that organism was a water form
and had no sanitary significance. The characters used
for the identification of the species in this case were fairly
exhaustive, but both Abba and Moroni used liter samples
for analysis.

Levy and Bruns (Levy and Bruns, 1899) gave a new
turn to the discussion by emphasizing the importance of
animal inoculation, already suggested by Blachstein (Blach-
stein, 1893) and others. They claimed that the existence of
numerous para-colon and para-typhoid organisms in air, in
dust, and in unpolluted water made it impossible to decide
by ordinary bacteriological methods whether true colon
bacilli were present in water or not. In no case, how-
ever, did representatives of the colon group isolated by
them from water kill a guinea-pig, even when i or 2 c.c.
were injected intraperitoneally; and the authors, there-
fore, considered pathogenicity as an attribute belonging
only to the true B. coU of the intestine. This paper
aroused Professor Kruse's pupil, Weissenfeld, to a pub-
lication, in which the position of the Bonn school was
carried to an extreme. Weissenfeld reported (Weissenfeld,

1900) the analysis of 30 samples of water supposedly pure,
and of 26 samples considered to be contaminated. In

THE SIGNIFICANCE OF B. COLI IN WATER. 79

each case a single centimeter sample was first incubated
in Parietti broth, and if no growth occurred, larger samples
of half a hter or a liter were examined. Colon bacilli
were found in all the samples examined; and the patho-
genicity varied independently of the source of the water.^
The author concluded that "the so-called Bacterium
coli may be found in waters from any source, good or bad,
if only a sufficiently large quantity of the water be taken
for analysis." In the first place it should be noted that
the characters used by this investigator for defining the
"so-called Bacterium coli" were absolutely inadequate.
He classed under that head all bacilli of medium size,
which formed grapevine-leaf colonies on gelatin and
gas in sugar agar, which were more or less motile, or
rarely non-motile, and which were decolorized by the
Gram method. As regards coagulation of milk and
formation of indol, "the bacteria isolated differed." In
the second place it is difficult to see how the author could
possibly have beheved that his experiments proved the
isolation of the colon bacillus to be "useless as an aid in
the sanitary examination of water," as the title of the
paper runs. Even his own work furnishes strong evi-
dence to the contrary. In 24 of the 26 samples from bad
sources, he isolated his imperfectly defined colon bacilH
from I c.c. of the water, while in only 8 of the 30 samples
of good waters could he find such organisms in that
quantity.

Of a series of 47 cultures of actic-acid bacteria

' Confirmed by Savage (Savage, 1903,.

»o ELEMENTS OF WATER BACTERIOLOGY.

recently examined by one of ourselves (Prescott, 1902 ^;
Prescott, 1903) 25 were found to give the reactions of B.
coli. These organisms were isolated chiefly from cereals
and products of milling, such as flour, bran, corn-meal,
oats, barley, etc., while others were in technical use for
producing the lactic fermentation. There is no evidence
that any of these organisms were of intestinal origin, and
yet they possess all the characters of typical colon bacilli,
even to the pathogenic action when inoculated into
guinea-pigs. These results explain the results of Klein
and Houston (Klein and Houston, 1900), who reported
the finding of typical colon bacilli in 3 out of 24 samples
of wheat and oats obtained from a wholesale house; rice,
flour, and oat- meal bought at two different retail shops
gave B. coU on all three cereals in i case and on none in
the other. In Germany, Papasotiriu (Papasotiriu, 1901)
was meanwhile carrying on almost exactly similar inves-
tigations to Prescott's, with identical results.

What then does a colon test prove? Obviously (i) the
presence of a colon bacillus which has come directly from
the intestine of some animal, or (2) of a colon bacillus
which has come indirectly from an animal, or (3) of a
saprophytic "lactic-acid bacillus" which gives the same
biochemical reactions. This immediately raises the
interesting questions. Is it possible that the lactic-acid
bacilh have been indirectly derived from animal intes-
tines, having "escaped from cultivation," as the botanists
say? Or is the converse true, namely, that all colon
bacilli are simply lactic-acid bacteria which have found

THE SIGNIFICANCE OF B. COLI IN WATER. 8i

in the warm intestinal canal richly supplied with food a
favorable habitat ?

The answer to these questions is of much theoretical
interest, but need not be further considered here. The
practical sanitary conclusions to be drawn are as foUpws:

1. Bacteria corresponding in every way to B. coli are
by no means confined to animal intestines, but are widely
distributed elsewhere in nature. "

2. The finding of a few colon bacilli in large samples
of water, or its occasional discovery in small samples,
does not necessarily have any special significance.

3- The detection of B. coh in a large proportion of
small samples (i c.c. or less) examined is imperatively
required as an indication of recent sewage pollution.

4. The number of colon baciUi in water rather than their
presence should be used as a criterion of recent sewage
pollution.

With these qualifications the value of the colon test
was never more firmly estabhshed than it is to-day.
Whether or not originally a domesticated form, it is clear
that the colon bacillus finds in the intestine of the higher
vertebrates an environment better suited to its growth
and multiplication than any other which occurs in nature
It IS almost certain that the only way in which large
numbers of these organisms gain access to natural waters
IS by pollution with the domestic, industrial, and agri-
cultural wastes of human life. If pollution has been
recent, colon baciUi will be found in comparatively smaU
samples of the waters. If pollution has been remote the

82 ELEMENTS OF WATER BACTERIOLOGY.

number of colon bacilli will be small, since there is good
evidence that the majority of intestinal bacteria die from
exposure in cold water. If derived from cereals or the
intestines of wild animals the number will be insignificant
except in the vicinity of great grain-fields or when the
water receives refuse from grist-mills, tanneries, dairies,
or lactic-acid factories.

The first recognition of the necessity for a quantitative
estimation of colon bacilli in water we owe to Dr. Smith,
who in 1892 (Smith, 1893^) outlined a plan for such a
study to be made by the New York Board of Health on the
Mohawk and Hudson Rivers. Burri (Burri, 1895) pointed
out that the use of so large a sample as a liter for exam-
ination would lead to the condemnation of many good
waters. Freudenreich (Freudenreich, 1895) at the same
time indicated the necessity for taking into account the
number of colon bacilli present. He recorded the isolation
of the organism from unpolluted wells, when as large a
quantity of water as 100 c.c. was used, and concluded that
it was entirely absent only from waters of great purity
and present in large numbers only in cases of high pollu-
tion. This author also quoted Miquel as having found
colon baciUi in almost every sample of drinking-water if
only a sufficient portion were taken for analysis, but gave
no reference.

The practical results of the application of the colon
test from this standpoint have always proved most instruc-
tive. As originally outUned by Dr. Smith, it consisted in
the inoculation of a series of dextrose tubes with small

THE SIGNIFICANCE OF B. COLI IN WATER. 83

portions of water, tenths or hundredths of the cubic centi-
meter. It was first used by Brown (Brown, 1893) in 1892
for the New York State Board of Heahh, and its results
showed from 22 to 92 fecal bacteria per c.c. in the water
of the Hudson River at the Albany intake, and from 3 to
49 at various points in the Mohawk River between Amster-
dam and Schenectady. In some previous work at St.
Louis, the colon bacilli in the Mississippi River were found
to vary from 3 to 7 per c.c.

Hammerl (Hammerl, 1897) used the presence of Bacillus
coU as a criterion of self-purification in the river Mur.
He considered, in spite of the position taken by Kruse,
that when a water contained large numbers of colon bacilli,
as well as an excess of bacteria in general, it might be
considered to be contaminated by human or animal excre-
ment. As, however, the organism would naturally be
pres-ent in large quantities of such a water as that of the
Mur, he used no enrichment process, but made plate cul-
tures direct; he defined the B. coli as a small bacillus,
non-motile or but feebly motile, growing rapidly at 37° C.,'
coagulating milk and forming gas in sugar media. In
general, Hammerl failed to find colon bacilU in the
river by this method, except immediately below the
various towns situated upon it; at these points of pollution
he discovered a few colon colonies upon his plates not
more than 4 to 6 per c.c. of the water. He concluded
that "the Bacterium coH, even when it is added to a
stream m great numbers, under certain circumstances
disappears very rapildy, so that it can no longer be detected

84 ELEMENTS OF WATER BACTERIOLOGY.

in the examination of small portions of the water." It
should be noted that Hammerl's method was much less
delicate than the use of the dextrose tube for preliminary
incubation.

A very important series of observations carried out in
England by the bacteriologists of the local government
board has led to similar conclusions. Dr. Houston in
particular (Houston, i8g8; Houston, 1899^; Houston,
igoo^) made an elaborate series of examinations of soils
from various sources to see whether the microbes con-
sidered to be characteristic of sewage could gain access
to water from surface washings free from human con-
tamination. In the three papers pubhshed on this sub-
ject the examination of 46 soils was recorded. In only
10 of the samples was B. coli found, and of these 10, 9
were obviously polluted, being derived from sewage fields,
freshly manured land, or the mud-banks of sewage-pol-
luted rivers. The author finally concluded that "as a
matter of actual observation, the relative abundance of
B. coh in pure and impure substances is so amazingly
different as to lead us to suspect that not only does B. coli
not flourish in nature under ordinary conditions, but that
it tends to even lose its vitahty and die." " In brief, I am
strongly of opinion that the presence of B. coH in any num-
ber, whether in soil or in water, impHes recent pollution of
animal sort." Pakes (Pakes, 1900) stated on the strength
of an examination of "about 300 different samples of
water," no particulars being pubhshed, that water from
a deep weU should not contain B. coli at all, but that water

THE SIGNIFICANCE OF B. COLI IN WATER. 85

from other sources need not be condemned unless the
organism was found in 20 c.c. or less. When colon bacilli
were found in only greater quantities than 100 c.c. the
water might be considered as probably safe. Horrocks
(Horrocks, 1901), after a general review of English prac-
tice, concluded that "when a water-supply has been
recently polluted with sewage, even in a dilution of one in
one hundred thousand, it is quite easy to isolate the B. coH
from I c.c. of the water." "I would say that a water
which contained B. coh so sparingly that 200 c.c. required
to be tested in order to find it had probably been polluted
with sewage, but the contamination was not of recent
date." Chick (Chick, 1900) found 6100 colon bacilli
per c.c. in the Manchester ship canal, 55-190 in the pol-
luted River Severn, and numbers up to 65,000 per gram in
roadside mud. On the other hand, of 38 unpolluted
streams and rivulets, 31 gave no Bacillus coH and the
other 7 gave i per c.c. or less. The Liverpool tap water,
snow, rain, and hail gave no colon bacilli.

In the United States the colon test has been exten-
sively apphed during the last few years to certain pol-
luted river- waters, in particular with the view of measuring
the purification attained by sand fikration and that
naturally occurring during the flow of a stream. A fairly
good idea has thus been obtained of the numerical dis-
tribution of the B. coh in the larger rivers. Fuller (Ful-
ler. 1899), for example, recorded the presence of colon
baciUi in 60 per cent of the i-c.c. samples taken from the
Ohio at Cincinnati. When this water was passed through

86 ELEMENTS OF WATER BACTERIOLOGY.

either slow sand or mechanical filters, the effluent gave
positive results about one-half the time in samples of

50 CO.

The last three reports of the Massachusetts State
Board of Health contain reports of the abundance of
B. coli in a more highly polluted stream, the Merrimac
at Lawrence. In 1898 (Massachusetts State Board of
Health, 1899) the number of organisms found by making
litmus-lactose-agar plates directly and inspecting the
colonies varied from 20 per c.c. in May and June to 92 per
c.c. in August and September, the average for the year
being 47. Of 117 samples of the water which had passed
through the city filter, only 9 showed the organism in a
single colony.

In 1899 (Massachusetts State Board of Health, 1900)
the study was considerably extended. The average num-
ber of colon bacilli in the river at the intake of the filter
was again 47, and in only i sample out of 180 was it absent ;
below the city at the Lawrence Experiment Station the
additional pollution raised the average number to 103.
The effluent as it came directly from the filter showed
B. coli in 24 per cent of the cubic centimeters examined,
but at the outlet of the reservoir, the proportion had
fallen to 7 per cent and at the Experiment Station, after
passage through the distribution pipes, to only 4 per cent.
The results obtained in the next year, 1900, were prac-
tically the same, but parallel tests were made in a larger
volume of water by incubating with the addition of phenol

THE SIGNIFICANCE OF B. COLI IN WATER. 87

broth. The results of these comparative tests may be
tabulated as follows:

Percentage of Samples of Water containing B. coli.
lawrence experiment station (massachusetts state board oj-

HEALTH, igoi).

Effluent o£ Outlet of Tap, Tap, Experiment

Filter. Reservoir. City Hall. Station.

In I c.c 18.14 8.57 4.07 1.87

Iniooc.c 38.12 23.30 15.54 15.54

It appeared that the use of the larger volume of water
gave very httle additional information, and indeed the
real difference between the waters examined is rather
obscured by the use of the large samples.

In 1900 Clark and Gage (Clark and Gage, 1900) re-
ported some specially instructive observations made when
certain of the underdrains of the Lawrence municipal
water-fiher were relaid in the autumn of 1898. In doing
this work the sand on some of the beds was seriously
disturbed; and in December, after the work was com-
pleted, B. coli was found in i c.c. of the filtered efflu-
ent in 72 per cent of the samples examined. In Janu-
ary and February the organisms were found in 54 per
cent and 62 per cent of the samples, respectively, while
in March the number fell to a normal value of 8 per cent.
Corresponding to this excess of B. coU in the city water,
there were 12 cases of typhoid fever in December, 59 cases
in January, 12 in February, and 9 in March, all during
the early part of the month. The authors conclude that
" when filtering a river-water as polluted as that of the

ELEMENTS OF WATER BACTERIOLOGY.

Merrimac, it is safe to assume that when B. coh is found
only infrequently in i c.c. of the effluent the typhoid germs,
necessarily fewer in number and more easily removed by
the filter, have been eliminated from the water."

Another interesting contribution to this question was
made by the Massachusetts State Board of Health
(Massachusetts State Board of Health, 1901) in connec-
tion with the examination of the spring-waters bottled
for sale in the State. Ninety-nine springs were included
in this study; and in almost every instance 4 samples
were examined, 2 taken directly from the spring by
the engineers of the board and 2 from the bottles as
delivered for sale to the public. In the water of one
spring B. coli was found twice, once in a sample from
the spring and once in the bottled sample. This spring
was situated in woodland, but was unprotected from
surface drainage, and the method of filling bottles sub-
jected it to possible contamination. In 5 other cases B.
coli was found once in the sample from the spring; all
were subject to pollution from dwellings or cultivated
fields, and 4 of the 5 were shown to be highly contam-
inated, chemically. In 7 other cases B. coU was found
in the bottled samples alone ; 3 of these sources were of
high purity, but the bottling process furnished opportunity
for contamination.

Probably the most elaborate application of the colon
test which has ever been attempted was made by Jordan
in his recent examinations of the fate of the Chicago sew-
age in the Desplaines and Illinois Rivers. At one time

THE SIGNIFICANCE OF B. COLI IN WATER. 89

Professor Jordan was himself somewhat sceptical as to
the value of the colon test, "for he stated in 1890 (Jordan,
1890) that he had found, "in spring- water which was
beyond any suspicion of contamination, bacteria which
in form, size, growth on gelatin, potato, etc., were indis-
tinguishable from B. coli commune." In his recent
studies of self-purification (Jordan, 1901) the analyses
were made quantitative by the examination of numerous
measured samples, fractions of the cubic centimeter; and
the method employed was the enrichment either in dex-
trose-broth fermentation- tubes or in phenol broth,
vidth the subsequent maldng of litmus-lactose-agar plates.
The cultures isolated on these plates were tested as to
their behavior in dextrose broth, peptone solution, milk,
and gelatin; of the dextrose tubes made directly from
the water all were considered positive which gave more
than 20 per cent gas in the closed arm, with an appreciable
excess of hydrogen. The results were very significant.
In fresh sewage a positive result was obtained about
one-third of the time in one one-hundred-thousandth
of a cubic centimeter and almost constantly in one ten-
thousandth of a cubic centimeter. The Illinois and
Michigan canal proved almost as bad, giving positive
results on seven days out of twenty-eight in dilution of
one in one hundred thousand and on twenty-eight days out
of thirty-two in a dilution of one in ten thousand. At
Morris, twenty-seven miles below Lockport, where the
canal enters the bed of the Desplaines River, and nine
miles below the entrance of the Kankakee, the principal

9° ELEMENTS OF WATER BACTERIOLOGY.

diluting factor, the numbers were so reduced that
positive results were obtained only on eleven days out
of twenty in one-thousandth of a cubic centimeter, on
twenty days out of thirty in one-hundredth of a cubic
centimeter, and on twenty days out of twenty-three in
one-tenth of a cubic centimeter. At Averyville, one hun-
dred and fifty-nine miles below Chicago, colon bacilli were
isolated on only four days out of twenty-seven in one-
tenth of a cubic centimeter, and on thirteen days out of
thirty-one in one cubic centimeter. A comparison with
certain neighboring rivers showed this to be about the
normal value for waters of that character, as the following
table extracted from Professor Jordan's paper vidll show.

NtTMBER OF B. COLI PRESENT IN CERTAIN RIVER WATERS.
(Jordan, igoi.)

No. Days

No. Days

No. Days

No. Day

SoTirce of Sample.

Water

B. CoH

Water

B. Colt

Examined.

Found.

Examined.

Found.

Illinois River, Averyville. ..

- 27

4

31

13

Mississippi River, Grafton.

• 34

10

35

23

22

2

23

6

- 25

27

21

- 32

13

31

21

These results harmonize rather closely with those pre-
viously recorded by Brown and Fuller and indicate that
in the larger rivers where the proportionate pollution is
not extreme, colon baciUi may be isolated in about half
the I -c.c. samples examined. Such rivers are of course
inadmissible as sources of water-supply, according to
modern sanitary standards, unless subjected to pre-
liminary treatment.

THE SIGNIFICANCE OF B. CO LI IN WATER. 9 1

More recently Hunnewell and one of ourselves (Wins-
low and Hunnewell, 1902 examined a considerable
series of normal waters for B. coli, testing i c.c.
from each by the dextrose-broth method and a larger
portion of 100 c.c. by incubation with phenol broth as
described in Chapter VI. The samples were obtained
from the public supplies of Taunton, Boston, Cam-
bridge, Braintree, Brookline, Needham, and Lynn in
Massachusetts, and of Newport, R. I., from the Sudbury
River, from the ocean, from the waters of springs bottled
for the market, from ponds, pools of rain and melted
snow, springs, brooks, shallow wells, and driven wells in
various towns near the city of Boston. For comparison
50 samples of polluted waters from the Charles, Mystic,
Neponset, and North Rivers were examined. The colon
bacillus was defined as outlined in Chapter VI; and
organisms which lacked the power t-o reduce nitrates or to
form indol were classed in the "Paracolon group." The
results are summarized in the following table :

Presence of B. Coli in Polluted and Unpolluted Waters.

(Winslow and Hunnewell, 1902''.)
UNPOLLUTED WATERS.

Samples examined. ....
Dextrose broth positive
Lactose plate positive..

Colon group

Paracolon group

B. cloaciE group

Streptococcus group. . .

I c.c.

■ IS7
. 40

■ 13

• 5

• S

3

153
76

31
II

5
S

92 ELEMENTS OF WATER BACTERIOLOGY.

POLLUTED WATERS.

I c.c. loo c.c.

Samples examined 50 48

Dextrose broth positive 50 37

Lactose plate positive 50 26

Colon group 18 4

Paracolon group 6

Streptococcus group 25 22

B. cloacae i

The authors pointed out that these tables indicate
that bacteria capable of growth at the body temperature
and fermenting dextrose and lactose are infrequently found
in unpolluted waters, and colon bacilH are very rarely
present, since in 157 samples typical colon bacilH only
appeared 5 times. In the polluted waters it is evident
that colon bacilli originally present were in many cases
killed out during the process of enrichment by the strep-
tococci, since every one of the dextrose-broth tubes showed
gas at the first incubation of i c.c.

The latest important contribution to this subject comes
from Dantzic. Petruschky and Pusch (Petruschky and
Pusch, 1903) examined a considerable series of waters
from different sources by incubating measured samples
with equal amounts of nutrient broth and isolating upon
agar. In 45 samples of well-waters they found B. coU
7 times in .01 c.c, 9 times in .1 c.c, and 7 times in i cc
In the other 22 cases it could not be found in i c.c. and in
4 cases not in 100 c.c. One sample showed it only in .600
c.c and i not in 750 c.c. Of 29 river-waters, only 2 failed
to give positive results in .1 c.c. and 14 showed B. coh in
.001 of a c.c. or less. In sewage the number varied from

THE SIGNIFICANCE OF B. CO LI IN WATER. 93

I to 1,000,000 per c.c. The authors conclude that a
quantitative estimation of the B. coli content furnishes a
good measure of the fecal pollution of water and that
the observed differences in the extent of B. coli contam-
ination of surface-waters are so great that they differ
from each other more than a million- fold.

Altogether the evidence is quite conclusive that the
absence of B. coh demonstrates the harmlessness of a
water as far as bacteriology can prove it. That when
present, its numbers form a reasonably close index of
the amount of pollution the authors above quoted
have proved beyond reasonable cavil. It may safely be
said that when the colon bacillus, as defined by the tests
above, is found in such abundance as to be isolated in a
large proportion of cases from i c.c. of water, it is reason-
able proof of the presence of serious pollution, a view
which is to-day accepted by most American bacteriolo-
gists.

CHAPTER VIII.

PRESUMPTIVE TESTS FOR B. COLI.

The isolation and identification of B, coli by the
methods which have been described is a time-consuming
and laborious operation, and one sometimes impossible
to apply in the practical supervision of a water-supply.

Hence it is of especial value to the engineer to have
certain tests which may be easily and quicldy carried out
and which will give a probably correct idea as to whether
pollution does or does not exist. Such tests are spoken of
as "presumptive tests." If positive, the water showing
such a test may be regarded as suspicious, but must be
further examined to prove the presence of fecal bacteria.
If the presumptive test is negative, no further examina-
tion need be made. Of the cultural reactions required
to establish the identity of the colon bacillus, the growth
and gas formation in dextrose broth, as originally sug-
gested by Smith (1893^) is so well marked and so strongly
typical of B. coH that this medium may be most con-
veniently used as a " presumptive test."

The details of the operation vary in individual labora-
tories, but the underlying principle in all is that B. coli
develops rapidly in dextrose broth v^dth gas formation of

94

PRESUMPTIVE TESTS FOR B. COLL 95

from 25 to 70 per cent of the capacity of the closed arm
of the fermentation- tube. Of this gas approximately
one-third is carbon dioxide and two-thirds hydrogen,

. H 2

that is, as the gas formula is generally expressed, -qq= ~-

In testing a water by this method a series of samples
of the water, in suitable dilution, .001, .01, .1, i.o, or 10 c.c,
is added directly to the dextrose-broth tubes and incu-
bated for twenty- four hours at 37°.

On measurement of the gas, if the results above given
are obtained, the reaction is considered typical. If the
amount of gas is between 10 and 25 per cent or more than
70 per cent, or if the percentage of carbon dioxide is
greater than 40, the reaction is considered atypical. If
no gas forms, or less than 10 per cent, the test is called
negative. It is recognized that the distinction is not
absolute, since even pure cultures of B. coli sometimes
fail to give typical reactions, and on the other hand, an
atypical organism sometimes gives a satisfactory positive
test. There can be little question, however, as to its
value to the sanitary engineer.

In recent years, Irons (Irons, 1901) was perhaps the
first to call attention to the value of this method, stating
that "when the dextrose tube yields approximately
33 per cent of CO2 Bacillus coH communis is almost
invariably present." In the next year the reliabiHty of
the fermentation test as an indication of B. coli was
worked out by Gage (Gage, 1902) as given in the follow-
ing table.

96 ELEMENTS OF WATER BACTERIOLOGY.

Number of samples tested

" giving preliminary fermentation.
Per cent of latter proved to contain coli. .

I c.c.

5^72
1036
70

100 c.c,

1375

474
71

The work of Hunnewell and one of ourselves (Wins-
low and Hunnewell, 1902^) showed that while 50 samples
of polluted waters all gave gas in the dextrose tube, only
40 out of 157 samples from presumably unpolluted sources
showed a similar fermentation. Whipple (Whipple, 1903)
examined a large number of surface-water supplies by
the "presumptive test" and obtained striking results,
shown in the following table. The waters are arranged
in six groups according to the results of sanitary inspec-
tion, group I including waters collected from almost
uninhabitated watersheds and group VI waters too much
polluted to be safely used for domestic purposes.

PERCENTAGE OF SAMPLES OF WATERS OF VARIOUS SANITARY GRADES
GIVING POSITIVE TESTS FOR B. COLI WHEN DIFFERENT AMOUNTS
WERE EXAMINED.

(Whipple. 1903.)

Group.

I.

IL
III..
IV-

V.

10 c.c.

100 c.c.

500 c.c.

0.0

3-S

20.8

50.0

50.0

S-o

7-3

15.0

60.0

60.0

0.0

7.0

50.0

50.0

60.0

4.0

6.8

41.7

67.0

75 -o

5.0

13.0

75-0

100.0

100.0

20.2

7S-0

80.0

100.0

In view of these results Whipple suggests the foUowing
provisional scheme of interpretation, which seems to the
authors to be, in general, sound.

PRESUMPTIVE TESTS FOR B. COLI. 97

Sanitary Quality.

Presumptive Test for Bacillus CoU.

lOO C.C.

Safe

0

0

o

0

+

Reasonably safe

o

0

0

+

+

Questionable

o

o

+

+

+

Probably unsafe

o

+

+

+

+

Un.safe

+

+

+

+

+

Results harmonizing well with the above were obtained
by Nibecker and one of us (Winslow and Nibecker, 1903),
as quoted in Chapter IV. Of 775 dextrose tubes inocu-
lated from 259 samples of water from apparently unpol-
luted sources, only 41 showed gas and only 3 gave the gas
formula characteristic of B. coH.

The Htmus-lactose-agar plate furnishes another pre-
sumptive test of considerable value as indicated in Chap-
ter IV, aUhough it is probably less deUcate than the dex-
trose-broth method. With polluted waters in particular
this test will be found of value, since streptococci may
interfere with the dextrose-broth method if dilution is
insufficient or incubation too prolonged.

Certain special media have also been suggested for
rapid routine water analysis of which those containing
" neutral red," one of the safranine dyes, has been most
fully studied. Rothberger (Rothberger, 1898) first pointed
out that B. coH reduces solutions of this substance, the
color changing to canary-yellow accompanied by green
fluorescence. MakgiU (MakgiU, 1901), Savage (Savage,
1901), and other English observers report favorable

98 ELEMENTS OF WATER BACTERIOLOGY.

results from the use of this test, but according to Ameri-
can standards, Irons (Irons, 1902) and Gage and Phelps
(Gage and Phelps, 1903) concmde that the group of
organisms giving a positive neutral red reaction is too
large a one to give very valuable sanitary information.
In a paper read at the Washington meeting of the Ameri-
can PubUc Health Association in 1903, Stokes suggested
a modification of this medium in which lactose is sub-
stituted for dextrose, since many saprophytic organisms
will attack the latter and not the former sugar.

The medium devised by MacConkey (MacConkey,
1900; MacConkey, 1901; MacConkey and Hill, 1901)
containing sodium taurocholate as its characteristic
ingredient has also proved successful in the hands of
some observers. The dextrose fermentation-tube, how-
ever, is the only presumptive test whose value has been
estabhshed by conclusive general investigations.

CHAPTER IX.

OTHER INTESTINAL BACTERIA.

It would be an obvious advantage if the evidence of
sewage contamination, furnished by the presence of B.
coU could be reinforced or confirmed by the discovery in
water of other forms equally characteristic of the intestinal
canal. The attention of a few bacteriologists in England
and America has been turned strongly in this direction
during the past few years; and two groups of organisms,
the sewage streptococci and the anaerobic spore-bearing
bacilli, have been described as being probably significant.

The term "sewage streptococci" covers an ill-defined
group including many organisms which do not actually
occur in well-marked chains. Those most commonly
found correspond, however, rather closely to the type of
Str. erysipelatos. They grow feebly on the surface of
ordinary nutrient agar, producing faint transparent,
rounded colonies, but under semi-anaerobic conditions
flourish better, giving a well-marked growth along the
gelatin stab and only a small circumscribed film on the
surface. They are favored by the presence of the sugars
and ferment dextrose and lactose, with the formation of
abundant acid but no gas. They are seen under the

99

lOO ELEMENTS OF WATER BACTERIOLOGY.

microscope as cocci, occurring as a rule in pairs, short
chains, or irregular groups. They do not show visible
growth in the standard peptone and nitrate solutions;
most of them do not Uquefy gelatin, though occasion-
ally forms are found which possess this power. No sys-
tematic study of the various species found in the intes-
tine has so far been made, and at present all cocci giving
the characteristic growth on agar and strongly fermenting
' lactose may be included as " sewage streptococci."

Although the significance of the streptococci as sewage
organisms is not established with the same definiteness
which marks our knowledge of the colon group, these
organisms have been isolated so frequently and with such
constant results that it now seems reasonable to regard
their presence as indicative of pollution. Although orig-
inally reported by Laws and Andrewes (Laws and
Andrewes, 1894), their importance was not emphasized
until 1899 and 1900, when Houston (Houston, 1899'',
1900'') laid special stress upon the fact that streptococci
and staphylococci seem to be characteristic of sewage and
animal waste, the former being, in his opinion, the more
truly indicative of dangerous pollution, since they are
" readily demonstrable in w.aters recently polluted and
seemingly altogether absent from waters above suspicion
of contamination." In the water of 6 rivers recently
extensively sewage-polluted he found streptococci in
from one-tenth to one ten-thousandth of a c.c. of the water
examined, although in some cases the chemical analysis
did not clearly indicate dangerous pollution. On the

OTHER INTESTINAL BACTERIA. lOi

Other hand, 8 rivers, polluted, but not recently and exten-
sively polluted, showed no streptococci in one-tenth of a
c.c, although the chemical and the ordinary bacteriological
tests gave results which would condemn the waters. Hor-
rocks (Horrocks, 1901) found these organisms in great
abundance in sewage and in waters known to be sewage-
polluted, but which contained no traces of Bacillus coli.
He found by experiment that B. coli gradually disappeared
from many specimens of sewage kept in the dark at the
temperature of an outside veranda, while the commonest
forms which persisted were varieties of streptococci and
staphylococci.

In America attention was j&rst called to these organisms
by Hunnewell and one of ourselves (Winslow and Hunne-
well, 1902''), and the same authors later (Winslow and
Hunnewell, 1902'') recorded the isolation of streptococci
from 25 out of 50 samples of polluted waters. Gage
(Gage, 1902), from the Lawrence Experiment Station
has reported the organisms present in the sewage of that
city, while one of us (Prescott, 1902'') has shown that
they are abundant in fecal matter and often overgrow
B. coU in a few hours when inoculations are made from
such material into dextrose broth. In the recent mono-
graph of Le Gros (Le Gros, 1902) of the many strep-
tococci described all without exception were isolated,
either from the body or from sewage. In the study of
259 samples of presumably unpolluted waters, by the
method of direct plating, Nibecker and one of the authors
(Winslow and Nibecker, 1903) found streptococci in

102 ELEMENTS OF WATER BACTERIOLOGY.

only I sample, while Baker and one of us (Prescott and
Baker, 1904) found the organism present in each of
50 samples of variously polluted waters when inoculation
was first made into dextrose broth from which Htmus-
lactose-agar plates were made after various intervals.

The isolation of these organisms either from plates or
liquid cultures is easy. On the lactose-agar plate, made
directly from a polluted water the colonies of the strep-
tococci may generally be distinguished from those of other
acid-formers by their small size, compact structure, and
deep-red color, which is permanent, never changing to blue
at a later period of incubation as the colonies of B. coli
may do. Developing somewhat slowly, however, they
may be overlooked if present only in small numbers. In
the dextrose-broth tube, streptococci will appear in abun-
dance after a suitable period of incubation. Prescott
and Baker in the work above mentioned found that with
mixtures of B. coli and streptococci in which the initial
ratios of the latter to the former varied from i : 94 to 208 : i ,
the colon bacilli developed rapidly during the early part
of the experiment, reaching a maximum after about
fourteen hours and then diminishing rapidly, while the
streptococci first became apparent after ten to fifteen
hours and reached their maximum after twenty to sixty
hours, according to the number originally present.

Applying the same method to polluted waters, similar
periodic changes were observed; pure cultures of B. coli
were first obtained, then the gradual displacement of one
form by the other took place, and at length streptococci

OTHER INTESTINAL BACTERIA. 103

were either present in pure culture or in great predomi-
nance as shown by the accompanying tables. The samples
of water were plated directly upon litmus-lactose-agar and
he plates were incubated at 37° for twenty-four hours,
when the red colonies were counted. At the time of plating,
I c.c. from each sample was also inoculated into dextrose
broth in fermentation-tubes, which were likewise incu-
bated at 37°. After various periods, as indicated by the
tables below, the tubes were shaken thoroughly and i c.c.
of the contents withdrawn. This was then diluted
(generally 1-1,000,000) with sterile water, plated with

Table I.

RELAXn^E GROWTH OF B. COLI AND SEWAGE STREPTOCOCCI FROM
POLLUTED WATERS IN DEXTROSE BROTH.
(Prescott and Baker, 1904.)

Sample Number

I

a

3

4

5

6

7

8

9

10

Red colonies developing
from I c.c. of original
sample on litmus-lac-

4

0

ID
20

9

5

8

55

35

460

1230

105

410
0

410

Number
found, in
millions
per cubic
centime-
ter, after'
growth in
dextrose
broth for
various
periods . .

II
hrs.

B. coli

68.4

200

185

400

130

332

420

Strept.

0

0

0

0

0

0

0

0

0

16
hrs.

B. coU

200

76

130

270

220

210

140

430

285

Strept.

40

20

10

45

30

20

210

75

145

23
hrs.

B. coli

280

150

385

370

300

570

200

405

320

300

Strept.

140

8s

280

170

300

1 700

110

350

370

3SO

30
hrs.

hrs.

B. coli

0

0

25

iioj 0

210

20

24

105

Strept.

474

420

480

300

390

1 70

400

105

2S0

B. coli

0

0

0

0

0
I

1 2
2

8

0

0

0

Strept.

2

0
10

0
0

45

45

150

86

170

First gas noted after (hrs) .

10

9

10

8

10

6

6

8

I04 ELEMENTS OF WATER BACTERIOLOGY.

Table II.

RELATIVE GROWTH OF B. COLI AND SEWAGE STREPTOCOCCI FROM
POLLUTED WATERS IN DEXTROSE BROTH.
(Prescott and Baker, 1904.)

18

19

20

21

22

23

24

2S

Red colonies developing from i c.c. 1
of original sample on litmus-lac- V

I

ISO

2S

30

SO

170

200

30

Number found, in
millions per cubic
centimeter, after,
growth in dextrose
broth for various

7

hrs.

B. coU

.04

. 1 2

• 55

I . 0

Strept.

0

0

0

0

0

0

17
hrs.

B. coli

266

100

88

3SO

Sio

380

330

160

Strept.

ISO

0

40

140

240

128

80

220

27
hrs.

B. coH

520

610

72

700

1000

740

100

300

Strept.

800

860

670

1080

2500

4380

3900

40
hrs.

B. coli

0

0

10

22

36

7

7

Strept.

252

330

260

22

66

60

S2

52
hrs.

B. coli

10

16

38

20

70

3S

10

27

Strept.

40

16

3.8

31

41

2S

10

30

litmus-lactose-agar in the usual way, and incubated for
twenty-four hours. The colonies of B. coli and strep-
tococci were distinguished microscopically, and by differ-
ence in color and general characters.

These facts suggest the following method for the detec-
tion of B. coli and sewage streptococci in polluted waters.

Inoculate the desired quantity of water, preferably i c.c,
into dextrose broth, in a fermentation-tube, and incubate
at 37°. After a few hours' incubation examine the cul-
tures for gas. Within two or three hours after gas for-
mation is first evident, plate from the broth in litmus-
lactose-agar, incubating for twelve to eighteen hours at

OTHER INTESTINAL BACTERIA. 105

37°, If at the end of this time no acid-producing colonies
are found, it is probably safe to assume that there were no
colon bacilli present. On the other hand, if red colonies
are developed, these must be further examined by the
regular diagnostic tests for B. coH. After the first plating
from the dextrose broth, replace the fermentation-tube
in the incubator and allow it to remain for twenty-four
to thirty-six hours, then plate again in litmus-lactose-agar.
This plating should give a nearly pure culture of strep-
tococci if these organisms were originally present in the
water. If colon bacilli are not found in the first set of
plates the streptococci may still be isolated by this method.

The relative relation of the streptococci and the colon
bacilU to sewage pollution is still uncertain. Houston
(Houston, 1900) held that the former microbes imply
" animal pollution of extremely recent and therefore
specially dangerous kind." Horrocks (Horrocks, 1901),
on the other hand, maintains, largely on the strength of
certain experiments with stored sewage, that the strep-
tococci persist after colon bacilU have disappeared and
indicate contamination with old sewage which is not
necessarily dangerous. On the whole, it seems probable
that the streptococci, like the colon bacilli, are primarily
parasitic organisms, the former being associated both
with the outer and inner surfaces of the body. Like the '
colon bacilli, their presence in water indicates contact
with the wastes of human Hfe, and their isolation from
a suspected sample furnishes valuable confirmatory evi-
dence of its dangerous character.

Io6 ELEMENTS OF WATER BACTERIOLOGY.

The English bacteriologists have ascribed much impor-
tance as indicators of sewage pollution to another group
of organisms, the anaerobic spore-forming bacilli, of which
the B. sporogenes is a type. This form was isolated by
Klein (Klein, 1898; Klein, 1899) in 1895, in the course
of an epidemic of diarrhoea at St. Bartholomew's Hos-
pital, and described under the name of B. enteritidis
sporogenes; it is apparently identical with the B. aero-
genes capsulatus of Welch (Welch and Nuttall, 1892).

Klein's procedure for isolating the B. sporogenes is
simple in the case of polluted waters. A portion of the
sample to be examined is added to a tube of sterile milk,
which is then heated to 80° C. for ten minutes to destroy
vegetative cells. The milk is then cooled and incubated
under an^robic conditions, which may be accomplished
most conveniently by Wright's method. A tight plug of
cotton is forced a quarter way down the test-tube, the
space above is loosely filled with pyrogallic acid, a few
drops of a strong solution of caustic potash are added,
and the tube is tightly closed with a rubber stopper. After
eighteen to thirty-six hours at 37° the appearance of the
tube will be very characteristic if the B. sporogenes is
present. "The cream is torn or altogether dissociated
by the development of gas, so that the surface of the
medium is covered with stringy, pinkish-white masses of
coagulated casein, enclosing a number of gas-bubbles.
The main portion of the tube formerly occupied by the
milk now contains a colorless, thin, watery whey, with a
few casein lumps adhering here and there to the sides of

OTHER INTESTINAL BACTERIA 107

the tube. When the tube is opened, the whey has a smell
of butyric acid and is acid in reaction. Under the micro-
scope the whey is found to contain numerous rods, some
motile, others motionless."

The B. sporogenes when isolated in pure culture on
glucose agar is a stout rod. It liquefies gelatin, forming
in this medium large oval spores. It is strongly patho-
genic for guinea-pigs, by which character it is distinguished
from the B. butyricus of Botkin.

The researches of Klein and Houston (Klein and Hous-
ton, 1898, 1899) have shown that the B. sporogenes occurs
in Enghsh sewage in numbers varying from 30 to 2000 per
c.c. and that it is often absent in considerable volumes of
pure water. In Boston sewage it may usually be isolated
from .01 or .001 of a c.c. (Winslow and Belcher, 1904),

Evidently in order to have any significance, an exam-
ination for this organism must be made with large samples
and the concentration of at least 2000 c.c. of water through
a Pasteur filter is recommended by Horrocks as a neces-
sary prelude (Horrocks, 1901). Since the spores of an
anaerobic bacillus may persist for an indefinite period in
polluted waters, their presence need not necessarily indi-
cate recent or dangerous pollution. Since the number
present even in sewage is so small and so variable, no
quantitative standard can be estabHshed; on the whole,
it does not appear that the practical application of the
anaerobic test will ever be a wide one.

There are numerous other sewage bacteria whose
presence is more or less constantly characteristic of pol-

lo8 ELEMENTS OF WATER BACTERIOLOGY.

luted waters. Organisms of the Proteus group are some-
times present, exhibiting marked morphological varia-
tions, from the coccus form to long twisted threads and
forming on gelatin irregular amoeboid colonies with filiform
processes extending into the surrounding gelatin. The
B. subtilis group of strongly aerobic spore-forming bacilli,
giving a brown wrinkled parchment-like growth on agar,
and moss-like liquefying colonies on gelatin, is usually
represented. Among the aUies of B. coli may be men-
tioned B. ffirogenes, which differs from it in being non-
motile, failing to produce indol, and forming spherical
drop-like colonies on gelatin. B. cloacs resembles B. coli
in most respects, but causes a Uquefaction of gelatin.
The property of liquefaction was formerly beheved to be
of general significance, inasmuch as the liquefying bacteria
were regarded as closely alUed to intestinal organisms,
and in themselves indicative of pollution. This position
is, however, no longer tenable, since many bacteria,
typical of the purest waters, may cause liquefaction.

While the organisms above mentioned, and many others
as well, deserve notice in the examination of gelatin plates
from I suspected water, none of them is of sufficient
importance to warrant any special procedure for their
isolation.

CHAPTER X.

THE SIGNIFICANCE AND APPLICABILITY OF THE BAC-
TERIOLOGICAL EXAMINATION.

The first attempt of the expert called in to pronounce
upon the character of a potable water should be to make
a thorough sanitary inspection of the pond, stream, or well
from which it is derived. Study of the possible sources
of pollution on a watershed, of the direction and velocity
of currents above and below ground, of the character of
soil and the liabiUty to contamination by surface-wash
are conceded to yield evidence of the greatest value.
Often, however, some opinion must be formed upon the
quality of water sent from a distance without the oppor-
tunity of examining its surroundings; and even when
sanitary inspection can be made, its results are by no
means conclusive. No reconnoissance can show cer-
tainly whether unpurified drainage from a cesspool does
or does not reach a given well; whether sewage discharged
into a lake does or does not find its way to a neighboring
intake; whether pollution of a stream has or has not been
removed by a certain period of flow. Evidence upon
these points must be obtained from a careful study of the

J09

no ELEMENTS OF WATER BACTERIOLOGY.

characteristics of the water in question, and this study
can be carried out along two lines, chemical and bac-
teriological.

A chemical examination of water for sanitary purposes,
is mainly useful in throwing light upon one point — the
amount of decomposing organic matter present. Humus-
like substances may be abundant in surface-waters quite
free from harmful pollution, but these are stable com-
pounds. Easily decomposable bodies, on the other hand,
must obviously have been recently introduced into the
water and mark a transitional state. "The state of
change is the state of danger," as Dr. T. M. Drown has
phrased it. Sometimes the organic matter has been
washed in by rain from the surface of the ground, some-
times it has been introduced in the more concentrated
form of sewage. In any case, it is a warning of possible
pollution, and the determination of free ammonia, nitrites,
carbonaceous matter, as shown by "oxygen consumed,"
and dissolved oxygen yield important evidence as to the
sanitary quaUty of a water.

Furthermore, nitrates, the final products of the oxida-
tion of organic matter, and the chlorine introduced as
common salt into all water which has been in contact
with the wastes of human life, furnish additional informa-
tion as to the antecedents of a sample. The results of
the chlorine determination are indeed perhaps more clear
than those of any other sanitary analysis, for chlorine
and sewage pollution vary together, due allowance being
made for the proximity of the sea and other geological

BACTERIOLOGICAL EXAMINATION.

Ill

and meteorological factors. Unfortunately, it is only
past history and not present conditions which these latter,
tests reveal, for in a ground-water completely purified
from a sanitary standpoint such soluble constituents
remain, of course, unchanged. Thus, in the last resort,
it is upon the presence and amount of decomposing
organic matter in the water studied that the opinion of
the chemist must be based.

The decomposition of organic matter may be measured
either by the material decomposed or by the number of
organisms engaged in carrying out the process of decom-
position. The latter method has the advantage of far
greater delicacy, since the bacteria respond by enormous
multiplication to very slight increase in their food-supply,
and thus it comes about that the standard gelatin-plate
count at 20° roughly corresponds, in not too heavily
polluted waters, to the free ammonia and " oxygen con-
sumed," as revealed by chemical analysis. If low num-
bers of bacteria are found, the evidence is highly reassuring,
for it is seldom that water could be contaminated under
natural conditions without the direct addition of foreign
bacteria or of organic matter which would condition a
rapid multiplication of those already present. The bac-
teriologist in such cases can declare the innocence of
the water with justifiable certainty. When high num-
bers are found the interpretation is less simple, since they
may exceptionally be due to the multiplication of certain
peciiliar water forms. Large counts, however, under
ordinary conditions, when including a normal variety of

112 ELEMENTS OF WATER BACTERIOLOGY.

forms indicate the presence of an excess of organic matter
derived in all probability either from sewage or from the
fresh washings of the surface of the ground. In either
case danger is indicated.

A still closer measure of polluting material may be
obtained from the numbers of colonies which develop
on litmus-lactose-agar at 37°, since organisms which
thrive at the body temperature, and particularly those
which ferment lactose, are characteristic of the intestinal
tract and but rarely occur in normal waters.

Finally, the search for the Bacillus coli furnishes the
most satisfactory of all single tests for fecal contamination.
This organism is pre-eminently a denizen of the alimen-
tary canal and may be isolated with ease from waters to
which even a small proportion of sewage has been added.
On the other hand, it is never found in abundance in
waters of good sanitary quality; and its numbers form
an excellent index of the value of waters of an interme-
diate grade. The streptococci appear to be forms of a
similar significance useful as yielding a certain amount of
confirmatory evidence. The full bacteriological analysis
should then consist of three parts— the gelatin-plate count,
as an estimate of the amount of organic decomposition
in process; the total count, and the count of red colonies,
on litmus-lactose-agar, as a measure of the organisms
which form acids and thrive at the body temperature;
and the study of a series of dextrose-broth tubes for the
isolation of colon bacilli and streptococci. The simple
examination of the dextrose-broth tube and the count on

BACTERIOLOGICAL EXAMINATION. 113

the litmus-lactose-agar plate serve for what Whipple has
well called presumptive tests.

The results of the bacteriological examination have, in
several respects, a pecuHar and unique significance.
First, this examination is the most direct method of sani-
tary water analysis. What we dread in drinking-water is
the presence of pathogenic bacteria, mainly from the
intestinal tract of man, and it is quite certam that the
related non-pathogenic bacteria from the same source
will behave more nearly as these disease germs do than
will any chemical compounds. In the second place, the
bacteriological methods are superior in delicacy to any
others. Klein and Houston (Klein and Houston, 1898)
showed by experiment with dilutions of sewage that the
colon test was from ten to one hundred times as sensitive
as the methods of chemical analysis; and studies of the
self-purification of streams have confirmed his results
on a practical scale. Thus in the Sudbury River it was
found that while the chemical evidences of pollution
persisted for six miles beyond the point of entrance,
the bacteria introduced could be detected for four miles
further (Woodman, Winslow, and Hansen, 1902).

The statement is sometimes made that while bac-
teriological methods may be more delicate for the detec-
tion of pollution in surface-waters^ contamination in
ground-waters may best be discovered by the chemical
analysis. That such is not the case has been well shown
by Whipple (Whipple, 1903), who cites the following

114 ELEMENTS OF WATER BACTERIOLOGY

two instances in which the presumptive test revealed con-
tamination not shown by the chemical analysis:

"A certain driven-well station was located in swampy
land along the shores of a stream, and the tops of the
wells were so placed that they were occasionally flooded
at times of high water. The water in the stream was
objectionable from the sanitary standpoint. The wells
themselves were more than loo feet deep; they pene-
trated a clay bed and yielded what may be termed arte-
sian water. Tests for the presence of Bacillus coli had
invariably given negative results, as might be naturally
expected. Suddenly, however, the tests became positive
and so continued for several days. On investigation it
was found that some of the wells had been taken up to
be cleaned, and that the workmen in resinking them had
used the water of the brook for washing them down.
This allowed some of the brook- water to enter the system.
It was also found that at the same time the water in the
brook had been high, and because of the lack of packing
in certain joints at the top of the wells the brook- water
leaked into the suction main. The remedy was obvious
and was immediately applied, after which the tests for
Bacillus coli once more became negative. During all
this time the chemical analysis of the water was not suffi-
ciently abnormal to attract attention. On another occa-
sion a water-supply taken from a small pond fed by
springs, and which was practically a large open well,
began to give positive tests for Bacillus coli, and on exam-
ination it was found that a gate which kept out the water

BACTERIOLOGICAL EXAMINATION. US

of a brook which had been formerly connected with the
pond was open at the bottom, although it was supposed
to have been shut, thus admitting a contaminated sur-
face-water to the supply." Whipple also calls attention
to the report on the Chemical and Bacteriological Exami-
nation of Chichester Well-waters by Houston (Houston,
1901), in which the results of chemical and bacteriologi-
cal examinations of 30 wells were compared. It was
found that the bacteriological results were in general
concordant and satisfactory. The wells which were high-
est in the number of bacteria showed also the greatest
amount of pollution, as indicated by the numbers of B.
coh, B. sporogenes, and streptococci. On the other
hand, the chlorine and the albuminoid ammonia showed
no correspondence with the bacteriological results.

Thirdly, negative tests for Bacillus coli and low bac-
terial counts may be interpreted as proofs of the good
quality of water, with a certainty not attainable by any
other method of analysis. Many a surface-water with
reasonably low chlorine and ammonias has caused epi-
demics of typhoid fever; but it is impossible under any
natural conditions that a water could contain the typhoid
bacillus without giving clear evidence of pollution in the
dextrose-broth tube or on the lactose-agar plate.

It seems to the writers that the real application of
chemistry begins where that of bacteriology ends. When
pollution is so gross that its existence is obvious and only
its amount needs to be determined, the bacteriological
tests will not serve, on account of their excessive dehcacy.

Il6 ELEMENTS OF WATER BACTERIOLOGY.

In Studying the heavy pollution of small streams, the
treatment of trades wastes, and the purification of sewage,
the relations of nitrogeneous compounds and of oxygen
compounds are of prime importance. In other words,
when pollution is to be avoided, because the decompo-
sition of chemical substances causes a nuisance, it must
be studied by chemical methods. When the danger is
sanitary and comes only from the presence of bacteria,
bacteriological methods furnish the true index of pollution.

In the study of certain special problems the paramount
importance of bacteriology is generally recognized. The
distribution of sewage in large bodies of water into which
it has been discharged may thus best be traced on account
of the ready response of the bacterial counts to slight
proportions of sewage, particularly since the ease and
rapidity with which the technique of plating can be carried
out make it possible to examine a large series of samples
with a minimum of time and trouble. The course of the
sewage carried out by the tide from the outlet of the
South MetropoUtan District of Boston was studied in
this way by E. P. Osgood in 1897, and mapped out by
its high bacterial content with greater accuracy than
could be attained by any other method. Some very
remarkable facts have been developed by similar studies
as to the persistence of separate streams of water in
immediate contact with each other. Heider showed
that the sewage of Vienna, after its discharge into the
Danube River, flowed along the right bank of the
stream, preserving its own bacterial characteristics, and

BACTERIOLOGICAL EXAMINATION.

117

not mixing perfectly witli the water of the river for a dis-
tance of more than twenty-four miles (Heider, 1893).
Jordan (Jordan, 1900), in studying the self-purification of
the sewage discharged from the great Chicago drainage
canal, found by bacteriological analyses that the Des
Plaines and the Kankakee Rivers could both be distin-
guished flowing along in the bed of the Illinois, the two
streams being in contact, yet each maintaining its own
individuahty. Finally, the quickness with which sUght
changes in the character of a water are marked by
fluctuations in bacterial numbers renders the bacterio-
logical methods invaluable for the daily supervision of
surface suppHes or of the effluents from municipal filtra-
tion plants.

In the commoner case, when normal values obtained
by such routine analyses are not at hand, the problem of
the interpretation of any sanitary analysis is a more difli-
cult one. The conditions which surround a source of
water-supply may be constantly changing. No engineer
can measure the flow of a stream in July and deduce the
amount of water which will pass in February; yet the
July gauging has its own value and significance. So a
single analysis of any sort is not sufficient for all past and
future time. If it gives a correct picture of the hygienic
condition of the water at the moment of examination it
has fulfilled its task, and this the bacteriological analysis
can do. The evidence furnished by inspection and by
chemical analysis should be sought for and welcomed
whenever it can be obtained, yet we are of the opinion

no ELEMENTS OF WATER BACTERIOLOGY.

that, on account of their directness, their delicacy, and
their certainty, the bacteriological methods should least
.of all be omitted, and, if necessary, they alone may fur-
nish conclusive testimony as to the safety of a potable
water.

APPENDIX.

It has been pointed out that the number of bacteria
developing from a given sample of water will be
largely dependent upon the composition and character
of the culture medium. As the nature of the medium
varies widely according to the method of preparation,
workers in different laboratories can have no rational
basis for comparison of results until their methods are
essentially uniform.

To make resuUs comparable as far as possible, the
Laboratory Section of the American Public Health Asso-
ciation has adopted standard methods for the preparation
of the commonly used nutrient media given in the follow-
ing extract from the report of the Committee (Fuller, 1902).

Standard Methods for Gelatin and Agar.

GELATIN.

AGAR.

^' gm. thread agar in 500 c c

wPi^h/T ^^^^ ^" make up

weight to spo gm. or digest for 10
minutes m the autoclavat no" C
Infuse 500 gm. lean meat ,4 hours Infuse 500 gm' ta'n °meat°\^- ^ '
with 1000 c.c. of distilled water with 500 c c of dl^tiSln , ^""'"^
m refrigerator. frigerator "'stilled water in re-

3- Make up any loss bv evaporation. ' ,

7 w infusion through cotton fiannel.

5- Weigh filtered infusion.

119

I20

APPENDIX.

GELATIN. AGAR.

6. Add I % Witte's peptone and io% Add 2 % of Witte's peptone,
gold label sheet gelatin.

7- . Warm on water-bath, stirring till peptone and gela-

tin are dissolved and not allowing the temperature to
rise above 60° C.
8. Neutralize.

9- To 500 gm. of the meat infusion

add 500 c c. of the 3% agar, keeping
the temperature below 60° C.

10. Heat over boiling water (or steam) bath 30 minutes.

11. Restore loss by evaporation.

12. Titrate, after boiling i minute to expel carbonic acid.

13. Adjust reaction to +1.0% by adding normal hydro-
chloric acid or sodium hydrate as required.

14. Boil 2 minutes over free flame, constantly stirring.
IS- Make up loss by evaporation.

x6. Filter through absorbent cotton and cotton flannel,

passing the filtrate through the filter until clear.

17. Titrate and record the final reaction.

18. Tube, using s c.c. in each tube in the case of gelatin,
and 7 c.c. in the case of agar.

19. Sterilize 15 minutes in the autoclav at 110°, or for
30 minutes in streaming steam on three successive days.

20. Store in the ice-chest in a moist atmosphere to pre-
vent evaporation.

Hill (Hill, 1899) has arranged the methods for prepa-
ration of broth, nutrient gelatin, and nutrient agar in
tabular form as given on the following page.

For titration, the following method, suggested by
FviUer (Fuller, 1895), was adopted by the PubUc Health
Association Committee in 1897 (Committee of Bacteriolo-
gists, 1898).

The medium to be tested, all ingredients being dis-
solved, is brought to the prescribed volume by the addition
of distilled water to replace that lost by boiling, and after
being thoroughly stirred, 5 c.c. are transferred to a 6-inch
porcelain evaporating-dish; to this 45 c.c. of distilled water
are added, and the 50 c.c. of fluid are boiled for three
minutes over a flame. One c.c. of a .5 per cent solution
of phenolphthalein in 50 per cent alcohol is then added

N

and the reaction is deternrined by titration with — sodium

APPENDIX.

121

TABLE SHOWING ANALOGY BETWEEN BROTH, NUTRIENT GELATIN, AND
NUTRIENT AGAR MADE BY METHODS HEREIN RECOMMENDED.

Boil 30 gm. thread agar
in I liter of water for half
fwi_ hour. Make up to a
weight of 1000 gm.

Cool and solidify.

NUTRIENT BROTH.

NUTRIENT GELATIN.

NUTRIENT AGAR.

1. Infuse lean meat 20
hours with twice its weight
of distilled water in refrig-
erator.

Say 1000 gm. meat.
2000 ■' water.

2. Make up weight of
meat infusion (and meat)
to original weight by add-
ing water, i.e. to 3000 gm.

3. Filter infusion through
cloth to remove meat.

4. Titrate and record re-
action of filtrate. Say re-
action-f- 2. 2%.

5. Weigh infusion. Say
1800 gm.

6. Set infusion on water-
bath, keeping temperature
below 60° C.

7. Add peptone, 1%, 18
gm.

8. After ingredients are
dissolved, titrate, reaction
probably-(-2.3 to +2.5.

9. Neutralize, F u 1 1 e r's
method.

Ditto.

Ditto.

Ditto.
Ditto.

Ditto.
Ditto.

Ditto and sheet
gelatin 10%, 180
gm.

Ditto.

Probably-!- 4.0 to
+ 0.5.

Ditto.

Infuse lean meat 20 hours
with its own weight of dis-
tilled water in refrigerator.

Say 1000 gm. meat.
" 1000 gm. water.

Ditto.

i.e. to 2000 gm.

Ditto.
Ditto.

Say reaction + 4.2%.

Ditto.

Say 900 gm.
Ditto.

Add peptone, 2%, 18 gm.

Ditto.

Prob. -(-4.5 to -1-4.7.

Ditto.

To the 900 gm. of meat
infusion (containing now
peptone and salt) add 900
gm. of the 3% agar jelly
described at the head of
this column.

10. Heat over boiling water (or steam) bath thirty minutes.
. Restore weight lost by evaporation to original weight of filtered meat
intusion, I.e. that on which the percentage of peptone and salt, etc., were cal-
culated, — 1800 gm. in each case.

12. Titrate, reaction probably 4-0.3 to +0.5.

13. Adjust reaction to final point desired, +i.s per cent.

14. Boil five minutes over free flame, with stirring.

water, if necessary, to make up loss by evaporation, to 1800 gm.
\ J ''".';°>i,S" absorbent cotton, passing the filtrate through the filter
repeatedly until clear.

tained'^'''^^*^ '° whether or not the desired reaction has been main-

18. Tube and sterilize.

122

APPENDIX.

hydroxid or — hydrochloric acid. As a rule, the reaction
20

will be acid and the alkali may be used. The determina-
tion should be made not less than three times, and the
average of the results taken.

LACTOSE AGAR.

This medium is made in the same manner as nutrient
agar except that 2 per cent of lactose is added after the
final filtration and the reaction is adjusted to — .5.

GLYCERINE AGAR.

For this medium, 5 per cent glycerine is added to nutri-
ent agar immediately after final filtration.

SUGAR BROTH.

The most important and widely used sugar broth is
that containing dextrose, although lactose and saccharose
and occasionally maltose are used for special experi-
mental work.

Dextrose broth may be easily prepared by adding
2 per cent of dextrose to ordinary broth just before the
final filtration, and making the reaction neutral. For
the preparation of lactose, saccharose, or maltose broth,
however, where it is necessary to ehminate dextrose, the
meat infusion after filtration is placed in an Erlenmeyer
flask, inoculated with an active broth culture of B. coh

APPENDIX.

123

and incubated for twenty- four hours at 37° C, whereby
the muscle sugar is removed by fermentation.

From this fermented infusion, broth is made in the
ordinary way, 2 per cent of the desired sugar added, and
the medium tubed and sterihzed. All sugar containing
media should be sterilized with great care to avoid break-
ing down the sugar, hence it is advisable to use the
discontinuous method, heating to 100° C. for twenty
minutes on three successive days, rather than to give a
single heating at the very high temperature. There is,
however, but httle action at 105° C.

It is the custom in some laboratories to prepare broth
and sugar solutions separately, and mix the two when a
sugar test is to be made.

MILK.

Fresh milk in a tall jar is placed in the refrigerator over
night, to allow the cream to rise. The skim-milk is then
siphoned off from below the cream, tubed, and sterilized
at 110° C. for fifteen minutes, or on three successive days
at 100° C. for twenty minutes.

LITMUS MILK.

This is prepared as above, with the addition of \ c.c.
blue litmus solution to give a faintly alkaline reaction.

If sterilized at a high temperature, reduction of the
htmus may take place with loss of color, but oxidation
will follow on cooling and exposure to air.

124

APPENDIX.

POTATO.

Large, sound potatoes are thoroughly washed and
brushed with a scrubbing-brush, then pared and cut into
cylinders with a cork-borer. After paring, the potato
should be kept under water as much as possible to pre-
vent darkening. The cylinders are then cut diagonally so
that each produces two pieces of the general shape of a
sohdified agar or serum slant. The pieces are then left
in cold, running water for several hours, after which they
are dropped, broad end down, into test-tubes containing
a small piece of glass rod or tubing at the bottom to keep
the potato out of the watery fluid which is produced by
steriHzing. Sterilize for twenty minutes at ioo° C. on
three successive days, or for fifteen minutes at iio° C.

NITRATE SOLUTION.

A stock solution is prepared by dissolving 2 grams C. P.
potassium nitrate in 100 c.c. sterile distilled water. This
should be kept in the ice-chest. Five c.c. of this solution
and I gram peptone are added to i liter of tap-water.
The solution is brought to a boil, filtered, and tubed.

Care must be taken that the stock solution does not
become reduced to nitrites or that nitrite is not present
in the original salt. Sterilize for fifteen minutes at 120°.

PEPTONE SOLUTION.

Ten grams peptone and 5 grams of salt are dissolved in
I litre of water; the solution is boiled, filtered, tubed, and
sterilized for fifteen minutes at 120° C.

APPENDIX.

loeffler's blood serum.

This medium consists of 3 parts blood serum and
I part of I per cent broth with reaction +0.8. The
serum is obtained from fresh beeves' blood, which is col-
lected in sterile jars and allowed to stand for twenty-four
hours in the refrigerator for coagulation. The serum
is then drawn off, filtered, and mixed with the dextrose
broth in the proportion above indicated. Hill finds that
filtering the serum through the coagulum obtained after
adjusting the reaction of the broth gives a filtrate which
is clear and almost colorless (HiU, 1899).

Tubes are filled with the mixture, placed in trays so
that the desired slant is obtained, and carefully heated
in a Koch coagulator containing cold water in the water-
jacket. This water is brought to a boil and kept boiling
for three hours. Repeating this process on three suc-
cessive days solidifies the serum so that it may be subse-
quently sterilized in flowing steam for twenty minutes
on three successive days.

PHENOL BROTH.

To 1000 c.c. w.ater add separately, and in the following
order, 100 grams dextrose, 50 grams peptone, 2.5 grams
phenol. Heat until all constituents are dissolved, boil
for fifteen minutes, and sterilize for fifteen minutes at
110-120° C.

126

APPENDIX.

NEUTRAL RED BROTH.

To neutral broth is added 0.5 per cent dextrose and
1 per cent of a 0.5 per cent aqueous solution of Griibler's
neutral red. Sterilize at 100°.

MacConkey's Media.

A. Agar.

Agar 1.5 grams

Sodium taurocholate (pure) 0.5 gram

Peptone 2.0 grams

Water 100.0 c.c.

This is boiled, clarified, and filtered as usual, then
i.o gram lactose is added, and the medium tubed and
sterilized for three successive days at ioo°.

B. Broth.

. Sodium taurocholate (pure) 0.5 gram

Peptone 2.0 grams

Glucose 0.5 gram

Water 100.0 grams

Boil, filter, and add sufficient neutral Htmus, fill fer-
mentation-tubes, and sterilize at ioo°.

Litmus Solution. — To one-half pound of litmus cubes
add enough water to more than cover, boil, and decant
off the solution. Repeat this operation with successive
small quantities of water until from 3 to 4 liters of water
have been used and the cubes are well exhausted of color-
ing matter. Pour the decantations together and allow
them to settle overnight. Siphon off the clear solution.
Concentrate to about i liter and make the solution de-
cidedly acid with glacial acetic acid. Boil down to about

APPENDIX.

127

^ liter and make exactly neutral with caustic soda or
potash. To test for the neutral point, place one drop of

N

the solution in a test-tube. One drop of — HCl should

20

N

turn the drop red, while one drop of ~ NaOH should turn

it blue. Filter the solution and sterilize at 110° C. This
solution should be added to the media just before use in
the proportion of about \ c.c. to 5 c.c. of medium.

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TORDAN E O. 1901. The Relative Abundance of Bacillus coli

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Journal of Hygiene, I, 295.
■Reith S C. See Dyar, H. G. . , t

Klein,' E. 1898. Report on the Morphology and Biology of

REFERENCES.

135

Bacillus enteritidis sporogenes; on the association of this
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Supplement to the Twenty-seventh Annual Report of the
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EIlein, E. 1899. Further Report on the Bacillus enteritidis
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Klein, E., and Houston, A. C. 1898. Report on Bacteriological
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Klein, E., and Houston, A. C. 1899. Further Report on Bac-
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Twenty-eighth Annual Report of the Local Government Board
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Klein, E., and Houston, A. C. 1900. Preliminary Account of
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Koch, R. 1893. Ueber den augenblicklichen Stand der bakterio-
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Kolle, W., and Gotschlich, E. 1903. Untersuchungen iiber die
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Kruse, W. 1894. Kritische und experimentelle Beitrage zur
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KuBLER and Neueeld, F. 1899. Ueber einen Befund von Typhus-
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KilsEL, G. C. See Pennington, M. E.

136

REFERENCES.

Laws, J. P., and Andeewes, F. W. 1894. Report on the Result

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The Thompson-Yates Laboratories Report, HI, 41-
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Maschek, J. 1887. Bakteriologische Untersuchungen des Leit-

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MiQXJEL, P. 1886. De la richesse en bacteries des eaux d'essan-

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Peteuschky, J., and Puscn, H. 1903. Bacterium coli als Indi-
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Phelps, E. B. See Gage, S. D.

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I40

REFERENCES.

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Smith, T. 1893. a. A New Method for Determining Quantita-
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Smith, T. 1893. b. The Fermentation-tube with Special Refer-
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Smith, T. 1895. Ueber den Nachweis des Bacillus coli com-
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Thomson, T. 1894. Report on an Epidemic of Enteric Fever in
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Wathetet, M. a. 1895. Recherches bactdriologiques sur les
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WEISSENPELCJ. 1900. DerBefund des Bactenumcoh im Wasser
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Whipple, G. C. 1896. Discussion of Paper on Water-borne
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Whipple, G. C. 1899. On the Necessity of Cultivating Water
Bacteria in an Atmosphere Saturated with Moisture. Tech-
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Whipple, G. C. 1901. Changes that take place in the Bacterial
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WiNSLOW, C.-E. A. 1903. a. Studies on Quantitative Variations
in Gas Production in the Fermentation-tube. Science N S
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WiNSLOW, C.-E. A. 1903. i. The Occurrence of the Colon Bacil-
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WmsLOW, C.-E. A., and Hunnewell, M. P. 1902. a. Strepto-
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WiNSLOW, C.-E. A., and Hunnewell, M. P. 1902. h. A Study of
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142

REFERENCES.

Sewage Streptococci of Houston in Polluted and Unpolluted
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WiNSLOw, C.-E. A., and Nibecker, C. P. 1903. The Significance
of Bacteriological Methods in Sanitary Water Analysis. Tech-
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WiNSLOW, C.-E. A. See Sedwick, W. T.

WiNSLOw, C.-E. A. See Woodman, A. G.

WolfphDgel, G. 1886. Erfahrungen uber den Keimgehalt
brauchbarer Trink- und Nutzwasser. Arbeiten aus dem
KaiserUchen Gesundheitsamte, I, 546.

WoLFFHUGEL, G., and Riedel, O. 1886. Die Vermehrung der
Bakterien im Wasser. Arbeiten aus dem Kaiseriichen Gesund-
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Woodman, A. G., Winslow, C.-E. A., and Hansen, P. 1902. A
Study of Self-purification in the Sudbury River. Technology
Quarterly, XV, 105.

Wright, F. R. See Moore, V. A.

WuRTZ, R. 1892. Note sur deux caracteres differentiels entre le

bacille d'Ebcrth et le B. coh commune. Archives de Medecine

Experimentale, IV, 85.
Zagari, G. 1887. Esperienze sulla concorrenza vitale dei microor-

ganismi e sopra un nuovo mezzo di profilassi carbonchiosa.

Giornale intemazionale della Scienze Mediche, IX, 617.

AUTHOR INDEX.

PAOB

Abba 78

Abbott 25

Amyot 75

Andiewes 51, 54, 100

Baker 102

Bassett 75

Beckmann 76

Belcher 107

Belitzer 75

Blachstein 78

Blunt 13

Bolton 27

Brotzu 7^

Brown

Bruns 78

Buchner

Burri 82

Cambier , ^ 25. i

Chick 45^ eo 8:

Clark 40, 87

Copeland

Cramer

Dombrowsky

Downes

Duclaux y

Dunbar '' * 55

143

144

AUTHOR INDEX.

T-v , PAGE

^""^^■^ 40, ss

^^"^ 74

Egger

EUms ' ' 25

Eisner

Escherich jg^

Fischer

Flatau ^2

Franklaiid 9, 10, 25, 54, 56

Fremlin

Freudenreich 12, 82

Frost 70

F'^ler 20, 29, 40, 73, 85, 119, 120

Gage 19, 21, 30, 61. 63, 66, 72, 73, 87, 95, 98, loi

Garre 12

Gartner 23, 37

Gotschlich 56

Hammerl 83

Hankin 50

Hansen 113

Heider 1 16, 117

Herseus 23

Hesse 19

Hill 25, 98, 120, 125

Hoag 75

Horrocks 12, 15, 85, loi, 105

Houston 80, 84, 100, 105, 107, 113, 115

Hunnewell 64, 91, 96, loi

Irons 61, 63, 9S, 98

Janowski 8

Johnson 73

Jordan 13. i4, 27> 39. 88, 89, 117

Keith 74

Klein 50, 80, 106, 107, 113

Koch 55

AUTHOR INDEX. 145

PAG3

Kolle 56

Kruse 7^

Kiibler S3

Kusel 73

Laws 51, 54, 100

Le Gros loi

Levy 78

Loeffler 55.

MacConkey , 98, 126

Makgill 97

Maschek 15, 23

Mathews 46, 59

Miquel S. 9- 25, 31, 35, 39, 82

Moore 75

Moroni 78

Neufeld 53

Nibecker 10, 46, 66, 97, loi

Niedner. . . iq

Nuttall 106

Osgood 116

84

Papasotiriu •

Parietti

rennington

Petruschky ^2

P'^^'P^ 19. 2i,"30,' 66', 72, 98

Puujol 77

Prausnitz

39

16, 17, 29, 61, 80, loi, 102

Procaccini

13

Pusch

92

^"PP 13, 14

Refik ^ ^

76

Keinsch

29

Remlinger

AUTHOR INDEX.

Reynolds ^'^^t2

Rideal

Riedel 26

Rothberger

Russell

Savage ^7

Scheurlen u

Schneider

Schottelius

Seflgwick 5, 12, 16, 17, 29, 57, 59

Sliuttleworth 41

Smith 60, 74, 82, 94

Sternberg 36

Stokes g8

Thomson 50

Tiemann 23, 37

Tissandier 7

Wathelet 51

Weissenfeld 78

Welch 106

Whipple 9, 20, 26, 30, 31, 58, 65, 68, 96, 113, IIS

Widal S3

Winslow 10, 12, 22, 41, 46, 64, 66, 73, 91, 96, 97, loi, 107, 113

WoUfhugel IS, 26

Woodman 1 13

Wright 75

Wurtz 44. 59

Zagari • 12

SUBJECT INDEX.

PAGE

Absorption of COj 70

Acid formers in Boston sewage 4^

production by B. subtilis 45

Acidity of media 29

Agar media.

plate count ^

preparation of , 120, 12 1

streak, types of growth 67

Agglutination

Air, dust in ^

American Public Health Association, standard procedure. . . 19, 20, 28,

30, 31, 119, 120

Ammes, formation of by B. coli gg

Ammonia, formation of by B. coli gg

Ammonium compounds, decomposition of e

Antagonism *

Bacillus serogenes

anthracis, isolation from water eg

cloacae I r ' ' „ o

,. 91, 92, 108

coll, as a saprophyte.

, as direct evidence of sewage

, atypical forms gg

, biochemical characters ^g

, characteristics of. '

, colonies on agar

, comparison of methods for isolation g'l g,

, decomposition of sugars * ' ^

, detection of, in presence of streptococci . . , [ 104

i4r

SUBJECT INDEX.

PAGE

Bacillus coli, determined by animal inoculation 78

, distribution of 81

, distribution of, in unpolluted waters 76-79

, distribution of, in various animals 74

, distribution in various waters 85

, effect of dilution on growth in litmus lactose agar. ... 66

, from cereals . . 82

, gas ratio 95

, general distribution of, in nature 76

, growth on agar streak 67

in effluents of Lawrence filter 87

in filter effluents 86

in large samples 82

in Merrimac River 86

in mud 45

in normal intestine 58

in polluted waters 89, 91

in refuse from tanneries, grist-mills, and dairies 82

in river water ^Si 92

in Severn River 45

in sewage 89, 92

in soil 84

in spring water 78, 88

in stored sewage loi

, in unpolluted waters 78, 91, 97

in well waters 755) 92

, isolation of, by Escherich 58

, isolation of pure cultures 65

, isolation of, by litmus lactose agar 59

, morphology of. 5^

, on cereals 80

, on hands 22

, overgrowth by streptococci 102, 103, 104

, overgrowth during preliminary incubation . ... 62, 64, 65

, pathogenesis for guinea-pigs , 59

, pathogenicity of • 7^

, percentage of cultures giving positive tests in sub-

culture 72

, positive isolations by different methods 63

, presumptive tests for 94

SUBJECT INDEX.

149

PAGE

Bacillus coli, presumptive tests in various amounts of water 96

, qualita^'ve analysis for 69, 70

, quantitative estimation of 35, 82

, relation to B. acidi lactici 80

, significance of 112

, significance of, in samples of dififerent size 87

, significance of, in water 74

, standards for drinking water 85

, standards for presumptive test 97

, sub-cultures of, in qualitative analysis 70

, susceptibility to phenol 59

, time of gas-production in dextrose broth by 6i

, variations in cultural reactions 73

, varieties of , 68

Bacillus dysenteriae ^3

enteritidis sporogenes 106

mycoides 66

, growth on agar streak 67

sporogenes, as index of pollution 106

, cultural characteristics 106, 107

in sewage 107

, method for detection of, in water 106

, significance of 107

typhi, biochemical characters of. 51

, distribution in the environment 54

in ice ' _

, isolation of.

, life of, in water

Bacteria, classes of, in relation to nutrition 27

, comparative numbers of, growing at 20° and 37° 47

, definition of species 57

, developing on various media

, distribution of _ ^

, distribution of, in sea-water jo

, distribution in water g

, diurnal variation

fermenting lactose, in normal walers 46, 48

, in polluted waters 4^^ 48

growing at body temperature '

in Boston tap-water o -iS

15° SUBJECT INDEX.

PAGB

Bacteria in Boston sewage 45

in Cambridge supply 10

in Chicago drainage canal 39

in Connecticut River 42

in deep wells , , . . 17, 40

in driven wells 17, 40

in filter effluents 40

in Framingliam supply 42

in ground water 15, 39

in ground waters, peculiar character of 18, 39

in Hartford supply 42

in ice 12

in Isar River 39

in Lake Champlain 10

in Lake of Lucerne 10

in Lake Zurich 38

in lakes and ponds 10

in Loch Katrine 10

in Loch of Lintralthen 10

in Lynn supply 10

in Medford supply 10

in Merrimac River 9

in Newport supply 42

in Ohio River 4i

in Ourcq River 9

in Peabody supply

in Plymouth supply 10

in polluted streams 39

in polluted waters, changes during storage 27

in polluted well 42

o

m ram "

in relation to food supply ^4

in Salem supply ^°

in sea-water at Naples

in sea-water at Wood's Hole 10

in Seine 39

in shallow wells ^S. 16

in snow ^

in springs

in stored samples, multiplication of 25-27

SUBJECT INDEX. ^S^

PAGE

Bacteria in surface wash

in surface waters 9

in Taunton supply ^°

in Thames River 9

in Wakefield supply ^°

in water, relation to organic matter 37) 3^)

, significance of number of 3^

in well water, effect of pumping on 23

, intestinal 99

, liquefying ^"-'^

, metabolism of 4

, multiplication of, in spring water 16

, peculiar groups in ground water 18, 39

, reduction of, in streams 8

, relation to environment.' 3) ^4-

organic matter in water 6

waste materials 4

, seasonal distribution in surface waters 9

which do not grow on ordinary media 19

Bacterial standards for filter plants 41

Bacteriological analyses, value of 4^

analysis of water, certainty of 42, 115

, comparison with chemical 113

, delicacy of 113

, directness of 113

, distribution of sewage in water. . . 116

groundwaters 113, 114, 115

examination of water, applicability of. 116

Berkefeld filter to concentrate organisms 50

Biochemical characters of B. coli 51

Blood serum, preparation of . . 125

Body temperature, count, Boston sewage 45

, Charles River 45

of various samples 47

, polluted water 45

, significance of 112

organisms growing at 43

Boston, B. spor6genes in sewage 107

, bacteria in deep wells. 17

f course of sewage in harbor n6

^52 SUBJECT INDEX.

Boston sewage, bacteria in ''^^^

tap-water, bacteria in ^8

Brookline tap-water, bacteria in 47

Brooks, bacteria in

Cambridge tap-water, bacteria in 10, 47

Carbon dioxide, absorption of yo

Cats, B. coli in 74) 75

Cereals, B. coli in 3o

Character of colonies, importance of, in ground waters 40

Charles River above Boston, body temperature, count 45

Chemical analysis of water, comparison with bacteriological 113

Chemical examination of water no

, applicability of 116

Chicago drainage canal, B. coli in 89

, bacteria in 39

, routine bacteriological analyses 41

Chichester, well waters in 115

Cleaning mixtures 22

Cold, action of, upon bacteria. , 12

Colon bacilli in Manchester Ship Canal 45

, overgrown by streptococci 92

Colon bacillus, as direct evidence of sewage 58

, biochemical characters 58, 59

, gas ratio 58

, importance of number of 82

, in normal intestine 58

, isolation of, by litmus lactose agar 59

, morphology of 58

, pathogenesis in guinea-pigs 59

, preliminary enrichment for 60

, quantitative estimation of. 60

, susceptibility to phenol 59

Comparability of quantitative results 20

Composition of media, effect on bacterial counts 30

Connecticut River, bacteria in 4-2

Constantinople, B. coli in water supplies 76

Counting S^j 33

Cows, B. coli in 74. 75

Cycle of organic nitrogen 5

SUBJECT INDEX.

PAGE

Daily analyses, at Chicago, etc ■

Danube River, distribution of sewage in

Dedham tap-water, bacteria in

Deep wells

, bacteria in

Depths below surface, taking samples from . .

Desplaines River, B. coli in

Dextrose broth, for enrichment of B. coli

, method for isolation of B. coli

, preparation of.

Dilution of samples in plating

Distribution of bacteria in sea-water

water

Diurnal variation in bacteria

Dogs, B. coli in

Driven wells, bacteria in

Dust in air

Egypt, cholera in 5

Elbe River, cholera bacilli in 55

Eisner's medium 54

Enrichment culture, for B. typhi 49

processes, disadvantage of. 5°

process, effect upon B. coli 5°

Environment, effect of ^4

Esmarch process 34

Examination of spring waters for B. coli t • • 88

Feces, streptococci in

Fermentation, characteristic gas formula of B. coli 75

, gas formula of B. coli 95

of sugars by B. subtilis 45

, variations in 73

Field, plating in 34

Filter effluent, B. coli in 86, 87

Filtration of water 4°

Fishes, B. coli in 75. 7^

Food supply, effect of, upon bacteria 11, 14

Fowls, B. coli in 75

Fox River, B. coli in 90

.. 17

.. 24

.. 89

. . 61

63
. . 122
.. 28
II

.. 6

.. 14
.74, 75
•40, 47
• • 7

SUBJECT INDEX.

Framingham- supply, bacteria in

Freezing of bacteria

12

Gas formula

tras measurement, in fermentation tube yo

Gasometer

70

Gas production in dextrose broth by B. coli, time of 61

Gelatin

119, 121

, liquefaction of

media

30

, physical condition of.

Glycerin, agar, preparation of. ' ' J22

Glycerin media

30

Goats, B. coli in

r A . 74, 75

Oround waters

, bacteria in 2 9

, bacteriological analysis of 113, n^.,

, character of bacteria in 18, 39

Hamburg, cholera epidemic

Hartford supply, bacteria in ^2

Hogs, B. coli in 74, 75

Horses, B. coli in

Hudson River, B. coli in

Ice, bacteria in j2

Illinois River ^ _ _ ^ _ j j

, B. coli in go

, distribution of sewage in 117

Incubation, conditions of

of agar plates at body temperature 44

, period of 31, 32

Indol reaction

test 71

Interpretation of analyses 117

Interval between sampling and examination 28

Intestinal bacteria gg

Isar, River, bacteria in 13, 14, 39

Isolation of B, coli, Chicago methods 89

, definition of species 67

SUBJECT INDEX.

PAGE

Isolation of B, coli, examination of subcultures 71

, from large samples 63, 64, 65

, growth on agar streak 67

, Mass. Inst. Tech. record blank 69

, Mass. State Board of Health methods 69

, necessity of standard methods 69

, percentage of cultures passing various tests 72

, period of preliminary enrichment 62

, phenol broth method 62

, preliminary enrichment 61

, separation of pure cultures 65

, significance of large samples 87, 91

, significance of quantitative results 81

, variations in cultural reactions 7^

streptococci ,

fewell filter, bacterial efficiency

Kiel, bacteria in wells at ,a

IS>

Lactic acid bacteria

79

Lactose-agar plate as a presumptive test

plates '

. 44

, use m isolation of B. coli 60 61;

, preparation of \ "' '^^^

broth, preparation of.

, fermentation of, by B. coli \\ ^" '0

-fermenting bacteria in polluted waters . '. '. . ' 48

-fermenting organisms in normal water

Lake Champlain, bacteria in

of Lucerne, bacteria in

Zurich, bacteria in

Lakes, bacteria in ^

Lawrence city filter, bacterial'efficiency of'. ". '. * ' ' 1°

Leitmeritz, bacteria in wells at 40, »7

Light, effect of, upon bacteria

: ".13

in streams

Liquefying bacteria ^3

' Phenolated, for B. coli isolation L
Litmus lactose agar plate, incubation of

plate, isolation of B. coli by

^ 59

156 SUBJECT INDEX.

PAGE

Litmus lactose agar plate, technique of 45

, red colonies in 66

Litmus milk, preparation of 123

Liverpool tap- water, B. coli in 85

Loch Katrine, bacteria in 10

Loch of Lintralthen, bacteria in 10

Loeffler's blood serum 125

Lynn tap-water, bacteria in 10, 47

MacConkey's media, preparation of 126

Mainz, bacteria in wells at IS

Maltose broth, preparation of 122

Manchester ship canal, B. coli in 45' ^5

Massachusetts Institute of Technology, blank for recording bacterio-
logical analysis ^9

Massachusetts State Board of Health 9, 72, 86, 88

, B. coli isolation 87

, bacteriological counts 40

, examination of springs 16

, methods for isolation of B. coli 69

Mechanical filtration 4°

Medford tap-water, bacteria in 10, 47

Media, alkaline, for isolation of Sp. cholerae 55

, bacteria developing on various 3°

, comparability 2°

, composition of ^9

, limitations of ordinary '9

, percentage of bacteria developing on various. 21

, practical requirements for 20

, reaction of

, standard methods "9> iz"

Merrimac River, bacteria in 9. 4°

Metabolism of different classes of bacteria ■ 37

Mice, B. coli in '^'^

Micro-organisms, effect of, upon bacteria ".12

Milk, preparation of ^^-^

test "^^

Milton tap-water bacteria in • '^7

. . „ ,. ■ 8-5, 90

Mississippi River, B. coli in

Missouri River, B. coli in 9°

SUBJECT INDEX.

PAGE

Mohawk River, B. coli in 83

Moisture in incubators 31

Montsouris, bacteria in air 8

Mud, colon bacilli in 45, 85

Multiplication of bacteria in spring waters 16, 25

in stored tamples 25-27

Mur River, B. coli in 83

Nahrstoflf Heyden agar iQi 20, 21, 30

Naples, bacteria in sea-water at 10

Neumark, typhoid bacillus in well at 53

Neutral-red _ _ g7

broth, preparation of 126

Newburyport tap-water, bacteria in 47

Newport supply, bacteria in 42

New York Board of Health 82

, examination of rivers for B. coli,

Nitrate solution, preparation of

test

, variations in

Nitrification

Nitrifying organisms

Nitrites

Nitrogen, cycle of organic

Normal waters, lactose fermenting bacteria in .

Ohio River, B. coli in

, bacteria in

Organic matter, decomposition of

in water, relation of bacteria to

, oxidation of

, relation of bacteria to

Osmotic pressure, effect of upon bacteria

Ourcq River, bacteria in

Overgrowth

Oxidation of organic matter

Oxygen, effect of, upon bacterial counts

Para-colon organisms

Para-typhoid organisms

Parietti's solution

3

5

48

8s

41

.... 4, III

37

5

6

II

9

. . . . 62, 64

5

31

78, 91, 92

78

SO

SUBJECT INDEX.

Paris, bacteria in air of.

Parma, B. coli in water supply of.

in wells and springs near

Peabody tap-water, bacteria in

Peptone method for isolation of cholera bacillus

solution, preparation of

test

Period of incubation

Phenol broth, composition of 62

, method for isolation of B. coli 62

, preparation of. 125

Phenolated gelatin, use of, for typhoid isolation 50

lactose litmus agar 60

Plating 28

in the field 24

Plymouth tap-water, bacteria in 10, 47

Polluted waters, bacterial changes during storage 27

, blood temperature count 45

, lactose fermenting bacteria in 48

, specific pathogenes in 49

Ponds, bacteria in lo, 47

Pools, bacteria in 47

Potato, preparation of 124

gelatin, use for typhoid bacteria 50

Presumptive tests for B. coli 94 if' •f^?-

Proteus organisms 108

Protozoa 12

Prussia, regulations regarding filter plants 41

Pseudocolon bacilli 68

Pump, sampling from 23

Pumping, effect of, upon bacterial content of well water 23

Quantitative analysis, arbitrary standards for 35

, composition of media 29

, conditions of incubation 31

, counting 33

, dilution 28

, general procedure 21

, interpretation of 35

, media for 19

,. 8
78

• 78

ID, 47

• 56
,. 124

SUBJECT INDEX.

159

PAGE

Quantitative analysis, period of incubation 31

, plating 28-29

, procedure for 21

, recording results 33

, sampling 21-24

, storage of samples 25

bacteriological analysis, significance of Ill

Rabbits, B. coli in 74, 75

Rain, bacteria in 8, 47

, contamination of surface water by 9

Reaction of media 29

Recording quantitative results 33

Red colonies in litmus lactose agar 66

Relation of B. coli to B. acidi lactic! 80

Rellingen, typhoid bacillus in well at 53

Saccharose broth, preparation of. 122

Salem tap-water, bacteria in I0> 47

Sample booties, cleaning of 22

, handling , 22

, size of, effect of, upon multiplication of bacteria. 27

, sterilization of 22

Sampling, for bacteriological analysis 21-25

, necessity for care in 27

Sand filtration

Sangamon River, B. coli in go

Sanitary inspection jq^

Research Laboratory, bacteria in Boston sewage 45

Sea- water, bacteria 111 jq

Seasonal distribution of bacteria in surface waters g

variation of bacterial content in surface waters 9, 38

Sedimentation of bacteria jj

Seine, bacteria in ^9

Self-purification of lakes and ponds 10

of streams 8, 13, 39, 113

Severn River, colon bacilli in

Sewage, B. coli in

, bacteria in

, distribution of, in water jjg^ ji^

pollution, bacteria in relation to ,o

loo SUBJECT INDEX.

PAGE

bewage, streptococci in 62, 100, loi

, typhoid bacillus in 51

Shallow wells, bacteria in 15^ i6

Sheep, B. coli in

Shiga bacillus

Significance of B. coli

in water >]() et seq., 93

, necessity for quantitative results 81

of blood temperature count 43

of quantitative analysis 38

of streptococci in water 105

Snow, bacteria in 8, 47

Sodium taurocholate medium 98

Specific pathogenes in polluted water 49

Spirillum cholerae in Elbe River 55

, isolation of 54, 55

of Asiatic cholera, isolation from polluted water 49

Spore-forming pathogenes in water, isolation of. 56

Spring waters, multiplication of bacteria in 16

Springs, bacteria in 15, 16, 47

Standard for interpreting presumptive test 97

Sterilization of gelatine, effect of 30

sample bottles 22

Storage 11

, effect on sample 37

, multiplication of bacteria during 25-27

of polluted waters 27

of samples, allowable maximum 28

Strassburg, B. coli in water of 7^

Streams, bacteria in 39

, sedimentation of bacteria in 11

Streams, self-purification of. 8, 13

Streptococci as indicative of pollution 100

, colonies on litmus lactose agar 66

, cultural characters 99

, decomposition of sugars by 44

, detection of, in presence of B. coli 104

, growth on agar streak 67

\ habitat of

SUBJECT INDEX.

i6i

PAGE

Streptococci in feces loi

in polluted waters 92, 102

in sewage loi

in sewage polluted rivers 100

in stored sewage loi

in unpolluted water.. . .■ 91, loi

, isolation of. 102

, overgrowth of B. coli by 102, 104

, relation to B. coli 102, 103, 104

sewage pollution 105

, sewage 62, 100

, significance of 112

, types of colonies on litmus lactose agar 66

Streptococcus erysipelatos

Sudbury River, chemical and bacteriological examination 113

Sugar broth, preparation of 122

Sugars, decomposition of. ^

Surface wash, bacteria in 8

water, sampling of. 24

, bacteria in 9, 39, 47

22

Tap-water sampling

Taunton tap- water, bacteria in jo, 47

Temperature, effect of, upon bacteria „ J2

, effect upon multiplication of bacteria in stored samples, '

26, 27

of incubation ^ x

Thames River, bacteria in

-*••••.... 9

Titration of media

120

loronto supply, bacteria in

Toxic products

Turin, B. coli in unpolluted waters near ' »g

Turkeys, B. coli in

Typhoid bacillus in ice . . .

12

in sewage

in well at Neumark

Rellingen

isolation from polluted water

, reported isolation from water

fever, spring epidemics o

30

SUBJECT INDEX.

Urea, decomposition of

Value of isolated analyses

Variation of B. coli gg

Variations in cultural reactions 72

Wakefield and Stoneham tap-water, bacteria in lo, 47

Water, B. coli in different grades of 96

in polluted and unpolluted 63

bacteria, peculiar metabolism of 37

, bacteriological analysis of 69

, chemical composition, relation of bacteria to 37, 38

, chemical examination of no

, complete bacteriological analysis 112

, distribution of bacteria in 6

purification 40

supplies, sanitary inspection of 109

Waters, bacteria in various classes of. 21

, classification of 6

Well, typhoid bacillus in, at Neumark 53

at Rellingen 53

water, bacteria in 42

Wells, bacteria in 47

Westerly tap-water, bacteria in 47

Widal reaction 53

Wood's Hole, bacteria in sea-water at 10

Wurtz litmus lactose agar 59

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Church's Mechanics of Engineering 8vo 6 00

Du Bois's Mechanics of Engineering. Vol. I Small 4to,' 7 so

Johnson's Materials of Construction Large 8vo, 6 00

Keep's Cast Iron g^^^'

Lanza's Applied Mechanics g^J

Martens's Handbook on Testing Materials. (Henning.) 2>oIs . . . . .8vo,' 7 30

Merrill's Stones for Building and Decoration ' gyo', 5 00

Merriman's Text-book on the Mechanics of Materials .8vo! 4 00

Strength of Materials t,,^„'

^_ i2mo, 1 00

Metcalf s Steel. A Manual for Steel-users x,~n -■

Patton s Practical Treatise on Foundations g^o .

Rockwell's Roads and Pavements in France i2mo'

Smith's Wire: Its Use and Manufacture SmVll At^' , ll

Materials of Machines. Small 4to, 300

Snow's Principal Species of Wood ' °°

Spalding's HydrauUc Cement. . . '

, „ . _ i2mo, 2 00

Text-book on:Roads and Pavements

i2mo, 3 00

7

2 so
7 so

Thurston's Materials of Engineering. 3 Parts 8vo 8 00

Part III.-A Treatise on Brasses, Bronzesraa'd Other AJloys and thlk ^
Constituents

Thurston's Text-book of the Materials oV Construction gvo
Iillson's Street Pavements and Paving Materials Bvo ' ^ 00

WaddeU's DePontibus. (A Pocket-book for Bridge EngiBeers.')'.'. '1600. mor ,' -

bpecificauons for Steel Bridges j^m

Wood's Treatise on the Resistance of Materials.'and 'an Appendu'on 'the P«s-

ervation of Timber ^

Elements of Analytical Mechanics g^o' ,

Wood's Rustless Coatings. {Shortly.)

3 00
i2mo, I 25

morocco, i as

4to, 5 00

morocco, i 50

RAILWAY ENGINEERING.

Andrews's Handbook for Street Railway Engineers. 3X5 inches.

Berg's Buildings and Structures of American Raihoads

Brooks's Handbook of Street Raihoad Location iemo

Butts's Civil Engineer's Field-book .'.i6mo, morocco.' 2 50

CrandaU's Transition Curve i6mo, morocco, i 50

Railway and Other Earthwork Tables 8vo, i 50

Dawson's "Engineering" and Electric Traction Pocket-book. i6mo, morocco! S 00

Dredge's History of the Pennsylvania Raihoad: (1879) Paper, 5 00

• Drinker's TunneUng, Explosive Compounds, and Rock DriUs, 4to, half mor ' 25 00

Fisher's Table of Cubic Yards Cardboard 25

Godwin's Raihoad Engineers' Field-book and Explorers' Guide i6mo, mor., 2 50

Howard's Transition Curve Field-book i6mo, morocco, i 50

Hudson's Tables for Calculating the Cubic Contents of Excavations and Em-
bankments 8vo, I 00

Molitor and Beard's Manual for Resident Engineers i6mo, i 00

Nagle's Field Manual for Raihoad Engineers i6mo, morocco. 3 00

Philbrick's Field Manual for Engineers i6mo, morocco, 3 00

Pratt and Alden's Street-railway Road-bed 8vo, 2 00

Searles's Field Engineering i6mo, morocco, 3 00

Railroad Spiral i6mo, morocco, 1 50

Taylor's Prismoidal Formulae and Earthwork 8vo, 1 50

• Trautwine's Method of Calculating the Cubic Contents of Excavations and

Embankments by the Aid of Diagrams 8vo, 2 00

The Field Practice of iLaying Out Circular Curves for Railroads.

i2mo, morocco, 2 50

• Cross-section Sheet Paper, 25

Webb's Railroad Construction. 2d Edition, Rewritten i6mn. morocco, s 00

WeUington's Economic Theory of the Location of Railways Small 8vo, 5 00

DRAWING.

Barr's Kinematics of Machinery 8vo, 2 50

* Bartlett's Mechanical Drawing 8vo, 3 00

♦ " " " Abridged Ed 8vo, i SO

Coolidge's Manual of Drawing 8vo, paper, i 00

Durley's Kinematics of Machines 8vo, 4 00

Hill's Text-book on Shades and Shadows, and Perspective 8vo, 2 00

Jones's Machine Design:

Part I. — Kinematics of Machinery 8vo, i SO

Partn. — Form, Strength, and Proportions of Parts 8vo, 3 00

8

MacCord's Elements of Descriptive Geometry 8vo, 3 00

Kinematics; or. Practical Mechanism 8vo, s 00

Mechanical Drawing 4to, 4 00

Velocity Diagrams 8vo, i 50

• Mahan's Descriptive Geometry and Stone-cutting 8vo, i 50

Industrial Drawing. (Thompson.) 8vo, 3 50

Reed's Topographical Drawing and Sketching 4to, 5 00

Reid's Course in Mechanical Drawing 8vo, 2 00

Text-book of Mechanical Drawing and Elementary Machine Design . . 8vo, 3 00

Robinson's Principles of Mechanism 8vo, 3 00

Smith's Manual of Topographical Drawing. (McMillan.) 8vo, 250

Warren's Elements of Plane and Solid Free-hand GeometricalDrawing. .l2mo, i 00

Drafting Instruments and Operations i2mo, 1 25

Manual of Elementary Projection Drawing i2mo, i 50

Manual of Elementary Problems in the Linear Perspective of Form and

Shadow j2mo, i 00

Plane Problems in Elementary Geometry i2mo, i 25

Primary Geometry i2mo, 75

Elements of Descriptive Geometry, Shadows, and Perspective 8vo, 3 50

General Problems of Shades and Shadows 8vo, 3 00

Elements of Machine Construction and Drawing 8vo, 7 So

Problems. Theorems, and Examples in Descriptive Geometrv 8vo, 2 50

Weisbach's Kinematics and the Power of Transimssion. (Hermaim and

Klein.) 8vo, 5 00

Whelpley's Practical Instruction in the Art of Letter Engraving i2mo, 2 00

Wilson's Topographic Surveying 8vo, 3 50

Free-hand Perspective 8vo, 2 so

Free-hand Lettering 8vo, i o»

Woolf's Elementary Course in Descriptive Geometry Large 8vo, 3 00

ELECTRICITY AND PHYSICS.

Anthony and Brackett's Text-book of Physics. (Magie.) SmaU 8vo. 3 00

Anthony's Lecture-notes on the Theory of Electrical Measurements i2mo',

Benjamin's History of Electricity '

Voltaic Cell o *

„, , „ 8vo, 3 00

Classen 8 Quantitative Chemical Analysis by Electrolysis. (Boltwood.). 8vo, 300

Crehore and Sauier's Polarizing Photo-chronograph gyo'

Dawson's "Eneineering" and Electric Traction Pocket-book. . iemo, morocco' 5 00
Dolezalek's Theory of the Lead Accumulator. (Storage Batterv )
(Shortly.) (VonEnde.)

Duhem's Thermodynamics and Chemistry. (Burgess.) 8vo

Flather's Dvnamometers, and the Measurement of Power lamo' ^ 00

Gilbert's Ue Magnete. (Mottelay.)

Hanchett's Alternating Currents Explained. (Shortly.) "'

Holman's Precision of Measurements . . g^^

Telescopic Mirror-scale Method. Adjustments,' kud Tests".'. '. '. '. Lai-ge ' 8vo' ' 7-^
Lanaauer's Spectrum Analysis. (Tingle.) 8 '

Le Chatelier's High-temperature Measuremenis.' (Bou'd'o'ua;d-Burgess.')r2mo: ^
Lttb s Electrolysis and Electrosynthesis of Organic Compounds. (Lorenz ) i2mo

• Lyons s Treatise on Electromagnetic Phenomena. Vols. I. and 11. 8vo each '

• Michie. Elements of Wave Motion Relating to Sound and Light. . . ' 8vo' 4 00
Niaudet's Elementary Treatise on Electric Batteries. (Fishoack , . . \'Zl' 2 ^o

• Parshall and Hobart's Electric Generators Small

.Rosenberg's EleCical Engineering. (Haldane GeelSztir )'"°^Z' Z
Ryan, Rorris, and Hoxie's Electrical Machinery Vol I .»vo, i 50

Thurston's Stationary Steam-engines.. ^ '°

• Tillman's Elementary Lessons in Heat ^ ^°

8vo, I 50

9

I 00
3 00

4 00
3 00
2 50

3 00
2mo, I 00
6 00

Tory and Pitcher's Manual of Laboratory Physics SmaU 8vo

Ulke's Modern Electrolytic Copper Reaning 8vo.'

2 OO

3 OO

LAW.

* Davis's Elements of Law . o

OVO, 2 50

on the MiUtary Law of United States 8vo, 7 00

Manual for Courts-martial i6mo. morocco. I 50

wait s Engmeermg and Architectural Jurisprudence 8vo. 6

* Treatise
*

00

. Sheep, 6 50

Law of Operations Preliminary to Construction in Engineering and Archi-

*«<=*^« Svo. 5 00

Sheep, 5 50

Law of Contracts g^^

Winthrop's Abridgment of MiUtary Law

3 00
. i2mo, 2 50

MANUFACTURES.

Bernadou's Smokeless Powder— Nitro-ceUulose and Theory of the CeUulose

Molecule 121110, 2 50

Bolland's Iron Founder i2mo, 2 50

"The Iron Founder," Supplement i2mo, 2 50

Encyclopedia of Founding and Dictionary of Foundry Terms Used in the

Practice of Moulding i2mo, 3 00

Eissler's Modern High Explosives i 8vo, 4 00

Effront's Enzymes and their Applications. (Prescott.) Svo, 300

Fitzgerald's Boston Machinist i8mo, i 00

Ford's Boiler Making for Boiler Makers i8mo, i 00

Hopkins's Oil-chemists' Handbook Svo, 3 00

Keep's Cast Iron 8vo, 2 so

Leach's The Inspection and Analysis of Food with Special Reference to State

Control. (In preparation.)

Metcalf's SteeL A Manual for Steel-users 4 i2mo, 200

Metcalfe's Cost of Manufactures— And the Administration of Workshops,

Public and Private Svo, s 00

Meyer's Modern Locomotive Construction 4to, 10 00

* Reisig's Guide to Piece-dyeing Svo, 25 00

Smith's Press-working of Metals Svo, 3 00

Wire: Its Use and Manufacture ' Small 410, 3 00

Spalding's Hydraulic Cement i2mo, 2 00

Spencer's Handbook for Chemists of Beet-sugar Houses i6mo, morocco, 3 00

Handbook tor sugar Manulacturers and their Chemists.. . i6mo, morocco, 2 00
Thurston's Manual of Steam-boilers, their Designs, Construction and Opera-
tion Svo, 5 00

♦ Walke's Lectures on Explosives Svo, 4 00

West's American Foundry Practice i2mo, 2 so

Moulder's Text-book i2mo, 2 50

Wiechmann's Sugar Analysis Small Svo, 2 50

Wolff's Windmill as a Prime Mover Svo, 300

Woodbury's Fire Protection of Mills Svo, 2 50

MATHEMATICS.

Baker's Elliptic Functions 8vo. 150

♦ Bass's Elements of Differential Calculus i2mo, 4 00

Briggs's Elements of Plane Analytic Geometry i2mo, i 00

10

Compton's Manual of Logarithmic Computations i2mo, i 50

Davis's Introduction to the Logic of Algebra 8vo, i 50

* Dickson's College Algebra Large i2mo, i 50

* Introduction to the Theory of Algebraic Equations Large i2mo, i 25

Halsted's Elements of Geometry 8vo, i 75

Elementary Synthetic Geometry 8vo. i 50

Rational Geometry. {Shorily.)

♦Johnson's Three-place Logarithmic Tables: Vest-pocket size paper, 15

100 copies for s 00

* , Mounted on heavy cardboard, 8 X lo inches, 25

10 copies for 2 00

Elementary Treatise on the Integral Calculus Small 8vo, i 50

Curve Tracing in Cartesian Co-ordinates i2mo, i 00

Treatise on Ordinary and Partial Differential Equations Small 8vo, 3 50

Theory of Errors and the Method of Least Squares i2mo, i so

* Theoretical Mechanics i2mo, 3 00

Laplace's Philosophical Essay on Probabilities. (Truscott and Emory.) i2mo, 2 00

* Ludlow and Bass. Elements of Trigonometry and Logarithmic and Other

Tables 8vo, 3 00

Trigonometry and Tables published separately Each, 2 00

Maurer's Technical Mechanics . 8vo, 4 00

Merriman and Woodward's Higher Mathematics 8vo, 5 00

Merriman's Method of Least Squares 8vo, 2 00

Rice and Johnson's Elementary Treatise on the Differential Calculus . Sm., 8vo, 3 00

Differential and Integral Calculus. 2 vols. In one 3mall 8vo, 2 50

Wood's Elements of Co-ordinate Geometry 8vo, 2 00

Trigonometry: Analytical, Plane, and Spherical i2mo, i 00

MECHANICAL ENGINEERING.
MATERIALS OF ENGINEERING, STEAM-ENGINES AND BOILERS.

Baldwin's Steam Heating for Buildings l2mo, 2 50

Barr's Kinematics of Machinery gvo

• Bartlett's Mechanical Drawing gyo'

* " " " Abridged Ed .%vo'.

2 so

3 00
I 50

. i2mo, 2 00

Benjamin's Wrinkles and Recipes .

Carpenter's Experimental Engineering "svo! 6 00

Heating and Ventilating Buildings ' gyg] ^

Cary's Smoke Suppression in Plants using Bituminous Coal. {In prep-
aralion.)

Clerk's Gas and Oil Engine Small Svo. 4 00

Coohdge s Manual of Drawing gvo, paper, 100

Cromwell's Treatise on Toothed Gearing i2mo 150

Treatise on Belts and PuI.eys . ' 12^0' ,

Durley's Kinematics of Machines o„„' , „„

— , . , ovo, 4 00

FUther s Dynamometers and the Measurement of Power. . . . i2mo i 00

Rope Driving ....... , j »/v.

/^.,„ „ i2mo, 2 00

GiU s Gas and Fuel Analysis for Engineers , „

Hall's Car Lubrication. '

Hutton's The Gas Engine . ' °°

Jones's Machine Design: »vo. 5 00

Part I.— Kinematics of Machinery g^^^

^ ^Ml Strength, and Proportions of Parts Svo' ^ no

Kent's Mechanical Engineer's Pocket-book rfi'mn ' ^^,

Kerr's Power and Power Transmission '"°^°==°' 5 °°

MacCord'sKinemaUcs; or. Practical Mechanism. Zl' l °°

Mechanical Drawing ^^°' ^ 00

Velocity Diagrams. ... 4to, 4 00

Svo, I 50

11

Mahan's Industrial Drawing. (Thompson.) o

Poole's Caloriac Power of Fuels . g ' ^ ^°

Reid's Course in Mechanical Drawing gy"' j °°

Text-book of Mechanical Drawing and Elementai-yMachinrDeVi^'/.Svo. 3 Z
Richards's Compressed Air . o *~

Robinson's Principles of Mechanism gvo' '

Smith's Press-working of Metals ...'.* gvn' 3 00

Thurston's Treatise on Friction and Lost Work in Machine^ " and Mu'l
Work ... o
. . .ovo, 3 00

Anunal as a Machine and Prime Motor, and the Laws of Energetics. i2mo, i 00

Warren s Elements of Machine Constructior and Drawing 8vo. 750

Weisbach's Kinematics and the Power of Trarsmission. Herrmann-
Klein.). . . . Q„„ „

_ OVO, 5 00

Machinery of Transmission and Governors. (Herrmann— Klein.). .8vo. 500

Hydraul.cs and Hydraulic Motors. (Du Bois.) 8vo, 500

Wolff's Windmill as a Prime Mover gvo 3 00

Wood's Turbines 1 "svo! 2 50

MATERIALS OF ENGINEERING.

Bovey's Strength of Materials and Theory of Structures 8vo, 7 50

Burr's Elasticity and Resistance of the Materials of Engineering. 6th Edition,

Reset gvo. 7 50

Church's Mechanics of Engineering gvo, 6 00

Johnson's Materials of Construction Large 8vo, 6 00

Keep's Cast Iron gvo, 250

Lanza's Applied Mechanics gvo, 7 50

Martens's Handbook on Testing Materials. (Henning.) 8vo, 7 so

Merriman's Text-book on the Mechanics of Materials 8vo, 4 00

Strength of Mater als izmo, i 00

Metcalf's Steel. A Manual for Steel-users i2mo 2 00

Smith's Wire: Its Use and Manufacture Small 4to, 3 00

Materials of Machines i i2mo i 00

Thurston's Materials of Engineering 3 vols , Svo, 8 00

Part n. — Iron and Steel Svo, 3 so

Part in. — A Treatise on Brasses, Bronzes, and Other Alloys and their

Constituents Svo 2 50

Text-book of the Materials of Construction Svo, 5 00

Wood's Treatise on the Resistance of Materials and an Appendix on the

Preservation of Timber Svo, 2 00

Elements of Analytical Mechanics Svo, 3 00

Wood's Rustless Coatings. (Shortly.)

STEAM-ENGINES AND BOILERS.

Carnot's Reflections on the Motive Power of Heat. (Thurston.) i2mo, 1 so

Dawson's "Engineering" and Electric Traction Pocket-book. . i6mo, mor., 5 00

Ford's Boiler Making for Boiler Makers iSmo, i 00

Goss's Locomotive Sparks 8vo, 2 00

Hemi-nway's Indicator Practice and Steam-engine Economy i2mo, 2 00

Hutton's Mechanical Engineering of Power Plants Svo, 5 00

Heat and Heat-engines 8vo. 5 00

Kent's Steam-bo'ler Economy 8vo, 4 00

Kneass's Practice and Theory of the Injector 8vo i 50

MacCord's Slide-valves 8vo, 2 00

Meyer's Modem Locomotive Construction 4to . i o 00

12

Peabody's Manuai of the Steam-engine Indicator i2mo, i So

Tables of the Properties of Saturated Steam and Other Vapors 8vo, i 00

Thermodynamics of the Steam-engine and Other Heat-engines 8vo, 5 00

Valve-gears for Steam-engines 8vo. 2 SO

Peabody and Miller's Steam-boilers 8vo, 4 00

Pray's Twenty Years with the Indicator Large 8vo, 2 so

Pupln's Thermodynamics of Reversible Cycles in Gases and Saturated Vapors.

(Osterberg.) ,..i2mo, i 25

Reagan's Locomotives : Simple, Compound, and Electric i2mo, 2 50

Rontgen's Principles of Thermodynamics. (Du Bois.) 8vo, s 00

Sinclair's Locomotive Engine Running and Management i2mo, 2 00

Smart's Handbook of Engineering Laboratory Practice i2mo, 2 so

Snow's Steam-boiler Practice 8vo, 3 00

Spangler's Valve-gears 8vo, 2 50

Notes on Thermodynamics i2mo, i 00

Spangler, Greene, and Marshall's Elements of Steam-engineering 8vo, 3 00

Thurston's Handy Tables 8vo. i 50

Manual of the Steam-engine 2 vols. 8vo, 10 00

Part L — History. Structuce, and Theory 8vo, 6 00

Part n. — Design, Construction, and Operation ... 8vo, 6 00

Handbook of Engine and Boiler Trials, and the Use of the Indicator and

the Prony Brake 8vOi s 00

Stationary Steam-engines 8vo, 2 50

Steam-boiler Explosions in ifieory and in Practice i2mo i 50

Manual of Steam-boilers , Their Designs, Construction, and Operation . 8vo, 5 00

Weisbach's Heat, Steam, a J Steam-engines. (Du Bois ) 8vo, s 00

Whitham's Steam-engine I isign 8vo, s 00

Wilson's Treatise on Steam- boilers. (Flather.) i6mo, 2 50

Wood's Thermodynamics. Heat Motors, and Refrigerating Machines. . . .8vo, 4 00

MECHANICS AND MACHINERY.

Barr's Kinematics ot Machinery. . 8vo, 2 so

Bovey's Strength of Materials and Theory of Structures 8vo, 7 so

Chase's The Art of Pattern-making i2mo, 2 so

Chordal. — Extracts from Letters i2mo 2 00

Church's Mechanics of Engineering gyo g

Notes and Examples in Mechanics 8vo 2 00

Compton's First Lessons in Metal-working i2mo, i 50

Compton and De Groodt's The Speed Lathe i2mo.' i so

Cromwell's Treatise on Toothed Gearing i2moi i so

Treatise on Belts and Pulleys T2mo,' i 50

Dana's Text-book of Elementary Mechanics for the Use of Colleges and

Schools ,

12 mo, I so

Dingey's Machinery Pattern Making i^mo 2 00

Dredge's Record of the Transportation Exhibits Building of the World's'

Columbian Exposition of i8q3" 4to, half morocco. 5 00

Du Bois's Elementary Principles of Mechanics :

Vol. I. — Kinematics q„

Vol n.-Statics 8vo, 3 so

Vol. iii.-Kinetics in-

Mechanics of Engineering. Vol. I V.V.VsmaU 4to,' 7 |o

n , , TT- ■ ° SmaU 4to, 1000

Durley's Kmematics of Machines o

Fitzgerald's Boston Machinist. ... ' '* °°

-y. , , T.. lomo, I 00

Flather s Dynamometers, and the Measurement of Power t-smn

Rope Driving * ^ °°

ry ,T . „ . I2m0, 2 00

<j0ss s Locomotive Sparks .... o

8vo, 2 00

13

Hall's Car Lubrication. . .

HoUy'sArtof SawFiUng...'..'.'.'.. ' °°
* Johnson's Theoretical Mechanics'. . .

Statics by Graphic and Algebraic Methods'.'.'.'. "c™"' ^ °°

2 00

00
2 00

1 'M.*^^uitu\, lucLnoas

Jones's Machine Design:

Part I. — Kinematics of Machinery

Part n.-Form. Strength, and Propo'riio'n's of 'parts Z°' \ ^°

Kerr's Power and Power Transmission g

Lanza's Applied Mechanics o^°' ^

MacCord's Kinematics; or, Practical Mechanism Jl' ' ^°

Velocity Diagrams g °' ^ O"

Maurer's Technical Mechanics ........'.'... gvo ' '

Merriman's Text-book on the Mechanics of Materials gvo' °°

* Michie's Elements of Analytical Mechanics 8vo ' "

Reagan's Locomotives: Simple, Compound, and Electric . . . . . . . . . . . . ' , 2mb 2 ^o

Reid's Course in Mechanical Drawing ' g^^'

Text-book of Mechanical Drawing and Elementary Machine Design 8vo
Richards's Compressed Air

, ^. i2mo, I so

Robmson's Prmciples of Mechanism g^^

Ryan, Norris, and Hoxie's Electrical Machinery 8vo' ^ °°

Sinclair's Locomotive-engine Running and Management lamo'

Smith's Press- working of Metals _ g.yp'

Materials of Machines. V,™„'

i2mo, I 00

Spangler, Greene, and Marshall's Elements of Steam-engineering Bvo, 3 00

Thurston's Treatise on Friction and Lost Work in Machinery and MiU '

Work s„„ ,

ovo, 3 00

Animal as a Machine and Prime Motor, and the Laws of Energetics. i2mo, i 00

Warren's Elements of Machine Construction and Drawing 8vo, 7 so

Weisbach's Kinematics and the Power of Transmission. (Herrmann—

^lein.) g^o^ 5 00

Machinery of Transmission and Governors. (Herrmann — Klein.). Svo, 5 00

Wood's Elements of Analytical Mechanics 8vo, 3 00

Principles of Elementary Mechanics i2mo, i 25

Turbines 8vo, 2 50

The World's Columbian Exposition of 1893 4to, i 00

o, 2 00
3 00

2 50

2 00

3 00

METALLURGY.

Egleston's Metallurgy of Silver, Gold, and Mercury:

VoL I. — Silver 8vo, 7 50

VoL II. — Gold and Mercury 8vo, 7 50

** Iles's Lead-smelting. (Postage 9 cents additional.) i2mo, 2 so

Keep's Cast Iron 8vo, 2 50

Kunhardt's Practice of Ore Dressing in Europe Svo, i 50

Le Chatelier's High-temperature Measurements. (Boudouard — Burgess.) . i2mo, 3 00

Metcalf's Steel. A Manual for Steel-users i2mo, 2 00

Smith's Materials of Machines i2mo, i 00

Thurston's Materials of Engineering. In Three Parts Svo, 8 00

Part II. — Iron and Steel Svo, 3 so

Part III. — A Treatise on Brasses, Bronzes, and Other Alloys and their

Constituents Svo, 2 50

Dike's Modern Electrolytic Copper Refining Svo, 3 00

MINERALOGY,

Barringer's Description of Minerals of Commercial Value. Oblong, morocco, 2 50

Boyd's Resources of Southwest Virginia Svo, 3 00

Map of Southwest Virginia Pocket-book form, 2 00

14

Brush's Manual of Determinative Mineralogy. (Penaeld.) 8vo, 4 00

Chester's Catalogue of Minerals 8vo, paper, i 00

Cloth, I 25

Dictionary of the Names of Minerals 8vo, 3 50

Dana's System of Mineralogy Large 8vo, half leather, 12 50

First Appendix to Dana's New "System of Mineralogy.". . . .Large 8vo, i 00

Text-book of Mineralogy 8vo, 4 00

Minerals and How to Study Them. . . : i2mo, i 50

Catalogue of American Localities of Minerals Large 8vo, i 00

Manual of Mineralogy and Petrography i2mo, 2 00

Eakle's Mineral Tables. (.Shorlly.)

Egleston's Catalogue of Minerals and Synonyms 8vo, 2 50

Hussak's The Determination of Rock-forming Minerals. (Smith.) Small 8vo, 2 00
Merrill's Non-Metallic Minerals. {Shortly.)

» Penfield's Notes on Determinative Mineralogy and Record of Mineral Tests.

8vo, paper, o 50

Rosenbusch's Microscopical Physiography of the Rock-making Minerals.

(Iddings.) 8vo, 5 00

» Tillman's Text-book of Important Minerals and Docks 8vo, 2 00

Williams's Manual of Lithology ..8vo, 3 00

'♦^ MINING.

Beard's Ventilation of Mines i2mo, 2 50

Boyd's Resources of Southwest Virginia 8vo, 3 00

Map of Southwest Virginia Pocket-book form, 2 00

• Drinker's Tunneling, Explosive Compounds, and Rock Drills.

4to, half morocco, 25 00

Eissler's Modem High Explosives 8vo, 4 00

Fowler's Sewage Works Analyses i2mo, 2 00

Goodyear's Coal-mines of the Western Coast of the United States i2mo, 2 50

Ihlseng's Manual of Mining 8vo, 4 00

** Iles's Lead-smelting. (Postage gc. additional.) i2mo, 250

Kunhardt's Practice of Ore Dressing in Europe 8vo, i 50

O'DriscoU's Notes on the Treatment of Gold Ores 8vo, 2 00

* Walke's Lectures on Explosives .' 8vo, 4 00

Wilson's Cyanide Processes l2mo, i 50

Chlorination Process i2mo, i 50

Hydraulic and Placer Mining i2mo, 200

Treatise on Practical and Theoretical Mine Ventilation i2mo i 25

SANITARY SCIENCE.

Copeland's Manual of Bacteriology. {In preparation.)

FolweU's Sewerage. (Designing, Construction and Maintenance.; 8vo,

Water-supply Engineering g^^, '

Fuertes's Water and Public Health i2mo'

Water-filtration Works ...........].. i2mo,' 2 so

Gerhard's Guide to Sanitary House-inspection i6mo'

Goodrich's Economical Disposal of Town's Refuse . .T)emy 8vo'

Hazen's Filtration of Public Water-supplies 8vo'

Kiersted's Sewage Disposal.. '

T ui T-u T . i2mo, I 25

Leach s The Inspection and Analysis of Food with Special Reference to State

Control. (In preparation.)
Mason's Water-supply. (Considered Principally from a Sanitary Stand-

pomt.) 3d Edition, Rewritten 8vo 4 00

Examination of Water. (Chemical and BacterioiogicaL ) . '. i2mo' i 25

15

3 00

4 00

I 00
3 50
3 00

Merriman's Elements of Sanitary Engineering o

Nichols's Water-supply. ( Considered Mainly from a Chemical and Sanitwy '

Standpomt.) (1883.).. .. - „
Ogden's Sewer Design .... "'°' ^
* Price's Handbook on Sanitation. . . "'"°' ' °°
liichards's Cost of Food. A Study in Dietaries H^l' [ H
Cost of Living as Modified by Sanitary Science . 12^0* x 00
Richards and Woodman's Air, Water, and Food from a Sanitary' Stand-
point

» Richards and WiUiams's The Dietary Computer . . , . . . '. 8vo' i so

Rideal's Sewage and Bacterial Purification of Sewage 8vo' ^ so

Turneaure and RusseU's PubUc Water-supplies .' .'svo.' 5 00

Whipple's Microscopy of Drinking-water 8vo' 3 so

WoodhuU's Notes and MiUtary Hygiene . . i6mo! i 50

MISCELLANEOaS.

Barker's Deep-sea Soundings 8vo, 2 00

Emmons's Geological Guide-book of the Rocky Mountain Excursion of the

International Congress of Geologists Large 8vo, i 50

Ferrel's Popular Treatise on the Winds 8vo, 4 oo

Haines's American Railway Management lamo, 2 ge

Mott's Composition, DigestibiUty, and Nutritive Value of Food. Mounted chart, i 25

Fallacy of the Present Theory of Sound i6mo, i 00

Ricketts's History of Rensselaer Polytechnic Institute, 1824-1894. Small 8vo, 3 00

Rotherham's Empbasized New Testament Large 8 vo, 2 00

Steel's Treatise on the Diseases of the Dog 8vo, 3 50

Totten's Important Question in Metrology 8vo, 2 so

The World's Columbian Exposition ot 1893 4to, i 00

Worcester and Atkinson. Small Hospitals, Establishment and Maintenance,
and Suggestions for Hospital Architecture, with Plans for a Small

Hospital i2mo, i 25

HEBREW AND CHALDEE TEXT-BOOKS.

Green's Grammar of the Hebrew Language Svo, 3 ao

Elementary Hebrew Grammar i2mo, i 25

Hebrew Chrestomathy Svo, 2 00

Gesenius's Hebrew and Chaldee Lexicon to the Old Testament Scriptures.

(Tregelles.) Small 4to, half morocco, Si«o

Letteris's Hebrew Bible Svo, 2 25

. 16

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