Standard methods for the examination of water and sewage American Public Health Association. Laboratory Section_ American Chemical Society_ and Association of Official Agricultural

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Title: Standard methods for the examination of water
and sewage

Author: American Public Health Association. Laboratory Section
American Chemical Society
Association of Official Agricultural Chemists

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*** START OF THE PROJECT GUTENBERG EBOOK
STANDARD METHODS FOR THE EXAMINATION OF
WATER AND SEWAGE ***

STANDARD METHODS
FOR THE

EXAMINATION
OF

WATER AND SEWAGE

FOURTH EDITION

Revised by committees of the American Public Health Association, American
Chemical Society, and referees of the Association of Official Agricultural
Chemists

AMERICAN PUBLIC HEALTH ASSOCIATION
169 Massachusetts Avenue
BOSTON
1920

Copyright, 1917 and 1920

By the American Public Health Association

CONTENTS.

PAGE
Preface to the Fourth Edition vii

Collection of Samples 1
Quantity of Water Required for Analysis 1
Bottles 1
Time Interval between Collection and Analysis 2
Representative Samples 3

Physical Examination 4
Temperature 4
Turbidity 4
Turbidity Standard 4
Platinum Wire Method 5
Turbidimetric Method 7
Coefficient of Fineness 8
Color 9
Comparison with Platinum-Cobalt Standards 9
Comparison with Glass Disks 10
Comparison with Nessler Standards 10
Lovibond Tintometer 11
Odor 12
Cold Odor 12
Hot Odor 12
Expression of Results 12

Chemical Examination 14
Expression of Results 14
Forms of Nitrogen 15
Ammonia Nitrogen 15
Determination by Distillation 15
Measurement of Ammonia Nitrogen 16
Comparison with Ammonia Standards 16
Comparison with Permanent Standards 17
Modification for Sewage 18
Determination by Direct Nesslerization 19
Albuminoid Nitrogen 20
Organic Nitrogen 21
Nitrite Nitrogen 22

Nitrate Nitrogen 23
Phenoldisulfonic Acid Method 23
Reduction Method 24
Total Nitrogen 25
Oxygen Consumed 25
Recommended Method 26
Other Methods 27
Residue on Evaporation 29
Total Residue 29
Fixed Residue and Loss on Ignition 29
Suspended Matter 30
Determination with Gooch Crucible 30
Determination by Filtration 30
Determination of Volume 30
Fixed Residue and Loss on Ignition 30
Hardness 30
Total Hardness by Calculation 31
Total Hardness by Soap Method 31
Total Hardness by Soda Reagent Method 34
Temporary Hardness by Titration with Acid 34
Non-Carbonate Hardness by Soda Reagent Method 34
Non-Carbonate Hardness by Soap Method 35
Alkalinity 35
Procedure with Phenolphthalein 36
Procedure with Methyl Orange 37
Procedure with Lacmoid 37
Procedure with Erythrosine 37
Bicarbonate 37
Normal Carbonate 38
Hydroxide 38
Alkali Carbonates 39
Acidity 39
Total Acidity 40
Free Carbon Dioxide 40
Free Mineral Acids 41
Mineral Acids and Sulfates of Iron and Aluminium 41
Chloride 41
Iron 43
Total Iron 44

Colorimetric Method 44
Comparison with Iron Standards 45
Comparison with Permanent Standards 46
Volumetric Method 46
Dissolved Iron 47
Suspended Iron 47
Ferrous Iron 47
Ferric Iron 48
Manganese 48
Persulfate Method 48
Bismuthate Method 49
Lead, Zinc, Copper, and Tin 50
Lead 51
Zinc 52
Copper 53
Tin 54
Mineral Analysis 56
Residue on Evaporation 56
Alkalinity and Acidity 56
Chloride 56
Nitrate Nitrogen 56
Separation of Silica, Iron, Aluminium, Calcium, and Magnesium 56
Silica 56
Iron and Aluminium 57
Calcium 57
Magnesium 57
Separation of Sulfate, Sodium, and Potassium 58
Sulfate 58
Sodium, Potassium and Lithium 58
Potassium 59
Lithium 60
Bromine, Iodine, Arsenic, and Boric Acid 61
Bromine and Iodine 61
Arsenic 63
Boric Acid 63
Hydrogen Sulfide 63
Chlorine 64
Dissolved Oxygen 65
Ether-Soluble Matter 69

Relative Stability of Effluents 69
Biochemical Oxygen Demand of Sewages and Effluents 71
Relative Stability Method 71
Sodium Nitrate Method 72

Analysis of Sewage Sludge and Mud Deposits 73
Collection of Sample 73
Reaction 73
Specific Gravity 74
Moisture 74
Volatile and Fixed Matter 74
Total Organic Nitrogen 74
Ether-Soluble Matter 75
Ferrous Sulfide 76
Biochemical Oxygen Demand 76

Analysis of Chemicals 77
Reagents 77
Sulfate of Aluminium 78
Insoluble Matter 78
Oxides of Aluminium and Iron 78
Total Iron 79
Ferric Iron 79
Ferrous Iron 80
Basicity Ratio 80
Lime 80
Sulfate of Iron 81
Insoluble Matter 81
Iron as Ferrous Sulfate 81
Acidity 81
Soda Ash 82
Insoluble Matter 82
Available Alkali 82

Chemical Bibliography 82

Microscopical Examination 89
Microscopical Bibliography 91

Bacteriological Examination 92
Apparatus 92
Sample Bottles 92
Pipettes 92
Dilution Bottles 92
Petri Dishes 92
Fermentation Tubes 92
Materials 93
Water 93
Meat Extract 93
Peptone 93
Sugars 93
Agar 93
Gelatin 93
Litmus 93
General Chemicals 93
Methods 93
Preparation of Culture Media 93
Titration 93
Sterilization 94
Nutrient Broth 95
Sugar Broths 95
Nutrient Gelatin 95
Nutrient Agar 96
Litmus or Azolitmin Solution 96
Litmus-Lactose-Agar 97
Endo’s Medium 97
Collection of Sample 98
Storage and Transportation of Sample 98
Dilutions 98
Plating 99
Incubation 99
Counting 99
The Test for the Presence of Members of the B. Coli Group 100
Presumptive Test 100
Partially Confirmed Test 101
Completed Test 102
Application of these Tests 102
Expression of Results 103

Summary of these Tests 104
Interpretation of Results 106
Differentiation of Fecal from Non-fecal Members of the B.
Coli Group 106
Methyl Red Test 107
Voges-Proskauer Test 108
Routine Procedure for Bacteriological Examination 108
Bacteriological Bibliography 110

Index 113

PREFACE TO FOURTH EDITION.

The Committee on Standard Methods of Bacteriological
Water Analysis was reorganized in 1918 with the following
membership: F. P. Gorham, chairman, L. A. Rogers, W. G.
Bissell, H. E. Hasseltine, H. W. Redfield, with M. Levine as
adjunct member. This committee made a report in 1918
which was not acted on by the Laboratory Section, and in
1919 made a revised report, recommending certain
changes in Standard Methods, which were adopted by the
section and which are now incorporated in this present
fourth edition.
Following are the more important changes:
New brands of peptone authorized.
Phenol Red Method of Hydrogen-ion Concentration.
Five-tenths per cent of sugar specified for broths instead
of 1 per cent.
Sterilization of sugar is media specified in greater detail.
Preparation of Endo Medium.
Synthetic Medium for the Methyl Red Test.
There are no changes in the chemical methods in this
edition.

AMERICAN PUBLIC HEALTH
ASSOCIATION.
LABORATORY SECTION.
STANDARD METHODS FOR THE
EXAMINATION OF WATER AND SEWAGE.

Compiled and revised by committees of the American
Public Health Association and the American Chemical
Society and referees of the Association of Official
Agricultural Chemists.

COLLECTION OF SAMPLES.

QUANTITY REQUIRED FOR ANALYSIS.

The minimum quantity necessary for making the ordinary
physical, chemical, and microscopical analyses of water or
sewage is 2 liters; for the bacteriological examination, 100
cc. In special analyses larger quantities may be required.

BOTTLES.

The bottles for the collection of samples shall have glass
stoppers, except when physical, mineral, or microscopical
examinations only are to be made. Jugs or metal containers
shall not be used.
Sample bottles shall be carefully cleansed each time
before using. This may be done by treating with sulfuric
acid and potassium bichromate, or with alkaline
permanganate, followed by a mixture of oxalic and sulfuric
acids, and by thoroughly rinsing with water and draining.
The stoppers and necks of the bottles shall be protected
from dirt by tying cloth, thick paper or tin foil over them.
For shipment bottles shall be packed in cases with a
separate compartment for each bottle. Wooden boxes may
be lined with corrugated fibre paper, felt, or similar
substance, or provided with spring corner strips, to prevent
breakage. Lined wicker baskets also may be used.
Bottles for bacteriological samples shall be sterilized as
directed on page 98.

INTERVAL BEFORE ANALYSIS.

In general, the shorter the time elapsing between the
collection and the analysis of a sample the more reliable
will be the analytical results. Under many conditions
analyses made in the field are to be commended, as data so
obtained are frequently preferable to data obtained in a
distant laboratory after the composition of the water has
changed.
The time that may be allowed to elapse between the
collection of a sample and the beginning of its analysis
cannot be stated definitely. It depends on the character of
the sample, the examinations to be made, and other
conditions. The following are suggested as fairly
reasonable maximum limits.
Physical and chemical analysis.

Ground waters 72 hours
Fairly pure surface waters 48 〃
Polluted surface waters 12 〃
Sewage effluents 6〃
Raw sewages 6〃

Microscopical examination.

Ground waters 72 hours
Fairly pure surface waters 24 〃
Waters containing fragile organisms Immediate examination

Bacteriological examination.

Samples kept at less than 10°C 24 hours

If a longer period elapses between collection and
examination the time should be noted. If sterilized by the

addition of chloroform, formaldehyde, mercuric chloride, or
some other germicide samples for sanitary chemical
examination may be allowed to stand for longer periods
than those indicated, but as this is a matter which will vary
according to circumstances, no definite procedure is
recommended. If unsterilized samples of sewage, sewage
effluents, and highly polluted surface waters are analyzed
after greater intervals than those suggested caution must
be used in interpreting analyses of the organic content,
which frequently changes materially upon standing.
Determinations of dissolved gases, especially oxygen,
hydrogen sulfide, and carbon dioxide, should be made at
the time of collection in order to be reasonably accurate, in
accordance with the directions given hereafter in
connection with each determination.

REPRESENTATIVE SAMPLES.

Care should be taken to obtain a sample that is truly
representative of the liquid to be analyzed. With sewages
this is especially important because marked variations in
composition occur from hour to hour. Satisfactory samples
of some liquids can be obtained only by mixing together
several portions collected at different times or at different
places—the details as to collection and mixing depending
upon local conditions.

PHYSICAL EXAMINATION.

TEMPERATURE.

The temperature of the sample, if taken, shall be taken at the
time of collection, and shall be expressed preferably in degrees
Centigrade, to the nearest degree, or closer if more precise data
are required. The thermophone[109] is recommended for obtaining
the temperature of water at various depths below the surface.

TURBIDITY.

The turbidity of water is due to suspended matter, such as clay,
silt, finely divided organic matter, microscopic organisms, and
similar material.

TURBIDITY STANDARD.[110]

The standard of turbidity shall be that adopted by the United
States Geological Survey, namely, a water which contains 100
parts per million of silica in such a state of fineness that a bright
platinum wire 1 millimeter in diameter can just be seen when the
center of the wire is 100 millimeters below the surface of the
water and the eye of the observer is 1.2 meters above the wire,
the observation being made in the middle of the day, in the open
air, but not in sunlight, and in a vessel so large that the sides do
not shut out the light so as to influence the results. The turbidity
of such water is arbitrarily fixed at 100 parts per million.
For preparation of the silica standard dry Pear’s “precipitated
fuller’s earth” and sift it through a 200–mesh sieve. One gram of
this preparation in 1 liter of distilled water makes a stock
suspension which contains 1,000 parts per million of silica and
which should have a turbidity of 1,000. Test this suspension,
after diluting a portion of it with nine times its volume of distilled
water, by the platinum wire method to ascertain if the silica has
the necessary degree of fineness and if the suspension has the
necessary degree of turbidity. If not, correct by adding more
silica or more water as the case demands.[A]
A. This method of correction very slightly alters the coefficient of fineness of the
standard, but does not noticeably affect its use.

Standards for comparison shall be prepared from this stock
suspension by dilution with distilled water. For turbidity readings
below 20, standards of 0, 5, 10, 15, and 20 shall be kept in clear
glass bottles of the same size as that containing the sample; for
readings above 20, standards of 20, 30, 40, 50, 60, 70, 80, 90,

and 100 shall be kept in 100 cc. Nessler tubes approximately 20
millimeters in diameter.
Comparison with the standards shall be made by viewing both
standard and sample sidewise toward the light by looking at
some object and noting the distinctness with which the margins
of the object can be seen.
The standards shall be kept stoppered, and both sample and
standards shall be thoroughly shaken before making the
comparison.
In order to prevent any bacterial or algal growths from
developing in the standards a small amount of mercury
bichloride may be added to them.

PLATINUM WIRE METHOD.[42]

This method requires a rod with a platinum wire 1 mm. in
diameter inserted in it about 1 inch from one end of the rod and
projecting from it at a right angle at least 25 mm. Near the other
end of the rod, at a distance of 1.2 meters from the platinum
wire, a small ring shall be placed directly above the wire through
which, with his eye directly above the ring, the observer shall
look when making the examination.
The rod shall be graduated as follows: The graduation mark of
100 shall be placed on the rod at a distance of 100 mm. from the
center of the wire. Other graduations shall be made according to
Table 1, which is based on the best obtainable data. The
distances recorded in Table 1 are intended to be such that when
the water is diluted the turbidity readings will decrease in the
same proportion as the percentage of the original water in the
mixture. These graduations are those on what is known as the U.
S. Geological Survey Turbidity Rod of 1902.[105]

Table 1.—Graduation of turbidity rod.

Turbidity
Vanishing depth of wire (mm.).
(parts per million).

7 1095
8 971
9 873
10 794
11 729
12 674
13 627
14 587
15 551
16 520
17 493
18 468
19 446
20 426
22 391
24 361
26 336
28 314
30 296
35 257
40 228
45 205
50 187
55 171
60 158
65 147
70 138
75 130
80 122
85 116
90 110
95 105
100 100
110 93
120 86
130 81
140 76
150 72
160 68.7
180 62.4
200 57.4

250 49.1
300 43.2
350 38.8
400 35.4
500 30.9
600 27.7
800 23.4
1000 20.9
1500 17.1
2000 14.8
3000 12.1
Procedure.—Lower the rod vertically into the water as far as
the wire can be seen and read the level of the surface of the
water on the graduated scale. This will indicate the turbidity.
The following precautions shall be taken to insure correct
results:
Observations shall be made in the open air, preferably in the
middle of the day and not in direct sunlight. The wire shall be
kept bright and clean. If for any reason observations cannot be
made directly under natural conditions a pail or tank may be
filled with water and the observation taken in that, but if this is
done care shall be taken that the water is thoroughly stirred
before the observation is made, and no vessel shall be used for
this purpose unless its diameter is at least twice as great as the
depth to which the wire is immersed. Waters which have a
turbidity greater than 500 shall be diluted with clear water
before the observations are made, but if this is done the degree
of dilution shall be reported.

TURBIDIMETRIC METHOD.

Several forms of turbidimeter or diaphanometer[73] have been
suggested for use. The simplest and most satisfactory form is the
candle turbidimeter.[116] This consists of a graduated glass tube
with a flat polished bottom, enclosed in a metal case. This is
supported over an English standard candle and so arranged that
one may look vertically down through the tube at the flame of
the candle. The observation is made by pouring the sample of
water into the tube until the image of the flame of the candle just

disappears from view. Care shall be taken not to allow soot or
moisture to accumulate on the lower side of the glass bottom of
the tube so as to interfere with the accuracy of the observations.
The graduations on the tube correspond to turbidities produced
in distilled water by certain numbers of parts per million of silica
standard. In order to insure uniform results it is necessary to
have the distance between the top rim of the candle and the
bottom of the tube constant, and this distance shall be 7.6 cm. or
3 inches. The observations shall be made in a darkened room or
with a black cloth over the head.
It is allowable to substitute for the candle an electric light.
Calibrate the apparatus to correspond with the United States
Geological Survey scale. The figures in Table 2 on page 8 are
believed to be approximately correct for the candle turbidimeter
but should be checked by the experimenter. It is allowable to
calibrate the tube of the instrument with waters of known
turbidity prepared by making a series of dilutions of the silica
standard with distilled water. From the figures obtained in
calibrating plot a curve from which the turbidity of a sample may
be read when the depth of water in the tube has been obtained.

Table 2.—Graduation of candle turbidimeter.

Depth of liquid Turbidity
(cm.). (parts per million of silica).
2.3 1000
2.6 900
2.9 800
3.2 700
3.5 650
3.8 600
4.1 550
4.5 500
4.9 450
5.5 400
5.6 390
5.8 380
5.9 370
6.1 360
6.3 350
6.4 340
6.6 330
6.8 320
7.0 310
7.3 300
7.5 290
7.8 280
8.1 270
8.4 260
8.7 250
9.1 240
9.5 230
9.9 220
10.3 210
10.9 200
11.4 190
12.0 180
12.7 170
13.5 160
14.4 150
15.4 140
16.6 130
18.0 120
19.6 110
21.5 100
The results of turbidity observations shall be expressed in
whole numbers which correspond to parts per million of silica

and recorded as follows:
Turbidity between 1 and 50 recorded to nearest unit
〃〃 51 〃 100 〃〃〃 5
〃〃 101 〃 500 〃〃〃 10
〃〃 501 〃 1000 〃〃〃 50
〃〃 1001 〃 greater 〃〃〃 100

COEFFICIENT OF FINENESS[80]

The quotient obtained by dividing the weight of suspended
matter in the sample by the turbidity, both expressed in the same
unit, shall be called the coefficient of fineness. If the quotient is
greater than unity the matter in suspension is coarser and if it is
less than unity it is finer than the standard.

COLOR.

The “color,” or the “true color,” of water shall be considered
the color that is due only to substances in solution; that is, it is
the color of the water after the suspended matter has been
removed. In stating results the word “color” shall mean the “true
color” unless otherwise designated.
The “apparent color” shall be considered as including not only
the true color but also any color produced by substances in
suspension. It is the color of the original unfiltered sample.
The platinum-cobalt method of measuring color shall be
considered as the standard, and the unit of color shall be that
produced by 1 part per million of platinum.

COMPARISON WITH PLATINUM-COBALT STANDARDS.[43]

Reagents.—Dissolve 1.246 grams of potassium platinic chloride
(PtCl42KCl), containing 0.5 gram platinum, and 1.00 gram
crystallized cobalt chloride (CoCl2.6H2O), containing 0.25 gram
of cobalt, in water with 100 cc. concentrated hydrochloric acid,
and dilute to 1 liter with distilled water. This solution has a color
of 500. Dilute this solution with distilled water in 50 cc. Nessler
tubes to prepare standards having colors of 0, 5, 10, 15, 20, 25,
30, 35, 40, 50, 60, and 70. Keep these standards in Nessler tubes
of such diameter that the graduation mark is between 20 and 25
cm. above the bottom and of such uniformity that they match
within such limit that the distance from the bottom to the
graduation mark of the longest tube shall not exceed that of the
shortest tube by more than 6 mm. Protect the tubes from dust
and light when not in use.
Procedure.—The color of a sample shall be observed by filling
a standard Nessler tube to the height equal to that in the
standard tubes with the sample and by comparing it with the
standards. The observation shall be made by looking vertically
downward through the tubes upon a white or mirrored surface

placed at such angle that light is reflected upward through the
column of liquid.
Water that has a color greater than 70 shall be diluted before
making the comparison, in order that no difficulties may be
encountered in matching the hues.
Water containing matter in suspension shall be filtered, before
the color observation is made, until no visible turbidity remains.
If the suspended matter is coarse, filter paper may be used for
this purpose; if the suspended matter is fine, the use of a
Berkefeld filter is recommended. The Pasteur filter shall not be
used as it exerts a marked decolorizing action.
The apparent color, if determined, shall be determined on the
original sample without filtration. The true and the apparent
color of clear waters or waters with low turbidities are
substantially the same.
The results of color determinations shall be expressed in whole
numbers and recorded as follows:
Color between 1 and 50 recorded to nearest unit
〃〃 51 〃 100 〃〃〃 5
〃〃 101 〃 250 〃〃〃 10
〃〃 251 〃 500 〃〃〃 20.

COMPARISON WITH GLASS DISKS.[105]

As the platinum-cobalt standard method is not well adapted for
field work, the color of the water to be tested may be compared
with that of glass disks held at the end of metallic tubes through
which they are viewed by looking toward a white surface. The
glass disks are individually calibrated to correspond with colors
on the platinum scale. Experience has shown that the glass disks
used by the U. S. Geological Survey give results in substantial
agreement with those obtained by the platinum determinations,
and their use is recognized as a standard procedure.

COMPARISON WITH NESSLER STANDARDS.

Inasmuch as the Nessler scale[62] and the natural water scale[22]
[49]
which agrees with it except for colors less than 20, have been
largely used in the past, the old results may be converted[117] into
terms of the platinum standard by means of the ratios in Table 3,
but they must not be considered as universally applicable as the
variable sensitiveness of the Nessler solution introduces an
uncertain factor.
Table 3.—Values for converting colors by the natural water scale into colors by
the platinum standard in parts per million.[B]

Modified
Nessler
or
0.00. 0.01. 0.02. 0.03. 0.04. 0.05. 0.06. 0.07. 0.08. 0.09.
natural
water
standard.
Platinum-cobalt standard color.
0.00 0 2 4 6 8 9 11 13 15 17
.10 18 19 20 20 21 22 23 24 24 26
.20 26 27 27 28 29 29 30 31 32 32
.30 33 34 34 35 35 36 37 37 38 38
.40 39 40 40 41 42 42 43 44 45 45
.50 46 47 47 48 48 49 50 50 51 51
.60 52 53 53 54 54 55 56 56 57 57
.70 58 58 59 59 60 60 61 61 62 62
.80 63 64 64 65 66 66 67 68 69 69
.90 70 71 72 73 74 75 77 78 79 80
1.00 81 82 82 83 84 84 85 86 87 87
1.10 88 89 89 90 91 91 92 93 94 94
1.20 95 96 96 97 98 98 99 100 101 101
1.30 102 103 103 104 105 105 106 107 108 108
1.40 109 110 110 111 112 112 113 114 115 115
1.50 116 117 117 118 118 119 120 120 121 121
1.60 122 123 123 124 125 125 126 127 128 128
1.70 129 130 130 131 132 132 133 134 135 136
1.80 136 137 137 138 139 139 140 141 142 142
1.90 143 144 144 145 146 146 147 148 149 149
2.00 150

B. Zero on the true Nessler scale is about 15 on the platinum scale.

LOVIBOND TINTOMETER.

The value of the readings of tint and shade by the Lovibond
tintometer[66][82][83] has not been commensurate with the labor
involved, but it is necessary to make a record of the reflected tint
and shade[50] of some waters. The standard color disks used in
teaching optics may be used for the purpose.
Procedure.—The white disk supports three movable standard
color sectors, red, yellow, and blue, and one movable black
sector. All are mounted on a device which can be revolved
rapidly, blending the colors into a uniform tint or shade. A scale
around the circumference of the disk is used to indicate the
percentage of each color or white or black in the blend.
Place the sample in a battery jar on a white ground; adjust the
sectors so that when blended the tint or shade will match the
reflected tint or shade of the sample. Report the percentages of
red, yellow blue, white, and black in the blended tint or shade.

ODOR.[4][14][53][72][92][114][115][121c]

The observation of the odor, cold and hot, of samples of surface
water is important as the odors are usually indicative of organic
growths or sewage contamination or both. The odor of some
ground waters is caused by the earthy constituents of the water-
bearing strata. The odor of a contaminated well water is often
contributory evidence of its pollution. A study of the organisms
as directed under Microscopical Examination (p. 90) is a
valuable adjunct to physical and chemical examination of water.
Certain odors distinguish or identify certain organisms, as, for
example, the “fishy” odor of Uroglena, the “aromatic” or “rose
geranium” odor of Asterionella and the “pig pen” odor of
Anabaena. Observe and record the odor, both at room
temperature and at just below the boiling point, as follows:

COLD ODOR.

Shake the sample violently in one of the collecting bottles,
when it is half to two-thirds full and when the sample is at room
temperature (about 20° C.). Remove the stopper and smell the
odor at the mouth of the bottle.

HOT ODOR.

Pour about 150 cc. of the sample into a 500 cc. Erlenmeyer
flask. Cover the flask with a well-fitting watch glass. Heat the
water almost to boiling on a hot plate. Remove the flask from the
plate and allow it to cool not more than five minutes. Then
agitate it with a rotary movement, slip the watch glass to one
side, and smell the odor.

EXPRESSION OF RESULTS.

Express the quality of the odor by a descriptive epithet like the
following, which may be abbreviated in the record:
a—aromatic
C—free chlorine
d—disagreeable
e—earthy
f—fishy
g—grassy
m—moldy
M—musty
P—peaty
s—sweetish
S—hydrogen sulfide
v—vegetable.

Express the intensity of the odor by a numeral prefixed to the
term expressing quality, which may be defined as follows:
Numerical Term. Definition.
value.

0 None. No odor perceptible.

1 Very An odor that would not be detected ordinarily by the
faint. average consumer, but that could be detected in the
laboratory by an experienced observer.

2 Faint. An odor that the consumer might detect if his attention
were called to it, but that would not attract attention
otherwise.

3 Distinct. An odor that would be detected readily and that might
cause the water to be regarded with disfavor.

4 Decided. An odor that would force itself upon the attention and
that might make the water unpalatable.

5 Very An odor of such intensity that the water would be
strong. absolutely unfit to drink. (A term to be used only in
extreme cases.)

CHEMICAL EXAMINATION.

EXPRESSION OF RESULTS.

The results of chemical analyses shall be expressed in
parts per million, which in most analyses is practically
equivalent to milligrams per liter. In some laboratories other
forms of expression have been used. Results expressed in
parts per 100,000 or in grains per gallon may be
transformed to parts per million, or conversely, by the use of
the following table:
Table 4.—Factors for transforming results of analyses.

Equivalent.
Unit. Grains per
Grains per Parts per Parts per
Imperial
U.S. gallon. 100,000. million.
gallon.
1 grain per U.
1.000 1.20 1.71 17.1
S. gallon
1 grain per
Imperial .835 1.00 1.43 14.3
gallon
1 part per
.585 .70 1.00 10.0
100,000
1 part per
.058 .07 .10 1.0
million

The following general rules shall govern the use of
significant figures in the expression of results:
1. If the results show quantities greater than 10 parts per
million use no decimals; record only whole numbers. If the
quantities reach hundreds and thousands of parts record
only two significant figures.
2. If the results are between 1 and 10 parts do not retain
more than one decimal place.
3. If the results are between 0.1 and 1 part do not retain
more than two decimal places.
4. Estimates of ammonia, albuminoid, and nitrite nitrogen
alone justify the use of three decimals.

5. If the results of analyses are tabulated ciphers should
not be added at the right of the decimal point to make the
column uniform.

FORMS OF NITROGEN.
Nitrogenous organic matter passes through several
intermediate compounds during its natural decomposition,
and that which does not gasify ultimately forms nitrate.
Nitrogen in organic matter is determined by the Kjeldahl
process.[13][14][58] An indication of the amount present is
obtained by the albuminoid nitrogen determination.[14][15][67]
[106][107]
It has not been found possible to differentiate the
nitrogen in the organic matter that readily decomposes from
that in stable or non-putrescible compounds. Decomposition
of organic matter produces nitrogen combined in ammonia,
which is the first step between nitrogenous organic matter
and the completely mineralized nitrate. Ammonia nitrogen
may be determined by distillation and Nesslerization or by
direct Nesslerization of the clarified sample. The next step is
oxidation to nitrite, and the final step, oxidation to nitrate. It
is recommended that all forms of nitrogen be reported as
the element nitrogen (N).

AMMONIA NITROGEN.

There are two methods for estimating ammonia nitrogen—
distillation and direct Nesslerization. Distillation is
recommended for most waters and direct Nesslerization is
recommended for sewages, sewage effluents, and highly
polluted surface waters.

DETERMINATION BY DISTILLATION.[38][68b][111][121]

Procedure.—Use a metal or a glass flask connected with a
condenser so that the distillate may drop from the condenser
tube directly into a Nessler tube or a flask. Free the
apparatus from ammonia by boiling distilled water in it until
the distillate shows no trace of ammonia. After this has been
done empty the distilling flask and measure into it 500 cc. of
the sample, or a smaller portion diluted to 500 cc. with
ammonia-free water. If the sample is acid or if the presence
of urea is suspected add about 0.5 gram of sodium
carbonate before distillation. Omit this if possible as it tends
to increase “bumping.” Apply heat so that the distillation
may proceed at the rate of not more than 10 cc. nor less
than 6 cc. per minute. Collect the distillate in four Nessler
tubes, 50 cc. to each tube, or if the nitrogen is high in a 200
cc. graduated flask. These receptacles contain the ammonia
nitrogen to be measured as hereafter described.
Use Nessler tubes of such diameter that the graduation
mark is between 20 and 25 cm. above the bottom and of
such uniformity of diameter that the distance from the
bottom to the graduation mark of the longest tube shall not
exceed that of the shortest tube by more than 6 mm. The
tubes must be of clear white glass with polished bottoms.

MEASUREMENT OF AMMONIA NITROGEN.

The amount of ammonia in the distillates may be measured
either by (1) comparison of the Nesslerized distillates with
Nesslerized solutions containing known quantities of
nitrogen as ammonium chloride, or by (2) comparison of the
Nesslerized distillates with permanent standard solutions in
which the colors of Nesslerized standard ammonia solutions
are duplicated by solutions of platinum and cobalt chlorides.

Comparison with ammonia standards.

Reagents.—1. Ammonia-free water.
2. Standard ammonium chloride solution. Dissolve 3.82
grams of ammonium chloride in ammonia-free water and
dilute to 1 liter; dilute 10 cc. of this to 1 liter with ammonia-
free water. One cc. equals 0.00001 gram of nitrogen.
3. Nessler reagent.[8] Dissolve 50 grams of potassium
iodide in a minimum quantity of cold water. Add a saturated
solution of mercuric chloride until a slight precipitate
persists permanently. Add 400 cc. of 50 per cent solution of
potassium hydroxide, made by dissolving the potassium
hydroxide and allowing it to clarify by sedimentation before
using. Dilute to 1 liter, allow to settle, and decant. This
solution should give the required color with ammonia within
five minutes after addition and should not produce a
precipitate with small amounts of ammonia within two
hours.
Procedure.—Prepare a series of 16 Nessler tubes
containing the following amounts of the standard ammonium
chloride solution, diluted to 50 cc. with ammonia-free water,
namely: 0.0, 0.1, 0.3, 0.5, 0.7, 1.0, 1.4, 1.7, 2.0, 2.5, 3.0, 3.5,
4.0, 4.5, 5.0, and 6.0 cc. These solutions will contain 0.00001
gram of nitrogen for each cubic centimeter of the standard
solution.

Nesslerize the standards and the distillates by adding
approximately 1 cc. of Nessler reagent to each tube. Do not
stir the contents of the tubes. The temperature of the tubes
should be practically the same as that of the standards;
otherwise the colors will not be directly comparable.[45] Allow
the tubes to stand at least 10 minutes after Nesslerizing.
Compare the color produced in the tubes with that in the
standards by looking vertically downward through them at a
white or mirrored surface placed at an angle in front of a
window so as to reflect the light upward.
If the color obtained by Nesslerizing the distillates is
greater than that of the darkest tube of the standards, mix
the contents of the tube thoroughly, pour out half of the
liquid, and dilute the remainder to the original volume with
ammonia-free water; then make the color comparison and
multiply the result by two. If the color is still too dark after
pouring out half the liquid, repeat this process of division
until a reading can be made. The process of dilution may be
shortened by mixing together the distillates from one sample
before making the comparison and comparing an aliquot
portion with the standards.
After the readings have been recorded add the results
obtained by Nesslerizing each portion of the entire distillate.
If 500 cc. of the sample is distilled this sum, expressed in
cubic centimeters and multiplied by 0.02, will give the
number of parts per million of ammonia nitrogen in the
sample. If x cc. of sample is used multiply the sum of the
readings by 10/x.
If the ammonia is known to be high the distillate may be
collected in 200 cc. flasks and an aliquot part Nesslerized.

Comparison with permanent standards.[62][65]

Reagents.—Platinum solution. Dissolve 2.00 grams of
potassium platinic chloride (PtCl4.2KCl) in a small amount of

distilled water, add 100 cc. of strong hydrochloric acid, and
dilute to 1 liter.
Cobalt solution. Dissolve 12 grams of cobaltous chloride
(CoCl2.6H2O) in distilled water, add 100 cc. of strong
hydrochloric acid, and dilute to 1 liter.
Prepare standards by putting various amounts of these
two solutions into Nessler tubes and diluting to the 50 cc.
mark with distilled water as indicated in Table 5. These
standards may be kept for several months if protected from
dust.
Table 5.—Preparation of permanent standards for the determination of
Ammonia.

Value in standard ammonium Solution of Solution of
chloride. platinum. cobalt.
cc. cc. cc.
0.0 1.2 0.0
.1 1.8 .0
.2 2.8 .0
.4 4.7 .1
.7 5.9 .2

1.0 7.7 .5
1.4 9.9 1.1
1.7 11.4 1.7
2.0 12.7 2.2
2.5 15.0 3.3

3.0 17.3 4.5
3.5 19.0 5.7
4.0 19.7 7.1
4.5 19.9 8.7
5.0 20.0 10.4

6.0 20.0 15.0
7.0 20.0 22.0
The amounts in Table 5 are approximate, and the actual
amount necessary will differ with the character of the
Nessler solution, the color sensitiveness of the analyst’s eye,

and other conditions. The final test of the standard is best
obtained by comparing it with Nesslerized standards and
modifying the tint accordingly. Such comparison should be
made for each new batch of Nessler solution and should be
checked by each analyst.
Procedure.—In comparison with permanent standards,
Nesslerize the distillates in the manner above described and
compare the resulting colors at the end of about 10 minutes
with the permanent standards. The method of calculating
results is precisely the same as with the ammonia standards.

MODIFICATION FOR SEWAGE.

Ammonia nitrogen and albuminoid nitrogen in sewages,
soils, and other materials of high nitrogen content may be
satisfactorily determined by diluting the sample with
ammonia-free distilled water and proceeding as described in
the preceding sections, but it is permissible to distill with
steam.[40]
Procedure.—Use a 200 cc. long-necked Kjeldahl flask
connected with a condenser so that the distillate may drop
from the condenser tube directly into a Nessler tube or a
flask. Connect the Kjeldahl flask with a steam generator by a
tube reaching almost to the bottom of the flask.
After the apparatus is freed from ammonia put the sample
to be tested into the flask. Use 10 to 100 cc. of the sample
according to its ammonia content. Pass ammonia-free steam
through the liquid in the Kjeldahl flask and collect the
distillate in the usual way. It is usually convenient to collect
the distillate in a 200 cc. flask and to take an aliquot part of
it for Nesslerization. Compare with standards and calculate
the nitrogen content in the usual manner.
This method has the advantage, when the sample is
treated with an alkaline solution of potassium
permanganate, of avoiding bumping, permitting the assay of

solid matter, and yielding the ammonia more rapidly than by
the ordinary process of distillation.

DETERMINATION BY DIRECT NESSLERIZATION.[21][75]

Reagents. 1. Ten per cent solution of copper sulfate
— (CuSO4.5H2O).
2. Ten per cent solution of lead acetate
(Pb(C2H3O2)2.3H2O).
3. Fifty per cent solution of sodium hydroxide
(NaOH) or potassium hydroxide (KOH).
Procedure.—To 50 cc. of the sample to be tested, diluted if
necessary with an equal volume of ammonia-free water, in a
short tube, add a few drops of the copper sulfate solution.
After thoroughly mixing, add 1 cc. of the alkali hydroxide
solution and again thoroughly mix. Allow the tube to stand
for a few minutes, when a heavy precipitate should fall to
the bottom, leaving a colorless supernatant liquid.
Nesslerize an aliquot part. Compare with standards and
compute the ammonia nitrogen in the same manner as in the
distillation procedure.
Samples containing hydrogen sulfide may require the use
of lead acetate in addition to the copper sulfate. Some
samples may require a few trials before the right
combination of the three solutions to bring about the best
results can be found.
Instead of adding copper sulfate to sewages of high
magnesium content satisfactory clarification of the sample
can be obtained by mixing it with the alkali hydroxide alone.
[54]

ALBUMINOID NITROGEN.
The addition of an alkaline permanganate solution to
liquids containing nitrogenous organic matter causes the
formation of ammonia, which can be distilled and
determined by Nesslerization of the distillate. The nitrogen
of the ammonia, thus obtained, is called albuminoid
nitrogen. As the ratio of nitrogenous organic matter to the
ammonia obtained by distillation is decidedly variable[6][30][75]
in sewages and other substances containing much
nitrogenous organic matter albuminoid nitrogen results on
such substances are less accurate[29] than organic (Kjeldahl)
nitrogen. Therefore in sewage work, including analysis of
influents and effluents of purification plants and the water of
highly polluted streams, it is recommended that
determinations of organic nitrogen be substituted for
determinations of albuminoid nitrogen. For ground waters
and surface waters containing but little pollution, the
albuminoid nitrogen is approximately one-half the organic
nitrogen; accordingly the continuance of albuminoid
nitrogen determinations for this class of work is approved.
Reagents.—Alkaline potassium permanganate. Pour 1,200
cc. of distilled water into a porcelain dish holding 2,500 cc.,
boil 10 minutes, and turn off the gas. Add 16 grams of C. P.
potassium permanganate and stir until solution is complete.
Then add 800 cc. of 50 per cent clarified solution of
potassium hydroxide or an equivalent amount of sodium
hydroxide and enough distilled water to fill the dish. Boil
down to 2,000 cc. Test this solution for ammonia by making
a blank determination. Correct determinations by the
amount of this blank.
Procedure.—After the collection of the distillate for
ammonia nitrogen described on page 15 add 50 cc. (or more
if necessary to insure the complete oxidation of the organic

matter) of alkaline potassium permanganate and continue
the distillation until at least four portions, and preferably
five portions, of 50 cc. each, of distillate have been collected
in separate tubes. Determine the albuminoid nitrogen in the
distillate by Nesslerization. If the albuminoid nitrogen is
known to be high it is convenient to collect the distillate in a
200 cc. flask and to Nesslerize an aliquot part of it.
Dissolved albuminoid nitrogen may be determined in a
sample from which suspended matter has been removed by
filtration either through filter paper or through a Berkefeld
filter. Suspended albuminoid nitrogen is the difference
between the total and the dissolved albuminoid nitrogen.

ORGANIC NITROGEN.[24b][69][71][76][84]

Procedure for water.—Boil 500 cc. of the sample in a
round-bottomed flask to remove ammonia nitrogen. This
usually causes the loss of 200 cc. of the sample, which may
be collected for the determination of ammonia nitrogen. Add
5 cc. of nitrogen-free concentrated sulfuric acid and a small
piece of ignited pumice. Mix by shaking and place over a
flame under a hood. Digest until copious fumes of sulfuric
acid are given off and the liquid finally becomes colorless or
pale straw color. Remove from the flame, and add potassium
permanganate crystals in small portions until a heavy green
precipitate persists in the liquid. Cool. Dilute to about 300
cc. with ammonia-free water. Make alkaline with 10 per cent
ammonia-free sodium hydroxide. Distill the ammonia, collect
the distillate in Nessler tubes, Nesslerize, and compare with
standards as described (pp. 16–18).
First procedure for sewage[76].—Distill the ammonia
nitrogen directly from 100 cc. or less of the sample, diluted
to 500 cc. with nitrogen-free water. Collect the distillate and
determine the ammonia nitrogen in it. Add 5 cc. of nitrogen-
free sulfuric acid and 1 cc. of 10 per cent nitrogen-free
copper sulfate, and digest the liquid for half an hour after it
has become colorless or pale straw color. Add 0.5 gram of
potassium permanganate crystals to the hot acid solution,
and dilute to 500 cc. with ammonia-free water. Dilute 10 cc.
or more of this liquid, in a Kjeldahl distilling flask, to about
300 cc. with ammonia-free water. Make alkaline with 10 per
cent sodium hydroxide, distill, and Nesslerize. With some
samples direct Nesslerization may be used. (See p. 19.)
In this determination care must be taken to digest
thoroughly, to add potassium permanganate to the point of
precipitation, to sample carefully after dilution, and to add
enough sodium hydroxide to insure the separation of the

ammonia from the precipitated manganese hydroxide.
Potassium permanganate should not be added during
digestion because it causes loss of nitrogen.
Second procedure for sewage.—Omit the separation of
ammonia nitrogen and determine the ammonia nitrogen and
organic nitrogen together. Determine the ammonia nitrogen
in a separate sample by direct Nesslerization as described
on page 19. The organic nitrogen is equal to the difference.

NITRITE NITROGEN.[51][63a][64][94c][108]

Reagents.—1. Sulfanilic acid solution. Dissolve 8.00 grams
of the purest sulfanilic acid in 1,000 cc. of 5 N acetic acid
(sp. gr. 1.041) or in 1,000 cc. of water containing 50 cc. of
concentrated hydrochloric acid. This is practically a
saturated solution.
2. α-naphthylamine acetate or chloride solution. Dissolve
5.00 grams solid α-naphthylamine in 1,000 cc. of 5 N acetic
acid or in 1,000 cc. of water containing 8 cc. of concentrated
hydrochloric acid. Filter the solution through washed
absorbent cotton or an alundum filter.
3. Sodium nitrite stock solution. Dissolve 1.1 gram silver
nitrite in nitrite-free water; precipitate the silver with
sodium chloride solution and dilute the whole to 1 liter.
4. Standard sodium nitrite solution. Dilute 100 cc. of
solution 3 to 1 liter, then dilute 50 cc. of this solution to 1
liter with sterilized nitrite-free water, add 1 cc. of
chloroform, and preserve in a sterilized bottle. One cc. =
0.0005 mg. nitrogen.
5. Fuchsine solution. 0.1 gram per liter.
Procedure.—Place in a standard Nessler tube 50 cc. of the
sample, decolorized if necessary with nitrite-free aluminium
hydroxide (see p. 42) or a smaller amount diluted to 50 cc.
At the same time prepare in Nessler tubes a set of
standards, by diluting to 50 cc. with nitrite-free water,
various amounts of the standard nitrite solution. The
following amounts of standard solution are suggested: 0.0,
0.1, 0.2, 0.4, 0.7, 1.0, 1.4, 1.7, 2.0, and 2.5 cc. Add 1 cc. of
the sulfanilic acid solution and 1 cc. of the α-naphthylamine
acetate or hydrochloride solution to the sample and to each
standard. Mix thoroughly and allow to stand 10 minutes;
then compare the sample with the standards. Do not allow

the sample to stand more than one-half hour before making
the comparison. If the color of the sample is deeper than
that of the highest standard repeat the test on a diluted
sample. If 50 cc. of the sample is used 0.01 times the
number of cc. of the standard matched equals parts per
million of nitrite nitrogen. Satisfactory results can be
obtained by using either hydrochloric or acetic acid in
preparing the test solutions, but the speed of the reaction is
more rapid if acetic acid is used.[112]
Permanent standards may be prepared by matching the
nitrite standards with dilutions of the fuchsine solution.
Fuchsine standards have been found to be sufficiently
accurate for waters high in nitrite and for sewage. The
standards should be checked once a month and kept out of
bright sunlight.

NITRATE NITROGEN.[16][36][90][100]

Two methods are recommended for the determination of
nitrate nitrogen in water, sewage, and sewage effluents.

PHENOLDISULFONIC ACID METHOD.[1][5][32]

Reagents.—1. Phenoldisulfonic acid. Dissolve 25 grams of
pure white phenol in 150 cc. of pure concentrated sulfuric
acid. Add 75 cc. of fuming sulfuric acid (15 per cent SO3),
stir well, and heat for 2 hours at about 100°C.
2. Potassium hydroxide solution. Prepare an approximately
12 N solution, 10 cc. of which will neutralize about 4 cc. of
the phenoldisulfonic acid.
3. Standard nitrate solution. Dissolve 0.72 gram of pure
recrystallized potassium nitrate in 1 liter of distilled water.
Evaporate cautiously to dryness 10 cc. of the solution on the
water bath. Moisten residue quickly and thoroughly with 2
cc. of phenoldisulfonic acid and dilute to 1 liter. This is the
standard solution, 1 cc. of which equals 0.001 mg. of nitrate
nitrogen.
4. Standard silver sulfate solution. Dissolve 4.4 grams of
silver sulfate free from nitrate in 1 liter of water. One cc. of
this solution is equal to 1 mg. of chloride.
Procedure.—The alkalinity, chloride, and nitrite content,
and color of the sample must first be determined. If the
sample is highly colored decolorize it with freshly
precipitated aluminium hydroxide. Measure into an
evaporating dish 100 cc. of the sample, or if nitrate is very
high such volume as will contain about 0.01 mg. of nitrate
nitrogen. Add sufficient N/50 sulfuric acid nearly to
neutralize the alkalinity. Then add sufficient standard silver

sulfate to precipitate all but about 0.1 mg. of chloride. The
removal of chloride may be omitted if the sample contains
less than 30 parts per million of chloride. Heat the mixture
to boiling, add a little aluminium hydroxide, stir, filter, and
wash with small amounts of hot water. Evaporate the filtrate
to dryness, and add 2 cc. of the phenoldisulfonic acid,
rubbing with a glass rod to insure intimate contact. If the
residue becomes packed or appears vitreous because of the
presence of much iron, heat the dish on the water bath for a
few minutes. Dilute the mixture with distilled water, and add
slowly a strong solution of potassium hydroxide or
ammonium hydroxide until the maximum color is developed.
Transfer the solution to a Nessler tube, filtering if necessary.
If nitrate is present a yellow color will be formed. Compare
the color with that of standards[52][55] made by adding 2 cc. of
strong potassium hydroxide or ammonium hydroxide to
various amounts of standard nitrate solution and diluting
them to 50 cc. in Nessler tubes. The following amounts of
standard nitrate solution are suggested: 0, 0.5, 1.0, 1.5, 2.0,
4.0, 6.0, 8.0, 10.0, 15.0, 20.0, and 40.0 cc. These standards
may be kept several weeks without deterioration. If 100 cc.
of water is used the number of cubic centimeters of the
standard multiplied by 0.01 is equal to parts per million of
nitrate nitrogen.
Standards that will remain permanent for several years if
stored in the dark may be prepared from tripotassium
nitrophenoldisulfonate.[5]
If nitrite nitrogen is present in excess of 1 part per million
it should be oxidized by heating the samples a few minutes
with a few drops of hydrogen peroxide free from nitrate
repeatedly added[95] or by adding dilute potassium
permanganate in the cold until a faint pink coloration
appears; the nitrogen equivalent of the nitrite thus oxidized
to nitrate is then subtracted from the final nitrate nitrogen
reading.

REDUCTION METHOD.[2][46]

Reagents.—1. Sodium or potassium hydroxide solution.
Dissolve 250 grams of the hydroxide in 1.25 liters of distilled
water. Add several strips of aluminium foil and allow the
evolution of hydrogen to continue over night. Concentrate
the solution to 1 liter by boiling.
2. Aluminium foil. Use strips of pure aluminium about 10
cm. long, 6 mm. wide, and 0.33 mm. thick and weighing
about 0.5 gram.
Procedure.—To 100 cc. of the sample in a 300 cc.
casserole add 2 cc. of the hydroxide solution and
concentrate by boiling to about 20 cc. Pour the contents of
the casserole into a test tube about 16 cm. long and 3 cm. in
diameter, or of approximately 100 cc. capacity. Rinse the
casserole several times with nitrogen-free water and add the
rinse water to the liquid already in the tube, thus making the
contents of the tube approximately 75 cc. Add a strip of
aluminium foil. Close the tube by means of a rubber stopper
through which passes a bent glass tube about 5 mm. in
diameter. Put the shorter arm of the tube flush with the
lower side of the rubber stopper and let the longer arm
extend below the surface of distilled water in another test
tube. This apparatus serves as a trap through which the
evolved hydrogen escapes freely. The small amount of
ammonia escaping into the trap may be neglected. Allow the
action to proceed for a minimum period of four hours or over
night. Pour the contents of the tube into a distilling flask,
dilute with 250 cc. of ammonia-free water, distill, collect the
distillate in Nessler tubes, and Nesslerize. If the nitrate
content is high collect the distillate in a 200 cc. flask and
Nesslerize an aliquot part. If the supernatant liquid in the
reduction tube is clear and colorless the solution may be
diluted to a definite volume and an aliquot part Nesslerized
without distillation.

TOTAL NITROGEN.[93]

In sewage work it is frequently of assistance to know the
total nitrogen content. This is ordinarily computed by adding
together the organic, ammonia, nitrite, and nitrate nitrogen,
each of which is determined as already described.

OXYGEN CONSUMED.[24][67][84a][85][94f][101][102]

Oxygen consumed means the oxygen that the oxidizable
compounds of sewage and water consume when treated in
an acid solution with potassium permanganate. The
expression is synonymous with oxygen required, oxygen
absorbed, and oxygen-consuming capacity. It should not be
confused with biochemical oxygen demand.
As the carbon, not the nitrogen, in organic matter is
oxidized by potassium permanganate, oxygen consumed is
considered by some an indication of the amount of
carbonaceous organic matter present. The determination
indicates, however, only part of the carbon, the proportion
varying in different samples because the carbon in
nitrogenous matter is not so readily oxidized as that in
carbonaceous organic matter. Furthermore, it does not
directly differentiate the carbon present in unstable organic
matter from that in fairly stable organic matter, such as is
sometimes referred to as residual humus matter. As nitrite
nitrogen, ferrous iron, sulfide, and other oxidizable mineral
substances reduce potassium permanganate, corrections for
them should be made in the determination.

RECOMMENDED METHOD.

Reagents.—1. Dilute sulfuric acid. Dilute 1 part of
concentrated sulfuric acid with 3 parts of distilled water and
free the solution from oxidizable matter by adding potassium
permanganate until a faint pink color persists after the
solution has stood several hours.
2. Standard ammonium oxalate. Dissolve 0.888 gram of
the pure salt in 1 liter of distilled water. One cc. is

equivalent to 0.1 mg. of oxygen. An equivalent quantity of
oxalic acid or sodium oxalate may be used.
3. Standard potassium permanganate. Dissolve 0.4 gram
of the crystallized salt in 1 liter of distilled water. Add 10 cc.
of the dilute sulfuric acid and 10 cc. of this solution of
potassium permanganate to 100 cc. of distilled water, and
digest 30 minutes. Add 10 cc. of the ammonium oxalate
solution, and then add potassium permanganate till a pink
coloration appears. This destroys the oxygen-consuming
capacity of the water used. Now add another 10 cc. of
ammonium oxalate solution and titrate with potassium
permanganate. Adjust the potassium permanganate solution
so that 1 cc. is equivalent to 1 cc. of ammonium oxalate
solution or 0.1 mg. of available oxygen.
Acid digestion.—Place in a flask 100 cc. of the water, or, if
the water is of high organic content, a smaller portion
diluted to 100 cc. Add 10 cc. of sulfuric acid solution and 10
cc. of standard potassium permanganate and digest the
liquid exactly 30 minutes in a bath of boiling water the level
of which is kept above the level of the contents of the flask.
[70][71a]
If the quantity of permanganate is insufficient for
complete oxidation repeat the digestion with a larger
quantity; at least 5 cc. excess of the standard permanganate
should be present when the ammonium oxalate solution is
added. Remove the flask, add 10 cc. of the ammonium
oxalate solution, and titrate with the standard permanganate
until a faint but distinct color is obtained. If 100 cc. of water
is used the number of cubic centimeters of potassium
permanganate solution in excess of the number of cubic
centimeters of ammonium oxalate solution is equal to parts
per million of oxygen consumed.
If oxidizable mineral substances, such as ferrous iron,
sulfide, or nitrite, are present in the sample corrections
should be applied as accurately as possible by suitable
procedures. Direct titration of the acidified sample in the
cold, using a three-minute period of digestion, serves this

purpose quite well for polluted surface waters and fairly well
for purified sewage effluents. Few raw sewages containing
no trade wastes need such a correction, but raw sewages
containing “pickling” liquors do need it. If the sample
contains both oxidizable mineral compounds and gaseous
organic substances the latter should be driven off by heat
and the sample allowed to cool before applying this test for
the correction factor. If such corrections are made the fact
should be stated with the amount of correction.
Period and temperature of digestion.—As the practice in
regard to the period and temperature of digestion has varied
widely it is difficult to compare the results obtained at one
laboratory with those obtained at another. None of the
methods gives absolute results. They are all relative[26][29][57]
at best. Digesting 30 minutes at the boiling temperature is
herein designated the recommended method. If samples are
analyzed by any other method the method should be noted,
and, representative results by the standard method should
be placed on record for purposes of comparison.

OTHER METHODS.

Additional reagents.—1. Potassium iodide solution. Ten per
cent solution, free from iodate.
2. Standard sodium thiosulfate. Dissolve 1.0 gram of the
pure crystallized salt in 1 liter of distilled water. Standardize
this solution against the standard potassium permanganate.
As the thiosulfate solution does not keep well determine its
actual strength at frequent intervals.
3. Starch indicator. Prepare as directed in the section on
dissolved oxygen (pp. 65–66).
4. Sodium hydroxide solution. Dissolve 1 part of pure
sodium hydroxide in 2 parts of distilled water.

Certain widely practiced deviations from the standard
procedure just described are noted in the following
paragraphs.
1. Heat the acidified sample to boiling, add the
permanganate solution, and digest for two minutes[16] at
boiling temperature. This procedure is facilitated by
agitating the liquid constantly with a small current of air to
guard against bumping.
2. Same method as No. 1 except that the period of
digestion is five minutes.[121a]
3. Same method as No. 2 except that the permanganate
solution is added to the acidified sample when cold, and
digestion is continued five minutes after the sample reaches
the boiling point. The advantage of this method is that there
is included the oxygen-consuming power of the volatile
matter present in some sewages and sewage effluents,
which is driven off by heat and thus escapes when the test is
made in accordance with procedures 1 and 2.
4. Same method as No. 3 except that the period of
digestion is 10 minutes.[63][68c]
5. Digestion of the sample after the acid and
permanganate solutions are added is carried out abroad,
especially in England, at approximately the room
temperature,[24a][69a][94f][100a] apparently to guard against
decomposition[17] of permanganate in the presence of high
chloride, for periods of three minutes, fifteen minutes, and
four hours; many observers record the oxygen consumed
after all three periods, while some record the result only for
the four-hour period. At the end of the period of digestion,
add 0.5 cc. of potassium iodide solution to discharge the
pink color; mix; titrate the liberated iodine with thiosulfate
until the yellow color is nearly destroyed, then add a few
drops of starch solution and continue titration until the blue
color is just discharged. The number of cubic centimeters of
potassium permanganate solution in excess of the number of

cubic centimeters of sodium thiosulfate solution is equal to
parts per million of oxygen consumed.
6. Digestion in alkaline solution[104] is preferable to
digestion in acid solution for brines or waters high in
chlorine. Place in a flask 100 cc. of the sample, or if it is of
high organic content a smaller portion diluted to 100 cc. Add
0.5 cc. of sodium hydroxide solution and 10 cc. of standard
potassium permanganate and digest exactly 30 minutes.
Remove the flask, add 5 cc. of sulfuric acid and 10 cc. of the
standard ammonium oxalate, and titrate with the standard
potassium permanganate as in the acid digestion.

RESIDUE ON EVAPORATION.

TOTAL RESIDUE.[16]

Ignite and weigh a clean platinum dish, and measure into
it 100 cc. of the thoroughly shaken sample. Evaporate to
dryness on a water bath. Then heat the dish in an oven at
103° C. or 180° C. for one hour. Cool in a desiccator and
weigh. The temperature of drying should be mentioned in
the report. The increase in weight gives the total solids or
residue on evaporation. If 100 cc. of the sample was taken
this weight expressed in milligrams and multiplied by 10 is
equal to parts per million of residue on evaporation. The
residue from waters low in organic matter but relatively
high in iron may be used, as a matter of convenience, for the
determination of iron.

FIXED RESIDUE AND LOSS ON IGNITION.[13][96]

The residue from sewages and waters high in organic
matter may be ignited to burn off the organic matter, which,
with some volatile inorganic matter, constitutes the loss on
ignition.
Procedure.—Ignite the residue in the platinum dish at a
low red heat. If great accuracy is desired this should be done
in an electric muffle furnace or in a radiator, which consists
of a platinum or a nickel dish large enough to allow an air
space of about half an inch between it and the dish within it,
the inner dish being supported by a triangle of platinum wire
laid on the bottom of the outer dish. A disc of platinum or
nickel foil large enough to cover the outer dish is suspended
over the inner dish to radiate the heat into it. The larger dish

is heated to bright redness until the residue is white or
nearly so. Allow the dish to cool, and moisten the residue
with a few drops of distilled water. Dry the residue in the
oven, cool in a desiccator, and weigh. The fixed residue on
evaporation is the difference between this weight and the
weight of the dish.
The loss on ignition is the difference between the total
residue on evaporation and the fixed residue on evaporation.
If the odor and color on ignition of some residues give
helpful clues to the character of the organic matter record
them.

SUSPENDED MATTER.[56][110]

DETERMINATION WITH GOOCH CRUCIBLE.

Reagent.—Prepare a dilute cream of asbestos fibre which
has been finely shredded, thoroughly ignited, treated with
strong hydrochloric acid for at least 12 hours, and washed
with distilled water till free from acid.
Procedure.—1. Prepare a mat of the asbestos fibre 1/16
inch thick in a Gooch crucible. Dry it in an oven at 103 or
180° C., cool and weigh. Filter 1,000 cc. of samples having a
turbidity of 50 parts per million or less. If the turbidity is
higher use sufficient water to obtain 50 to 100 mg. of
suspended matter. Dry for one hour at 103 or 180° C., cool
and weigh. Report the temperature at which the residue was
dried. If 1,000 cc. is filtered the increase in weight
expressed in milligrams is equal to parts per million of
suspended matter.

DETERMINATION BY FILTRATION.

The difference between the total solids in filtered and
unfiltered portions of a sample may be used as a basis for
calculating suspended matter.

DETERMINATION OF VOLUME.

The determination of the volume[9][69b] of suspended matter
in sewages has received considerable attention abroad.
Imhoff recommends the use of conical glass vessels holding
1 liter with the lower portions graduated in cubic

centimeters. Others recommend centrifuges with sediment
tubes.

FIXED RESIDUE AND LOSS ON IGNITION.

Treat the total residue from a filtered sample in the same
manner as described for the total residue, and obtain the
loss on ignition due to dissolved matter, and by difference
the loss on ignition due to suspended matter.

HARDNESS.[94e]

A water containing certain mineral constituents in
solution, chiefly calcium and magnesium, which form
insoluble compounds with soap, is said to be hard. Carbon
dioxide in water increases the solubility of calcium and
magnesium carbonates, forming bicarbonate. If carbon
dioxide is removed from the water by boiling the
bicarbonate is decomposed and calcium and magnesium are
partly precipitated. The proportion of calcium or magnesium
carbonate that a water can hold in solution depends on the
concentration of carbon dioxide, which in turn depends on
the temperature of the water and the proportion of carbon
dioxide in the atmosphere with which the water has been in
contact. Consequently, when the carbon dioxide is removed
from the water by boiling or otherwise the carbonates of
calcium and magnesium are partly, but not completely,
precipitated, and the hardness of the water is thus
diminished and the water is softened to the extent to which
these substances are precipitated. The hardness thus
removed is called temporary hardness. The hardness which
still remains after boiling is due mainly to calcium and
magnesium in equilibrium with sulfate, chloride, and nitrate,
and residual carbonate, and it is called permanent hardness.
Non-carbonate hardness is the hardness caused by sulfates,
chlorides, and nitrates of calcium, magnesium, iron, and
other metals that form insoluble soaps.

TOTAL HARDNESS BY CALCULATION.

The most accurate method of ascertaining total hardness
is to compute it from the results of determinations of
calcium and magnesium in the sample. (See methods, pp.

57–58.) Iron and other metals must be included in the
calculation if they are present in significant amounts. Total
hardness as CaCO3 equals 2.5 Ca plus 4.1 Mg.

TOTAL HARDNESS BY SOAP METHOD.[121b]

The determination of hardness by the soap method roughly
approximates the amount of calcium and magnesium in a
water, though it actually measures the soap-consuming
power of the water.
Reagents.—1. Standard calcium chloride solution. Dissolve
0.2 gram of pure calcite (calcium carbonate) in a little dilute
hydrochloric acid, being careful to avoid loss of solution by
spattering. Evaporate the solution to dryness several times
with distilled water to expel excess of acid. Dissolve the
residue in distilled water and dilute the solution to 1 liter.
One cc. of this dilution is equivalent to 0.2 mg. of calcium
carbonate.
2. Standard soap solution. Dissolve 100 grams of dry white
Castile soap in 1 liter of 80 per cent alcohol, and allow this
solution to stand several days before standardizing. Pure
potassium oleate made from lead plaster and potassium
carbonate may be used in place of Castile soap.
First method of standardization.—Dilute 20 cc. of the
calcium chloride solution in a 250 cc. glass-stoppered bottle
to 50 cc. with distilled water which has been recently boiled
and cooled. Add soap solution from a burette, 0.2 or 0.3 cc.
at a time, shaking the bottle vigorously after each addition
until a lather remains unbroken for five minutes over the
entire surface of the water while the bottle lies on its side.
Then adjust the strength of the stock solution with 70 per
cent alcohol so that the resulting diluted soap solution will
give a permanent lather when 6.40 cc. of it is properly added
to 20 cc. of standard calcium chloride solution diluted to 50
cc. Usually 75 to 100 cc. of the stock soap solution is

required to make 1 liter of the standard soap solution. The
quantity of calcium carbonate equivalent to each cubic
centimeter of the standard soap solution consumed in the
titration is indicated in Table 6.
Table 6.—Total hardness in parts per million of CaCO3 for each tenth of a
cubic centimeter of soap solution when 50 cc. of the sample is titrated.

Cubic
centimeters
0.0. 0.1. 0.2. 0.3. 0.4. 0.5. 0.6. 0.7. 0.8. 0.9.
of soap
solution.
0.0 0.0 1.6 3.2
1.0 4.8 6.3 7.9 9.5 11.1 12.7 14.3 15.6 16.9 18.2
2.0 19.5 20.8 22.1 23.4 24.7 26.0 27.3 28.6 29.9 31.2

3.0 32.5 33.8 35.1 36.4 37.7 38.0 40.3 41.6 42.9 44.3
4.0 45.7 47.1 48.6 50.0 51.4 52.9 54.3 55.7 57.1 58.6
5.0 60.0 61.4 62.9 64.3 65.7 67.1 68.6 70.0 71.4 72.9

6.0 74.3 75.7 77.1 78.6 80.0 81.4 82.9 84.3 85.7 87.1
7.0 88.6 90.0 91.4 92.9 94.3 95.7 97.1 98.6 100.0 101.5
This table does not provide for the use of so large volume
of soap solution for a single determination as former ones
because the end-point becomes somewhat obscured in the
presence of magnesium if more than 7 cc. is used.
Second method of standardization.—Dilute 100 cc. of the
stock soap solution to 1 liter with 70 per cent alcohol. This
dilute solution should be of such strength that approximately
6.4 cc. of it will give a permanent lather when 20 cc. of
standard calcium chloride solution diluted to 50 cc. with
distilled water is titrated with it. Determine the amount of
soap solution required to give a permanent lather with 50
cc. of distilled water and with 5, 10, 15, and 20 cc. of
standard calcium chloride solution diluted to 50 cc. with
distilled water. Finally plot on cross-section paper a curve
showing the relation of various quantities of soap solution to
corresponding quantities of standard calcium carbonate
solution and therefore to parts per million of hardness.

Procedure.—Measure 50 cc. of the water into a 250 cc.
bottle and add to it soap solution in small quantities in
precisely the same manner as described under the
standardization of the soap solution. From the number of
cubic centimeters of soap solution used obtain from Table 6
or from the plotted curve the total hardness of the water in
parts per million of calcium carbonate.
To avoid mistaking the false or magnesium end-point for
the true one[35] when adding the soap solution to waters
containing magnesium salts, read the burette after the
titration is apparently finished, and add about 0.5 cc. more
of soap solution. If the end-point was due to magnesium the
lather will disappear. Soap solution must then be added until
the true end-point is reached. Usually the false lather
persists for less than five minutes.
If more than 7 cc. of soap solution is required for 50 cc. of
the water take less of the sample and dilute it to 50 cc. with
distilled water which has been recently boiled and cooled.
This step reduces somewhat the disturbing influence of
magnesium,[107a] which consumes more soap than an
equivalent weight of calcium.
At best the soap method is not a precise test on account of
the different relative amounts of calcium and magnesium in
different waters. For hard waters, especially in connection
with processes for purification and softening, it is advised
that this method be not exclusively used. If the same water
is frequently analyzed it may be of assistance to standardize
the soap solution against a mixture of calcium and
magnesium salts, the relative proportions of which
approximate those found in the water.
The strength of the soap solution should be determined
from time to time, to make sure that it has not materially
changed. Record all results in parts per million of calcium
carbonate.

One English degree of hardness, Clark’s scale, is
equivalent to 1 grain per Imperial gallon of calcium
carbonate. One French degree of hardness is equivalent to 1
part per 100,000 of calcium carbonate. One German degree
of hardness is equivalent to 1 part per 100,000 of calcium
oxide, and multiplied by 17.9 gives parts per million of
calcium carbonate. The relations of these various scales are
indicated in Table 7.
Table 7.—Conversion table for hardness.

Equivalent.
Unit. Parts per Clark French German
million. degrees. degrees. degrees.
One part per
1.00 0.07 0.10 0.056
million
One Clark
14.3 1.00 1.43 .80
degree
One French
10.0 .70 1.00 .56
degree
One German
17.9 1.24 1.78 1.00
degree

TOTAL HARDNESS BY SODA REAGENT METHOD.[47][74]
[81][94d]

Add standard sulfuric acid to 200 cc. of the sample until
the alkalinity is neutralized. (See Procedure with methyl
orange, p. 37.) Then apply the non-carbonate hardness
method (pp. 34–35). This method gives fairly satisfactory
estimates of total hardness of hard waters.

TEMPORARY HARDNESS BY TITRATION WITH ACID.

Determine the alkalinity in presence of methyl orange (see
p. 37) in the original sample and also in the sample after
boiling, cooling, restoring to the original volume with boiled
distilled water, and filtering. The difference between the

two, if any, is the temporary hardness. This is the most
accurate method of determining the temporary hardness of
ordinary waters. Iron bicarbonate is included as a part of the
temporary hardness.

NON-CARBONATE HARDNESS BY SODA REAGENT
METHOD.[47][74][81][94d]

The use of soda reagent does not avoid entirely the error
due to solubility of the salts of calcium and magnesium;
consequently, if much depends on the results, as in water
softening, gravimetric determinations of the calcium and
magnesium that remain in solution should be made and a
correction should be applied for those amounts.
Reagent.—Prepare soda reagent from equal parts of
sodium hydroxide and sodium carbonate. It should be
approximately tenth normal.
Procedure.—Measure 200 cc. of the sample and 200 cc. of
distilled water into 500 cc. Jena or similar glass Erlenmeyer
flasks. Treat the contents of each flask in the following
manner. Boil 15 minutes to expel free carbon dioxide. Add
25 cc. of soda reagent. Boil 10 minutes, cool, rinse into 200
cc. graduated flasks, and dilute to 200 cc. with boiled
distilled water. Filter, rejecting the first 50 cc., and titrate 50
cc. of each filtrate with N/50 sulfuric acid in the presence of
methyl orange or erythrosine indicator. The non-carbonate
hardness in parts per million of calcium carbonate is equal
to 20 times the difference between the number of cubic
centimeters of sulfuric acid required for the soda reagent in
distilled water and the number of cubic centimeters of N/50
sulfuric acid required for the soda reagent in the sample.
Water naturally containing bicarbonate and carbonate in
excess of calcium and magnesium requires a larger amount
of acid to neutralize the sample after it has been treated

than is required to neutralize the volume of soda reagent
originally added. (See p. 39.)

NON-CARBONATE HARDNESS BY SOAP METHOD.

Non-carbonate hardness may be calculated for waters
which are soft or moderately hard in a fairly satisfactory
manner by deducting the total alkalinity from the total
hardness by the soap method (pp. 31–34). For waters that
are very hard, and particularly those that contain much
magnesium, this method is not advised.

ALKALINITY.[11][18][47][97]

The alkalinity of a natural water represents its content of
carbonate, bicarbonate, borate, silicate, phosphate, and
hydroxide. Alkalinity is determined by neutralization with
standard sulfuric acid or potassium bisulfate in the presence
of phenolphthalein and either methyl orange, erythrosine, or
lacmoid as indicators. Methyl orange may be used except in
waters containing aluminium sulfate or iron sulfate. The
relations between estimates in presence of these indicators
and the carbonate, bicarbonate, and hydroxide radicles are
indicated in Table 8. The alkalinity of carbonates in the
presence of phenolphthalein is different from that in the
presence of methyl orange, partly because of loss of carbon
dioxide and partly because of defects in phenolphthalein as
an indicator in such conditions.
Table 8.—Relations between alkalinity to phenolphthalein and that to
methyl orange, erythrosine, or lacmoid, in presence of bicarbonate,
carbonate, and hydroxide.

Value of radicle expressed in terms of calcium
Result of
carbonate.
titration.[C]
Bicarbonate. Carbonate. Hydroxide.
P=0 T 0 0
P < 1/2T T − 2P 2P 0
P = 1/2T 0 2P 0
P > 1/2T 0 2(T − P) 2P − T
P=T 0 0 T

C. T = Total alkalinity in presence of methyl orange, erythrosine, or lacmoid.
P = Alkalinity in presence of phenolphthalein.
Reagents.—1. Sulfuric acid or potassium bisulfate. A N/50
solution.
2. Phenolphthalein indicator. Dissolve 5 grams of a good
quality of phenolphthalein in 1 liter of 50 per cent alcohol.

Neutralize with N/10 potassium hydroxide. The alcohol
should be diluted with boiled distilled water.
3. Methyl orange indicator. Dissolve 0.5 gram of a good
grade of methyl orange in 1 liter of distilled water. Keep the
solution in the dark.
4. Lacmoid indicator. Dissolve 2.0 grams of lacmoid in 1
liter of 50 per cent alcohol. Dilute the alcohol with freshly
boiled distilled water.
5. Erythrosine indicator. Dissolve 0.5 gram of erythrosine
(the sodium salt) in 1 liter of freshly boiled distilled water.

PROCEDURE WITH PHENOLPHTHALEIN.

Add 4 drops of phenolphthalein indicator to 50 or 100 cc.
of the sample in a white porcelain casserole or an
Erlenmeyer flask over a white surface. If the solution
becomes colored, hydroxide or normal carbonate is present.
Add N/50 sulfuric acid from a burette until the coloration
disappears.
The phenolphthalein alkalinity in parts per million of
calcium carbonate is equal to the number of cubic
centimeters of N/50 sulfuric acid used multiplied by 20 if 50
cc. of the sample was used, or by 10 if 100 cc. was used.

PROCEDURE WITH METHYL ORANGE.

Add 2 drops of methyl orange indicator to 50 or 100 cc. of
the sample, or to the solution to which phenolphthalein has
been added, in a white porcelain casserole or an Erlenmeyer
flask over a white surface. If the solution becomes yellow,
hydroxide, normal carbonate, or bicarbonate is present. Add
N/50 sulfuric acid from a burette until the faintest pink
coloration appears. The methyl orange alkalinity in parts per
million of calcium carbonate is equal to the total number of

cubic centimeters of N/50 sulfuric acid used multiplied by 20
if 50 cc. of the sample was used, or by 10 if 100 cc. was
used.

PROCEDURE WITH LACMOID.

Add 4 drops of lacmoid indicator to 50 or 100 cc. of the
sample in a porcelain casserole or an Erlenmeyer flask. Add
N/50 sulfuric acid from a burette until within 1 or 2 cc. of
the amount necessary for neutralization has been added.
Heat the solution until bubbles of steam begin to break at
the surface. Remove the dish from the source of heat and
continue the titration until a drop of the acid striking the
surface of the liquid and sinking to the bottom of the vessel
produces no change in the uniform reddish or purple color of
the solution. The calculation is the same as for
phenolphthalein alkalinity.

PROCEDURE WITH ERYTHROSINE.

Add 5 cc. of neutral chloroform and 1 cc. of erythrosine
indicator to 50 or 100 cc. of the sample in a 250 cc. clear
glass-stoppered bottle. If the chloroform becomes rose
colored on shaking, hydroxide, bicarbonate, or normal
carbonate is present. Add N/50 sulfuric acid from a burette
until the chloroform becomes colorless. A white surface
behind the bottle facilitates detection of a trace of color as
the end-point is approached. The calculation is the same as
with phenolphthalein alkalinity.

BICARBONATE.

Bicarbonate is present if the alkalinity to phenolphthalein
is less than one-half the alkalinity to methyl orange,

erythrosine, or lacmoid. The alkalinity to methyl orange,
erythrosine, or lacmoid is due entirely to bicarbonate if
there is no phenolphthalein alkalinity. If there is
phenolphthalein alkalinity the bicarbonate, in terms of
calcium carbonate, is equal to the methyl orange,
erythrosine, or lacmoid alkalinity minus twice the
phenolphthalein alkalinity. Bicarbonate, carbon dioxide as
bicarbonate, and half-bound carbon dioxide can be
calculated as follows:
Bicarbonate (HCO3) = 1.22 times the bicarbonate
expressed in terms of calcium carbonate.
Carbon dioxide (CO2) as bicarbonate = 0.88 times the
bicarbonate expressed in terms of calcium carbonate.
Half-bound carbon dioxide (CO2) = 0.44 times the
bicarbonate expressed in terms of calcium carbonate.

NORMAL CARBONATE.[20][94]

Normal carbonate is present if the alkalinity to
phenolphthalein is greater than zero but less than the
alkalinity to methyl orange, erythrosine, or lacmoid. If the
phenolphthalein alkalinity is exactly equal to one-half the
methyl orange, erythrosine, or lacmoid alkalinity the
alkalinity is due entirely to normal carbonate. If the
phenolphthalein alkalinity is less than one-half the methyl
orange, erythrosine, or lacmoid alkalinity normal carbonate
expressed in terms of calcium carbonate is equal to twice
the phenolphthalein alkalinity. If the phenolphthalein
alkalinity is greater than one-half the methyl orange,
erythrosine, or lacmoid alkalinity the normal carbonate is
equal to twice the difference between the methyl orange,
erythrosine, or lacmoid alkalinity and the phenolphthalein
alkalinity. The carbonate, carbon dioxide as carbonate, and
bound carbon dioxide can be calculated as follows:

Carbonate (CO3) = 0.6 times the normal carbonate
expressed in terms of calcium carbonate.
Carbon dioxide as carbonate (CO2) = 0.44 times the
normal carbonate expressed in terms of calcium carbonate.
Bound carbon dioxide (CO2) is the sum of the carbon
dioxide as carbonate and one-half that as bicarbonate.

HYDROXIDE.[20][94]

If hydroxide, or caustic alkalinity, is present the alkalinity
to phenolphthalein is greater than one-half the alkalinity to
methyl orange, erythrosine, or lacmoid; the alkalinity is due
entirely to hydroxide if the phenolphthalein alkalinity is
equal to the methyl orange, erythrosine, or lacmoid
alkalinity. If the phenolphthalein alkalinity is more than half
and less than all the methyl orange, erythrosine, or lacmoid
alkalinity, hydroxide, expressed in terms of calcium
carbonate, is equal to twice the phenolphthalein alkalinity
minus the methyl orange, erythrosine, or lacmoid alkalinity.

ALKALI CARBONATES.

Waters which contain sodium or potassium carbonates or
bicarbonates contain all of their calcium and magnesium as
carbonates or bicarbonates. That is, they possess no non-
carbonate hardness (sulfates, nitrates or chlorides of
calcium and magnesium).
The most accurate method is to determine the total
alkalinity by titration with N/50 sulfuric acid, using methyl
orange, erythrosine, or lacmoid as an indicator; then
determine the calcium and magnesium content; and subtract
from the total alkalinity the computed alkalinity due to the
calcium and magnesium expressed in terms of calcium

carbonate. The remainder is the alkalinity due to carbonates
and bicarbonates of sodium and potassium.
This determination may also be made by applying the
method, for non-carbonate hardness with soda reagent (see
p. 35), and by noting the excess of acid required to
neutralize the alkaline carbonates originally present.
With present information as to solubilities of the normal
carbonates of calcium and magnesium, it is difficult in their
presence to measure slight quantities of carbonates of
sodium or potassium.

ACIDITY.[24d][37]

Waters may have an acid reaction because of the presence
of free carbon dioxide, mineral acids, or some of their salts,
especially those of iron and aluminium.
Reagents.—1. N/50 sodium carbonate. Dissolve 1.06 grams
of anhydrous sodium carbonate in 1 liter of boiled distilled
water that has been cooled in an atmosphere free from
carbon dioxide. Preserve this solution in bottles of resistant
glass protected from the air by tubes filled with soda-lime.
One cc. is equivalent to 1 mg. of CaCO3.
2. N/22 sodium carbonate. Dissolve 2.41 grams of
anhydrous sodium carbonate in 1 liter of boiled distilled
water that has been cooled in an atmosphere free from
carbon dioxide. Preserve this solution in bottles of resistant
glass protected from the air by tubes filled with soda-lime.
One cc. is equivalent to 1 mg. of CO2.
3. Phenolphthalein indicator (see p. 36).
4. Methyl orange indicator (see p. 36).

TOTAL ACIDITY.

Procedure.—Add 4 drops of phenolphthalein indicator to
50 or 100 cc. of the sample in a white porcelain casserole or
an Erlenmeyer flask over a white surface. Add N/50 sodium
carbonate until the solution turns pink. The total acidity in
parts per million of calcium carbonate is equal to the
number of cubic centimeters of N/50 sodium carbonate used
multiplied by 20 if 50 cc. of the sample was used, or by 10 if
100 cc. was used.

FREE CARBON DIOXIDE.[20][23][61][87][88][94a][118]

Carbon dioxide may exist in water in three forms—free
carbon dioxide, bicarbonate (pp. 37–38), and carbonate (p.
38). One-half the carbon dioxide as bicarbonate is known as
the half-bound carbon dioxide. The carbon dioxide as
carbonate plus one-half that as bicarbonate is known as the
bound carbon dioxide.
Procedure.—Pour 100 cc. of the sample into a tall narrow
vessel, preferably a 100 cc. Nessler tube. Add 10 drops of
phenolphthalein indicator, and titrate rapidly with N/22
sodium carbonate, stirring gently, until a faint but
permanent pink color is produced. The free carbon dioxide
(CO2) in parts per million is equal to 10 times the number of
cubic centimeters of N/22 sodium carbonate used.
Because of the ease with which free carbon dioxide
escapes from water, particularly when the gas is present in
large amount, a special sample should be collected for this
determination, which should preferably be made at the time
of collection. If the analysis cannot be made at the time of
collection approximate results with water not too high in
free carbon dioxide may be obtained on samples collected in
bottles completely filled so as to leave no air space under the
stopper. Bottled samples should be kept, until tested, at a
temperature lower than that of the water when collected. If
mineral acids or certain salts are present correction must be
made. At best, the results of the titration are uncertain
because the proper end-point for correct results differs in
color with different types of water.

FREE MINERAL ACIDS.

Procedure.—Add 2 drops of methyl orange indicator to 50
or 100 cc. of the sample in a white porcelain casserole or an
Erlenmeyer flask over a white surface. Add N/50 sodium
carbonate from a burette until the pink coloration of the
solution disappears. The acidity due to free mineral acids,

expressed in terms of calcium carbonate, is equal to the
number of cubic centimeters of N/50 sodium carbonate used
multiplied by 20 if 50 cc. of the sample was used, or by 10 if
100 cc. was used.

MINERAL ACIDS AND SULFATES OF IRON AND
ALUMINIUM.[24d][37]

Procedure.—Modify the method for free mineral acids by
titrating the water at boiling temperature in the presence of
phenolphthalein indicator. The acidity due to free mineral
acids and sulfates of iron and aluminium, expressed in terms
of calcium carbonate, is equal to the number of cubic
centimeters of N/50 sodium carbonate used multiplied by 20
if 50 cc. of the sample was used, or by 10 if 100 cc. was
used.
The acidity due to sulfates of iron and aluminium is equal
to the acidity due to mineral acids and sulfates minus the
acidity due to mineral acids. The acidity due to ferrous and
ferric sulfate can be calculated from the determined amount
of these salts (pp. 43–48). The acidity due to aluminium
sulfate is equal to the acidity due to total acid sulfates minus
that due to iron sulfates.
Acidity shall be reported in parts per million of calcium
carbonate (CaCO3). Sulfate (SO4) equals parts per million of
calcium carbonate multiplied by 0.96.
Carbon dioxide (CO2) equals parts per million of calcium
carbonate multiplied by 0.44.

CHLORIDE.[16]

Chloride in water and sewage has its origin in common
salt, from mineral deposits in the earth, from ocean vapors
carried inland by the wind, or from polluting materials like
sewage and trade wastes, which contain the salt used in the
household and in manufacturing. Comparison of the chloride
content of a water with that of other waters in the vicinity
known to be unpolluted frequently affords useful information
as to its sanitary quality. If, however, the chloride normally
exceeds 20 parts per million because of chloride-bearing
mineral deposits the chloride content of a water has little
sanitary significance.
Reagents.—1. Standard sodium chloride solution. Dissolve
16.48 grams of pure fused sodium chloride in 1 liter of
distilled water. Dilute 100 cc. of this stock solution to 1 liter
in order to obtain a standard solution each cubic centimeter
of which contains 0.001 gram of chloride.
2. Standard silver nitrate solution. Dissolve about 2.40
grams of silver nitrate crystals in 1 liter of distilled water.
Standardize this with the standard salt solution, and adjust,
correcting for volume (see p. 43), so that 1 cc. will be exactly
equivalent to 0.0005 gram of chloride.
3. Potassium chromate indicator. Dissolve 50 grams of
neutral potassium chromate in a little distilled water. Add
enough silver nitrate to produce a slight red precipitate.
Filter and dilute the filtrate to 1 liter with distilled water.
4. Aluminium hydroxide. Electrolyze ammonia-free water,
using aluminium electrodes. Wash the precipitate until it is
free from chloride, ammonia, and nitrite. Or dissolve 125
grams of potassium or ammonium alum in 1 liter of distilled
water. Precipitate the aluminium by adding cautiously
ammonium hydroxide. Wash the precipitate in a large jar by

successive additions and decantations of distilled water until
free from chloride, nitrite, and ammonia.
Procedure.—Add 1 cc. of potassium chromate indicator to
50 cc. of the sample in a 6–inch white porcelain evaporating
dish or a 150 cc. Erlenmeyer flask over a white surface.
Titrate with the silver nitrate solution under similar
conditions of volume, light, and temperature as were used in
standardizing the silver nitrate until a faint reddish
coloration is perceptible. The detection of the end-point is
facilitated by comparison of the contents of the porcelain
dish with those of another dish containing the same quantity
of potassium chromate indicator in 50 cc. of distilled water.
Some analysts prefer to make the titration in a dark-room
provided with a yellow light. The end-point is very sharp by
electric light and also by daylight with photographic yellow
glass. The titration may be made in Nessler tubes[68a] if the
solutions are standardized under similar conditions.
If the amount of chloride is very high use 25 cc., or even a
smaller quantity, dilute the volume taken to 50 cc. with
distilled water. If the amount of chloride is very low
concentrate 250 cc. of the sample to 50 cc. by evaporation.
Rotate the liquid to make sure that no residue remains
undissolved on the walls of the dish, and, if necessary, use a
rubber-tipped glass rod to assist in this operation.
Chloride is determined by some observers by extracting
with hot distilled water the residue in the platinum dish in
the determination of the residue on evaporation and
proceeding as just described. This is permissible if a little
sodium carbonate is added before evaporation to prevent
loss of chloride through decomposition of magnesium
chloride in the residue.
If the sample has a color greater than 30 it should be
decolorized by shaking it thoroughly with washed aluminium
hydroxide (3 cc. to 500 cc. of the sample) and allowing the
precipitate to settle. Make the determination on a portion of

the clarified sample, filtered if necessary. If the sample is
acid, neutralize it with sodium carbonate; if hydroxide is
present, add dilute sulfuric acid until the cold liquid will just
discharge the color of phenolphthalein. If the presence of
sulfide and sulfocyanate renders it necessary, make proper
corrections[24c][100b] or modifications in treatment.
Make correction for the error due to variations in the
volume of the liquid and precipitate by means of the
formula[39] X = 0.003V + 0.02, in which X = the correction in
cubic centimeters of silver nitrate solution and V = cubic
centimeters of liquid at the end of the titration. If 50 cc. of
the sample is titrated chloride (Cl) in parts per million is
equal to the number of cubic centimeters of silver nitrate
solution multiplied by 10. The correction to be applied is 0.2
cc. unless unusual accuracy is required.

IRON.[94b][98]

Iron occurs in natural waters in both ferrous and ferric
condition, depending on the source of the sample. In ground
waters the iron is usually in an unoxidized and soluble
condition, sometimes combined with carbonic or sulfuric
acid, and also in combination with organic matter. Many
waters, especially those that have been exposed to the air,
contain the iron in the form of a colloidal hydroxide. Silt-
bearing waters often contain much iron in suspension,
usually in an oxidized form. Sewages and sewage effluents,
particularly those receiving manufacturing wastes, contain
various forms of iron of different degrees of solubility,
oxidation, and coagulation.

TOTAL IRON.[59][63b]

COLORIMETRIC METHOD.

Reagents.—1. Standard iron solution. Dissolve 0.7 gram of
crystallized ferrous ammonium sulfate in 50 cc. of distilled
water to which 20 cc. of dilute sulfuric acid has been added.
Warm the solution slightly and add potassium permanganate
until the iron is completely oxidized. Dilute the solution to 1
liter. One cc. of the standard solution equals 0.1 mg. Fe.
2. Potassium sulfocyanide solution. Dissolve 20 grams of
the salt in 1 liter of distilled water.
3. Dilute hydrochloric acid. One volume of acid (Sp. gr.
1.2) and one volume of distilled water. This shall be free
from nitric acid.
4. N/5 potassium permanganate. Dissolve 6.30 grams of
the salt in distilled water and dilute to 1 liter.
5. Hydrochloric acid. Concentrated, free from iron.

6. Nitric acid. Concentrated, free from iron.
7. Nitric acid. 5N, free from iron.
First procedure.—Evaporate 100 cc. of the water to
dryness, or use the residue left after the determination of
residue on evaporation (p. 29). Ignite the residue at a low
red heat taking care not to heat it hot enough to make the
iron difficultly soluble. Cool the dish and add 5 cc. of
concentrated hydrochloric acid. Moisten the inner surface of
the dish. Warm the solution for two or three minutes, and
again moisten the inner surface of the dish by permitting the
hot acid to flow over it. Wash the hot solution from the dish
into a 50 cc. Nessler tube, filtering if necessary through
paper that has been washed with hot water. Dilute to 50 cc.,
and add 3 drops of potassium permanganate solution. Add 5
cc. of potassium sulfocyanide solution, mix, and compare
with standards.
If it is not convenient to use the residue on evaporation
and if the sample is relatively free from organic matter, boil
50 cc. of the sample with 5 cc. of 5N nitric acid for five
minutes. Add a few drops of permanganate and 5 cc. of
potassium sulfocyanide and compare with standards, using
nitric acid in place of hydrochloric acid in the standards.
This method is excellent for ground waters. The
permanganate and acid liberate chlorine in water high in
chloride, and produce a permanent yellow color which
interferes with the determination, unless the sample is first
diluted to 50 cc. An excess of permanganate, reacting with
hydrochloric acid, causes similar trouble. The amounts of
hydrochloric acid, 5 cc., and of sulfocyanide, 5 cc., should be
approximately measured because more acid lightens the
color whereas more sulfocyanide deepens it. This is
especially important if permanent standards are used.
Second procedure.—For surface waters containing small
amounts of organic matter, the method of Klut[59] is
recommended. Samples containing small amounts of iron

should be concentrated, if possible, until at least 0.5 mg. of
iron is present in the volume tested. Boil the sample in a
beaker with 2 to 3 cc. of concentrated nitric acid free from
iron, adding permanganate if necessary to destroy the
organic matter. To the hot liquid add ammonia in slight
excess and warm until the smell of ammonia is hardly
discernible. Filter and wash with water at 70° to 80° C.
containing a little ammonia. Dissolve the iron in the beaker
and on the filter paper in 5 cc. of concentrated hydrochloric
acid, and wash with hot water until the iron is all dissolved,
collecting the filtrate in a 50 cc. Nessler tube. Dilute to 50
cc. Add potassium sulfocyanide and determine the iron by
comparison with standards.

Comparison with iron standards.

First procedure.—Prepare standards containing amounts
of standard iron solution ranging from 0.05 to 4 cc.
according to the quantity of iron in the sample. Dilute these
amounts with water to about 40 cc. Add 5 cc. of dilute
hydrochloric acid and 3 drops of potassium permanganate to
each tube and dilute to 50 cc. Add 5 cc. of the potassium
sulfocyanide to each of the standard solutions at the same
time that it is added to the samples of water under
examination, and compare immediately after mixing. If 100
cc. of the sample is used the iron in parts per million is equal
to the number of cubic centimeters of the standard iron
solution in the standard that the sample matches.
Second procedure.—For a small number of determinations
it is more convenient to run the standard iron solution into a
Nessler tube containing the acid, distilled water, and
potassium sulfocyanide until the color matches that of the
sample tested. When determining iron in three or four
samples the colors may be matched in the order of their
intensity and the volumes of standard iron solution required
for each tube may be read from the burette.

Comparison with permanent standards.

Reagents.—1. Platinum solution. Dissolve 2 grams of
potassium platinic chloride (PtCl4.2KCl) in distilled water,
add 100 cc. of concentrated hydrochloric acid, and dilute to
1 liter with distilled water.
2. Cobalt solution. Dissolve 24 grams of dry cobaltous
chloride crystals (CoCl2.6H2O) in a small amount of distilled
water, add 100 cc. of strong hydrochloric acid, and dilute to
1 liter with distilled water.
Procedure.—Prepare a series of permanent standards by
diluting to 50 cc. with distilled water the amounts of
platinum and cobalt solutions, in 50 cc. Nessler tubes,
indicated in Table 9. Compare the sample with these
standards, and calculate the parts per million of iron.
Table 9.—Preparation of permanent standards for the determination of iron.

Value in standard iron solution. Platinum solution. Cobalt solution.
cc. cc. cc.
0.0 0 0.0
.1 2 1.0
.3 6 3.0
.5 10 5.0
.7 14 7.5
1.0 20 11.0
1.5 28 17.0
2.0 35 24.0
2.5 39 32.0
3.0 39 43.0
3.5 40 55.0

VOLUMETRIC METHOD.[24f]

Some samples of sewage and water mixed with trade
wastes and mine drainage contain so much iron that it is
preferable to use the volumetric method described on page
57 for the determination of both total and dissolved iron,

rather than to work with quantities small enough to permit
application of the colorimetric methods just described. If
iron is present in large quantities in suspension, as in some
sewages and septic tank effluents, it may be filtered off and
the residue washed, ignited, and fused with potassium and
sodium carbonate. The fusion is then extracted with
hydrochloric acid and the iron determined as on page 57.
Samples containing much organic matter should be
evaporated to dryness with 0.5 cc. of concentrated sulfuric
acid and the residue then ignited before estimation of iron.

DISSOLVED IRON.

Determine, by the method described for total iron, the iron
in the sample after filtration. Iron may precipitate from some
samples during filtration.

SUSPENDED IRON.

The suspended iron is the difference between total iron in
the unfiltered sample and dissolved iron in the filtered
sample.

FERROUS IRON.[24e]

Determine the total ferrous iron in an unfiltered sample
and the dissolved ferrous iron in a filtered sample.
Reagents.—1. Standard iron solution. Dissolve 0.7 gram of
crystallized ferrous ammonium sulfate in a large volume of
freshly boiled distilled water to which 10 cc. of dilute
sulfuric acid has been added and dilute to 1 liter. This
solution should be freshly prepared when needed. One cc. of
this standard solution contains 0.1 mg. of Fe.

2. Potassium ferricyanide solution. Dissolve 5 grams of the
salt in 1 liter of distilled water. Use a freshly prepared
solution.
3. Dilute sulfuric acid. Dilute 1 part of sulfuric acid,
specific gravity 1.84, with 5 parts of distilled water.
Procedure.—Add 10 cc. of dilute sulfuric acid to 50 cc. of
the sample, remove the suspended matter by filtration if
necessary, and add 15 cc. of potassium ferricyanide solution.
Dilute the solution to 100 cc. with distilled water. Compare
the color developed in the sample with that in standards
made at the same time from the ferrous iron solution. Place
in 100 cc. Nessler tubes, in the following order, 75 cc. of
distilled water, 10 cc. of dilute sulfuric acid, and 15 cc. of
potassium ferricyanide solution, and mix well the contents of
each tube. Prepare as many tubes in this way as are needed.
Add various quantities of standard ferrous iron solution to
several tubes, mix well, and compare the resulting colors
with the samples immediately.

FERRIC IRON.

The amount of ferric iron in solution and suspension is
equal to the difference between the total iron and the
ferrous iron obtained by the methods described.

MANGANESE.

If the sample contains less than 10 parts per million of
manganese, use a colorimetric method in which the
manganous salt is oxidized to permanganate and the color
produced thereby is compared with that of a standard
solution similarly treated. The persulfate method and the
bismuthate method are suitable. If the sample contains more
than 10 parts per million of manganese it is sometimes
preferable to use a volumetric or gravimetric method.

PERSULFATE METHOD.

Reagents.—1. Nitric acid. Dilute concentrated nitric acid
with an equal volume of distilled water. Free the diluted acid
from brown oxides of nitrogen by aeration.
2. Silver nitrate. Dissolve 20 grams of silver nitrate in 1
liter of distilled water.
3. Standard manganous sulfate. Dissolve 0.288 gram of
purest potassium permanganate in about 100 cc. of distilled
water. Acidify the solution with sulfuric acid and heat to
boiling. Add slowly a sufficient quantity of dilute solution of
oxalic acid to discharge the color. Cool and dilute to 1 liter.
One cc. of this solution contains 0.1 mg. of manganese.
4. Ammonium persulfate. Crystals, free from chloride.
Procedure.—Use an amount of the sample that contains
not more than 0.2 mg. of manganese. Add 2 cc. of nitric acid
and boil down to about 50 cc. Precipitate the chloride with
silver nitrate solution, adding at least 1 cc. in excess. Shake
and heat to coagulate the precipitate, and filter. A sample
that contains much chloride should be evaporated with a few
drops of sulfuric acid until white fumes appear and then
diluted before the nitric acid and silver nitrate are added as

directed above. If the sample is highly colored by organic
matter it should be evaporated with sulfuric acid, and the
residue ignited and dissolved in dilute nitric acid. Add about
0.5 gram of ammonium persulfate crystals and warm the
solution until the maximum permanganate color is
developed. This usually takes about ten minutes. At the same
time prepare standards by diluting portions of 0.2, 0.4, 0.6
cc., etc. of the standard manganous sulfate solution to about
50 cc. and treating them exactly as the sample was treated.
Transfer the sample and the standards to 50 cc. Nessler
tubes, and compare the colors immediately. Manganese in
parts per million is equal to the number of cubic centimeters
of standard manganous sulfate solution in the tube that the
sample matches multiplied by 100, divided by the number of
cubic centimeters of the sample used.

BISMUTHATE METHOD.[2a][113]

Reagents.—1. Nitric acid. Dilute 1 part of concentrated
nitric acid with 4 parts of distilled water. Free the dilute acid
from brown oxides of nitrogen by aeration.
2. Sulfuric acid. Dilute 1 part of concentrated sulfuric acid
with 3 parts of distilled water.
3. Dilute sulfuric acid. Dilute 25 cc. of concentrated acid to
1 liter with distilled water. Add enough permanganate
solution to color faintly the dilute acid.
4. Standard manganous sulfate. The standard solution of
manganous sulfate prepared as described under persulfate
method (p. 48) should be used and the standards should be
prepared by following the same procedure as is used for the
sample. This solution is more permanent than a solution of
potassium permanganate, which may, however, be used. To
prepare it dissolve 0.288 gram of potassium permanganate
in distilled water and dilute the solution to 1 liter.
5. Sodium bismuthate. Purest dry salt.

Procedure.—Use an amount of the sample that contains
not more than 0.2 mg. of manganese. Add 0.5 cc. of sulfuric
acid and evaporate to dryness. Heat until the sulfuric acid is
volatilized and ignite the residue. Dissolve in 40 cc. of nitric
acid, add about 0.5 gram of sodium bismuthate, and heat
until the permanganate color disappears. Add a few drops of
a solution of ammonium or sodium bisulfate to clear the
solution and again boil to expel oxides of nitrogen. Remove
from the source of heat, cool to 20° C., again add 0.5 gram
of sodium bismuthate, and stir. When the maximum
permanganate color has developed, filter through an
alundum or Gooch crucible containing an asbestos mat
ignited and washed with potassium permanganate. Wash the
precipitate with dilute sulfuric acid until the washings are
colorless. Transfer the filtrate to a 50 cc. Nessler tube and
compare the color of it with that of standards prepared from
the potassium permanganate solution. To prepare the
standards, dilute portions of 0.2, 0.4, 0.6 cc., etc. of the
permanganate solution to 50 cc. with dilute sulfuric acid.
The content of manganese is calculated as described under
persulfate method (p. 49).

LEAD, ZINC, COPPER, AND TIN.[7][60]

Determinations of lead, zinc, copper, and tin are important
in certain mining regions and in places where the water has
a solvent action on pipes and other containers. The use of
certain “germicides” also makes it necessary to test for
some of these metals.
Lead, zinc, and copper may be determined colorimetrically
or electrolytically. The colorimetric methods are not so
accurate as a combination of both, and are chiefly of value
as qualitative tests.
It is possible to make a rough estimation of the amount of
lead in clear waters by acidifying with acetic acid, saturating
with hydrogen sulfide, and comparing the color produced
with that produced by standard lead solutions in Nessler
tubes, treated in similar manner. This method, however, is
not applicable if the water is colored or contains iron.
Reagents.—1. Standard lead solution. Dissolve 1.60 grams
of lead nitrate (Pb(NO3)2) in 1 liter of distilled water. One cc.
of this solution contains 1 mg. of lead (Pb). As a check it is
desirable to determine lead as sulfate in a measured portion
of this solution.
2. Standard copper solution. Dissolve about 0.8 gram of
copper sulfate crystals (CuSO4.5H2O) in water and, after the
addition of 1 cc. of concentrated sulfuric acid, dilute the
solution to 1 liter. Determine the copper in 100 cc. of this
solution in the usual way by electrolytic deposition. Dilute
the solution so that 1 cc. contains 0.2 milligram copper (Cu).
This solution is permanent.
3. Ammonium chloride. Twenty-five per cent solution.
4. Ammonium acetate. Fifty per cent solution.
5. Ammonium hydroxide. (Sp. gr. 0.96.)

6. Hydrogen sulfide. Saturated solution.
7. Potassium sulfide. An alkaline solution of potassium
sulfide made by mixing equal volumes of 10 per cent
potassium hydroxide and a saturated aqueous solution of
hydrogen sulfide.
8. Potassium oxalate. Crystals.
9. Potassium sulfate. Crystals.
10. Alcohol. Ninety-five per cent.
11. Alcohol. Fifty per cent.
12. Acetic acid. Fifty per cent.
13. Nitric acid. Concentrated acid (Sp. gr. 1.42).
14. Nitric acid. Dilute 1 part of the concentrated acid to 10
parts with distilled water.
15. Hydrochloric acid. (Sp. gr. 1.20.)
16. Sulfuric acid. Concentrated acid (Sp. gr. 1.84).
17. Sulfuric acid. Dilute the concentrated acid with an
equal volume of distilled water.
18. Urea. Crystals.

LEAD.

Concentrate (1)[D] rapidly by boiling in a 7–inch porcelain
dish over a free flame 3 or 4 liters of the sample to be tested,
or more if very small amounts of the metals are present, to a
volume of about 30 cc. Add 10 or 15 cc. of ammonium
chloride solution to assist in the separation of the sulfides,
then add a few drops of concentrated ammonium hydroxide,
and saturate with hydrogen sulfide. Allow to stand some
time, preferably over night, add a little more ammonium
hydroxide and hydrogen sulfide, boil the contents of the dish
a few minutes, and filter. The precipitate (2) may consist of
lead, zinc, copper, and iron sulfides and the suspended
organic matter. The soluble coloring matter is in the filtrate

(3). Wash the precipitate a few times with hot water, place
the precipitate and the filter paper in the original dish and
boil with dilute nitric acid, rubbing down the sides of the
dish, if necessary, to detach any adhering sulfide precipitate.
After again filtering and washing several times with hot
water, evaporate the filtrate and washings in the original
dish to a bulk of 10 to 15 cc., cool, add 5 cc. of concentrated
sulfuric acid, and heat until copious fumes of sulfuric acid
are evolved.
D. The numbers in parentheses refer to tables 10–12, pages 55–56.

If lead is present dilute the contents of the dish slightly
with water, and treat them with 150 cc. of 50 per cent
alcohol, in which the lead sulfate is insoluble. Allow to stand
some time, preferably over night, filter off the lead sulfate,
and wash it with 50 per cent alcohol. Save the filtrate for the
determination of zinc.
Dissolve the precipitate of lead sulfate by boiling the filter
containing it in ammonium acetate solution in a porcelain
dish. (4). Filter into a 50 cc. Nessler tube and wash the filter
with boiling water containing a little ammonium acetate.
Divide this filtrate in halves and treat one-half with
saturated hydrogen sulfide water in order to get an
approximation of the amount of lead present. To the other
half, or an aliquot portion, if a large amount of lead is
present, add a few drops of acetic acid, then an excess of
saturated hydrogen sulfide solution, and compare the color
with that of standards made by treating known amounts of
the standard lead solution with a little acetic acid,
ammonium acetate, and hydrogen sulfide.

ZINC.

If zinc is present and copper is absent concentrate the
filtrate from the lead sulfate to expel the alcohol, and

remove the iron by adding an excess of ammonium
hydroxide. Filter, wash, and acidify the filtrate with sulfuric
acid. Concentrate the filtrate to about 150 cc. and transfer
to a weighed platinum dish. Add 2 grams of potassium
oxalate and 1.5 grams of potassium sulfate. Deposit the zinc
electrolytically by means of a current of about 0.3 ampere
for three hours. After deposition is complete and while the
current is on, siphon off the solution and at the same time
run into the dish a stream of distilled water in order to expel
the free sulfuric acid, which might dissolve some of the zinc
if the circuit were broken. After the acid has been removed
break the circuit, wash the dish with water, then with 95 per
cent alcohol, dry at 70° C., cool, and weigh it. The difference
between this weight (10) and the weight of the platinum dish
equals the amount of metallic zinc. Some difficulty has been
experienced in this determination in obtaining pure
reagents. It is therefore advisable to make blank
determinations with each new lot of reagents and to correct
the results if necessary.
If copper also is present (5) concentrate the filtrate from
the lead sulfate until the alcohol is expelled, and add an
excess of ammonium hydroxide. (6) Remove any iron
precipitate by filtration. Neutralize the filtrate (7) with
sulfuric acid, and add 2 cc. of concentrated sulfuric acid and
1 gram of urea. Electrolyze the solution and determine
copper colorimetrically as described in the procedure for
copper (p. 54). After the copper has been deposited add
ammonium hydroxide to the solution containing the zinc
until nearly all the sulfuric acid has been neutralized,
concentrate to slightly less than the capacity of the platinum
dish, add 1.5 grams of potassium sulfate and 2 grams of
potassium oxalate, and electrolyze for zinc. As this solution
is usually saturated with ammonium salts due to neutralizing
the large quantity of sulfuric acid, it is frequently impossible
to get the zinc deposited firmly on the dish before the salts
interfere by crystallization. To avoid this difficulty, dilute half

the solution and electrolyze it for zinc; or, if the amount of
zinc is very small, precipitate the zinc as sulfide in acetic
acid solution, wash, ignite to oxide, and weigh the
precipitate. This difficulty will not be encountered if copper
is absent as there will then be no excess of ammonium salts.
If lead and copper are known to be absent and zinc alone
is to be determined (13), after treating with sulfuric acid for
separation of lead, slightly dilute the contents of the dish.
Add an excess of ammonium hydroxide to precipitate iron
and filter. Make the filtrate slightly acid with sulfuric acid,
concentrate to about 150 cc., transfer to a weighed platinum
dish, add potassium oxalate and sulfate, and electrolyze the
solution as described for deposition of zinc.

COPPER.[77]

Use 1 liter of a sample containing 0.1 to 1.0 part per
million of copper, and proportionate amounts for other
concentrations. Evaporate to about 75 cc., and wash into a
100 cc. platinum dish. Add 2 cc. of dilute sulfuric acid for
clear and soft waters; add more acid to very alkaline waters
to offset the alkalinity; add 5 cc. of acid to waters carrying
much organic matter or clay to insure the formation of a
soluble copper salt. Then place the dish as the anode in a
direct current circuit, suspend a spiral wire cathode in the
solution so that it is parallel to and about half an inch from
the bottom of the dish, and close the circuit.
Electrolyze for about four hours with occasional stirring,
or over night, if convenient. The current may be supplied by
two gravity cells in series, yielding a current through the
solution of about 0.02 ampere. Lift out the cathode without
previously opening the circuit, and immerse the spiral in a
small amount of dilute nitric acid previously heated to
boiling. Wash off the wire and evaporate the nitric acid
solution to dryness on the water bath. If the presence of

silver is suspected add a few drops of hydrochloric acid
before evaporation. Dissolve the residue in water and wash
it into a 50 cc. Nessler tube. Dilute to 50 cc. and add 10 cc.
of the potassium sulfide solution. The color of the copper
sulfide develops at once and is fairly permanent, lasting at
least several hours. Add 10 cc. of the potassium sulfide
solution to a similar tube containing 50 cc. of distilled water,
and then add to it standard copper solution in 0.2 cc.
portions until the colors of the two tubes match. If 1 liter of
the sample is used copper in parts per million is equal to the
number of cubic centimeters of standard copper solution
required to match the color of the sample multiplied by 0.2.

TIN.

Small quantities of tin are occasionally found in waters
that have passed through tin or tin-lined pipes. This metal, if
present, is precipitated with the iron by ammonia in the
lead, zinc, and copper separations. In the method for copper
alone, it is removed in the same way and may be further
avoided by dissolving the sulfides in concentrated nitric
acid. Any tin present will then separate as an insoluble
compound, which may be ignited and weighed as the oxide
(SnO2).
The following schematic tables illustrate the procedures
given.

Table 10.—Scheme for the separation of lead, zinc, and copper.

1. Concentrate sample. Add 10 cc. NH4Cl, a few drops NH4OH and saturate
with H2S. Allow to stand, add more NH4OH and H2S. Boil, filter, and wash.
2. Dissolve the precipitate in dilute HNO3. Filter 3. Reject the filtrate
and wash. Evaporate to 10 or 15 cc. Cool. Add 5 cc. which contains the
concentrated H2SO4, and heat until white fumes are coloring matter.
given off. Dilute slightly and treat with 150 cc. of 50
per cent alcohol. Allow to stand; filter, and wash with
50 per cent alcohol.
4. The precipitate contains 5. The filtrate
the Pb. Dissolve in NH4C2H3O2 contains the Zn
solution. Filter into a 50 cc. and Cu.
Nessler tube and wash with Concentrate to
water containing NH4C2H3O2. expel alcohol. Add
excess of NH4OH,
Divide filtrate in halves.
Saturate one-half with H2S. filter and wash
precipitate.
Determine the Pb in the other
half by adding HC2H3O2 and 6. Reject the 7. The filtrate
precipitate which contains the Zn and Cu.
H2S and comparing with
contains the Fe. Neutralize with H2SO4.
standards containing known
Add 10 cc.
amounts of Pb.
concentrated H2SO4
and 1 g. urea.
Electrolyze for two
hours with a current of
0.5 ampere. Break
circuit, empty dish and
wash.
8. The deposit is Cu. Immerse 9. The solution contains the Zn. Nearly
the cathode in a small amount neutralize with NH4OH. Concentrate to less
of hot, dilute HNO3; wash off than the capacity of the dish. Add 2 g.
and evaporate to dryness. Take K2C2O4 and 1.5 g. K2SO4. Electrolyze for 3
up in water and wash into a hours with a current of 0.3 ampere. Siphon
Nessler tube. Make up to mark, off solution, break circuit, wash with water,
and add 10 cc. of potassium then alcohol, dry at 70° C., cool and weigh.
sulfide solution. Compare with
standard. If large amount is
present, dry and weigh as Cu. 10. The weighed residue is metallic Zn.

Table 11.—Scheme for determination of copper only.

11. Concentrate sample to 75 cc. Add 2 cc. conc. H2SO4 for clear, soft
waters and 5 cc. for alkaline or turbid waters. Electrolyze following procedure
in 7 and 8.

Table 12.—Scheme for determination of zinc only.

13. Follow scheme for all three metals as given in Table 10 through section 5.
Nearly neutralize the filtrate with H2SO4, concentrate to less than the capacity
of the dish and electrolyze as directed in section 9.

MINERAL ANALYSIS.

RESIDUE ON EVAPORATION.

See description of method (p. 29). The residue should be
dried one hour at 180° C. Turbid waters should be filtered,
and the composition of the suspended matter should be
determined separately or the amount of it reported as
suspended matter.

ALKALINITY AND ACIDITY.

See description of method (pp. 35–41).

CHLORIDE.

See description of method (pp. 41–43).

NITRATE NITROGEN.

See description of method (pp. 23–25).

SEPARATION OF SILICA, IRON, ALUMINIUM,
CALCIUM, AND MAGNESIUM.[10][48]

SILICA.

Evaporate in platinum 100 to 1,000 cc. of the sample or
sufficient if possible to form a residue weighing 0.4 to 0.6
gram, and preferably containing 0.1 to 0.2 gram of calcium.

When the residue is nearly dry add 1 cc. of hydrochloric acid
(1 to 1) and, after moistening the sides of the dish,
evaporate to dryness. Dry at 180° C. and if much organic
matter is present char it in a radiator. Moisten the residue
with dilute hydrochloric acid and expel the excess of acid by
heating on the water bath. Add a few drops of hydrochloric
acid, dissolve in hot water, and filter. Wash the residue with
hot water. Evaporate the filtrate to dryness, repeat the
filtration, and combine the two residues. If great accuracy is
not required the second evaporation with hydrochloric acid
may be omitted. Ignite and weigh the insoluble residue. Add
2 drops of concentrated sulfuric acid and a little hydrofluoric
acid, volatilize the acids, ignite, and weigh again. Report the
loss in weight as silica (SiO2). A weight of non-volatile
matter exceeding 0.5 mg. should be analyzed.

IRON AND ALUMINIUM.

Heat to boiling the filtrate from the insoluble residue,
oxidize with concentrated nitric acid or bromine, and
concentrate to about 25 cc. Add ammonium hydroxide in
slight excess, boil one minute, and filter. Dissolve the
precipitate on the filter in a small amount of hot dilute
hydrochloric acid. Reprecipitate with ammonium hydroxide,
filter, and wash. Unless very accurate results are necessary
this solution and reprecipitation may be omitted. Unite the
two filtrates for determination of calcium. Ignite and weigh
the precipitate. It will comprise oxides of iron and
aluminium and phosphate. If much phosphate is present it
should be determined in a separate sample and a correction
for the amount applied; otherwise it may be neglected.
Determine the iron in the ignited precipitate by fusion with
sodium or potassium pyrosulfate, reduction with zinc, and
titration with potassium permanganate. Aluminium (Al) is
calculated as follows:

Al = 0.53[(Fe2O3 + Al2O3) − 1.43 Fe]

CALCIUM.

Concentrate the filtrate from the separation of iron and
aluminium to about 100 cc., and add an excess of
concentrated solution of ammonium oxalate, little by little,
to the hot ammoniacal solution. Keep the solution warm and
stir at intervals till the precipitate settles readily and leaves
a clear supernatant liquid. Filter, dissolve the precipitate in
a little hot dilute hydrochloric acid, and reprecipitate with
ammonium hydroxide and ammonium oxalate. If great
accuracy is not required this solution and reprecipitation
may be omitted, and the first precipitate may be washed
clean with hot water[64a]. Save the filtrate for determination
of magnesium. Ignite the precipitate and weigh it as calcium
oxide, 71.5 per cent of which is the equivalent of calcium
(Ca); or dissolve the precipitate in hot 2 per cent sulfuric
acid and titrate with a standard solution of potassium
permanganate.

MAGNESIUM.

Acidify with hydrochloric acid the filtrate from the
separation of calcium and concentrate it to about 100 cc.
Add 20 cc. of a saturated solution of microcosmic salt, cool,
and make slightly but distinctly alkaline by adding
ammonium hydroxide, drop by drop. Allow the solution to
stand four hours, then filter and wash with 3 per cent
ammonium hydroxide. Dissolve the precipitate, especially in
the presence of large amounts of sodium or potassium, in a
slight excess of dilute hydrochloric acid and reprecipitate
the magnesium with ammonium hydroxide and a few drops
of microcosmic salt solution. If great accuracy is not
required this solution and reprecipitation may be omitted.
Ignite the precipitate and weigh it as magnesium

pyrophosphate (Mg2P2O7), 21.9 per cent of which is the
equivalent of magnesium (Mg.). If manganese is present[64a]
it is precipitated with the magnesium and a correction for it
should be applied after having determined manganese in a
separate sample. The weight of manganese pyrophosphate
(Mn2P2O7) is 2.58 times the weight of manganese.

SEPARATION OF SULFATE, SODIUM, AND
POTASSIUM.

SULFATE.

Evaporate to dryness 100 to 1,000 cc. of the sample, or
sufficient to obtain a residue weighing 0.4 to 0.6 gram and
containing preferably 0.05 to 0.2 gram of sodium. Acidify the
residue with hydrochloric acid and remove the silica, iron,
and aluminium (pp. 56–57). Make acid and add a hot
solution of barium chloride in slight excess to the hot
filtrate, and warm it, stirring at intervals for one-half hour,
until the precipitate settles readily and leaves a clear
supernatant liquid. Dry, ignite, and weigh the precipitate of
barium sulfate, 41.1 per cent of which is equal to the content
of sulfate (SO4).

SODIUM, POTASSIUM, AND LITHIUM.

Evaporate to dryness the filtrate from the precipitation of
barium sulfate. Add a few cubic centimeters of hot water
and then a saturated solution of barium hydroxide until a
slight film collects on the top of the solution. Filter and wash
the precipitate with hot water. Add to the filtrate an excess
of ammonium hydroxide and ammonium carbonate solution.
Filter, evaporate the filtrate to dryness, dry, and ignite at low
red heat to expel ammonium salts. Repeat the operations
including the addition of barium hydroxide until no

precipitate is obtained by barium hydroxide or by
ammonium hydroxide and ammonium carbonate. Evaporate
the final filtrate to dryness in a weighed platinum dish, dry,
cool, and weigh the residue. Dissolve the residue in a few
cubic centimeters of water, filter, wash the filter paper twice
with hot water, then ignite the filter paper in the platinum
dish. Cool and weigh the residue. Subtract this weight from
the first weight including the residue. The difference is the
weight of the chlorides of sodium and potassium and lithium.
If it is not desired to separate sodium and potassium the
weight of sodium and potassium as sodium may be
calculated from this difference by multiplying it by 0.394.

POTASSIUM.

First procedure.—Add to the solution of the chlorides of
sodium and potassium a few drops of dilute hydrochloric
acid (1 to 3) and 1 cc. of 10 per cent platinic chloride (PtCl4)
for each 30 mg. of the combined chlorides. Evaporate to a
thick syrup on the water bath, then remove dish and allow it
to come to dryness at laboratory temperature. Treat the
residue cold with 80 per cent alcohol and filter. Wash the
precipitate with 80 per cent alcohol until the filtrate is no
longer colored. Dry the precipitate and dissolve it in hot
water. Evaporate the solution to dryness in a platinum dish
and weigh it as potassium platinic chloride (K2PtCl6). The
weight of potassium (K) is 16.1 per cent of this weight and
the equivalent of potassium chloride (KCl) is 30.7 per cent of
this weight. Subtract the equivalent weight of potassium
chloride from the weight of the combined chlorides. The
weight of the sodium is 39.4 per cent of the difference.
Second procedure.[86][103a]—Add to the hot solution of the
combined chlorides 20 per cent perchloric acid (HClO4)
slightly in excess of the amount required to combine with
the bases. One cubic centimeter of 20 per cent perchloric

acid is equivalent to 90 mg. of potassium. Evaporate the
solution to dryness, dissolve the residue in 10 cc. of hot
water and a small amount of perchloric acid, and again
evaporate to dryness. Repeat the addition of water,
perchloric acid, and evaporation until white fumes appear on
evaporating to dryness. Add to the residue 25 cc. of 96 per
cent alcohol containing 0.2 per cent of perchloric acid (1 cc.
of 20 per cent perchloric acid in 100 cc. of 98 per cent
alcohol). Break up the residue with a stirring rod. Decant the
supernatant liquid through a weighed Gooch crucible that
has been washed with 0.2 per cent perchloric acid in
alcohol. If the precipitate is unusually large dissolve it in hot
water and repeat the evaporation with perchloric acid. Wash
the precipitate once by decantation with the 0.2 per cent
perchloric acid in alcohol, transfer the precipitate to the
crucible, and wash it several times with a 0.2 per cent
perchloric acid in alcohol. Dry the crucible at 120–130° C.
for one hour, cool, and weigh it. The increase in weight is
potassium perchlorate (KClO4). The equivalent weight of
potassium is 28.2 per cent and the equivalent weight of
potassium chloride is 53.8 per cent of the potassium
perchlorate. Calculate the content of sodium by difference.

LITHIUM.[34]

Use a large quantity of the sample. Obtain the combined
chlorides of sodium, potassium, and lithium (see pp. 58–59).
Transfer the combined chlorides to a small Erlenmeyer flask
(50 or 100 cc. capacity) and evaporate the solution nearly,
but not quite, to dryness. Add about 30 cc. of redistilled
amyl alcohol. Connect the flask, the stopper of which carries
a thermometer, with a condenser[E] and boil until the
temperature rises approximately to the boiling point of amyl
alcohol (130° C.), showing that all the water has been driven
off. Cool slightly and add a drop of hydrochloric acid to

convert small amounts of lithium hydroxide to lithium
chloride. Connect with the condenser and continue the
boiling to drive off again all water and until the temperature
reaches the boiling point of amyl alcohol. The content of the
flask at this time is usually 15 to 20 cc. Filter through a
small paper or a Gooch crucible into a graduated cylinder
and note exact quantity of filtrate, which determines the
subsequent correction. Wash the precipitate with small
quantities of dehydrated amyl alcohol. Evaporate the filtrate
and washings in a platinum dish to dryness on the steam
bath, dissolve the residue in water, and add a few drops of
sulfuric acid. Evaporate on a steam bath and expel the
excess of sulfuric acid by gentle heat over a flame. Repeat
until carbonaceous matter is completely burned off. Cool and
weigh the dish and contents. Dissolve in a small quantity of
hot water, filter through a small filter, wash, and return filter
to dish, ignite, and weigh. The difference between the
original weight of dish and contents and the weight of the
dish and small amount of residue equals the weight of
impure lithium sulfate. The purity of the lithium sulfate
should be tested by adding small amounts of ammonium
phosphate and ammonium hydroxide, which will precipitate
any magnesium present with the lithium sulfate. Any
precipitate appearing after standing over night should be
collected on a small filter and weighed as magnesium
pyrophosphate, calculated to sulfate, and subtracted from
the weight of impure lithium sulfate. From this weight
subtract 0.00113 gram for every 10 cc. of amyl alcohol
filtrate exclusive of the amyl alcohol used in washing residue
because of the slight solubility of solid mixed chlorides in
amyl alcohol. Calculate lithium from the corrected weight of
lithium sulfate. Dissolve the mixed chlorides from flask and
filter with hot water, evaporate to dryness, ignite gently to
remove amyl alcohol, filter and thoroughly wash;
concentrate the filtrates and washings to 25 to 50 cc.

E. The amyl alcohol may be boiled off without the use of a condenser, but
the vapors are very disagreeable.

To the weight of potassium chloride add 0.00051 gram
for every 10 cc. of amyl alcohol used in the extraction of
the lithium chloride, which corrects for the solubility of the
potassium chloride in amyl alcohol. Calculate to potassium.
The weight of sodium chloride is found by subtracting the
combined weights of lithium chloride and potassium
chloride (corrected) from the total weight of the three
chlorides. Calculate sodium chloride to sodium.

BROMINE, IODINE, ARSENIC, AND BORIC ACID.

Evaporate to dryness a large quantity of the sample to
which a small amount of sodium carbonate has been added.
Boil the residue with distilled water, transfer it to a filter,
and thoroughly wash it with hot water. Dilute the alkaline
filtrate to a definite volume, and determine bromine and
iodine, arsenic, and boric acid in aliquot portions of it.

BROMINE AND IODINE.[10]

Reagents.—1. Sulfuric acid. 1 to 5.
2. Potassium nitrite or sodium nitrite. Two per cent
solution.
3. Carbon bisulfide. Freshly purified by distillation.
4. Iodine standards. Acidify with dilute sulfuric acid
measured quantities of a standard solution of potassium
iodide in small tubes. Add 3 or 4 drops of the potassium
nitrite solution and extract with carbon bisulfide as in the
actual determination. Transfer to small flasks the standards
from which the iodine has been removed.
5. Chlorine water. Saturated solution.

6. Bromine standards. Add measured quantities of a
standard solution of a bromide to the liquid in each of the
small flasks from which the iodine has been removed. Add
to each 5 cc. of purified carbon bisulfide, and proceed
exactly as with the sample.
Procedure.—Evaporate to dryness an aliquot portion of
the alkaline filtrate. Dissolve the residue in 2 or 3 cc. of
water, and add enough absolute alcohol to make the
percentage of alcohol about 90. Boil and filter and repeat
the extraction of the residue with alcohol once or twice.
Add 2 or 3 drops of sodium hydroxide to the combined
alcoholic filtrates and evaporate to dryness. Dissolve the
residue in 2 or 3 cc. of water and repeat the extraction with
alcohol and the filtration. Add a drop of sodium hydroxide
to the filtrate and evaporate it to dryness. Dissolve the
residue in a little water. Acidify this solution with dilute
sulfuric acid, adding 3 or 4 drops excess, and transfer it to
a small flask. Add 4 drops of the solution of potassium
nitrite or sodium nitrite and about 5 cc. of carbon bisulfide.
Shake the mixture until all the iodine is extracted. Separate
the acid solution from the carbon bisulfide by filtration.
Wash the flask, filter, and contents with cold distilled water,
and transfer the carbon bisulfide containing the iodine in
solution to Nessler tubes by means of about 5 cc. of pure
carbon bisulfide. In washing the filter, dilute the contents of
the tube to a definite volume, usually 12 or 15 cc., and
compare the color with that of known amounts of iodine
dissolved in carbon bisulfide in other tubes.
Transfer to a small flask the sample from which the
iodine has been removed. Add saturated chlorine water, 1
cc. at a time, shaking after each addition until all the
bromine is freed. Care must be taken not to add much more
chlorine than that necessary to free the bromine, since an
excess of reagent may form a bromochloride that spoils the
color reaction. Separate the water solution from the carbon

bisulfide by filtration through a moistened filter, wash the
contents of the filter two or three times with water, and
then transfer them to a Nessler tube by means of about 1
cc. of carbon bisulfide. Repeat this extraction of the filtrate
twice, using 3 cc. of carbon bisulfide each time. The
combined carbon bisulfide extracts usually amount to 11.5
to 12 cc. Add enough carbon bisulfide to the tubes to bring
them to a definite volume, usually 12 to 15 cc., and
compare the sample with the standards. If much bromine is
present it is not always completely extracted by the
amounts of carbon bisulfide recommended. If the extraction
is incomplete, therefore, make one or two extra extractions
with carbon bisulfide, transfer the extracts to another tube,
and compare the color with that of the standards.

ARSENIC.[31]

Evaporate to dryness an aliquot portion of the alkaline
filtrate (p. 61). Acidify the residue with arsenic-free sulfuric
acid, and subject it to the action of arsenic-free zinc and
sulfuric acid in a Marsh-Berzelius apparatus. Compare the
mirror obtained with a mirror obtained from an arsenious
oxide solution of known strength.

BORIC ACID.

Evaporate to dryness an aliquot portion of the alkaline
filtrate (p. 61), treat the residue with 1 or 2 cc. of water,
and slightly acidify the solution with hydrochloric acid. Add
about 25 cc. of absolute alcohol, boil, filter, and repeat the
extraction of the residue. Make the filtrate slightly alkaline
with sodium hydroxide, and evaporate it to dryness. Add a
little water, slightly acidify with hydrochloric acid, and
place a strip of turmeric paper in the liquid. Evaporate to
dryness on the steam bath, and continue the heating until

the turmeric paper is dry. If boric acid is present the
turmeric paper becomes cherry red. It is not usually
necessary to determine quantitatively boric acid; the
quantitative method devised by Gooch[33] is recommended.

HYDROGEN SULFIDE.[103]

Hydrogen sulfide should be determined preferably in the
field; the procedure as far as the final titration with sodium
thiosulfate must be carried out in the field.
Reagents.—1. N/100 sodium thiosulfate.
2. Standard iodine. A N/100 solution containing
potassium iodide standardized against the N/100 sodium
thiosulfate. To standardize, add 10 cc. of the iodine solution
to 500 cc. of boiled distilled water. Add about 1 gram of
potassium iodide, and titrate with N/100 sodium thiosulfate
in the presence of starch indicator. One cc. of N/100 iodine
is equivalent to 0.17 mg. H2S.
3. Potassium iodide. Crystals.
4. Starch. A freshly prepared solution for use as indicator.
Procedure.—Add 500 cc. of the sample to 10 cc. of the
standard iodine solution and 1 gram of potassium iodide in
a large glass-stoppered bottle or flask. If the sample is to
be collected from a tap lead the water into the bottle
through a rubber tube extending to the bottom of the bottle
so as to eliminate errors due to aeration. Shake the bottle,
allow it to stand for a few minutes, and then titrate the
excess of iodine with sodium thiosulfate in the presence of
starch indicator. Hydrogen sulfide (H2S) in parts per million
is equal to 0.34 times the difference in cubic centimeters
between the amount of iodine solution added and the
amount of N/100 thiosulfate used in the titration.

CHLORINE.

In waters that have been treated with calcium
hypochlorite or liquid chlorine it is frequently advisable to
ascertain the presence or absence of chlorine. As the
reagents which have been proposed for its detection are
not specific for chlorine but give similar or identical
reactions with oxidizing agents or reducible substances
care must be exercised in interpreting the results of such
tests: nitrites and ferric salts are of common occurrence,
and chlorates also may lead to misinterpretation in waters
treated with calcium hypochlorite.
Reagents.—1. Tolidin solution. One gram of o-tolidin,
purified by being recrystallized from alcohol, is dissolved in
1 liter of 10 per cent hydrochloric acid.
2. Copper sulfate solution. Dissolve 1.5 grams of copper
sulfate and 1 cc. of concentrated sulfuric acid in distilled
water and dilute the solution to 100 cc.
3. Potassium bichromate solution. Dissolve 0.025 gram of
potassium bichromate and 0.1 cc. of concentrated sulfuric
acid in distilled water and dilute the solution to 100 cc.
Procedure.—Mix 1 cc. of the tolidin reagent with 100 cc.
of the sample in a Nessler tube and allow the solution to
stand at least 5 minutes. Small amounts of free chlorine
give a yellow and larger amounts an orange color.
For quantitative determination compare the color with
that of standards in similar tubes prepared from the
solutions of copper sulfate and potassium bichromate. The
amounts of solution for various standards are indicated in
Table 13.

Table 13.—Preparation of permanent standards for content of chlorine.

Solution of copper Solution of potassium
Chlorine.
sulfate. bichromate.
Parts per
cc. cc.
million.

0.01 0.0 0.8
.02 .0 2.1
.03 .0 3.2
.04 .0 4.3
.05 .4 5.5
.06 .8 6.6
.07 1.2 7.5
.08 1.5 8.7
.09 1.7 9.0
.10 1.8 10.0
.20 1.9 20.0
.30 1.9 30.0
.40 2.0 38.0
.50 2.0 45.0

DISSOLVED OXYGEN.[16][65][68][71b][99][100c][120]

Reagents.—1. Sulfuric acid, concentrated. (Sp. gr. 1.83–
1.84.)
2. Potassium permanganate. Dissolve 6.32 grams of the
salt in water and dilute the solution to 1 liter.
3. Potassium oxalate. A 2 per cent solution.
4. Manganous sulfate. Dissolve 480 grams of the salt in
water and dilute the solution to 1 liter.
5. Alkaline potassium iodide. Dissolve 700 grams of
potassium hydroxide and 150 grams of potassium iodide in
water and dilute the solution to 1 liter.
6. Hydrochloric acid. Concentrated (Sp. gr. 1.18–1.19).
7. Sodium thiosulfate. A N/40 solution. Dissolve 6.2
grams of chemically pure recrystallized sodium thiosulfate
in water and dilute the solution to 1 liter with freshly boiled
distilled water. Each cc. is equivalent to 0.2 mg. of oxygen
or to 0.1395 cc. of oxygen at 0°C. and 760 mm. pressure.
Inasmuch as this solution is not permanent it should be
standardized occasionally against a N/40 solution of
potassium bichromate. The keeping qualities of the
thiosulfate solution are improved by adding to each liter 5
cc. of chloroform and 1.5 grams of ammonium carbonate
before diluting to the prescribed volume.
8. Starch solution. Mix a small amount of clean starch
with cold water until it becomes a thin paste and stir this
mass into 150 to 200 times its weight of boiling water. Boil
for a few minutes, then sterilize. It may be preserved by
adding a few drops of chloroform.
Collection of sample.—Collect the sample in a narrow-
necked glass-stoppered bottle of 250 to 270 cc. capacity.
The following procedure should be followed in order to

avoid entrainment or absorption of atmospheric oxygen. In
collecting from a tap fill the bottle through a glass or
rubber tube extending well into the tap and to the bottom
of the bottle. To avoid air bubbles allow the bottle to
overflow for several minutes, and then carefully replace the
glass stopper so that no air bubble is entrained. In
collecting from the surface of a pond or tank connect the
sample bottle to a bottle of 1 liter capacity. Provide each
bottle with a two-hole rubber stopper having one glass tube
extending to the bottom and another glass tube entering
but not projecting into the bottle. Connect the short tube of
the sample bottle with the long tube of the liter bottle.
Immerse the sample bottle in the water and apply suction
to the outlet of the liter bottle. To collect a sample at any
depth arrange the two bottles so that the outlet tube of the
liter bottle is at a higher elevation then the inlet tube of the
sample bottle. Lower the two bottles, in any convenient
form of cage properly weighted, to the desired depth.
Water entering during the descent will be flushed through
into the liter bottle. When air bubbles cease rising to the
surface raise the bottles. Finally replace the perforated
stopper of the sample bottle with a glass stopper in such
manner as to avoid entraining bubbles of air.
Procedure.—Remove the stopper from the bottle and add,
first, 0.7 cc. of the concentrated sulfuric acid, and then 1
cc. of the potassium permanganate solution. These and all
other reagents should be introduced by pipette under the
surface of the liquid. Insert the stopper and mix by
inverting the bottle several times. After 20 minutes have
elapsed destroy the excess of permanganate by adding 1
cc. of the potassium oxalate solution, the bottle being at
once restoppered and its contents mixed. If a noticeable
excess of potassium permanganate is not present at the
end of 20 minutes, again add 1 cc. of the potassium
permanganate solution. If this is still insufficient use a

stronger potassium permanganate solution. After the liquid
has been decolorized by the addition of potassium oxalate
add 1 cc. of the manganous sulfate solution and 3 cc. of the
alkaline potassium iodide solution. Allow the precipitate to
settle. Add 2 cc. of the hydrochloric acid and mix by
shaking.
The procedure to this point must be carried out in the
field, but after the acid has been added and the stopper
replaced there is no further change, and the rest of the test
may be performed within a few hours, as convenient.
Transfer 200 cc. of the contents of the bottle to a flask and
titrate with N/40 sodium thiosulfate, using a few cubic
centimeters of the starch solution as indicator toward the
end of the titration. Do not add the starch solution until the
color has become faint yellow, and titrate until the blue
color disappears.
The use of potassium permanganate is made necessary
by high nitrite or organic matter. The procedure outlined
must be followed in all work on sewage and partly purified
effluents or seriously polluted streams or samples whose
nitrite nitrogen exceeds 0.1 part per million. In testing
other samples the procedure may be shortened by
beginning with the addition of the manganous sulfate
solution and proceeding from that point as outlined, except
that only 1 cc. of alkaline potassium iodide need be added.
Calculation of Results.—Oxygen shall be reported in parts
per million by weight. It is sometimes convenient to know
the number of cubic centimeters per liter of the gas at 0°C.
temperature and 760 mm. pressure and also to know the
percentage which the amount of gas present is of the
maximum amount capable of being dissolved by distilled
water at the same temperature and pressure. If 200 cc. of
the sample is taken the number of cubic centimeters of
N/40 thiosulfate used is equal to parts per million of
oxygen. Corrections for volume of reagents added amount

to less than 3 per cent and are not justified except in work
of unusual precision. To obtain the result in cubic
centimeters per liter multiply the number of cubic
centimeters of thiosulfate used by 0.698. To obtain the
result in percentage of saturation divide the number of
cubic centimeters of thiosulfate by the figure in Table 14
opposite the temperature of the water and under the
proper chlorine figure. The last column of Table 14 permits
interpolation for intermediate chlorine values. At elevations
differing considerably from mean sea level and for accurate
work attention must be given to barometric pressure, the
normal pressure in the region being preferable to the
specific pressure at the time of sampling. The term
“saturation” refers to a condition of equilibrium between
the solution and an oxygen pressure in the atmosphere
corresponding to 158.8 millimeters, or approximately one-
fifth atmosphere. The true saturation or equilibrium
between the solution and pure oxygen is nearly five times
this value, and consequently values in excess of 100 per
cent saturation frequently occur in the presence of oxygen-
forming plants.

Table 14.—Solubility of oxygen in fresh water and in sea water of stated
degrees of salinity at various temperatures when exposed to an atmosphere
containing 20.9 per cent of oxygen under a pressure of 760 mm.[F]

(Calculated by G. C. Whipple and M. C. Whipple from measurements of
C. J. Fox.)[27][119]

Chloride in sea water (milligrams per Difference
liter). per 100
Temperature.
parts of
0. 5000. 10000. 15000. 20000. chloride.
Parts per
°C. Dissolved oxygen in milligrams per liter.
million.
0 14.62 13.79 12.97 12.14 11.32 0.0165
1 14.23 13.41 12.61 11.82 11.03 .0160
2 13.84 13.05 12.28 11.52 10.76 .0154
3 13.48 12.72 11.98 11.24 10.50 .0149
4 13.13 12.41 11.69 10.97 10.25 .0144
5 12.80 12.09 11.39 10.70 10.01 .0140

6 12.48 11.79 11.12 10.45 9.78 .0135
7 12.17 11.51 10.85 10.21 9.57 .0130
8 11.87 11.24 10.61 9.98 9.36 .0125
9 11.59 10.97 10.36 9.76 9.17 .0121
10 11.33 10.73 10.13 9.55 8.98 .0118

11 11.08 10.49 9.92 9.35 8.80 .0114
12 10.83 10.28 9.72 9.17 8.62 .0110
13 10.60 10.05 9.52 8.98 8.46 .0107
14 10.37 9.85 9.32 8.80 8.30 .0104
15 10.15 9.65 9.14 8.63 8.14 .0100

16 9.95 9.46 8.96 8.47 7.99 .0098
17 9.74 9.26 8.78 8.30 7.84 .0095
18 9.54 9.07 8.62 8.15 7.70 .0092
19 9.35 8.89 8.45 8.00 7.56 .0089
20 9.17 8.73 8.30 7.86 7.42 .0088

21 8.99 8.57 8.14 7.71 7.28 .0086
22 8.83 8.42 7.99 7.57 7.14 .0085
23 8.68 8.27 7.85 7.43 7.00 .0083
24 8.53 8.12 7.71 7.30 6.87 .0083
25 8.38 7.96 7.56 7.15 6.74 .0082

26 8.22 7.81 7.42 7.02 6.61 .0080
27 8.07 7.67 7.28 6.88 6.49 .0079
28 7.92 7.53 7.14 6.75 6.37 .0078
29 7.77 7.39 7.00 6.62 6.25 .0076
30 7.63 7.25 6.86 6.49 6.13 .0075

F. Under any other barometric pressure, B, the solubility can be obtained
from the corresponding value in the table by the formula:
B
S´ = S760 B´
= S29.92 in which S´ = Solubility at B or B´,

S = Solubility at 760 mm. or 29.92 inches,
B = Barometric pressure in mm.,
and B´ = Barometric pressure in inches.

ETHER-SOLUBLE MATTER.[44]

Evaporate 500 cc. of the sample in a porcelain
evaporating dish to a volume of about 50 cc. By means of a
rubber-tipped glass rod remove to the bottom of the dish
the solid matter attached to the sides, and add normal
sulfuric acid to neutralize the alkalinity. Do not use an
excess of acid. Then evaporate the contents of the dish to
dryness. Treat the dry residue with boiling ether, rubbing
the bottom and sides of the dish to insure complete solution
of fat. Three extractions with ether are required. Filter the
ether solution through a 5 cm. filter into a weighed flask
having a wide mouth. Evaporate the ether slowly, and dry
the flask at 100° C. for 30 minutes. The increase in weight
of the flask gives the amount of fats, or, in more precise
language, the ether-soluble matter.
An excess of acid gives too high results because of the
formation of fatty-acid residues.

RELATIVE STABILITY OF EFFLUENTS.[78]

Reagent.—Methylene blue solution. A 0.05 per cent
aqueous solution of methylene blue, preferably the double
zinc salt or commercial variety.[60b]
Collection of sample.—Collect the sample in a glass-
stoppered bottle holding approximately 150 cc. If the
dissolved oxygen is low observe precautions similar to
those used in collecting samples for dissolved oxygen (p.
66).
Procedure.—Add 0.4 cc. of the methylene blue solution to
the sample in the 150 cc. bottle. As methylene blue has a
slightly antiseptic property be careful to add exactly 0.4 cc.
Add the methylene blue solution preferably below the
surface of the liquid after filling the bottle with the sample.
If the methylene blue is added first do not allow the liquid
to overflow as coloring matter will thus be lost. Incubate
the sample at 20° C. for ten days. Four days’ incubation
may be considered sufficient for all practical purposes in
routine plant-control work. If quick results are desired
incubate the sample at 37° C. for five days using suitable
stoppers[1a][2a] to prevent the loss and reabsorption of
dissolved oxygen. The bacterial flora at 37° C. is different
from that at 20° C. The lower temperature is more nearly
the average temperature of surface waters and therefore
the higher temperature should be used only when quick
approximate results are essential. Observe the sample at
least twice a day during incubation. Give a sample in which
the methylene blue becomes decolorized a relative stability
corresponding to the time required for reduction (see Table
15). For routine filter control ordinary room or cellar
temperature gives fairly satisfactory results. For accurate
studies, room temperature incubation is very undesirable,

as the fluctuations in temperature which are ordinarily not
noticed are responsible for appreciable deviations from the
true values of relative stability. If the samples are incubated
less than 10 days at 20° C. and are not decolorized place a
plus sign after the stability value in order to indicate that
the stability might have been higher if more time had been
allowed. In applying this test to river waters it often
happens that the blue coloring matter is removed either
partly or completely through absorption by the clay which
many rivers carry in suspension. True relative stabilities
cannot be obtained for such waters except by determining
the initial available oxygen at the start and the biochemical
oxygen demand on incubation at 20° C. for 10 days (pp. 71–
73). Germicides, such as hypochlorite of lime, if present in
sufficient quantity, vitiate the results. If a sample contains
free chlorine, therefore, store it about 2 hours, or until the
chlorine is gone, and then add methylene blue.
Table 15[78] gives the relation between the time in days to
decolorize methylene blue at 20° C. (t20) and the relative
stability number or ratio of available oxygen to oxygen
required for equilibrium, expressed in percentage (S).

Table 15.—Relative stability numbers.

Time required for decolorization at 20° C. Relative stability.
Days. Percentage.
0.5 11
1.0 21
1.5 30
2.0 37
2.5 44
3.0 50
4.0 60
5.0 68
6.0 75
7.0 80
8.0 84
9.0 87
10.0 90
11.0 92
12.0 94
13.0 95
14.0 96
16.0 97
18.0 98
20.0 99

The theoretical relation is, S = 100 (1 − 0.794t20)
The relation between the time of reduction at 20° C. and
that at 37° C. is approximately two to one, but if an
observer incubates at 37° C. he should work out his own
comparative 37° C. table or factor.
A relative stability of 75 signifies that the sample
examined contains a supply of available oxygen equal to 75
per cent of the amount of oxygen which it requires in order
to become perfectly stable. The available oxygen is
approximately equivalent to the dissolved oxygen plus the
available oxygen of nitrate and nitrite. Nitrite in sewage is
usually so low as to be negligible.

BIOCHEMICAL OXYGEN DEMAND OF SEWAGE
AND EFFLUENTS.[60a][60c][60d]

RELATIVE STABILITY METHOD.

The relative stability method may be employed to obtain
a measure of the putrescible material in sewages and
effluents in terms of oxygen demand.
Procedure for effluents.—Divide the total available
oxygen, including the oxygen of nitrite and nitrate, by the
relative stability expressed as a decimal.
Procedure for sewages.—Make one or two dilutions with
fully aerated distilled water of known dissolved oxygen
content. Tap water may be employed if it is free from
nitrates. Vary the relative proportions of sewage and water
to be employed to give a relative stability of 50 to 75.
Unless seals[1b][2b][52a] are used bring the water as well as the
sewage to the temperature at which the mixtures are to be
incubated before preparing the dilutions. During the
manipulation avoid aeration. Having made the proper
dilutions, determine the relative stability of each.
Calculate the oxygen demand in parts per million by the
formula:
Oxygen demand = O(1 − p)/Rp
In this formula, O is the initial dissolved oxygen of the
diluting water, p is the proportion of sewage; and R is the
relative stability of the mixture. Ordinarily the available
oxygen in crude sewages, septic tank effluents, settling
tank effluents, and trade wastes can be neglected.

SODIUM NITRATE METHOD.

For the determination of the biochemical oxygen demand
the sodium nitrate method may be used[60a][60c][60d][52a]. The
method is based on the biochemical consumption of oxygen
from sodium nitrate by a sewage or polluted water during
an incubation period of ten days at 20° C. A reasonable
excess of sodium nitrate does not give a higher oxygen
demand, as do higher dilutions with aerated water. The
oxygen absorbed from the air in applying the method to
sewages is negligible.
Reagent.—Sodium nitrate solution. Dissolve 26.56 grams
of pure sodium nitrate in 1 liter of distilled water. One cc.
of this solution in 250 cc. of sewage represents 50 parts per
million of available oxygen. The strength of the sodium
nitrate solution may be varied to suit conditions.
Procedure for sewages.—Ordinarily disregard the initial
available oxygen as it is very small compared with the total
biochemical oxygen demand. Add measured amounts of the
sodium nitrate solution to the sewage in bottles holding
approximately 250 cc. which have been completely filled
and stoppered. Incubate for 10 days at 20° C. A seal is not
required during incubation. The appearance of a black
sediment and the development of a putrid odor during
incubation indicates that too little sodium nitrate has been
added. Methylene blue solution in proper proportion may
be added at the start to serve as an indicator during the
incubation. Domestic sewage usually varies in its oxygen
demand from 100 to 300 parts per million, approximately
30 per cent of which is used up at 20° C. in the first 24
hours. At the end of the incubation period determine the
residual nitrite and nitrate. Determine the nitrate by the
aluminium reduction method and direct Nesslerization. To
convert the nitrogen into oxygen equivalents, multiply the
nitrite nitrogen by 1.7 and the nitrate nitrogen by 2.9. The

difference between the available oxygen added as sodium
nitrate and that found as nitrite and nitrate at the end of
the incubation period is the biochemical oxygen demand.
Procedure for industrial wastes.—Employ the same
procedure using larger quantities of the sodium nitrate
solution. Make the reaction alkaline to methyl orange and
acid to phenolphthalein. Adjust an acid reaction with
sodium bicarbonate and a caustic alkaline reaction with
weak hydrochloric acid. If the liquid is devoid of sewage
bacteria seed it with sewage after adjusting the reaction.
Procedure for polluted river waters.—Determine the
initial available oxygen. Unless the river water is badly
polluted add 10 parts per million of sodium nitrate oxygen.
Collect carefully, avoiding aeration, three samples in 250
cc. bottles. To one sample add a definite quantity of sodium
nitrate solution and incubate. Incubate the other two
samples for the determination of the residual free oxygen,
nitrite, and nitrate. If there is free oxygen left, the bottle
containing the sodium nitrate solution may be discarded. If
there is no free oxygen determine residual nitrite and
nitrate as directed under the procedure for sewage (p. 72)
and calculate the oxygen demand.

ANALYSIS OF SEWAGE SLUDGE AND
MUD DEPOSITS.

COLLECTION OF SAMPLE.

Collect a representative sample of the material. In
general more than one sample should be taken from a spot
and a large number of samples should be collected rather
than a few large samples. If the surface layer is darker and
a lower layer consists of pure clay sample only the surface
layer. Samples may be analyzed either separately or as
composites of careful mixtures. After the sample has
settled a few minutes roughly drain or siphon the excess
water. Allow sewage sludge to stand for one hour before
draining it free from excess water unless it is essential to
determine the moisture content of the sample originally
collected. If sludge cannot be analyzed within twenty-four
hours it is better not to use air-tight bottles and to add
small quantities of chloroform and keep in the ice box to
retard decomposition. At the time of collection carefully
examine mud from the bottom of surface water for
evidence of sewage pollution and macroscopic and
microscopic animal and plant organisms. Record the
predominant species. Note the physical appearance of the
material, particularly its color, odor, and consistency.
Express all analytical results in percentage on a dry basis.

REACTION.

Determine the reaction by diluting a definite quantity of
the wet sludge and titrating by the methods given under
alkalinity and acidity (pp. 35–39 and 39–41).

SPECIFIC GRAVITY.
Weigh to the nearest tenth of a gram a wide-mouthed
flask of 100 to 300 cc. capacity, according to the quantity of
material available. Then completely fill the flask with
distilled water to the brim and weigh it again. Empty the
flask and fill it completely with fresh sewage sludge or
mud. If the material is of such consistency that it flows
readily fill the flask to the brim and weigh. The specific
gravity is equal to the weight of the sludge or mud divided
by the weight of an equal volume of distilled water.
If the material does not flow readily fill the weighed flask
as completely as possible without exerting pressure during
the procedure. Weigh and then fill the flask to the brim
with distilled water. Let it stand for a few minutes, until
trapped air has escaped, then add more water if necessary
and weigh. Subtract the weight of the added water from
the weight of the water that completely fills the flask; the
specific gravity is equal to the weight of the material
divided by this difference. Record the specific gravity only
to the second decimal place.

MOISTURE.

Heat approximately 25 grams of sludge or mud in a
weighed nickel dish on the water bath until it is fairly dry.
Dry the residue in an oven at 100° C., cool, and weigh.
Repeat to approximate constant weight. The loss in weight
is moisture.

VOLATILE AND FIXED MATTER.

Ignite, at dull red heat in a hood, the residue from the
determination of moisture until all the carbon has
disappeared. Cool the residue in a desiccator and weigh it.
The residue is the fixed matter. The volatile matter is the
difference in weight between the original dried sludge and
the ignited sludge.

TOTAL ORGANIC NITROGEN.

Preparation of sample.—For the determination of organic
nitrogen and fats dry approximately 50 to 75 grams of the
sludge or mud in a porcelain dish first on the water bath
and finally in the hot-water oven until all the moisture has
disappeared. Grind the dry material to a fine powder and
keep it in a glass-stoppered bottle.
Reagents.—1. Sulfuric acid. Concentrated, nitrogen-free.
2. Copper sulfate solution. Ten per cent.
3. Potassium permanganate. Crystals.

First procedure.—Weigh accurately 0.5 gram of dried
sludge or 5.0 grams of dried mud and put it into a 500 cc.
Kjeldahl flask. Digest it with 20 cc. of sulfuric acid, or more
if necessary, and 1 cc. of copper sulfate solution to assist
the oxidation. Boil for several hours until the liquid
becomes colorless or slightly yellow. Oxidize the residue
with 0.5 gram of potassium permanganate, and follow the
“Procedure for Sewage” (pp. 21–22).
Second procedure.—The following method is convenient
for routine work at sewage disposal plants. After digestion
as described in the first procedure, cool, transfer to a glass-
stoppered 100 cc. flask, dilute with distilled water to 100
cc., and mix well. Transfer 50 cc. with a pipette into
another 100 cc. volumetric flask, and make this portion
alkaline with 50 per cent sodium hydroxide, testing a drop
of the liquid on a porcelain plate with phenolphthalein to
insure neutralization. The formation of a floc usually
indicates that neutralization is complete. Dilute the solution
to 100 cc., pour it into a small glass-stoppered bottle and
permit it to stand until the next day. Nesslerize an aliquot
portion of the clear supernatant liquid, and calculate the
percentage of nitrogen in the material.

ETHER-SOLUBLE MATTER.

Fats are usually determined only on sewage sludge, but
some mud deposits contain small quantities due to the
presence of trade wastes.
Procedure.—Weigh 0.5 to 25 grams of dry material
according to the quality of the sludge or mud. Add water to
the weighed portion in a porcelain dish and acidify the
mixture with N/50 sulfuric acid in the presence of litmus
tincture or azolitmin solution as indicator. Avoid adding too
much acid as an excess gives too high results on account of
fatty acid residues. Evaporate the acidified mixture to
dryness on the water bath, and heat it in the hot air oven at
100° C. two to three hours. Extract the dry residue with
boiling ether, rubbing the sides and bottom of the dish to
insure complete solution of the fat. Three extractions with
ether are usually sufficient. Filter the ether solution
through a 5 cm. filter paper into a small flask. Evaporate
the ether slowly, dry the fatty extract for half an hour at
100° C., cool in a desiccator, and weigh. If it is desirable,
particularly with certain industrial wastes, to determine the
quantity of saponified fat determine the fats with and
without the addition of acid. The difference between the
quantities found by the two determinations is the content of
saponified fat.

FERROUS SULFIDE.

The liberation of hydrogen sulfide on adding dilute
hydrochloric acid to a sludge indicates the presence of
ferrous sulfide. As ferrous sulfide quickly oxidizes on
exposure to air a quantitative determination of this
constituent must be made immediately after collection of
the sample.
Procedure.—Heat a definite portion of the sludge with
hydrochloric acid in a flask. Pass the liberated gas through
bromine water or hydrogen peroxide. Determine
gravimetrically the sulfate in the oxidizing solution, and
calculate the equivalent of ferrous sulfide by multiplying
the weight of barium sulfate by 0.376.

BIOCHEMICAL OXYGEN DEMAND.

The quantity of river mud most suitable for the
determination of the biochemical oxygen demand ranges
within certain limits, largely according to the amount of
oxidizable matter present. For examinations of river mud
prepare a 1 per cent stock suspension in distilled water or
tap water saturated with oxygen and free from nitrate; use
in the test a dilution of this stock suspension equivalent to
a concentration of 1 to 10 grams per liter of mud. For
examinations of fresh sewage sludge prepare a 1 per cent
stock suspension in a similar manner, but use in the test a
dilution equivalent to only 0.1 to 1.0 gram per liter of wet
material. For examinations of dried sludges, which have
undergone more or less oxidation higher concentrations
may be required.
Procedure.—Place a measured portion of the sample, or
the proper amount of the 1 per cent stock suspension of the
sample, in a 300 cc. narrow-mouth glass-stoppered bottle,
and dilute it to the desired concentration with water
saturated with oxygen. Determine the oxygen content at
20° C. of the waters that are used for dilution. This
determination must be made before the mud or sludge is
added because iron sulfide in the mud or sludge rapidly
consumes part of the dissolved oxygen. Incubate at 20° C.
for five days.
Shortly before the determination of the oxygen remaining
in solution at the end of five days rotate the bottle once or
twice to mix its contents and allow sedimentation for about
30 minutes. Siphon the greater part of the liquid through a
narrow-bore siphon into a 150 cc. bottle, which has been
filled with carbon dioxide. Reject the first 25 cc. of the
siphoned liquid and allow a little to overflow at the end of

siphoning. Determine the oxygen content of the solution in
the bottle in the usual way (pp. 65–68). Report the oxygen
demand in percentage of the dried mud or sludge.

ANALYSIS OF CHEMICALS.

The following sections describe the accepted methods for
the analysis of the chemicals commonly used in the
treatment of water.

REAGENTS.

1. Distilled water. In practically all the tests of chemicals
it is necessary to use exclusively distilled water that has
been freshly boiled to free it from carbon dioxide and
oxygen.
2. Concentrated hydrochloric acid. Sp. gr. 1.20.
3. Hydrochloric acid, N/2.
4. Hydrochloric acid, N/10.
5. Ammonium hydroxide. Redistilled; sp. gr. 0.90.
6. Dilute sulfuric acid. Dilute 1 part of concentrated
sulfuric acid with 3 parts of freshly boiled distilled water.
7. Methyl orange indicator. See page 36.
8. Phenolphthalein indicator. See page 36.
9. Bromine water.
10. Stannous chloride, N/20. This should be frequently
standardized by titration against a standard iron solution.
One cc. of N/20 stannous chloride is equal to 0.0028 gram
of iron (Fe) estimated in the ferrous state.
11. Sodium hydroxide, N/1. Free from carbonate. This
should be frequently standardized by titration against a
standard acid solution in presence of phenolphthalein
indicator. One cc. of N/1 sodium hydroxide is equal to 0.049
gram of sulfuric acid (H2SO4), or to 0.03645 gram of
hydrochloric acid (HCl).
12. Sodium hydroxide, N/20. Free from carbonate.
13. Standard potassium permanganate. A N/10 solution.
One cc. of N/10 potassium permanganate is equal to 0.0056
gram of iron (Fe) estimated in the ferrous state.
14. Alcohol. Ethyl alcohol, 95 per cent.

15. Sugar. Solid granulated cane sugar.

SULFATE OF ALUMINIUM.

Determine and report insoluble matter, aluminium oxide
(Al2O3), ferric oxide (Fe2O3), ferrous oxide (FeO), basicity
ratio, and, if present, free acid as H2SO4. If the material is
what is known as “granular” sulfate mix it well before
sampling. If it is in lump form crush it to ⅛ to ¼ inch size,
mix, and sample it. It is unnecessary to grind the sample to
a fine powder, but it is preferable to have the particles
fairly uniform in size.

INSOLUBLE MATTER.

Treat 10 grams of the sample with 100 cc. of distilled
water and digest one hour at boiling temperature. Filter
through a weighed Gooch crucible and wash the insoluble
matter with hot water freshly boiled to free it from carbon
dioxide. Dry the crucible to constant weight at 100° C.,
cool, and weigh. Report the percentage of insoluble matter.

OXIDES OF IRON AND ALUMINIUM.

Dilute the filtrate from the determination of insoluble
matter to 500 cc. with water free from carbon dioxide and
thoroughly mix the solution. Transfer 50 cc. of the solution
to a 250 cc. beaker, add about 150 cc. of water and 5 cc. of
concentrated hydrochloric acid, and heat to boiling. Add
ammonium hydroxide in slight excess; when the solution
has been almost neutralized it is convenient to add a drop
of methyl orange indicator and then to add about 0.5 cc. of
ammonium hydroxide after the solution is neutral to the
indicator. Digest at about 100° C. for a few minutes and

filter. Some analysts prefer to wash this gelatinous
precipitate with hot water by decantation, and some to
wash it evenly distributed over the surface of a filter paper;
either method may be used. It is difficult to free it
completely from impurities and it is not necessary to do so
unless unusual quantities of calcium, magnesium, sodium,
or potassium are present. While the precipitate is being
washed do not allow it to become dry, as it then packs and
can not be washed clean. After most of the water has
drained drying the filter may be hastened by placing it on a
sheet of blotting paper. If much iron is present completely
dry the precipitate, remove it from the paper, and ignite the
paper separately. Finally, blast the precipitate, with free
access of air to the crucible, for five or ten minutes, cool,
and weigh as oxides of iron and aluminium (Fe2O3 + Al2O3).
Subtract the content of total iron, expressed as ferric
oxide (Fe2O3), from the weight of the combined oxides and
report the difference as aluminium oxide (Al2O3), in
percentage.

TOTAL IRON.

As filter alum usually contains 0.2 to 0.3 per cent of iron
use a 10 gram sample for the determination of total iron.
Treat the sample with 50 cc. of freshly boiled distilled
water and add 5 cc. of concentrated hydrochloric acid and
1 cc. of bromine water. Evaporate the solution to dryness,
dissolve the residue in water, and wash it into a flask with
sufficient water to make the volume about 50 cc. Add 50 cc.
of concentrated hydrochloric acid, boil to expel oxygen, and
titrate, as hot as possible, with N/20 stannous chloride.
If a 10 gram sample is used the percentage of iron (Fe) is
equal to the number of cubic centimeters of stannous
chloride used multiplied by 0.028, and the percentage of

iron expressed as ferric oxide is equal to the number of
cubic centimeters of stannous chloride used multiplied by
0.040.

FERRIC IRON.

As filter alum usually contains 0.02 to 0.04 per cent of
ferric iron use a 20 gram sample. Boil 50 cc. of distilled
water to expel oxygen, add 50 cc. of concentrated
hydrochloric acid, and add the sample while the solution is
boiling. Keep it boiling till the sample is dissolved. The flask
should be kept filled with carbon dioxide during this
process by dropping in occasionally small amounts of
sodium carbonate. When solution of the sample is complete
titrate it hot immediately with N/20 stannous chloride.
If a 20 gram sample is used the percentage of ferric oxide
(Fe2O3) is equal to the number of cubic centimeters of
stannous chloride used multiplied by 0.020.

FERROUS IRON.

The content of ferrous iron is the difference between
total and ferric iron. The percentage of ferrous oxide (FeO)
is, therefore, equal to 0.90 times the difference between
the percentage of total iron expressed as ferric oxide and
the percentage of ferric iron expressed as ferric oxide.
Report the percentage of ferrous oxide (FeO).

BASICITY RATIO.

Transfer 50 cc. of the filtrate from the determination of
insoluble matter to a 200 cc. casserole and dilute it to 100
cc. Boil the solution and titrate it at boiling temperature

with N/1 sodium hydroxide in presence of phenolphthalein
indicator. The percentage of acidity in equivalent of sulfuric
acid (H2SO4) is equal to the number of cubic centimeters of
sodium hydroxide used multiplied by 4.9. In this titration
iron and aluminium are precipitated as hydroxides and any
free acid is neutralized.
Calculate the percentage of sulfuric acid equivalent to
the determined percentages of aluminium oxide, ferric
oxide, and ferrous oxide by the following formula:
2.88 Al2O3 + 1.83 Fe2O3 + 1.36 FeO.
If this percentage of acid equivalent is less than that
found by titration report the difference as percentage of
free acid. If the percentage of acid equivalent is greater
than that found by titration the difference divided by 2.88 is
the percentage equivalent to the excess of aluminium oxide
present. Divide this excess by the percentage of total
aluminium oxide and report the quotient as the basicity
ratio.

LIME.

Mix well the sample, which should contain no lumps. If
foreign matter is present grind the sample to pass a 100–
mesh sieve.
Place 20 grams of granulated cane sugar and 1 gram of
the sample in a 250 cc. glass-stoppered bottle, tightly
stopped, and mix the mass by rolling. Do not shake hard as
much of the lime could thus be lost as dust. Then add 187.4
cc. of distilled water freshly boiled to expel carbon dioxide.
This makes 200 cc. of sugar solution. The lime is mixed dry
with the sugar and the water added later to keep the lime
from lumping. After shaking the sugar solution one hour
titrate 50 cc. of it with N/2 hydrochloric acid in presence of
methyl orange indicator. The acid used is equivalent to the
carbonate and hydroxide in 0.25 gram of the sample.
Filter the remainder of the sugar solution, discarding the
first 25 cc. of filtrate. Titrate 50 cc. of the filtrate with N/2
hydrochloric acid in presence of methyl orange indicator.
The acid used is equivalent to the hydroxide in 0.25 gram
of the sample.
If a 1 gram sample is used the percentage of calcium
oxide (CaO) is equal to 5.6 times the number of cubic
centimeters of hydrochloric acid used in the second
titration; and the percentage of calcium carbonate (CaCO3)
equivalent to the carbonate present is equal to 10 times the
difference in cubic centimeters between the results of the
two titrations.

SULFATE OF IRON.

INSOLUBLE MATTER.

Treat 10 grams of the sample with 100 cc. of freshly
boiled distilled water cooled to 30° C. or less. When
solution is complete filter through a weighed Gooch
crucible, wash, dry, cool, and weigh. Report the weight of
the residue, in percentage, as insoluble matter.

IRON AS FERROUS SULFATE.

Dissolve 1 gram of the sample and dilute to 200 cc. with
freshly boiled distilled water cooled to 30° C. or less. Add 5
cc. of dilute sulfuric acid (1 to 3) to a 50 cc. portion of the
solution and titrate with N/10 potassium permanganate.
The percentage of ferrous sulfate (FeSO4.7H2O) is equal to
11.12 times the number of cubic centimeters of potassium
permanganate used.

ACIDITY.

Shake 12.25 grams of the sample in a 150 cc. bottle with
75 cc. of 95 per cent alcohol for ten minutes. Run a blank.
Filter rapidly both sample and blank and wash rapidly with
alcohol sufficient to make 100 cc. of filtrate. Titrate with
N/20 sodium hydroxide in presence of phenolphthalein and
subtract the result of titrating the blank from that of
titrating the solution of the sample. The percentage of
acidity, expressed as sulfuric acid (H2SO4), is equal to 0.02

times the number of cubic centimeters of sodium hydroxide
used.

SODA ASH.

INSOLUBLE MATTER.

Treat 5.305 grams of the sample with 200 cc. of freshly
boiled and cooled distilled water. When solution is complete
filter through an asbestos mat in a weighed Gooch crucible,
dry, cool, and weigh. Report the weight of the residue, in
percentage, as insoluble matter.

AVAILABLE ALKALI.

Dilute the filtrate from the determination of insoluble
matter to 1,000 cc. and thoroughly mix. Titrate 25 cc. of
this dilution with N/10 hydrochloric acid in presence of
methyl orange indicator. The percentage of available alkali,
expressed as sodium carbonate (Na2CO3), is equal to 4
times the number of cubic centimeters of hydrochloric acid
used.

CHEMICAL BIBLIOGRAPHY.

The subjoined bibliography comprises the publications
cited in the text of this report. The references are arranged
alphabetically by authors’ names and under each author in
order of dates of publication. When different pages of a
single work are cited letters are used in connection with
the number that refers to the work.
1. Andrews, L. W. Sprengel’s method for colorimetric determination of
nitrates: J. Am. Chem. Soc., Vol. 26, pp. 388–91, 1904.

1a. Assoc. Off. Ag. Chemists. Determination of iodine and bromine: J. A. O.
A. C., Vol. 1, No. 4, pt. 1, pp. 47–8, 1916.

1b. Bachmann, Frank. A new seal for the prevention of aeration in
deaerated liquids: J. Ind. Eng. Chem., Vol. 6, pp. 764–5, 1914.

2. Bartow, Edward, and Rodgers, J. S. Determination of nitrates by reduction
with aluminium: Am. J. Public Hygiene, new ser., Vol. 5, pp. 536–44, 1909; also
Illinois Univ. Bull., Vol. 7, No. 2 (Water Survey Ser. No. 7), pp. 14–27, 1909.

2a. Blinn, William. Determination of manganese as sulfate and by the
sodium bismuthate method: J. Am. Chem. Soc., Vol. 34, pp. 1379–98, 1912.

2b. Buswell, A. M. Modified apparatus for the putrescibility test: J. Ind.
Eng. Chem., Vol. 6, p. 325, 1914.

3. Caldwell, G. C. A method in part for the sanitary examination of water
and for the statement of results, offered for general adoption: J. Anal. Chem.,
Vol. 3, pp. 398–403, 1889.

4. Calkins, G. N. A study of odors observed in the drinking waters of
Massachusetts: Report Mass. State Board of Health, pp. 355–80, 1892.

5. Chamot, E. M., and Pratt, D. S. A study on the phenoldisulfonic acid
method for the determination of nitrates in water: J. Am. Chem. Soc., Vol. 31,

pp. 922–8, 1909; Vol. 32, pp. 630–7, 1910; and Redfield, H. W., Vol. 33, pp. 366–
81, 381–4, 1911.

6. Clark, H. W. Experiments upon the purification of sewage and water at
the Lawrence Experiment Station: Report Mass. State Board of Health, pp.
427–578, 1896.

7. Clark, H. W., and Forbes, F. B. Methods for the determination of lead, tin,
zinc, and copper in drinking waters: Report Mass. State Board of Health, pp.
577–85, 1898; pp. 498–506, 1900.

8. Cohn, A. I. Tests and reagents, 1st ed., p. 216, John Wiley & Sons, New
York, 1903.

9. Dibdin, W. J. The purification of sewage and water, 3d ed., pp. 345–51, D.
Van Nostrand Co., New York, 1903.

10. Dole, R. B. The quality of the surface waters in the United States: U. S.
Geol. Survey Water-Supply Paper 236, pp. 15–9, 1909.

11. Draper, H. N. Lacmoid and carminic acid as reagents for alkalies:
Chem. News, Vol. 51, pp. 206–7, 1885.

13. Drown, T. M., and Martin, Henry. Determination of organic nitrogen in
natural waters by the Kjeldahl method: Tech. Quart., Vol. 2, No. 3; Chem. News,
Vol. 59, pp. 272–6, 1889.

14. Drown, T. M. The chemical examination of waters and the interpretation
of analyses: Examinations by the State Board of Health of water supplies of
Mass. 1887–90, pt. 1, Examination of water supplies, pp. 519–78, 1890.

15. ——. Report upon the examination of the outlets of sewers and the
effect of sewage disposal in Massachusetts: Report Mass. State Board of
Health, pp. 285–452, 1902.

16. ——, and Hazen, Allen. A report of the chemical work done at the
Lawrence Experiment Station: Examinations by the State Board of Health of
water supplies of Mass. 1887–90, pt. 2, Purification of sewage and water, pp.
707–34, 1890.

17. Dupre, Dr. Some observations on the permanganate test in water
analysis: Analyst, Vol. 10, pp. 118–22, 1885.

18. Ellms, J. W. A study of the relative value of lacmoid, phenacetolin, and
erythrosine as indicators in the determination of the alkalinity of water by
Hehner’s method: J. Am. Chem. Soc., Vol. 21, pp. 359–69, 1899.

20. ——, and Beneker, J. C. The estimation of carbonic acid in water: J. Am.
Chem. Soc., Vol. 23, pp. 405–31, 1901.

21. Farnsteiner, Buttenburg, and Korn, Leitfaden für die chemische
Untersuchung von Abwasser, Berlin, p. 20, 1902.

22. Fitzgerald, and Foss, Report Boston Water Board, p. 86, 1893.

23. Forbes, F. B., and Pratt, G. H. The determination of carbonic acid in
drinking water: J. Am. Chem. Soc., Vol. 25, pp. 742–56, 1903.

24. Fowler, G. J. Sewage works analyses, pp. 21–37, John Wiley & Sons,
New York, 1902;

24a. pp. 31–4;

24b. pp. 58–60;

24c. pp. 86–9;

24d. pp. 89–95;

24e. pp. 96–7;

24f. pp. 98–100.

26. Fowler, G. J. Univ. of Manchester Lecture, March, 1904, Pamphlet, p. 7.

27. Fox, Charles J. J. On the coefficients of absorption of nitrogen and
oxygen in distilled water and sea water and of atmospheric carbonic acid in sea
water: Trans. Faraday Soc., Vol. 5, pp. 68–87, 1909.

29. ——. The composition of sewage in relation to problems of disposal:
Tech. Quart., Vol. 16, pp. 132–160, 1903.

30. ——. Experiments upon the purification of sewage and water at the
Lawrence Experiment Station: Report Mass. State Board of Health, pp. 447–
700, 1894.

31. Haywood, J. K., and Warner, H. J. Arsenic in papers and fabrics: U. S.
Agri. Dept. Bur. Chem. Bull. 86, pp. 25–7, 1904.

32. Gill, A. H. On the determination of nitrates in potable water: J. Am.
Chem. Soc., Vol. 16, pp. 122–32, 193–7, 1894.

33. Gooch, F. A. A method for the separation and estimation of boric acid:
Am. Chem. J., Vol. 9, pp. 23–33, 1887.

34. ——. A method for the separation of sodium and potassium from lithium
by the action of amyl alcohol on the chlorides: Am. Chem. J., Vol. 9, pp. 33–51,
1887; also U. S. Geol. Survey Bull. 422, p. 175; also U. S. Agri. Dept. Bur.
Chem., Bull. 152, p. 80, 1911.

35. Gottschalk, V. H., and Roesler, H. A. Action of soap on calcium and
magnesium solutions: J. Am. Chem. Soc., Vol. 26, pp. 851–6, 1904.

36. Grandval, Al., and Lajoux, H. Nouveau procédé pour la recherche et le
dosage rapide de faibles quantités d’ acide nitrique dans l’air, l’eau, le sol, etc.,
Comptes rend., Vol. 101, pp. 62–5, 1885.

37. Handy, J. O. Determination of acidity or alkalinity: Proc. Engineers Soc.
West. Pa., Vol. 19, p. 705, 1903.

38. Harrington, Charles, and Richardson, M. W. A manual of practical
hygiene, 5th ed., pp. 457–62, Lea & Febiger, Phila. and New York, 1914.

39. Hazen, Allen. On the determination of chlorine in water: Am. Chem. J.,
Vol. 11, pp. 409–14, 1889.

40. ——. Apparatus for the determination of ammonias in sand sewage: Am.
Chem. J., Vol. 12, pp. 427–8, 1890.

42. ——. Report on the chemical precipitation of sewage: Examinations by
the State Board of Health of water supplies of Mass., 1887–90, pt. 2,
Purification of water and sewage, pp. 735–91, 1890.

43. ——. A new color standard for natural waters: Am. Chem. J., Vol. 14, pp.
300–10, 1892.

44. ——. Experiments on the purification of sewage at the Lawrence
Experiment Station: Report Mass. State Board of Health, pp. 393–448, 1892.

45. ——, and Clark, H. W. On the effect of temperature upon the
determination of ammonia by Nesslerization: Am. Chem. J., Vol. 12, pp. 425–6,
1890.

46. ——, and ——. On the determination of nitrates in water: Chem. News,
Vol. 64, pp. 162–4, 1891.

47. Hehner, Otto. Estimation of hardness without soap solution: Analyst,
Vol. 8, pp. 77–81, 1883.

48. Hillebrand, W. F. The analysis of silicate and carbonate rocks: U. S.
Geol. Survey Bull. 422, pp. 113–8, 127–8, 141–6, 219–20, 221, 222, and 230,
1910.

49. Hollis, F. S. Methods for the determination of color and the relation of
the color to the character of the water: J. N. E. Water Works Assoc., Vol. 13, pp.
94–118, 1898.

50. Howe, Freeland, Jr. A new method for determining the color of the
turbidity of water: Eng. Rec., Vol. 50, pp. 720–1, 1904.

51. Ilosvay, L. L’acide azoteux dans la salive et dans l’air exhalé: Bull. de la
Sociéte Chimique, ser. 3, Vol. 2, pp. 388–91, 1889.

52. Jackson, D. D. Permanent standards for use in the analysis of water:
Tech. Quart., Vol. 13, pp. 314–26, 1900.

52a. ——, and Horton, W. A. Experiments on the putrescibility test for
sewage and sewage effluents: J. Ind. Eng. Chem., Vol. 1, pp. 328–33, 1909.

53. ——, and Ellms, J. W. On odors and tastes of surface waters, with
special reference to anabaena, a microscopical organism found in certain water
supplies of Massachusetts: Tech. Quart., Vol. 10, pp. 410–20, 1897.

54. Johnson, G. A. Report on sewage purification at Columbus, Ohio, made
to the chief engineer of the Board of Public Service, p. 47, 1905.

55. Kendall, L. M., and Richards, E. H. Permanent standards in water
analysis: Tech. Quart., Vol. 17, pp. 277–80, 1904.

56. Kimberley, A. E., and Hommon, H. B. The practical advantages of the
Gooch crucible in the determination of the total and volatile suspended matter
in sewage: Pub. Health Papers and Repts., Am. Pub. Health Assoc., Vol. 31, pt.
2, pp. 123–35, 1905.

57. Kinnicutt, L. P. Quoted by Gage, J. Am. Chem. Soc., Vol. 27, p. 339,
1905.

58. Kjeldahl, J. Neue Methode zur Bestimmung des Stickstoffs in
organischen Körpern: Z. anal. Chem., Vol. 22, pp. 366–82, 1883.

59. Klut, Mitt. a. d. König. Prüfungs, Vol. 12, p. 186.

60. Leach, A. E. Food inspection and analysis, pp. 493, 495, and 497, John
Wiley & Sons, New York, 1904.

60a. Lederer, Arthur. A new method for determining the relative stability of
sewage, effluent, or polluted river water: J. Infect. Diseases, Vol. 14, pp. 482–
97, 1914.

60b. ——. A serious fallacy of the “standard” methylene blue putrescibility
test: Am. J. Pub. Health, Vol. 4 (old series Vol. 10), pp. 241–8, 1914.

60c. ——. Notes on the practical application of the “saltpeter method” for
determining the strength of sewages: Am. J. Pub. Health, Vol. 5, pp. 354–61,
1915.

60d. ——. Determination of the biochemical oxygen demand by the
saltpeter method in stockyards, tannery, and corn products wastes: J. Ind. Eng.
Chem., Vol. 7, pp. 514–6, 1915.

61. Leeds, A. R. Estimation by titration of dissolved carbon dioxide in water:
J. Am. Chem. Soc., Vol. 13, pp. 98–9, 1891.

62. ——. The alteration of standard ammonium solutions when kept in the
dark: Proc. Am. Chem. Soc., Vol. 2, p. 1, 1878.

63. Leffman, Henry. Examination of water, 3d ed., pp. 46–50, P. Blakiston’s
Son & Co., Philadelphia, 1895;

63a. pp. 44–6;

63b. pp. 57–8.

64. ——. Examination of water, 7th ed., pp. 35–7, P. Blakiston’s Son & Co.,
Philadelphia, 1915;

64a. pp. 64–7.

65. Levy, D. D. Ann. de l’Observatoire de Mont-Souris, 1883 et seq.

66. Lovibond, J. W. A description of the tintometer with some remarks on its
application to chemical analysis: J. Soc. Chem. Ind., Vol. 7, pp. 424–6, 1888.

67. Mallet, J. W. Water analysis: Annual Report National Board of Health,
pp. 189–354, 1882.

68. Mason, W. P. Examination of water, 4th ed., pp. 85–9, John Wiley & Sons,
New York, 1910;

68a. pp. 33–41;

68b. pp. 59–74;

68c. pp. 106–9.

69. McGowan, George. Kjeldahl process for the estimation of total nitrogen
and (indirectly) of total organic nitrogen: Royal Commission on Sewage
Disposal, Vol. 4, pt. 5, pp. 24–31, 1904;

69a. pp. 37–41;

69b. pp. 47–8.

70. Palmer, A. W. Chemical survey of the waters of Illinois, Report for years
1897–1902, pp. 27–8, Univ. Ill., 1903.

71. ——. Report of the University of Illinois [in Report of streams
examination, Sanitary Dist. Chicago], p. 60, Chicago, 1903;

71a. p. 56;

71b. pp. 61–4.

72. Parker, G. H. Report of the biologist: Examinations by the State Board
of Health of water supplies of Mass., 1887–90, pt. 1, Examination of water
supplies, pp. 583–7, 1890.

73. Parmelee, C. L., and Ellms, J. W. On rapid methods for the estimation of
the weight of suspended matters in turbid waters: Tech. Quart., Vol. 12, pp.
145–64, 1899.

74. Pfeifer, J., and Wartha, Prof. Kritische Studien über Untersuchung und
Reinigung des Kesselspeisewassers: Z. angew. Chem., Vol. 15, pp. 193–207,
1902.

75. Phelps, E. B. A critical study of the methods in current use for the
determination of free and albuminoid ammonia in sewage, Public Health Papers
and Reports, Am. Pub. Health Assoc., Vol. 29, p. 354, 1904; J. Infect. Dis., Vol.
1, p. 327, 1904.

76. ——. The determination of the organic nitrogen in sewage by the
Kjeldahl process: J. Infect. Dis., Supp. 1, pp. 255–72, 1905.

77. ——. The determination of small quantities of copper in water: J. Am.
Chem. Soc., Vol. 28, pp. 368–72, 1906.

78. ——. Putrescibility and stability of sewage effluents: Contrib. Sanit.
Research Lab., Mass. Inst. Tech., Vol. 5, p. 87, 1909; also The disinfection of
sewage and sewage filter effluents: U. S. Geol. Survey Water-Supply Paper 229,
pp. 74–88, 1909.

79. Preface to report of committee on the pollution of water supplies:
Public Health Papers and Reports, Am. Pub. Health Assoc., Vol. 23, pp. 56–7,
1897. Report, pp. 58–100.

80. Report of committee on standard methods of water analysis: Public
Health Papers and Reports, Am. Pub. Health Assoc., Vol. 27, pp. 377–91, 1901.

81. Proctor, H. R. Some recent methods of technical water analysis: J. Soc.
Chem. Ind., Vol. 23, pp. 8–11, 1904.

82. ——. On a modified form of tintometer or colorimeter: J. Soc. Chem.
Ind., Vol. 14, pp. 122–4, 1895.

83. Richards, E. H., and Ellms, J. W. The coloring matter of natural waters,
its source, composition, and quantitative measurement: J. Am. Chem. Soc., Vol.
18, pp. 68–81, 1896.

84. Rideal, Samuel. Sewage, 2d ed., pp. 38–40, John Wiley & Sons, New
York, 1901;

84a. pp. 31–4.

85. Royal Commission on Sewage Disposal, Testimony, Vol. 2, pp. 326–37,
1902.

86. Scholl, Clarence. The perchloric method of determining potassium as
applied to water analysis: J. Am. Chem. Soc., Vol. 36, pp. 2985–9, 1914.

87. Seyler, C. A. Notes on water analysis: Chem. News, Vol. 70, pp. 82–3,
104–5, 112–4, 140–1, 151–2, and 187, 1894.

88. ——. The estimation of carbonic acid in natural waters: Analyst, Vol. 22,
pp. 312–9, 1897.

89. Smart, Chas. Report of the committee on pollution of water supplies:
Public Health Papers and Reports, Am. Pub. Health Assoc., Vol. 20, pp. 72–82,
1895;

89a. pp. 459–516.

90. Sprengel, Hermann. Ueber die Erkennung der Salpetersaüre: Ann.
Physik und Chemie, Vol. 121, pp. 188–91, 1864.

91. Standard methods of water analysis: Science, new ser., Vol. 12, pp. 906–
15, 1900.

92. Stearns, F. P., and Drown, T. M. Discussion of special topics relating to
the quality of public water supplies: Examinations by the State Board of Health
of water supplies of Mass., 1887–90, pt. 1, Examination of water supplies, pp.
740–9, 1890.

93. Street, J. P. Report on nitrogen: [In Proc. Assoc. Off. Agri. Chemists]; U.
S. Agri. Dept. Bur. Chem. Bull. 49, pp. 12–25, 1897.

94. Sutton, Francis. Volumetric analysis, 10th ed., pp. 72–4, P. Blakiston’s
Son & Co., Philadelphia, 1911;

94a. pp. 99–101;

94b. pp. 239 and 477;

94c. pp. 470–1;

94d. pp. 479–83;

94e. p. 473, 479–83;

94f. pp. 484–8.

95. Tatlock, R. R., and Thompson, R. T. The analysis of waters and their
changes in composition when employed in steam raising: J. Soc. Chem. Ind.,

Vol. 23, pp. 428–31, 1904.

96. Thomas, G. E., and Hall, C. A. New apparatus in water analysis: J. Am.
Chem. Soc., Vol. 24, pp. 535–9, 1902.

97. Thomson, R. T. Use of litmus, methyl orange, phenacetolin, and
phenolphthalein as indicators: Chem. News, Vol. 47, pp. 123–7, 1883.

98. Thomson, Andrew. Colorimetric method for determining small quantities
of iron: J. Chem. Soc., Vol. 47, pp. 493–7, 1885.

99. Thresh, J. C. A new method of estimating the oxygen dissolved in water:
J. Chem. Soc., Vol. 57, pp. 185–95, 1890.

100. ——. The examination of water and water supplies, p. 200,
Philadelphia, 1904;

100a. p. 219;

100b. p. 195;

100c. p. 282.

101. Tidy, C. M. The process for determining the organic purity of potable
waters: J. Chem. Soc., Vol. 35, pp. 46–106, 1879.

102. Tiemann, Ferdinand, and Gärtner, August. Handbuch der Wässer, 4th
ed., pp. 255–8, Friedrich Vieweg und Sohn, Braunschweig, 1895.

103. Treadwell, F. P. [translated by Hall, W. T.], Analytical Chemistry, 3d ed.,
Vol. 2, pp. 687–688, John Wiley & Sons, New York, 1911;

103a. pp. 50–3.

104. Trommsdorff, Hugo. Bestimmung der Organischen Substanzen: Zeit.
Anal. Chem., Vol. 8, p. 344, 1869.

105. U. S. Geological Survey. Measurement of color and turbidity of water,
Form 9–182, Washington, 1902.

106. Wanklyn, J. A. Verification of Wanklyn, Chapman, and Smith’s water
analyses on a series of artificial waters: J. Chem. Soc., Vol. 20, pp. 591–5, 1867.

107. ——. Water analysis, 10th ed., pp. 33–5, Kegan, Paul, Trench, Trübner,
& Co., Ltd., London, 1896;

107a. pp. 106–7.

108. Warington, Robert. Note on the appearance of nitrous acid during
evaporation of water: J. Chem. Soc., Vol. 39, pp. 229–34, 1881.

109. Warren, H. E., and Whipple, G. C. The thermophone, a new instrument
for determining temperatures: Tech. Quart., Vol. 8, pp. 125–52, 1895.

110. West, F. D. The preparation of standards for the determination of
turbidity of water: Proc. Ill. Water Supply Assoc., Vol. 6, pp. 49–51, 1914.

111. Weston, R. S. Apparatus for the determination of ammonia in water by
the Wanklyn method, and total nitrogen by the Kjeldahl method: J. Am. Chem.
Soc., Vol. 22, pp. 468–73, 1900.

112. ——. The determination of nitrogen as nitrites in waters: J. Am. Chem.
Soc., Vol. 27, pp. 281–7, 1905.

113. ——. The determination of manganese in water: J. Am. Chem. Soc., Vol.
29, pp. 1074–8, 1907.

114. Whipple, G. C. The observation of odor as an essential part of water
analysis: Public Health Papers and Reports, Am. Pub. Health Assoc., Vol. 25,
pp. 587–93, 1899.

115. ——. The microscopy of drinking water, 3d ed., pp. 186–205, John
Wiley & Sons, New York, 1914.

116. ——, and Jackson, D. D. A comparative study of the methods used for
the measurement of the turbidity of water: Tech. Quart., Vol. 13, pp. 274–94,
1900.

117. ——, and others. The decolorization of water: Trans. Am. Soc. Civil
Eng., Vol. 46, pp. 141–81, 1901.

118. ——, and Parker, H. N. On the amount of oxygen and carbonic acid
dissolved in natural waters and the effect of these gases upon the occurrence of
microscopic organisms: Trans. Am. Microscopical Soc., Vol. 23, pp. 103–44,
1901.

119. ——, and Whipple, M. C. Solubility of oxygen in sea water: J. Am.
Chem. Soc., Vol. 33, pp. 362–5, 1911.

120. Winkler, L. W. Die Bestimmung des im Wasser gelösten Sauerstoffes:
Ber., pp. 2843–54, 1888.

121. Woodman, A. G., and Norton, J. F. Air, water, and food, 4th ed., pp. 72–
8, John Wiley & Sons, New York, 1914;

121a. pp. 85–7;

121b. pp. 90–1, 216, and 231;

121c. pp. 106–8.

MICROSCOPICAL EXAMINATION.

The microscopical examination of water consists of the
enumeration of the kinds of microscopic organisms
(Plankton), and an estimation of their quantity.
It may serve any one or more of the following purposes:

(1) To explain the presence of objectionable odors and
tastes.
(2) To indicate the progress of the self purification of
streams.
(3) To indicate the presence of sewage contamination.
(4) To explain the chemical analysis.
(5) To identify the source of a water.
(6) To aid in the study of the food of fish, shellfish, and
other aquatic organisms.
The term “Microscopic Organisms” shall include all
organisms microscopic or barely visible to the naked eye,
with the exception of the bacteria. It includes the
diatomaceae, chlorophyceae, cyanophyceae, fungi,
protozoa, rotifera, crustacea, bryophyta, and spongidae
found in water.
Fragments of organic matter, silt, mineral matter,
zoöglea, etc., shall be considered as amorphous matter. The
recording of amorphous matter usually serves no useful

purpose and shall not be considered a part of the standard
method.
Apparatus.—1. A cylindrical funnel about two inches in
diameter at the top, with a straight side for nine inches,
narrowed over a distance of three inches to a bore of one-
half inch in diameter, and terminating in a straight portion
of this diameter two and one-half inches in length. The
capacity of this funnel is 500 cc. It shall be provided at the
bottom with a tightly fitting rubber stopper with a single
perforation and a disk of silk bolting cloth over the hole
about three eighths of an inch in diameter.
2. A counting cell consisting of a brass rim closely
cemented to a plate of optical glass. The shape and size of
this cell are not essential but its depth shall be one
millimeter. A convenient capacity is about one cubic
centimeter.
3. An ocular micrometer ruled as follows: The ocular
micrometer is commonly of such a size that with a 16 mm.
objective and a suitable tube length, the largest square
cuts off one square millimeter on the stage.
Procedure.—Filter 250 cc. of the water (more or less
according to the clearness of the sample) through a one-
half inch layer of quartz sand (washed and screened
between 60 and 120 mesh sieves) supported by the disk of
bolting cloth and rubber stopper at the bottom of the
funnel. Suction may be applied to hasten the filtration.
Remove the stopper and catch the plug of sand and its
entrained organisms in a small beaker or test tube,
washing down the inside of the funnel into the beaker with
5 cc. of clean (preferably distilled) water. Agitate the
mixture of sand, water, and organisms to detach the latter
from the sand grains, and quickly decant the water and the
organisms in suspension to a test tube. If desired the sand

may then be again washed with 5 cc. water and the wash
water added to the first portion.
Cover the cell partially with a cover glass, and by means
of a pipette run the concentrate under the cover glass until
the cell is completely filled.
Cover and place on the microscope stage in a horizontal
position for examination.
Count the organisms in twenty fields, i. e., twenty cubic
millimeters, estimating their areas in terms of Standard
Units.
The Standard Unit is the smallest square in the ocular
micrometer, and represents an area 20µ × 20µ, or 400
square microns on the stage.
Results shall be expressed in the number of Standard
Units of each kind of micro-organism per cc. and also the
total number of standard units of all kinds per cc. The
general directions as to significant figures given under
Turbidity shall apply also to the microscopical examination.
Caution.—Many micro-organisms, especially some of
those causing odors, are so fragile that they are broken up
in filtration, especially if the agitation of the filtrate is too
vigorous. A direct examination of a fresh sample is
therefore a useful supplementary procedure. For the same
reason the concentrate should not stand long before
examination. Also some organisms are carried by specific
gravity to the top of the cell which should be scrutinized as
well as the bottom layer each time.
It is always better to examine the micro-organisms in the
field when possible, and for this purpose the sling filter has
been devised consisting of a metal funnel slung to a pivoted
handle, with a disk of wire gauze in the detachable lower
end to support the sand. Filtration is hastened by imparting
a whirling motion to the whole and utilizing the centrifugal
force thus generated.

THE OCULAR MICROMETER.

MICROSCOPICAL BIBLIOGRAPHY.

a. Kean, A. L. A new method for the microscopical examination of water:
Science, Vol. 13, p. 132, 1889; Eng. News, pp. 21, 276, 1889.
b. Sedgwick, W. T. Recent progress in biological water analysis: J. N. E. Water
Works Assoc., Vol. 4, pp. 50–64, 1889.
c. ——. A report of the biological work of the Lawrence Experiment Station:
Examinations by the State Board of Health of water supplies of Mass.,
1887–90, pt. 2, Purification of sewage and water, pp. 793–862, 1890.
d. Parker, G. H. Report upon the organisms, excepting the bacteria found in
the waters of the State: Examinations by the State Board of Health of
water supplies of Mass., 1887–90, pt. 1, Examination of water supplies,
pp. 579–620, 1890.
e. Rafter, G. W. The microscopical examination of potable water, D. Van
Nostrand Co., New York, 1910. (Contains bibliography.)
f. Calkins, G. N. The microscopical examination of water: Report Mass. State
Board of Health, pp. 397–421, 1892.
g. Jackson, D. D. On an improvement in the Sedgwick-Rafter method for the
microscopical examination of drinking water: Tech. Quart., Vol. 9, pp.
271–4, 1896.
h. Whipple, G. C. Experience with the Sedgwick-Rafter method at the
Biological Laboratory of the Boston Water Works: Tech. Quart., Vol. 9,
pp. 275–9, 1896.
i. ——. Microscopy of drinking water, 3d ed., John Wiley & Sons, New York,
1914. (Contains bibliography.)

BACTERIOLOGICAL EXAMINATION.

I. APPARATUS.

1. Sample Bottles.—Any size, shape or quality of bottle
may be used for a bacterial sample, provided it holds a
sufficient amount to carry out all the tests required and is
such that it may be properly washed and sterilized and will
keep the sample uncontaminated until the analysis is made.
Four- or eight-ounce, ground-glass-stoppered bottles are
recommended. These should be protected by being
wrapped in paper, or their necks covered with tin foil, and
should be placed in proper boxes for transportation.
2. Pipettes.—Pipettes may be of any convenient size or
shape provided it is found by actual test that they deliver
accurately the required amount in the manner in which
they are used. The error of calibration shall in no case
exceed 2 per cent. Protecting the pipettes with a cotton
stopper is recommended.
3. Dilution Bottles.—Bottles for use in making dilutions
should preferably be of tall form, of such capacity as to
hold at least twice the volume of water actually used.
Close-fitting ground-glass stoppers are preferable, but tight
fitting cotton stoppers may be used, provided due care is
taken to prevent contamination and to avoid loss of volume
through wetting of the stopper before mixing has been
accomplished.
4. Petri Dishes.—Petri dishes ten centimeters in diameter
shall be used with glass or porous tops[211] as preferred. The
bottoms of the dishes shall be as flat as possible so that the
medium shall be of uniform thickness throughout the plate.
5. Fermentation Tubes.—Any type of fermentation
tube[203] may be used provided it holds at least three times
as much medium as the amount of water to be tested.

II. MATERIALS.
1. Water.—Distilled water shall be used in the preparation
of all culture media and reagents.
2. Meat Extract.—Liebig’s meat extract shall be used in
place of meat infusion. Other brands may be substituted for
Liebig’s when comparative tests have shown that they give
equivalent results.
3. Peptone.—Armour’s, Digestive Ferments Company’s,
Fairchild’s, or any other peptone which gives equivalent
results may be used.
4. Sugars.—All sugars used shall be of the highest purity
obtainable.
5. Agar.—The agar used shall be of the best quality and
shall be dried for one-half hour at 105° C. before weighing.
Much of the agar on the market contains considerable
amounts of sea salts.[221][225][228] These may be removed by
soaking in water and draining before use.
6. Gelatin.—The gelatin used shall be of light color, shall
contain not more than a trace of arsenic, copper, sulfides,
and shall be free from preservatives, and of such a melting
point that a 10 per cent. standard nutrient gelatin shall
have a melting point of 25° C. or over. Gelatin shall be
dried for one-half hour at 105° C. before weighing.
7. Litmus.—Reagent litmus of highest purity (not litmus
cubes) or azolitmin (Kahlbaum) shall be used for all media
requiring a litmus indicator.
8. General Chemicals.—Special effort shall be made to
have all the other ingredients used for culture media
chemically pure.

III. METHODS.

1. PREPARATION OF CULTURE MEDIA.

a. Adjustment of Reaction.

aa. Phenol Red Method for adjustment to a hydrogen-ion
concentration of PH+ = 6.8–8.4. Withdraw 5 cc. of the
medium, dilute with 5 cc. of distilled water, and add 5
drops of a solution of phenol red (phenol sulphone
phthalein). This solution is made by dissolving 0.04 grams
of phenol red in 30 cc. of alcohol and diluting to 100 cc.
with distilled water.
Titrate with a 1:10 dilution of a standard solution of
NaOH (which need not be of known normality) until the
phenol red shows a slight but distinct pink color. Calculate
the amount of the standard NaOH solution which must be
added to the medium to reach this reaction. After the
addition check the reaction by adding 5 drops of phenol red
to 5 cc. of the medium and 5 cc. of water.
bb. Titration with phenolphthalein. (For the convenience
of those who wish to retain the use of this method for the
present it is given here, but it is recommended that as soon
as possible the more accurate method of determining the
hydrogen-ion concentration be substituted.)
In a white porcelain dish put 5 cc. of the medium to be
tested, add 45 cc. of distilled water. Boil briskly for one
minute. Add 1 cc. of phenolphthalein solution (5 grams of
commercial salt to one liter of 50 per cent. alcohol). Titrate
immediately with a n/20 solution of sodium hydrate. A faint
but distinct pink color marks the true end-point. This color
may be precisely described as a combination of 25 per cent.

of red (wave length approximately 658) with 75 per cent. of
white as shown by the disks of the standard color top made
by the Milton Bradley Educational Co., Springfield, Mass.
All reactions shall be expressed with reference to the
phenolphthalein neutral point and shall be stated in
percentages of normal acid or alkali solutions required to
neutralize them. Alkaline media shall be recorded with a
minus (-) sign before the percentage of normal acid needed
for their neutralization and acid media with a plus (+) sign
before the percentage of normal alkali solution needed for
their neutralization.
The standard reaction for culture media for water
analysis shall be +1.0 per cent., as determined by tests of
the sterilized medium. As ordinarily prepared, broth and
agar will be found to have a reaction between +0.5 and
+1.0. For such media no adjustment shall be made. The
reaction of media containing sugar shall be neutral to
phenolphthalein. Whenever reactions other than the
standard are used, it shall be so stated.

b. Sterilization.

All media and dilution water shall be sterilized in the
autoclav at 15 lbs. (120° C.) for 15 minutes after the
pressure reaches 15 lbs. All air must be forced out of the
autoclav before the pressure is allowed to rise. As soon as
possible after sterilization the media shall be removed from
the autoclav and cooled rapidly. Rapid and immediate
cooling of gelatin is imperative.
Media shall be sterilized in small containers, and these
must not be closely packed together. No part of the medium
shall be more than 2.5 cm. from the outside surface of the
glass. All glassware shall be sterilized in the dry oven at
170° C. for at least 1½ hours.

c. Nutrient Broth. To Make One Liter.

1. Add 3 grams of beef extract and 5 grams of peptone to
1,000 cc. of distilled water.
2. Heat slowly on a steam bath to at least 65° C.
3. Make up lost weight and adjust the reaction to a faint
pink with phenol red, or if the phenolphthalein titration is
used, and the reaction is not already between +0.5 and +1,
adjust to +1.
4. Cool to 25° C. and filter through filter paper until clear.
5. Distribute in test-tubes, 10 cc. to each tube.
6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15
minutes after the pressure reaches 15 lbs.

d. Sugar Broths.

Sugar broths shall be prepared in the same general
manner as nutrient broth with the addition of 0.5 per cent.
of the required carbohydrate just before sterilization. The
removal of muscle sugar is unnecessary as the beef extract
and peptone are free from any fermentable carbohydrates.
The reaction of sugar broths shall be a faint pink with
phenol red or, if on titration with phenolphthalein the
reaction is not already between neutral and +1, adjust to
neutral. Sterilization shall be in the autoclav at 15 lbs.
(120° C.) for 15 minutes after the pressure reaches 15 lbs.,
provided the total time of exposure to heat is not more than
one-half hour; otherwise a 10 per cent. solution of the
required carbohydrate shall be made in distilled water and
sterilized at 100° C. for 1½ hours, and this solution shall be
added to sterile nutrient broth in amounts sufficient to
make a 0.5 per cent. solution of the carbohydrate and the
mixture shall then be tubed and sterilized at 100° C. for 30
minutes, or it is permissible to add by means of a sterile
pipette directly to a tube of sterile neutral broth enough of

the carbohydrate to make the required 0.5 per cent. The
tubes so made shall be incubated at 37° C. for 24 hours as
a test for sterility.

e. Nutrient Gelatin. To Make One Liter.

1. Add 3 grams of beef extract and 5 grams of peptone to
1,000 cc. of distilled water and add 100 grams of gelatin
dried for one-half hour at 105° C. before weighing.
2. Heat slowly on a steam bath to 65° C. until all gelatin
is dissolved.[G]
G. The solution of the gelatin will be facilitated by allowing it to soak in the
cold one-half hour before heating.

3. Make up lost weight and adjust the reaction to a faint
pink with phenol red, or if the phenolphthalein titration is
used, and the reaction is not already between +0.5 and +1,
adjust to +1.
4. Filter through cloth and cotton until clear.
5. Distribute in test-tubes, 10 cc. to each tube, or in
larger containers as desired.
6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15
minutes after the pressure reaches 15 lbs.

f. Nutrient Agar. To Make One Liter.

1. Add 3 grams of beef extract, 5 grams of peptone and
12 grams of agar, dried for one-half hour at 105° C. before
weighing, to 1,000 cc. of distilled water. Boil over a water
bath until all agar is dissolved, and then make up the loss
by evaporation.
2. Cool to 45° C. in a cold water bath, then warm to 65°
C. in the same bath, without stirring.
3. Make up lost weight and adjust the reaction to a faint
pink with phenol red, or if the phenolphthalein titration is

used, and the reaction is not already between +0.5 and +1,
adjust to +1.
4. Filter through cloth and cotton until clear.
5. Distribute in test-tubes, 10 cc. to each tube, or in
larger containers, as desired.
6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15
minutes after the pressure reaches 15 lbs.

g. Litmus or Azolitmin Solution.

The standard litmus solution shall be a 2 per cent.
aqueous solution of reagent litmus. Powder the litmus, add
to the water and boil for five minutes. The solution usually
needs no correction in reaction and may be at once
distributed in flasks or test-tubes and sterilized as is
culture media. It should give a distinctly blue plate when 1
cc. is added to 10 cc. of neutral culture medium in a Petri
dish.
The standard azolitmin solution shall be a 1 per cent.
solution of Kahlbaum’s azolitmin. Add the azolitmin powder
to the water and boil for five minutes. The solution may
need to be corrected in reaction by the addition of sodium
hydrate solution so that it will be approximately neutral
and will give a distinctly blue plate when 1 cc. is added to
10 cc. of neutral culture medium in a Petri dish. It may be
distributed in flasks or test-tubes and sterilized as is
culture media.

h. Litmus-lactose-agar.

Litmus-lactose-agar shall be prepared in the same
manner as nutrient agar with the addition of 1 per cent. of
lactose just before sterilization. The reaction shall be a
faint pink with phenol red, or, if on titration with
phenolphthalein the reaction is not already between

neutral and +1, adjust to neutral. One cc. of sterilized
litmus or azolitmin solution shall be added to each 10 cc. of
the medium just before it is poured into the Petri dish, or
the mixture may be made in the dish itself.

i. Endo’s Medium.[209][214][215] To Make One Liter.

1. Add 5 grams of beef extract, 10 grams of peptone and
30 grams of agar dried for one-half hour at 105° C. before
weighing, to 1,000 cc. of distilled water. Boil on a water
bath until all the agar is dissolved and then make up the
loss by evaporation.
2. Cool the mixture to 45° C. in a cold water bath, then
warm to 65° C. in the same bath without stirring.
3. Make up lost weight, titrate, and if the reaction is not
already between neutral and +1 adjust to neutral.
4. Filter through cloth and cotton until clear.
5. Distribute 100 cc. or larger known quantities in flasks
large enough to hold the other ingredients which are to be
added later.
6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15
minutes after the pressure reaches 15 lbs.
7. Prepare a 10 per cent. solution of basic fuchsin in 95
per cent. alcohol, allow to stand 20 hours, decant and filter
the supernatant fluid. This is a stock solution.
8. When ready to make plates melt 100 cc. of agar in
streaming steam or on a water bath. Dissolve 1 gram of
lactose in 15 cc. of distilled water, using heat if necessary.
Dissolve 0.25 gram anhydrous sodium sulphite in 10 cc.
water. To the sulphite solution add 0.5 cc. of the fuchsin
stock solution. Add the fuchsin-sulphite solution to the
lactose solution and then add the resulting solution to the
melted agar. The lactose used must be chemically pure and
the sulphite solution must be made up fresh.

9. Pour plates and allow to harden thoroughly in the
incubator before use.

2. COLLECTION OF SAMPLE.

Samples for bacterial analysis shall be collected in bottles
which have been cleansed with great care, rinsed in clean
water, and sterilized with dry heat for at least one hour and
a half at 170° C., or in the autoclav at 15 lbs. (120° C.) for
15 minutes or longer after the pressure reaches 15 lbs.
Great care must be exercised to have the samples
representative of the water to be tested and to see that no
contamination occurs at the time of filling the sample
bottles.

3. STORAGE AND TRANSPORTATION OF SAMPLES.

Because of the rapid and often extensive changes which
may take place in the bacterial flora of bottled samples
when stored even at temperatures as low as 10° C., it is
urged, as of importance, that all samples be examined as
promptly as possible after collection.
The time allowed for storage or transportation of a
bacterial sample between the filling of the sample bottle
and the beginning of the analysis should be not more than
six hours for impure waters and not more than twelve
hours for relatively pure waters. During the period of
storage, the temperature shall be kept as near 10° C. as
possible. Any deviation from the above limits shall be so
stated in making reports.

4. DILUTIONS.

Dilution bottles shall be filled with the proper amount of
tap water so that after sterilization they shall contain
exactly 9 cc. or 99 cc. as desired. The exact amount of
water can only be determined by experiment with the
particular autoclav in use. If desired, the 9 cc. dilution may
be measured out from a flask of sterile water with a sterile
pipette.
Dilution bottles shall be sterilized in the autoclav at 15
lbs. (120° C.) for 15 minutes after the pressure reaches 15
lbs.
The sample bottle shall be shaken vigorously 25 times
and 1 cc. withdrawn and added to the proper dilution
bottles as required. Each dilution bottle after the addition
of the 1 cc. of the sample, shall be shaken vigorously 25
times before a second dilution is made from it or before a
sample is removed for plating.

5. PLATING.

All sample and dilution bottles shall be shaken vigorously
25 times before samples are removed for plating. Plating
shall be done immediately after the dilutions are made. One
cc. of the sample or dilution shall be used for plating and
shall be placed in the Petri dish, first. Ten cc. of liquefied
medium at a temperature of 40° C. shall be added to the 1
cc. of water in the Petri dish. The cover of the Petri dish
shall be lifted just enough for the introduction of the
pipette or culture medium, and the lips of all test-tubes or
flasks used for pouring the medium shall be flamed. In
making litmus-lactose-agar plates, 1 cc. of sterile litmus or
azolitmin solution shall be added to each 10 cc. of culture
medium either in the Petri dish or before pouring into the
Petri dish. The medium and sample in the Petri dish shall
be thoroughly mixed and uniformly spread over the bottom

of the Petri dish by tilting or rotating the dish. All plates
shall be solidified as rapidly as possible after pouring and
gelatin plates shall be placed immediately in the 20° C.
incubator and the agar plates in the 37° C. incubator. Endo
plates shall be made by placing one loopful of the material
to be tested on the surface of the plate and distributing the
material with a sterile loop or glass rod.

6. INCUBATION.

All gelatin plates shall be incubated for 48 hours at 20 C.
in a dark, well-ventilated incubator in an atmosphere
practically saturated with moisture.[227]
All agar plates shall be incubated for 24 hours at 37° C.
in a dark, well-ventilated incubator in an atmosphere
practically saturated with moisture. Glass covered plates
shall be inverted in the incubator. Any deviation from the
above described method shall be stated in making reports.

7. COUNTING.

In preparing plates, such amounts of the water under
examination shall be planted as will give from 25 to 250
colonies on a plate;[202] and the aim should be always to
have at least two plates giving colonies between these
limits. Where it is possible to obtain plates showing
colonies within these limits, only such plates should be
considered in recording results, except where the same
amount of water has been planted in two or more plates, of
which one gives colonies within these limits, while the
others give less than 25 or more than 250. In such case, the
result recorded should be the average of all the plates
planted with this amount of water. Ordinarily it is not
desirable to plant more than 1 cc. of water in a plate;

therefore, when the total number of colonies developing
from 1 cc. is less than 25, it is obviously necessary to
record the results as observed, disregarding the general
rule given above.
Counting shall in all cases be done with a lens of 2½
diameter’s magnification, 3½X. The Engraver’s Lens No.
146 made by the Bausch & Lomb Optical Company fills the
requirements, and is a convenient lens for the purpose.

8. THE TEST FOR THE PRESENCE OF MEMBERS OF
THE B. COLI GROUP.

It is recommended that the B. coli group be considered as
including all non-spore-forming bacilli which ferment
lactose with gas formation and grow aërobically on
standard solid media.
The formation of 10 per cent. or more of gas in a
standard lactose broth fermentation tube within 24 hours
at 37° C. is presumptive evidence of the presence of
members of the B. coli group, since the majority of the
bacteria which give such a reaction belong to this group.
The appearance of aërobic lactose-splitting colonies on
lactose-litmus-agar or Endo’s medium plates made from a
lactose broth fermentation tube in which gas has formed
confirms to a considerable extent the presumption that gas-
formation in the fermentation tube was due to the presence
of members of the B. coli group.
To complete the demonstration of the presence of B. coli
as above defined, it is necessary to show that one or more
of these aërobic plate colonies consists of non-spore-
forming bacilli which, when inoculated into a lactose broth
fermentation tube, form gas.
It is recommended that the standard tests for the B. coli
group be either (A) the Presumptive, (B) the Partially

Confirmed, or (C) the Completed test as hereafter defined,
each test being applicable under the circumstances
specified.

A. PRESUMPTIVE TEST.

1. Inoculate a series of fermentation tubes with
appropriate graduated quantities of the water to be tested.
In every fermentation tube there must always be at least
three times as much medium as the amount of water to be
tested. When necessary to examine larger amounts than 10
cc. as many tubes as necessary shall be inoculated with 10
cc. each.
2. Incubate these tubes at 37° C. for 48 hours. Examine
each tube at 24 and 48 hours, and record gas-formation.
The records should be such as to distinguish between:
(a) Absence of gas-formation.
(b) Formation of gas occupying less than ten per cent.
(10%) of the closed arm.
(c) Formation of gas occupying more than ten per cent.
(10%) of the closed arm.
More detailed records of the amount of gas formed,
though desirable for purposes of study, are not necessary
for carrying out the standard tests prescribed.
3. The formation within 24 hours of gas occupying more
than ten per cent. (10%) of the closed arm of fermentation
tube constitutes a positive presumptive test.
4. If no gas is formed in 24 hours, or if the gas formed is
less than ten per cent. (10%), the incubation shall be
continued to 48 hours. The presence of gas in any amount
in such a tube at 48 hours constitutes a doubtful test,
which in all cases requires confirmation.
5. The absence of gas formation after 48 hours’
incubation constitutes a negative test. (An arbitrary limit of

48 hours’ observation doubtless excludes from
consideration occasional members of the B. coli group
which form gas very slowly, but for the purposes of a
standard test the exclusion of these occasional slow gas-
forming organisms is considered immaterial.)

B. PARTIALLY CONFIRMED TEST.

1. Make one or more Endo’s medium or lactose-litmus-
agar plates from the tube which, after 48 hours’ incubation,
shows gas formation from the smallest amount of water
tested. (For example, if the water has been tested in
amounts of 10 cc., 1 cc., and 0.1 cc., and gas is formed in
10 cc., and 1 cc., not in 0.1 cc., the test need be confirmed
only in the 1 cc. amount.)
2. Incubate the plates at 37° C., 18 to 24 hours.
3. If typical colon-like red colonies have developed upon
the plate within this period, the confirmed test may be
considered positive.
4. If, however, no typical colonies have developed within
24 hours, the test cannot yet be considered definitely
negative, since it not infrequently happens that members of
the B. coli group fail to form typical colonies on Endo’s
medium or lactose-litmus-agar plates, or that the colonies
develop slowly. In such case, it is always necessary to
complete the test as directed under “C” 2 and 3.

C. COMPLETED TEST.

1. From the Endo’s medium or lactose-litmus-agar plate
made as prescribed under “B,” fish at least two typical
colonies, transferring each to an agar slant and a lactose
broth fermentation tube.
2. If no typical colonies appear upon the plate within 24
hours, the plate should be reincubated another 24 hours,

after which at least two of the colonies considered to be
most likely B. coli, whether typical or not, shall be
transferred to agar slants and lactose broth fermentation
tubes.
3. The lactose broth fermentation tubes thus inoculated
shall be incubated until gas formation is noted; the
incubation not to exceed 48 hours. The agar slants shall be
incubated at 37° C. for 48 hours, when a microscopic
examination shall be made of at least one culture, selecting
one which corresponds to one of the lactose broth
fermentation tubes which has shown gas-formation.
The formation of gas in lactose broth and the
demonstration of non-spore-forming bacilli in the agar
culture shall be considered a satisfactory completed test,
demonstrating the presence of a member of the B. coli
group.
The absence of gas-formation in lactose broth or failure
to demonstrate non-spore-forming bacilli in a gas-forming
culture constitutes a negative test.

APPLICATION OF PRESUMPTIVE, PARTIALLY CONFIRMED, AND
COMPLETED TESTS.

A. The Presumptive Test.

1. When definitely positive, that is showing more than 10
per cent. (10%) of gas in 24 hours, is sufficient:
(a) As applied to all except the smallest gas-forming
portion of each sample in all examinations.
(b) As applied to the smallest gas-forming portion in
the examination of sewage or of water showing
relatively high pollution, such that its fitness for use
as drinking water does not come into consideration.
This applies to the routine examinations of raw

water in connection with control of the operation of
purification plants.
2. When definitely negative, that is showing no gas in 48
hours, is final and therefore sufficient in all cases.
3. When doubtful, that is showing gas less than 10 per
cent. (10%) (or none) in 24 hours, with gas either more
or less than 10 per cent. in 48 hours, must always be
confirmed.

B. The Partially Confirmed Test.

1. When definitely positive, that is, showing typical plate
colonies within 24 hours, is sufficient:
(a) When applied to confirm a doubtful presumptive
test in cases where the latter, if definitely positive,
would have been sufficient.
(b) In the routine examination of water supplies where
a sufficient number of prior examinations have
established a satisfactory index of the accuracy and
significance of this test in terms of the completed
test.
2. When doubtful, that is, showing colonies of doubtful or
negative appearance in 24 hours, must always be
completed.

C. The Completed Test.

The completed test is required as applied to the smallest
gas-forming portion of each sample in all cases other
than those noted as exceptions under the “presumptive”
and the “partially confirmed” tests.
The completed test is required in all cases where the
result of the confirmed test has been doubtful.

9. EXPRESSION OF RESULTS.

In order to avoid fictitious accuracy and yet to express
the numerical results by a method consistent with the
precision of the work, the numbers of colonies of bacteria
per cubic centimeter shall be recorded as follows:[212]
Number of bacteria per cc.
From 1 to 50 shall be recorded as found
〃 51 〃 100 〃 〃 〃 to the nearest 5
〃 101 〃 250 〃〃〃〃〃〃 10
〃 251 〃 500 〃〃〃〃〃〃 25
〃 501 〃 1,000 〃〃〃〃〃〃 50
〃 1,001 〃 10,000 〃〃〃〃〃〃 100
〃 10,001 〃 50,000 〃〃〃〃〃〃 500
〃 50,001 〃 100,000 〃〃〃〃〃〃 1,000
〃 100,001 〃 500,000 〃〃〃〃〃〃 10,000
〃 500,001 〃 1,000,000 〃〃〃〃〃〃 50,000
〃 1,000,001 〃 10,000,000 〃〃〃〃〃〃 100,000

This applies to the gelatin count at 20° C. and to the agar
count at 37° C.

Summary of steps involved in making presumptive, partially confirmed and
completed tests for b. coli.

Steps in procedure. Further
procedure
required.
I. Inoculate lactose broth fermentation tubes; incubate 24
hours at 37° C.; observe gas-formation in each tube.
1. Gas-formation, 10 per cent. or more; constitutes
positive presumptive test.
(a) For other than smallest portion of any sample
showing gas at this time, and for all portions,
including smallest, of sewage and raw water this
test is sufficient. None
(b) For smallest gas-forming portion, except in
examinations of sewage and raw water. III
2. Gas-formation less than 10 per cent. in 24 hours;
inconclusive. II
II. Incubate an additional 24 hours, making a total of 48
hours’ incubation; observe gas-formation.
1. Gas-formation, any amount; constitutes doubtful test,
which must always be carried further. III
2. No gas-formation in 48 hours; constitutes final
negative test. None
III. Make plate from smallest gas-forming portion of sample
under examination; incubate 18 to 24 hours; observe
colonies.
1. One or more colonies typical in appearance.
(a) If only “partially confirmed” test is required None
(b) If completed test is required, select two typical
colonies for identification. V
2. No typical colonies. IV
IV. Replace plate in incubator for an additional 18 to 24
hours; then, whether colonies appear typical or not, select
at least two of those which most nearly resemble B. coli. V
V. Transfer each colony fished to:
1. Lactose broth fermentation tube; incubate not more
than 48 hours at 37° C. Observe gas-formation. None
2. Agar slant; incubate 48 hours at 37° C.
(a) If gas formed in lactose broth tube inoculated with
corresponding culture VI
(b) If no gas formed in corresponding lactose broth
tube, test is completed and negative. None
VI. Make stained cover-slip or slide preparation, and
examine microscopically.
1. If preparation shows non-spore-forming bacilli in
apparently pure culture, demonstration of B. coli is
completed. None

2. If preparation fails to show non-spore-forming bacilli
or shows them mixed with spore-bearing forms or
bacteria of other morphology. VII
VII. Replate, to obtain assuredly pure culture, select several
colonies of bacilli and repeat steps V and VI.

In order that tests for B. coli may have quantitative
significance, the following general principles and rules
should be observed:
Ordinarily not less than three portions of each sample
should be tested, the portions being even decimal multiples
or fractions of a cubic centimeter; for example, 10 cc., 1
cc., 0.1 cc., .01 cc., etc. It is essential that the dilutions
should be such that the largest amount gives a positive test
(unless the water is such as to give negative tests in 10
cc.), and the smallest dilution, a negative result. To insure
this result, it is often necessary to plant four or five
dilutions, especially in the examination of a sample of
entirely unknown quality. The quantitative value of a series
of tests is lost, unless all or at least a large proportion of
the smallest dilutions tested have given negative results.
In reporting a single test, it is preferable merely to
record results as observed, indicating the amounts tested
and the result in each, rather than to attempt expression of
the result in numbers of B. coli per cc. In summarizing the
results of a series of tests, however, it is desirable, for the
sake of simplicity, to express the results in terms of the
numbers of B. coli per cc., or per 100 cc. To convert results
of fermentation tests to this form, the result of each test is
recorded as indicating a number of B. coli per cc. equal to
the reciprocal of the smallest decimal or multiple fraction
of a cubic centimeter giving a positive result. For example,
the result: 10 cc. +; 1 cc. +; 0.1 cc. -; would be recorded as
indicating one B. coli per cc. An exception should be made
in the case where a negative result is obtained in an
amount larger than the smallest portion giving a positive
result; for example, in a result such as: 10 cc. +; 1 cc. -; 0.1

cc. +. In such case, the result should be recorded as
indicating a number of B. coli per cc. equal to the
reciprocal of the dilution next larger than the smallest one
giving a positive test, this being a more probable result.
Where tests are made in amounts larger than 1 cc.,
giving average results less than one B. coli per cc., it is
more convenient to express results in terms of the numbers
of B. coli per 100 cc.
The following table illustrates the method of recording
and averaging results of B. coli tests:
Result of Tests in Amounts Designated. Indicated Number of B. coli.
10 cc. 1 cc. 0.1 cc. .01 cc. per cc. per 100 cc.
+ − − − 0.1 10.
+ + − − 1.0 100.
+ + + − 10.0 1,000.
+ + + + 100.0 10,000.
+ + − + 10.0 1,000.

Totals (for estimating averages) 121.1 12,110.
Average of 5 tests 24.0 2,422.

The above method of expressing results is not
mathematically altogether correct. The average number of
B. coli per cc., as thus estimated, is not precisely the most
probable number calculated by application of the theory of
probability.[220] To apply this theory to a correct
mathematical solution of any considerable series of results
involves, however, mathematical calculations so complex as
to be impracticable of application in general practice. The
simpler method given is therefore considered preferable,
since it is easily applied and the results so expressed are
readily comprehensible.
In order that results as reported may be checked and
carefully valuated, it is necessary that the report should
show not only the average number of B. coli per cc., but
also the number of samples examined; and, for each

dilution, the total number of tests made, and the number
(or per cent.) positive.

10. INTERPRETATION OF RESULTS.

While it is not within the province of this report to
suggest the proper interpretation of results obtained by the
use of the methods herein specified as standard, the
committee feels that a word of caution should be given
regarding the significance of the presence in a water of
members of the B. coli group as defined in this report.
Recent work seems to indicate that the B. coli group as
herein defined consists of organisms of both fecal and non-
fecal origin. Therefore care must be exercised in judging
the sanitary quality of a water solely from the
determination of the presence of members of the group.

11. DIFFERENTIATION OF FECAL FROM NON-FECAL
MEMBERS OF THE B. COLI GROUP.

(1) At least 10 cultures should be used. If possible these
should be subcultured from plates made direct from the
water since all of the cultures obtained by plating from
fermentation tubes may be descendants of a single cell in
the water. If cultures from water plates are not available
those obtained from plates made as prescribed under B (p.
101) may be used.
(2) Inoculate each culture into dextrose potassium
phosphate broth,[H] adonite broth, and gelatin. For
additional confirmatory evidence inoculation may be made
into tryptophane broth,[I] and saccharose broth. The
dextrose broth must be incubated at 30°. Other sugar
broths may be incubated at 30° or 37° as convenient.
Gelatin should be incubated at 20°.

H. (a) Peptone Medium for the Methyl Red Test. To Make One Liter.
1. To 800 cc. of distilled water add 5 grams of Proteose-Peptone, Difco., or
Witte’s Peptone (other peptones should not be substituted), 5 grams c. p.
dextrose, and 5 grams dipotassium hydrogen phosphate (K2HPO4). A dilute
solution of the K2HPO4 should give a distinct pink with phenolphthalein.
2. Heat with occasional stirring over steam for twenty minutes.
3. Filter through folded filter paper, cool to 20° C. and dilute to 1,000 cc.
with distilled water.
4. Distribute 10 cc. portions in sterilized test-tubes.
5. Sterilize by the intermittent method for 20 minutes on three successive
days.

I. Tryptophane Broth for Indol Test.
To 1,000 cc. of distilled water add 0.3 gram tryptophane, 5 grams
dipotassium hydrogen phosphate (K2HPO4), and 1 gram peptone. Heat until
ingredients are thoroughly dissolved, tube (6 to 8 cc.), and sterilize in autoclave
for 15 minutes after the pressure reaches 15 pounds. Some American peptones
are standardized to contain a uniform amount of tryptophane. If such peptone
is used the tryptophane in the above formula may be omitted and the peptone
increased to 5 grams.

(3) After 48 hours record gas formation in adonite and
saccharose broths. Determine indol formation in
tryptophane broth by adding drop by drop, to avoid mixing
with the medium, about 1 cc. of a 2 per cent. alcoholic
solution of p-dimethyl amido-benzaldehyd, then a few drops
of concentrated hydrochloric acid. The presence of indol is
indicated by a red color which is soluble in chloroform.
There may be some unconverted tryptophane still present
which will give a distinctly blue color which is insoluble in
chloroform. A mixture of the two will be either blue or
violet. If from such a mixture of colors the red of indol be
extracted with chloroform proof of the presence of indol
will be complete.
(4) After 5 days apply methyl red test and Voges-
Proskauer test to dextrose broth.

Methyl Red Test.[J]

Indicator solution.—Dissolve 0.1 gram methyl red in 300
cc. alcohol and dilute to 500 cc. with distilled water.
J. (b) Synthetic Medium for the Methyl Red Test. To Make One Liter.
Dissolve 7 grams Na2HPO4 (anhydrous) or 8.8 grams Na2HPO4.2H2O, 2
grams KHphthalate, 1 gram aspartic acid, and 4 grams dextrose in about 800
cc. of warm distilled water. When solution is complete, cool and make up to 1
liter at room temperature. Heat in an autoclave for 15 minutes after the
pressure has reached 15 pounds, provided the total time of exposure to heat is
not more than one-half hour. The hydrogen-ion concentration of the medium is
fixed by the composition. It should be very close to PH 7.0, slightly red with
phenol red. All materials should be recrystallized or if used from stock
furnished by manufacturers, should be carefully examined. The di-sodium
hydrogen phosphate may be used either as the anhydrous salt obtained by
dessication in vacuo at 100° C. or else as the salt containing two molecules of
water of crystallization. This is obtained by exposing the recrystallized
Na2HPO4.12H2O for two weeks. Use 0.88 per cent. of Na2HPO4.2H2O.

Procedure in test.—1. To 5 cc. of each culture add 5
drops of methyl red solution.
2. Record distinct red color as methyl red +, distinct
yellow color as methyl red -, and intermediate colors as ?.

Voges-Proskauer Test.[216]

To the remaining 5 cc. of medium add 5 cc. of a 10 per
cent. solution of potassium hydroxide. Allow to stand over
night. A positive test is indicated by an eosin pink color.
(5) Gelatin tubes should not be pronounced negative until
they have been incubated at least 15 days.
The following group reactions indicate the source of the
culture with a high degree of probability:

Methyl red +
Voges-Proskauer −
Gelatin −
B. coli of fecal origin.
Adonite −
Indol, usually +
Saccharose, usually −

Methyl red −
Voges-Proskauer +
Gelatin −
B. aërogenes of fecal origin.
Adonite +
Indol, usually −
Saccharose +

Methyl red −
Voges-Proskauer +
Gelatin −
B. aërogenes, probably not of fecal origin.
Adonite −
Indol, usually −
Saccharose +

Methyl red −
Voges-Proskauer +
Gelatin +
B. cloacae, may or may not be of fecal origin.
Adonite +
Indol, usually −
Saccharose +

12. ROUTINE PROCEDURE FOR EXAMINATION OF
SAMPLES OF WATER.

First Day:
1. Prepare dilutions as required.
2. Make two (2) gelatin plates from each dilution, and
incubate at 20° C.
3. Make two (2) agar plates from each dilution, and
incubate at 37° C.

4. Inoculate lactose broth fermentation tubes with
appropriate amounts for B. coli tests, inoculating two
(2) tubes with each amount.
Note:—Where repeated tests are made of water from the
same source, as is customary in the control of public
supplies, it is not necessary to make duplicate plates or
fermentation tubes in each dilution. It is sufficient, in such
circumstances, to make duplicate plates only from the
dilution which will most probably give from 25 to 250
colonies per plate.
Second Day:
1. Count the agar plates made on the first day.
2. Record the number of lactose broth fermentation
tubes which show 10 per cent. (10%) or more of gas.
Note:—In case only the presumptive test for B. coli is
required, fermentation tubes showing more than 10 per
cent. (10%) of gas at this time may be discarded.
Third Day:
1. Count gelatin plates made on first day.
2. Record the number of additional fermentation tubes
which show 10 per cent. (10%) or more of gas.
3. Make a lactose-litmus-agar or Endo’s medium plate
from the smallest portion of each sample showing gas.
Incubate plate at 37° C.
Note:—In case the smallest portion in which gas has been
formed shows less than 10 per cent. (10%) of gas, it is well
to make a plate also from the next larger portion, so that, in
case the smallest portion gives a negative end result it may
still be possible to demonstrate B. coli in the next larger
dilution.
Fourth Day:
1. Examine Endo’s medium or lactose-litmus-agar
plates. If typical colonies have developed, select two

and transfer each to a lactose broth fermentation tube
and an agar slant, both of which are to be incubated at
37° C.
2. If no typical B. coli colonies are found, incubate the
plates another 24 hours.
Fifth Day:
1. Select at least two colonies, whether typical or not,
from the Endo’s medium or lactose-litmus-agar plates
which have been incubated an additional 24 hours;
transfer each to a lactose broth fermentation tube and
an agar slant, and complete the test as for typical
colonies.
2. Examine lactose broth fermentation tubes inoculated
from plates on the previous day. Tubes in which gas
has been formed may be discarded after the result has
been recorded. Those in which no gas has formed
should be incubated an additional 24 hours.
Sixth Day:
1. Examine lactose broth fermentation tubes
reincubated the previous day.
2. Examine microscopically agar slants corresponding
to lactose fermentation tubes inoculated from plate
colonies and showing gas-formation.

BACTERIOLOGICAL BIBLIOGRAPHY.
201. Bovie, W. T. A Direct Reading Potentiometer for Measuring and
Recording both the Actual and the Total Reaction of Solutions. Jour. Med.
Research, 33, 1915–16, 295.

202. Breed, R. S. and Dotterrer, W. D. The Number of Colonies Allowable
on Satisfactory Agar Plates. Jour. of Bact., 1, 1916, 321.

203. Browne, W. W. A Comparative Study of the Smith Fermentation Tube
and the Inverted Vial in the Determination of Sugar Fermentation. Amer. Jour.
of Public Health, 3, 1913, 701.

204. Clark, W. M. An Hydrogen Electrode Vessel. Jour. Biol. Chem., 23,
1915, 475.

205. Clark, W. M. The “Reaction” of Bacteriological Culture Media. Jour. of
Inf. Diseases, 17, 1915, 109.

206. Clark, W. M. The Final Hydrogen Ion Concentrations of Cultures of
Bacillus Coli. Science, n. s. 42, 1915, 71.

207. Clark, W. M. and Lubs, H. A. Hydrogen Electrode Potentials of
Phthalate, Phosphate, and Borate Buffer Mixtures. Jour. Biol. Chem., 25, 1916,
479.

208. Clark, W. M. and Lubs, H. A. The Differentiation of Bacteria of the
Colon-Aërogenes Family by the Use of Indicators. Jour. of Inf. Diseases, 17,
1915, 160.

209. Endo, S. Ueber ein Verfahren zum Nachweis der Typhusbacillen
Centbl. f. Bakt. Erste Abt., 35, 1903–4, 109.

210. Gillespie, L. J. The Reaction of Soil and Measurements of Hydrogen
Ion Concentration. Jour. Wash. Acad. of Sciences, 6, 1916, 7.

211. Hill, H. W. Porous Tops for Petri Dishes. Jour. Med. Research, 13,
1904, 93.

212. Hill, H. W. The Mathematics of the Bacterial Count. Public Health
Reports and Papers, 33, 1907, 110.

213. Itano, A. The Relation of Hydrogen Ion Concentration of Media to the
Proteolytic Activity of Bacillus Subtilis. Bulletin 167, 1916, Mass. Agric. Ex.
Station.

214. Kendall, A. I. and Walker, A. W. The Isolation of Bacillus Dysenteriae
from Stools. Jour. Med. Research, 23, 1910, 481.

215. Kinyoun, J. J. and Deiter, L. V. On the Preparation of Endo’s Medium.
Amer. Jour. Public Health, n. s. 2, 1912, 979.

216. Levine, M. On the Significance of the Voges-Proskauer Reaction. Jour.
of Bacteriology, 1, 1916, 153.

217. Lubs, H. A. and Clark, W. M. On Some New Indicators for the
Colorimetric Determination of Hydrogen-ion Concentration. Jour. Wash. Acad.
of Sciences, 5, 1915, 609.

218. McClendon, J. F. New Hydrogen Electrodes and Rapid Methods of
Determining Hydrogen Ion Concentrations. Amer. Jour. of Physiology, 38, 1915,
180.

219. McClendon, J. F. A Direct Reading Potentiometer for Measuring
Hydrogen Ion Concentrations. Amer. Jour. of Physiology, 38, 1915, 186.

220. McCrady, M. H. The Numerical Interpretation of Fermentation-tube
Results. Jour. Inf. Diseases, 17, 1915, 183.

221. Noyes, H. A. Agar Agar for Bacteriological Use. Science, n. s. 44, 1916,
797.

222. Rogers, L. A., Clark, W. M. and Davis, B. J. The Colon Group of
Bacteria. Jour. of Inf. Diseases, 14, 1914, 411.

223. Rogers, L. A., Clark, W. M. and Evans, A. C. The Characteristics of
Bacteria of the Colon Type Found in Bovine Feces. Jour. of Inf. Diseases, 15,
1914, 99.

224. Rogers, L. A., Clark, W. M. and Evans, A. C. The Characteristics of
Bacteria of the Colon Type Occurring on Grains. Jour. of Inf. Diseases, 17, 1915,
137.

225. Smith, H. M. The Seaweed Industries of Japan. Bulletin of the Bureau
of Fisheries, 24, 1904, 135.

226. Sörensen, S. P. L. Enzymstudien. Biochem. Ztschr., 21, 1909, 131 and
201.

227. Whipple, G. C. On the Necessity of Cultivating Water Bacteria in an
Atmosphere Saturated with Moisture. Tech. Quart., 12, 1899, 276.

228. Whittaker, H. A. The Source, Manufacture and Composition of
Commercial Agar-agar. Jour. Amer. Pub. Health Assoc., n. s. 1, 1911, 632.

229. Clark, W. M. and Lubs, H. A. The colorimetric determination of the
hydrogen-ion concentration of bacteriological culture media. Jour. Wash. Acad.
Sciences, 6, 1916, 483.

230. Clark, W. M. and Lubs, H. A. The colorimetric determination of
hydrogen-ion concentration and its application in bacteriology. Jour. of Bact., 2,
1919, 1 and 109.

231. Cohen, B. and Clark, W. M. The growth of certain bacteria in media of
different hydrogen-ion concentrations. Jour. of Bact., 4, 1919, 409.

232. Fennel, E. A. and Fisher, M. A. Adjustment of culture medium
reactions. Jour. of Inf. Diseases, 25, 1919, 444.

233. Jones, H. M. A rapid hydrogen-ion electrode method for the
determination of hydrogen-ion concentrations in bacterial cultures or other
turbid or colored solutions. Jour. of Inf. Diseases, 25, 1919, 262.

INDEX.

A.
Acidity, determination of, 39.
Acids, mineral, 41.
Agar, nutrient, 94, 96.
lactose-litmus, 97.
Alkali carbonates, 39.
Alkalinity, determination of, 35.
Albuminoid nitrogen, 20.
Aluminium sulfate, determination of, 41.
analysis of, 78.
Aluminium and iron, determination of, 57.
Ammonia nitrogen, determination of, 15.
Apparatus, bacteriological, 93.
Application of colon group tests, 102.
Arsenic, determination of, 63.
Azolitmin solution, 96.

B.
Bacillus aërogenes, reactions, 108.
cloacae, reactions, 108.

coli, reactions, 108.
B. coli group, tests, 100.
application of, 102.
fecal and non-fecal, 106.
summary of tests, 104.
Bacteriological examination, 93.
bibliography, 110.
Basicity ratio, 80.
Bibliography,
bacteriological, 110.
chemical, 82.
microscopical, 91.
Biochemical oxygen demand, 71.
in sludge and mud, 76.
Bismuthate method (Mn), 49.
Boric acid, 63.
Bottles, sample, 1, 93.
dilution, 93.
Bromine and iodine, determination of, 61.
Broth, nutrient, 95.
sugar, 95.

C.
Calcium, determination of, 57.
Carbon dioxide, determination of, 40.
Chemical analysis, water and sewage, 1.
bibliography, 82.
Chemicals, analysis of, 77.
Chloride, determination of, 41.

Chlorine, determination of, 64.
Coefficient of fineness, 8.
Collection of samples, bacteriological, 93.
chemical, 1.
Colon group, tests (see “B. coli”), 100.
Color, determination of, 9.
Copper, determination of, 53, 55.
Counting (bacterial), 99.
Cultural characters of colon group, 108.
Culture media, 94.
azolitmin solution, 96.
Endo’s medium, 97.
litmus-lactose-agar, 97.
litmus solution, 96.
methyl red test, 107.
nutrient agar, 96.
nutrient broth, 95.
nutrient gelatin, 96.
sterilization, 95.
sugar broth, 95.
titration, 94.
tryptophane broth, 107.

D.
Dilution (bacteriological), 98.
Dissolved oxygen, 65.

E.
Effluents, relative stability of, 69.
biochemical, oxygen, demand of, 71.

Endo’s medium, 97.
Erythrosine indicator, 36.
Ether—soluble matter, 69.
in sludge and mud, 75.
Evaporation, 29.
Expression of results (see under “Results”).

F.
Fat, determination of, 69, 75.
Fecal and non-fecal members, colon group, 106.
Fermentation tubes, 93.
Ferrous sulfide in sludge and mud, 76.
Ferrous iron, determination of, 47.
Ferric iron, determination of, 48.
Fineness, coefficient of, 8.

G.
Gelatin media, 94, 96.

H.
Hardness, determination of, 30.
bicarbonate, 37.
carbonate, 38.
hydroxide, 38.
non-carbonate, 34.
temporary, 34.
Hydrogen sulfide, determination of, 63.

Hydrogen-ion determination, 94.

I.
Ignition, loss on, 30.
Incubation, 99.
Indol test, broth for, 107.
Indicators, 36, 94, 107.
Iodine and bromine, determination of, 61.
Iron and Aluminium, separation, 57.
analysis of, 79.
Iron, determination of, 43.
standards, 45.
sulfate, determination of, 41.
analysis of, 81.

L.
Lacmoid indicator, 36.
Lead, determination of, 51, 55.
Lime, analysis of, 80.
Lithium, determination of, 60.
Litmus reagent, 94.
lactose-agar, 97.
solution, 96.

M.
Manganese, determination of, 48.
Materials, bacteriological, 93.

Meat extract, 93.
Media, culture (see “Culture media”), 94–7, 107.
Methyl orange indicator, 37.
Methyl red media, 107.
test, 107.
Microscopical bibliography, 91.
examination, 89.
Mineral analysis, 56.
Moisture in sludge and mud, 74.
Mud deposits, analysis, 73.

N.
Nessler’s reagent,
color standards, 10.
ammonia determination, 19.
Nitrogen, 15.
ammonia, 15.
albuminoid, 20.
Nitrogen, in sludge and mud, 74.
nitrate, 23.
nitrite, 22.
organic, 21.
total, 25.
Nutrient media (see “Culture media”), 94, 107.

O.
Odor, 12.
Organic nitrogen, 21.

Oxygen consumed, 25.
demand, biochemical, 71.
dissolved, 65.
in fresh and sea water (table), 68.

P.
Peptone, authorized brands, 93.
Persulfate method (Mn), 48.
Petri dishes, 93.
Phenoldisulfonic acid method (nitrate), 23.
Phenolphthalein indicator, 36, 94.
Physical examination, 4.
Pipettes, bacteriological, 93.
Plating, bacteriological, 99.
Platinum-cobalt color standard, 9.
wire turbidity, 5.
Potassium, determination of, 59.
Presumptive tests, colon group, 102.

R.
Reactions of colon group, 108.
Reaction of culture media, 94.
of sludge and mud, 73.
Reduction method (nitrate), 24.
Relative stability method, 71.
Residue on evaporation, 29.
Results, expression of,

bacteriological, 103.
chemical examination, 14.
color, 8.
odor, 12.
Results, interpretation of (bacteriological), 106.
Routine procedure (bacteriological), 108.

S.
Samples,
bacterial, 93.
bottles, 1.
chemical, 1.
interval before analysis of, 2.
quantity required, 1.
representative, 3.
sludge and mud, 73.
Sewage sludge, analysis, 73.
Silica, determination of, 56.
Soda ash, analysis of, 82.
Soap method (hardness), 31.
Sodium and potassium, 58.
Solids, total, fixed, volatile, 29.
Specific gravity of sludge and mud, 74.
Stability, relative, of effluents, 69.
method, relative, 71.
Standards,
ammonia, 17.
chlorine, 65.
color, 9.
hardness, 32.

iron, 45.
Nessler, color, 10.
platinum-cobalt, 10.
turbidity, 4.
Sterilization of media, 95.
Storage of samples, 2, 98.
Sugars for media, 94.
Sugar broths, 95.
Sulfate, K and Na, 58.
Suspended matter, 30.

T.
Tin, determination of, 54, 55.
Tintometer, Lovibond, 11.
Titration of media, 94.
Total nitrogen, 25.
residue on evaporation, 29.
Tryptophane broth, 107.
Turbidity, 4.
coefficient of fineness, 8.
platinum wire method, 5.
rod, graduation, 6.
standard, 4.
turbidimetric method, 7.
turbidometer, graduation, 8.

V.
Voges-Proskauer test, 107.

Volatile matter, 29.
in sludge and mud, 74.

Z.
Zinc, 52.

TRANSCRIBER’S NOTES
1. Silently corrected typographical errors and variations in spelling.
2. Archaic, non-standard, and uncertain spellings retained as printed.
3. The Chemical Bibliography was reformatted in footnote style.
4. The Bacteriological Bibliography was reformatted in footnote style and
the numbering was increased by 200.

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