Chapter 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°.

Footnote 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
(K_{2}HPO_{4}). A dilute solution of the K_{2}HPO_{4} 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.

Footnote I:

_Tryptophane Broth for Indol Test._

To 1,000 cc. of distilled water add 0.3 gram tryptophane, 5 grams
dipotassium hydrogen phosphate (K_{2}HPO_{4}), 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.

Footnote J:

(b) _Synthetic Medium for the Methyl Red Test._ To Make One Liter.
Dissolve 7 grams Na_{2}HPO_{4} (anhydrous) or 8.8 grams
Na_{2}HPO_{4}.2H_{2}O, 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 P_{H} 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 Na_{2}HPO_{4}.12H_{2}O for two weeks.
Use 0.88 per cent. of Na_{2}HPO_{4}.2H_{2}O.

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

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

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

Methyl red − │B. cloacae, may or may not be of fecal
│ origin.
Voges-Proskauer + │
Gelatin + │
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.

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Bibliography 202:

BREED, R. S. and DOTTERRER, W. D. The Number of Colonies Allowable on
Satisfactory Agar Plates. _Jour. of Bact._, 1, 1916, 321.

Bibliography 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.

Bibliography 204:

CLARK, W. M. An Hydrogen Electrode Vessel. _Jour. Biol. Chem._, 23,
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Bibliography 205:

CLARK, W. M. The “Reaction” of Bacteriological Culture Media. _Jour.
of Inf. Diseases_, 17, 1915, 109.

Bibliography 206:

CLARK, W. M. The Final Hydrogen Ion Concentrations of Cultures of
Bacillus Coli. _Science_, n. s. 42, 1915, 71.

Bibliography 207:

CLARK, W. M. and LUBS, H. A. Hydrogen Electrode Potentials of
Phthalate, Phosphate, and Borate Buffer Mixtures. _Jour. Biol. Chem._,
25, 1916, 479.

Bibliography 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.

Bibliography 209:

ENDO, S. Ueber ein Verfahren zum Nachweis der Typhusbacillen Centbl.
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Bibliography 210:

GILLESPIE, L. J. The Reaction of Soil and Measurements of Hydrogen Ion
Concentration. _Jour. Wash. Acad. of Sciences_, 6, 1916, 7.

Bibliography 211:

HILL, H. W. Porous Tops for Petri Dishes. _Jour. Med. Research_, 13,
1904, 93.

Bibliography 212:

HILL, H. W. The Mathematics of the Bacterial Count. _Public Health
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Bibliography 213:

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Bibliography 214:

KENDALL, A. I. and WALKER, A. W. The Isolation of Bacillus Dysenteriae
from Stools. _Jour. Med. Research_, 23, 1910, 481.

Bibliography 215:

KINYOUN, J. J. and DEITER, L. V. On the Preparation of Endo’s Medium.
_Amer. Jour. Public Health_, n. s. 2, 1912, 979.

Bibliography 216:

LEVINE, M. On the Significance of the Voges-Proskauer Reaction. _Jour.
of Bacteriology_, 1, 1916, 153.

Bibliography 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.

Bibliography 218:

MCCLENDON, J. F. New Hydrogen Electrodes and Rapid Methods of
Determining Hydrogen Ion Concentrations. _Amer. Jour. of Physiology_,
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Bibliography 219:

MCCLENDON, J. F. A Direct Reading Potentiometer for Measuring Hydrogen
Ion Concentrations. _Amer. Jour. of Physiology_, 38, 1915, 186.

Bibliography 220:

MCCRADY, M. H. The Numerical Interpretation of Fermentation-tube
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Bibliography 221:

NOYES, H. A. Agar Agar for Bacteriological Use. _Science_, n. s. 44,
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Bibliography 222:

ROGERS, L. A., CLARK, W. M. and DAVIS, B. J. The Colon Group of
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Bibliography 223:

ROGERS, L. A., CLARK, W. M. and EVANS, A. C. The Characteristics of
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Bibliography 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.
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Bibliography 225:

SMITH, H. M. The Seaweed Industries of Japan. _Bulletin of the Bureau
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WHITTAKER, H. A. The Source, Manufacture and Composition of Commercial
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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.

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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. 5. Enclosed italics font in _underscores_.