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BIOLOGY

LIBRARY

A LABORATORY MANUAL

SOIL BACTERIOLOGY

EDWIN B. FRED, Ph. D.

r- /

ASSOCIATE PROFESSOR OF AGRICULTURAL BACTERIOLOGY IN THE COLLEGE
OF AGRICULTURE, UNIVERSITY OF WISCONSIN

ILLUSTRATED

PHILADELPHIA AND LONDON

W. B. SAUNDERS COMPANY

1916

a®

PRINTED IN AMERICA

PRESS OF

W. B. SAUNDERS COMPANY
PHILADELPHIA

PREFACE

THE exercises described in this laboratory manual are
arranged primarily for students of soil bacteriology, soil
chemistry and physics, and plant pathology. As far as
possible the experiments are planned to give quantitative
results. It is assumed that the student has had previous
training in general bacteriology and chemistry.

The section entitled "Formulae and Methods" is intended
to present in a convenient form some of the more impor-
tant media and methods used in a study of soil bacteria.
The chemical methods employed are, with few exceptions,
those given in standard text-books. Much of the material
was collected and arranged by Mr. C. Hoffmann.

In addition to the references given in the text, frequent
use has been made of the various manuals in bacteriology.

Any suggestions in regard to improvement of the manual
will be welcomed.

E. B. FRED.

MADISON, WISCONSIN,
October, 1916.

345373

CONTENTS

INTRODUCTION n

Apparatus for One Student 1 1

Literature 12

Laboratory Rules 14

EXERCISES IN SOIL BACTERIOLOGY 16

Number of Microorganisms in Soil 16

Relation of Microorganisms to the Nitrogen Cycle 35

Relation of Microorganisms to the Carbon Cycle 75

Relation of Microorganisms to the Sulphur Cycle 81

Relation of Microorganisms to the Iron Cycle 84

Relation of Microorganisms to the Physical Properties of Soil.. . . 87

FORMULA AND METHODS 88

Cleaning Glassware 88

Preparation of Culture-media 89

Media for the Determination of the Number and for the

Separation of Soil Bacteria 89

Counting Soil Protozoa 97

Ammonification 98

Nitrification 100

Denitrification 106

Nitrogen Assimilating Organisms 108

Cellulose Destroying Organisms in

Sulphur Organisms 116

Iron Organisms 118

Yeasts 119

Fungi 1 20

Actinomycetes 122

Algae 123

Higher Plants 124

Preparation of Stains 127

Preparation of Reagents and Qualitative Methods of Analysis. . . 133

Quantitative Methods of Analysis I41

Special Methods i5S

INDEX 165

SOIL BACTERIOLOGY

INTRODUCTION
APPARATUS FOR ONE STUDENT

THE following apparatus should be in each desk. Any
omission must be reported to the instructor at once.

1 Bunsen burner and tubing $ .40

2 Wire baskets 50

1 Metal cup. 25

2 4-inch funnels 24

1 Graduate cylinder (100 c.c.) 35

2 Erlenmeyer flasks (1000 c.c.) 80

4 Erlenmeyer flasks ( 500 c.c.) i.oo

2 Erlenmeyer flasks (150 c.c.) 30

50 Test-tubes (small) 75

5 Test-tubes (large) 15

5 Petri dishes i.oo

20 Glass tumblers : i.oo

10 Pipets (i c.c.) 80

2 Pipets (10 c.c.) 30

i Hanging-drop slide 20

i Thermometer 75

i Platinum needle 35

50 Object slides (not returnable) 50

50 Cover glasses (not returnable) 25

i Aluminum weighing dish 20

6 Evaporating dishes 60

i Test plate .20

i Wash bottle 10

Filter-paper (8-inch) 10

i Forceps (steel) 25

i Spatula 25

i Trowel - 10

i Mixing cloth 10

i Slide box 10

i Test-tube brush 05

i Towel 05

i Box of matches 01

i Box of labels

$12.00
ii

Sit BACTERIOLOGY

LITERATURE

The following list includes some of the more important
books and journals that treat of bacteriology:

A. General Bacteriology:

Benecke, W ............... Bau und Leben der Bakterien, 1912.

Charpentier, P. G ......... Les Microbes, 1909.

Frost and McCampbell. . . .Text-Book of General Bacteriology, 1912.

Hiss and Zinsser .......... Text-Book of Bacteriology, 1915.

Jordan, E. O .............. General Bacteriology, 5th ed., 1916.

Kayser, E ................ Microbiologie Agricole, 3d ed., 1914.

Kruse, W ................. Allgemeine Mikrobiologie, 1910.

Meyer, A ........... ...... Die Zelle der Bakterien, 1912.

B. Agricultural Bacteriology:

Conn, H. W .............. Agricultural Bacteriology, 2d ed., 1909.

Fuhrmann, F ............. Vorlesungen iiber Technische Mykologie,

Kossowicz, A ............. Einfiihrung in die Agrikulturmykologie,

Teil I, Bodenbakteriologie, 1912.

Lipman, J. G ............. Bacteria in Relation to Country Life, 1911.

Lohnis, F ................. Vorlesungen iiber landwirtschaftliche Bak-

teriologie, 1913.

Marshall, C. E ............. Microbiology, 1911.

Percival, J ................. Agricultural Bacteriology, 1910.

Russell and Hastings ...... Agricultural Bacteriology, 1915.

C. Reference Books in Agricultural Bacteriology:

Duclaux, E Traite de Microbiologie, 1898-1901.

Lafar, F Handbuch der Technischen Mykologie,

Bd. Ill, 1904-1906; Mykologie des

Bodens, des Wassers und des Dungers.
Lohnis, F Handbuch der landwirtschaftlichen Bak-

teriologie, 1910.
Smith, E. F Bacteria in Relation to Plant Diseases,

vols. i, 1905; ii, 1911; iii, 1914.

LITERATURE 13

D. Manuals of Bacteriologic Technic:

American Public Health

Association. . .' Standard Methods for the Examination of

Water and Sewage, 1915.

Burgess, P. S Soil Bacteriology Laboratory Manual, 1914.

Eyre, J. W. H Bacteriological Technique, ad ed., 1913.

Giltner, Ward Laboratory Manual in General Micro-
biology, 1916.

Heinemann, P. G A Laboratory Guide in Bacteriology, 3d

ed., 1915.

Kiister, E Kultur der Mikroorganismen, 1913.

Lohnis, F Landwirtschaftlich-bakteriologisches Prak-

tikum, 1911.

Moore and Fitch Bacteriology and Diagnosis, 1914.

Muir and Ritchie Manual of Bacteriology, 6th ed., 1913.

Reed, H. S A Manual of Bacteriology, 1914.

E. Classification of Bacteria:

Chester, F. D A Manual of Determinative Bacteriology,

1901.
Lehmann und Neumann. . .Bakteriologie und bakteriologische Diag-

nostik., Teil I, Atlas, 1910; Teil II,

Text. 1912.
Migula, W System der Bakterien, Bd. I, 1897; Bd. II,

1900.
Winslow and Winslow Systematic Relationships of the Coccaceae,

1908.

F. Journals of Bacteriology and Related Subjects'.

Annales de ITnstitute Pasteur, T. I., 1887.

Arb. Biol. Abt. Landw.-und Forstw. K. Gsndhtsamt., Bd. I, 1900.

Biedermann's Centralblatt fur Agrikulturchemie, Bd. I, 1872.

Botanical Gazette, vol. i, 1876.

Centralblatt fur Bakteriologie, Abt. I, Orignale, Bd. i, 1887.

Centralblatt fur Bakteriologie, Abt. I, Referate, Bd. 31, 1902.

Centralblatt fur Bakteriologie (etc.), Abt/2, Bd. i, 1892.

Comptes Rendus Academic des Sciences, T., 1835.

Folia Mikrobiologica, i, 1912.

Jahresbericht iiber Fortschritte, Garungs Organism en, Bd. i, 1890.

Jahresbericht uber die Fortschritte der Agrikulturchemie, Bd. i, 1858.

14 SOIL BACTERIOLOGY

Jahresbericht iiber die Landwirtschaft, Bd. i, 1886.
Journal of Agricultural Science, vol. i, 1906.
Journal of American Society of Agronomy, vol. i, 1910.
Journal of Bacteriology, vol. i, 1916.
Journal of Biological Chemistry, vol. i, 1906.
Journal of Industrial and Engineering Chemistry, vol. i, 1909.
Journal of Infectious Diseases, vol. i, 1904.
Journal fur Landwirtschaft, Bd. i, 1853.
Landwirtschaftliche Jahrbiicher, Bd. i, 1872.
Landwirtschaftliches Jahrbuch der Schweiz, Bd. i, 1887.
* Landwirtschaftliche Versuchs-Stationen, Bd. i, 1859.
Phytopathology, vol. i, 1911.
Soil Science, vol. i, 1916.
Zeitsch. fur Garungs-Physiologie, Bd. i, 1912.
Zeitschrift fiir das landw. Versuchswesen in Oesterreich, Bd. i, il

Agricultural Index, vol. i, 1916.

Experiment Station Record, vol. i, 1888.

International Catalogue of Scientific Literature, 1901.

Journal of Agricultural Research, vol. i, 1913.

State Experiment Station Bulletins.

United States Department of Agriculture Bulletins.

LABORATORY RULES

Read Carefully the Following Rules:

1. Before pouring plates or making transfers, wash off
the desk with a i : 1000 mercuric chlorid solution. Hold
the test-tube cultures to be transferred as nearly in a
horizontal position as possible. Avoid opening cultures
in a current of air.

2. All cultures are to be grown in the incubator at 28° C.
unless otherwise stated.

3. After using balances, always return weights to their
proper places. Do not leave any dust or dirt on balances.

4. All solid material, as soil, agar, cotton or filter-
paper, must be emptied into waste jars and not into the
sinks.

LABORATORY RULES 15

5. Soil should not be sieved in the laboratory. The
greenhouse or potting room may be used for this purpose.

6. At the end of the laboratory period return all stock
bottles and chemicals to their proper places on the shelves.
See that all apparatus is replaced in the lockers and that all
gas-burners are shut off. Wipe off the table top before
leaving.

7. Anything left on the desk will be collected after the
laboratory period and returned to the store-room.

EXERCISES IN SOIL BACTERIOLOGY

SECTION I

NUMBER OF MICROORGANISMS IN SOIL
Directions for Drawing Soil Samples

WHEN it is necessary to secure accurate samples, dig a
ditch to the desired depth. By means of a sterile trowel
representative samples may be drawn from the sides of the
ditch. In this way outside contamination is largely pre-
vented.

Samples from the surface to i foot deep may be taken as
follows: Remove the coarse surface debris and sink a
large, sterile test-tube or metal cylinder to the desired
depth. Samples of surface soil may be taken with a sterile
spatula*. Draw several samples and empty into sterilized
paper bags or other vessels. Mix and pulverize the sample.
This may be done with a sterile spatula upon a large piece
of sterile paper. From the well-mixed sample remove a
representative portion for dilution, and at the same time
make a moisture determination. As soon as possible
after samples are drawn arrange to count.

Exercise i
Number of Bacteria According to the Dilution Method

i. Add 50 grams of soil to 500 c.c. of sterile water or
500 c.c. of physiologic salt solution.

PROTOZOA ACCORDING TO THE DILUTION METHOD 17

2. Shake the suspension vigorously for five minutes.

3. Allow the coarse particles to settle and dilute in the
following manner :

(a) Add i c.c. of the soil extract to 99 c.c. of sterile water; equal to

i : 1000.
(&) Add i c.c. of dilution (a) to 99 c.c. of sterile water; equal to

i : 100,000.

i : 1,000,000.

(d) Add 10 c.c. of dilution (c) to 90 c.c. of sterile water; equal to

i : 10,000,000.

(e) Add 10 c.c. of dilution (d) to 90 c.c. of sterile water; equal to

i : 100,000,000.

4. Shake thoroughly between each dilution.

5. Inoculate three tubes of bouillon with i c.c. from each
dilution.

6. Incubate these at 28° C. for one week. At the end
of two-day periods examine the tubes for evidence of
growth, as shown by turbidity, pellicles, or sediment. If
all cultures in dilutions from (a) to (d) show growth,
there must be 10,000,000 or more bacteria present in i
gram of soil.

Note. — If it is desired to determine the numbers of specific types of bac-
teria present, prepare additional liquid media. Use urea bouillon for urea
fermenters, peptone solution for ammonifiers, etc. In this way it is possible
to secure an approximate idea of the number of the various groups of organ-
isms present in a sample of soil.

Exercise 2
Number of Protozoa According to the Dilution Method

i. Add 50 grams of soil to 500 c.c. of sterile water, as
given in the preceding exercise.

1 8 SOIL BACTERIOLOGY

2. After the coarse particles have settled, dilute as fol-
lows:

(a) Add i c.c. of the soil suspension to 9 c.c. of sterile water; equal

to i : 100.

(b) Add i c.c. of dilution (a) to 9 c.c. of sterile water; equal to i : 1000.

(d) Add i c.c. of dilution (c) to 9 c.c. of sterile water; equal to i : 100,000.

3. Inoculate two tubes of protozoa media (hay-soil
extract, soil extract, or any medium adapted to protozoa)
with i c.c. of each of the above dilutions (see p. 97).

4. Incubate the protozoa cultures at room temperature.

5. At regular intervals of two days each make a micro-
scopic examination of the cultures. Since the protozoa
are usually larger than bacteria — the 16 mm. two- thirds
and 4 mm. one-sixth — objectives will be found desirable.

6. By means of a large-mouthed pipet or loop transfer a
small portion of the protozoa culture to a slide and examine.
A wet or hanging-drop mount may be used. In certain
cases the small flagellates become so numerous that it is
difficult to distinguish between the bacteria and protozoa.

Note. — An abundant growth of large protozoa may be obtained if mannit
solution (m. 39) is inoculated with a small amount of field soil and the
culture incubated for one week or longer at room temperature. A drop of
the culture treated with Gram's iodin solution will show the presence of
numerous ciliates. In certain cases the ciliates are marked by numerous
small, deep golden bodies within their cell v- a M ^apparently Azotobacter
cells.

Exercise 3
Number of Bacteria According to Plate Method

In order to reduce the error common to determinations
of this character, it is well to use a large sample of soil.
All weighings should be made as rapidly as possible to
avoid errors due to loss of moisture by evaporation. Bal-

NUMBER OF BACTERIA ACCORDING TO PLATE METHOD IQ

ances sensitive to 10 milligrams are satisfactory for this
work. Analytic balances may be used, but are not neces-
sary.

1. Weigh 20 to 30 grams of soil on a piece of sterile
paper or scoop, or weigh the entire soil sample, bottle,
and contents; then remove about 20 grams with a sterile
spatula. Re weigh sampling bottle and contents and record
loss in weight. Transfer the soil to a 2Oo-c.c. sterile water
blank.

Note. — Two hundred c.c. of water in a 500-0.0. Erlenmeyer flask allows
ample space for shaking. Tap-water will be found very satisfactory for
soil counts. The water blanks may be sterilized in the autoclave for fifteen
minutes at 15 pounds' pressure. For ordinary work, provided blanks are not
stored for a long time, thirty minutes in the steamer will be sufficient.

2. Shake this suspension vigorously for five minutes
and allow the coarse particles to settle.

3. Add 10 c.c. of this first dilution, equivalent to i gram
of soil, to a go-c.c. sterile water blank. One c.c. from this
dilution is equal to o.oi gram of soil.

4. After shaking, add i c.c. to a gg-c.c. sterile water
blank. (Dilution i : 10,000.)

5. Transfer i c.c. of the above to a g-c.c. sterile water
blank. As a rule, this dilution, which represents i : 100,000
of a gram of soil to ;?ftph cubic centimeter, is the one from
which to pour plates. If the soil is very poor, use a dilution
of i : 10,000; if very rich, i : 1,000,000. The number of
dilutions will depend on the type of soil. Garden or well-
cultivated soil rich in organic matter requires a higher
dilution than poor, sandy soil.

6. Pour plates from the following dilutions in triplicate:
i : 10,000, i : 100,000, and i : 1,000,000.

7. Add about 10 c.c. of an agar medium, melted and cooled

2O SOIL BACTERIOLOGY

to 40° C., to each plate. A blank plate or control should
be poured with each series. In case the medium is turbid,
heat slowly, allowing the deposit to settle. Use only the
clear portion of the medium for pouring plates,

8. Immediately after adding the culture-medium rotate
each plate to secure a uniform mixture. Allow agar plates
to harden on a level surface.

Fig. i. — Agar plate showing a common form of spreading colonies found

in soil.

9. Agar plates should be inverted and incubated under
a moist chamber at 28° C. The time of incubation will
depend upon the different culture-media. After five to
ten days count the number of colonies on each plate. If
the colonies are not too thick, it is well to dot each one with
a pen. When the colonies are too thick to count easily,
use a hand lens and counting plate.

NUMBER OF BACTERIA ON DIFFERENT CULTURE-MEDIA 21

10. Reduce all results to number of bacteria in i gram of
soil.

Exercise 4

Comparison of the Number of Bacteria on Different
Culture-media

1. Weigh out a representative sample of field soil,
about 20 grams, and transfer to a sterile 2oo-c.c. water
blank.

2. Carry through dilutions as given in the previous
exercise. In the last dilution use 2 c.c. to 18 c.c. of water
instead of i c.c. to 9 c.c.

3. At the same time the count is made weigh a sample
of soil and determine the moisture.

4. Pour triplicate plates of the following media: Heyden-
Nahrstoff, sodium asparaginate, soil-extract, casein agars,
and soil-extract gelatin. After melting, the gelatin may
be cooled to 30° C. before pouring plates.

5. Gelatin should be incubated at a constant temperature,
about 20° C., for one week. At regular intervals of two
days each remove the plates and count the number of
colonies, differentiating between the liquefiers and non-
liquefiers. In order to prevent a rapid liquefaction of the
gelatin, the peptonizing organisms may be killed by touch-
ing them with the point of a silver nitrate pencil.

6. Compare the number of peptonizing colonies on casein
agar with the number of liquefiers on gelatin.

Note. — After counting the total number of colonies on casein agar, flood
the plates with N/2O lactic acid. After the medium turns white the acid
may be poured off. The lactic acid precipitates the casein, which produces
an opaque white medium except around the peptonizing colonies, where the
casein has been digested. These colonies may be distinguished by the
clear zone.

SOIL BACTERIOLOGY

7. The results of the plate counts may be tabulated as
follows :

TABLE i. — Comparison of the Number of Bacteria on Different Culture-media

Bacteria per plate.

Bacteria in i gm. of soil.

Medium.

Total.

Aver-

Liquefiers.

Average.

f-iquefiers.

age.

Total.

Aver-
age.

Average.

Heyden Nahrstoff.

do.

Sodium asparaginate.

do.

Soil-extract agar.

do.

Casein agar.

do.

Soil-extract gelatin.

do.

Exercise 5
Effect of Depth on Number of Bacteria

i. The samples for this exercise should be drawn from
virgin soil well removed from any source of contamination.
Save some of this soil for Exercise 8, p. 42. The type of
soil will determine to a certain degree the number of
organisms at different depths.

EFFECT OF DEPTH ON NUMBER OF BACTERIA

2. Divide the sample of soil, taking one portion for
plate count, the other for moisture determination. For
virgin field soil the following dilutions have been found
satisfactory.

3. Take soil samples and plate as follows-

(a) Surface soil i

(fe) Soil i foot deep i

(d) Soil 4 feet deep i

100,000.

10,000 and i : 100,000.
1000 and i : 10,000.
100 and i : 1000.

Follow the method given in previous exercises.

4. Pour triplicate plates, using the medium that gave
the highest count. (See preceding exercise.)

5. Tabulate results as follows:

TABLE 2. — Effect of Depth on Number of Bacteria

Position.

Moisture.

Bacteria per plate.

Bacteria in i
gm. of dry soil.

Per cent.

Total-

Average.

Surface.

do.

i foot.

do.

2 feet.

do.

4 feet.

do.

SOIL BACTERIOLOGY

Exercise 6
Effect of Moisture on Number of Bacteria

i. Weigh out i kilogram of air-dry field soil and mix
thoroughly.

TABLE 3. — Effect of Moisture on Number of Bacteria

Time.

Moisture.

Dilution.

Bacteria per plate.

Bacteria in
i gm. of dry soil.

Days.

Per cent.
Air dry.
do.

Total.

Average.

do.

•

3°

Air dry.
do.

do.

EFFECT OF MANURES ON NUMBER OF BACTERIA 25

2. Place 2oo-gram portions of the air-dry soil into four
small glass jars. One pint Mason jars may be used.

3. Adjust the moisture content as follows:

(a) Air dry.

(6) 15 per cent, moisture.

(d) 45 per cent, moisture.

4. Incubate the soil samples at room temperature.

5. After intervals of one and three weeks determine the
number of bacteria according to the plate method. Pour
triplicate plates from the dilution of i : 100,000.

6. Arrange results in tabular form (see p. 24).

Exercise 7

Effect of Manures on Number of Bacteria

1. Prepare three tumblers or beakers with 100 grams
each of field soil.

2. Arrange as follows:

(a) Control.1

(b) Treat with i per cent, of finely chopped green clover.

3. Since these substances contain great numbers of
bacteria, especially the stable manure, plate counts should
be made of the manures at the time the soils are treated.
For this purpose shake 2o-gram portions of the manures
with 200 c.c. of sterile water. Dilute as given in the
previous exercises. Pour plates from the dilutions
i : 100,000 and i : 1,000,000.

4. After mixing thoroughly the soil and manure in
tumblers, raise the moisture to two- thirds saturation.

1 Control or blank is equivalent to no treatment.

SOIL BACTERIOLOGY

5. • Cover the soils with Petri dishes and incubate at room
temperature.

6. Determine the number of bacteria after one and
three weeks.

7. Before drawing the sample for counts mix the contents
of the tumblers thoroughly. This may be done with a
sterile spatula. In the case of treated soils plate from the
dilutions i : 100,000 and i : 1,000,000.

8. Tabulate results.

TABLE 4. — Effect of Manures on Number of Bacteria

Time.

Treatment.

Dilution.

Bacteria per plate.

Bacteria in i gm.
of dry soil.

Days.

Per cent.

Total.

Average.

None.

do.

i clover.

do.

. do.

i manure.

do.

None.

do.

i clover.

do.

i manure.

do.

EFFECT OF LIMESTONE ON NUMBER OF BACTERIA 27

Exercise 8
Effect of Limestone on Number of Bacteria

1. Fill three tumblers with 100 grams each of field soil.

2. Arrange as follows:

(a) Control.

(b) Treat with 0.5 per cent, of calcium carbonate or one-half enough

calcium carbonate to neutralize the soil acidity.

carbonate to neutralize all soil acidity.

TABLE 5. — E/ect of Limestone on Number of Bacteria

Time.

Treatment.

Dilution.

Bacteria per plate.

Bacteria in i gm.
of dry soil.

Days.

Per cent.

Total.

Average.

Total.

None.

do.

\ lime.

do.

i lime.

do.

None.

do.

\ lime.

do.

dp.

i lime.

do.

28 SOIL BACTERIOLOGY

3. Use a clean spatula to mix the soil and limestone.

4. Add water to bring the moisture to two- thirds satura-
tion. Cover the tumblers with Petri dishes.

5. Allow the soils to incubate at room temperature.

6. Make plate counts after one and three weeks.

7. Arrange the results in a table (see p. 27).

Exercise 9
Effect of Partial Sterilization on Number of Bacteria

1. Weigh out three portions, 200 grams each, of garden
or field soil into small jars (pint Mason). The field soil
should be mixed thoroughly before sampling.

2. Treat the jars of soil as follows:

(a) Control.

(6) Heat to 100° C. for one hour.

(c) Treat the soil with i per cent, of carbon bisulphid. Pour the CS2
into small holes (use a glass rod) in the soil and cover imme-
diately.

3. After the soil in jar (c) has been exposed to the action
of carbon bisulphid for one hour, and the soil in (b) heated
for one hour, determine the number of bacteria in all three
soils. In the case of the untreated soil, plaj:e from the
dilution i : 100,000, the heated soil dilution i : 100, and
the carbon bisulphid soil dilution i : 10,000.

4. The carbon bisulphid jar. should be left uncovered for
one day.

5. Incubate all of the samples at room temperature.

6. The second plate count should be made one week
after treatment. Here again, untreated soil, use the dilu-
tion i : 100,000, and the treated series, dilutions i : 100,000
and i : 1,000,000.

7. A third count should be made two weeks after treat-

EFFECT OF PLANT ROOTS ON NUMBER OF BACTERIA 2p

ment, plating from the same dilutions as in the second
count.
8. Tabulate the data.

TABLE 6. — Effect of Partial Sterilization on Number of Bacteria

Time.

Treatment.

Dilution.

Bacteria per plate.

Bacteria in i gm.
of dry soil.

Days.

I 2 3

Average.

Beg.

None.

Beg.

100° C.

Beg.

i%CS2

None.

100° C.

i% CS2

None.

100 ' C.

i%CS2

Exercise 10
Effect of Plant Roots on Number of Bacteria

1. Collect soil samples from the immediate vicinity of
the roots of various plants (alfalfa, clover, etc.), and similar
samples i or. 2 feet away from the plants.

2. Prepare plate counts and moisture determinations of
these soils.

3. Tabulate results as follows:

TABLE 7. — Effect of Plant Roots on Number of Bacteria

No.

Position.

Bacteria per plate.

Bacteria in i gm.
of dry soil.

i 2 3

Average.

Near roots.

Away from roots.

Increase or decrease ....

3°

SOIL BACTERIOLOGY

Exercise n
Effect of Season on Number of Bacteria

1. In order to study the relation of bacteria to season,
select an isolated plot of soil high enough to prevent drain-
age from above.

2. Draw samples of soil from this plot at intervals of
two weeks each during the fall and winter. If it is not
possible to make counts so often, plan to take samples
when there is a decided change in temperature. If the
soil is frozen, it will be necessary to use a pick or hatchet
in securing samples.

TABLE 8. — E/ect of Season on Number of Bacteria

No.

Date.

Moisture.

Temperature of —

Bacteria per plate.

Bacteria in
i gm. of

dry soil.

Air.

Soil.

Per cent.

°C.

0 C.

123

Average-

Average.

OCCURRENCE OF THERMOPHILIC BACTERIA 31

3. Prepare plates immediately on arrival at laboratory.

4. At the same time plates are poured make a moisture
determination.

5. Record the outside temperature and soil temperature
at the time sample is drawn.

6. Tabulate results (see p. 30).

Exercise 12

Effect of Cultivation on Number of Bacteria

1. Prepare two tumblers with 100 grams each of clay
soil.

2. Arrange as follows:

(a) Untreated.

(b) Cultivated every day by stirring with a sterile spatula.

3. Add water to two- thirds saturation. Cover with
Petri dishes and keep in the incubator at 28° C.

4. Count the number of bacteria after one, two, and
three weeks, pouring plates from the dilution i : 100,000.

Exercise 13

Occurrence of Thermophilic Bacteria

1. Incubate several samples of soil and some fresh stable
manure at 60° C.

2. In order to prevent evaporation all samples must be
kept in a moist chamber. A large glass beaker or metal
container may be used. Avoid glass bell jars, since the
high temperature may cause them to crack.

32 SOIL BACTERIOLOGY

3. After one week in the incubator prepare agar plates
from the different samples. Pour from dilutions i : 1000
and i : 10,000.

4. The plates must be incubated at 60° C.

5. Determine the number of thermophilic bacteria in
i gram of soil.

6. If desirable, a study may be carried on of the bacteria
growing at low temperatures.

Exercise 14

Catalytic Power of Soils

1. Arrange three large, heavy walled glass tubes with 5
grams of soil in each.

2. Treat as follows:

(a) Fresh soil, no treatment.

(6) Heat in the steamer for thirty minutes.

3. Set up the following apparatus (Fig. 2): Insert a
two-holed rubber stopper into a large test-tube. In one
hole fit a piece of short, straight glass tubing closed at
one end with a rubber tube and clamp; in the other, a right
angle tube of glass connected with a rubber tube 8 to 10
inches long. This rubber tubing should be fitted to a
tube of glass bent as shown in Fig. 2 and connected to the
gas-measuring tube.

4. Fill the loo-c.c. gas-measuring tube with water
and invert mouth under water. Clamp the tube in
place.

5. Then add 10 c.c. of a 1.5 per cent solution of hydrogen

-i

-3

— 4

—s

— 6

— 7

— sr

— 9
— /(

— n
-i

-u

•1'.
—u

— ii
-if

-2

^
-J

r "*^ i^^\j

— ^

V:
^»«*.

^— .

m^&

ig. 2. — Apparatus for determining catalytic power of soils.
3 33

34 SOIL BACTERIOLOGY

peroxid to the soil in large test-tube. Shake the tube at
regular intervals.

Note. — For this test 3 per cent, hydrogen peroxid should be made neutral
or faintly alkaline to phenolphthalein with dilute sodium hydroxid and
diluted to 1.5 per cent.

6. Record the volume of oxygen evolved from a definite
quantity of peroxid after ten, thirty, and sixty minutes.

Sullivan, M. X., and Reid, F. R., Bui. 86, U. S. Dept. Agr., Bur. Soils,
1912.

Lohnis, F., Landwirtschaftlich-bakteriologisches Praktikum, p. 120,
1911.

SECTION II

RELATION OF MICROORGANISMS TO THE NITROGEN

CYCLE

Exercise i
Ammonification of Urea

1. Prepare six 2oo-c.c. Erlenmeyer flasks with 50 c.c.
each of urea solution (m. 19).

2. After sterilizing, arrange as follows:

(a) i and 2, control.

(&) 3 and 4, inoculate with i gm. of soil.

It is not necessary to weigh accurately the soil or manure.

3. Incubate the cultures at 28° C.

4. After two days remove from each flask 5 -c.c. portions
of the solution, with a sterile pipet, to a 5oo-c.c. Erlenmeyer
flask.

5. Add about 50 c.c. of distilled water to the urea solu-
tion in the large flask, a few drops of methyl red or cochineal,
and titrate against N/i4 sulphuric acid.

6. From the results of the titrations calculate the amount
of ammonia nitrogen in 100 c.c. of the different urea solu-
tions. In order to find the amount of ammonia formed by
bacterial action, subtract the untreated from the treated
series.

7. Determine the percentage of urea transformed into
ammonia.

SOIL BACTERIOLOGY

8. Similar samples may be drawn after three or four
days and the amount of ammonia titrated.

9. Tabulate the results.

TABLE 9. — Ammonification of Urea.

Ammonia nitrogen in TOO c.c. of solution.

1 ,

After two days.

After three days.

Total.

Per cent.

Total.

Per cent.

None.

do.

Soil.

do.

Manure.

do.

Exercise 2
Isolation of Urea-fermenting Organisms

1. Inoculate duplicate tubes of urea solutions as follows:

(a) Medium 19 soil.

(6) Medium 19 manure.

(d) Medium 20 manure.

A small inoculum is sufficient. In place of tubes (a)
and (6), cultures from the preceding exercise may be used.

2. Two or three days after inoculation examine the test-
tube cultures for ammonia production (Nessler's reagent,

P- i3S)-

3. From one tube of each medium showing abundant
growth make transfers into tubes of sterile urea solution or
water blanks. A wide range of dilutions should be made.

4. Pour plates from the different dilutions with the
same medium as in the tube culture plus gelatin (m. 23).

PREPARATION OF UREASE FROM UREA BACTERIA 37

5. Incubate the gelatin plates at 20° C.

6. Examine the plates every forty-eight hours for a
period of ten days. The urea organisms are often character-
ized by a distinct halo around the colonies. Under the
low power of the microscope the halo is composed of dumb-
bell-shaped crystals.

7. Make transfers to tubes of urea gelatin and incubate
for two days.

8. Now test the ammonia-producing power of the pure
cultures by inserting sterilized strips of Nessler's paper
or turmeric paper in the upper part of the tube.

9. Prepare a stained mount of these two organisms.

Exercise 3
Preparation of Urease From Urea Bacteria

1. Inoculate two large Erlenmeyer flasks, looo-c.c.
capacity, containing 100 c.c. each of urea bouillon (m. 19)
with a pure culture of urea fermenter. A very active
culture should be used.

2. Incubate at 30° C. until ammonia formation is very
evident.

3. In order to secure the urease free of bacteria, filter
aseptically through a Berkefeld filter.

4. Prepare a 10 per cent, solution of urea in distilled
water. Now mix equal volumes of the nitrate and the
urea- water solution.

5. Incubate the enzyme culture at 48° to 50° C. until a
large part of the urea nitrogen is converted into ammonia.

6. The action of the urease may be measured by Nessler-
izing aliquot portions of the treated and untreated urea-
water.

SOIL BACTERIOLOGY

Exercise 4
Ammonification of Gelatin in Solution

1. Prepare eight large Erlenmeyer flasks with 100 c.c.
each of gelatin solution (m. 17).

2. Stopper loosely with cotton and sterilize in the auto-
clave at 15 pounds' pressure for ten minutes.

3. Inoculate six of the flasks with 5 c.c. of a soil suspen-
sion.

Note. — Shake 100 gm. of field soil with 200 c.c. of sterile water and allow
the coarse particles to settle.

The two remaining flasks keep as controls.

4. Incubate all of the flasks at 28° C.

5. At intervals of two, four, and six days remove duplicate
flasks from the incubator and analyze the contents for
ammonia (see p. 141). At the time of the last analysis
determine the ammonia in controls and subtract from the
total amount in the cultures.

6. Tabulate results.

TABLE 10. — Ammonification of Gelatin in Solution

Ammonia nitrogen in 100 c.c. of
solution.

No.

Time.

Average.

Nitrogen
ammonified.

Total.

Blank
subtracted.

Days.

Mgm.

Per cent.

AMMONIFICATION OF VARIOUS SUBSTANCES 39

Exercise 5
Isolation of Ammonifying Organisms

1 . As soon as the cultures of the preceding exercise begin
to show a vigorous ammonia production, make loop trans-
fers to tubes of sterile gelatin solution or sterile water.

2. Repeat the dilution four times.

3. Pour gelatin (m. 2) or agar (m. 3) plates from dilu-
tions i : 10,000 and i : 1,000,000, using i-c.c. portions
for each plate.

4. After colonies have developed, pick several pure
cultures. These should be transferred to gelatin solution
and their ammonifying power studied. For this purpose
use Nessler's reagent.

5. Select two of the organisms that produce the largest
amount of ammonia. These should be kept for a later
study.

6. Prepare stained mounts of these organisms.

Exercise 6
Ammomfication of Various Substances

1. Prepare twelve 5o-gram portions of field-soil in clean,
dry tumblers. The soil should be mixed thoroughly be-
fore samples are drawn.

2. Treat the soils as follows:

(a) i, 2, 3, and 4, add i per cent, of casein.

(&) 5> 6> 7> an(l 8> a(ld i per cent, of blood-meal.

3. Add these substances in the powdered form and mix
thoroughly.

SOIL BACTERIOLOGY

4. Bring the moisture content of the soil to two-thirds
saturation.

Note. — In order to secure the proper moisture content it is necessary to
take into account the water-holding capacity of the added substances, e. #.,
i gram of casein or blood-meal requires about 2 c.c. of water; i gram of
clover, about 5 c.c. of water.

Allow the soil to stand for one hour or more and remix
with a sterile spatula.

5. Determine the amount of ammonia nitrogen in samples
!> 2> 5) 6) 9) and 10 at once. The ammonia nitrogen of
these cultures or controls is to be subtracted from the am-
monia nitrogen found at the final analysis. This gives the
figures "blank substracted."

6. Cover the remaining tumblers with Petri dishes and
incubate for four to six days at 28° C. Now determine the
ammonia content of all of the soils. The percentage of
nitrogen in the different substances is given on the label.

7. Tabulate results.

TABLE n. — Ammonification of Various Substances

Ammonia nitrogen in 100 gm. of

soil.

No.

Treatment.

Average.

Nitrogen
ammonified.

Total.

Blank
subtracted.

Mgm.

Mgm

Per cent.

Casein.

do.

Blood-meal.

do.

Clover.

do.

EFFECT OF SOIL TYPE ON RATE OF AMMONIFICATION 41

Exercise 7
Effect of Soil Type on Rate of Ammonification

1. Weigh out four 5o-gram portions of garden soil,
field soil, and acid soil into clean, dry tumblers.

2. Mix thoroughly i per cent, of ground clover or
powdered casein with each soil.

3. Determine at once the ammonia nitrogen in duplicate
tumblers of the various soils.

4. Add water to the soil of the s^x remaining tumblers
until it is two- thirds saturated. After standing for one
hour stir with a spatula.

5. Cover with Petri dishes and incubate for six days or
longer. If casein is used, the time of incubation may be
shortened to two or three days. In the case of clover
no definite date need be followed, one week or longer will
not seriously change the results.

6. Determine the amount of ammonia nitrogen.

7. Arrange the data in a table.

TABLE 12. — Effect of Soil Type on Rate of Ammonification

Ammonia nitrogen in 100 gm. of
soil.

No.

Soil type.

Average.

Nitrogen
ammonified.

Total.

Blank
subtracted.

Mgm.

Per cent.

SOIL BACTERIOLOGY

Exercise 8
Effect of Depth on Rate of Ammonification

1. A portion of the soil from Exercise 5, page 22, may be
used for this study.

2. Prepare sixteen tumblers with 50 grams of soil in
each tumbler.

3. In order to prevent contamination of the soil the
tumblers and clover must be sterilized.

4. Arrange thus:

(a) i and 2, surface soil.

3 and 4, surface soil plus i per cent, clover.
(&) 5 and 6, i foot deep.

7 and 8, i foot deep plus i per cent, clover.

ii and 12, 2 feet deep plus i per cent, clover.

(d) 13 and '14, 4 feet deep.

15 and 16, 4 feet deep plus i per cent, clover.

5. After six days' incubation at 28° C. determine the
amount of ammonia.

6. Tabulate.

TABLE 13. — Effect of Depth on. Rate of Ammonification

Ammonia nitrogen in 100 gm. of soil.

No.

Position.

Nitrogen
ammonified.

Total.

Blank
subtracted.

Average.

Mgm.

Per cent.

Surface.

do.

i foot.

do.

2 feet.

do.

4 feet.

do.

EFFECT OF MOISTURE ON RATE OF AMMONIFICATION 43

Exercise 9
Effect of Moisture on Rate of Ammonification

1. Weigh into tumblers eight portions, 50 grams each,
of air-dry field soil.

2. Add i per cent, of clover or casein to each tumbler.

3. Arrange thus:

(a) i and 2, air dry.

(b) 3 and 4, 15 per cent, of moisture.

(d) 7 and 8, 45 per cent, of moisture.

Note. — The amount of moisture will depend on the soil type. In this
case 45 per cent, represents saturation.

4. Incubate at 28° C. for seven days. If casein is used,
four days is long enough.

5. Analyze and compare the results of this test with
those of Exercise 6, page 24. Prepare a combination table
from the results of these two exercises.

TABLE 14. — Effect of Moisture on Rate of Ammonification

Ammonia nitrogen in 100 gm. of soil.

No.

Moisture.

Nitrogen,
ammonified.

Total.

Blank
subtracted.

Average.

Per cent.

Mgm.

Per cent.

Air dry.

do.

SOIL BACTERIOLOGY

Exercise 10
Effect of Limestone on Rate of Ammonification

1. Prepare eight tumblers with 50 grams each of soil.
Use two types, a neutral and an acid soil.

2. Add to each soil i per cent, of clover or casein.

3. Bring the moisture content up to optimum for am-
monincation.

4. Arrange as follows:

(a) i and 2, neutral field soil.

(6) 3 and 4, neutral field soil plus i per cent, of CaCO3.

(d) 7 and 8, acid field soil plus i per cent, of CaCO3.

5. Determine the amount of ammonia after four or six
days.

TABLE 15. — Effect of Limestone on Rate of Ammonification

No.
I

Soil type.

Calcium
carbonate.

Ammonia nitrogen in 100 gm. of soil.

Total.

Average.

Gain or loss
from limestone.

Per cent.
None.

Mgm.

do.

i CaC03

do.

None.

do.

i CaCO3

do.

POTASSIUM PHOSPHATE ON RATE OF AMMONIFICATION 45

Exercise 1 1

Effect of Dibasic Potassium Phosphate on Rate of Am-
monification

1. Prepare ten 5o-gram portions of soil in tumblers.

2. Add i per cent, clover and hold the moisture at
two-thirds saturation.

3. Arrange as follows:

(a) i and 2, analyze at once.
(6) 3 and 4, control.

(d) 7 and 8, plus 0.2 per cent. K2HPO4.

(e) 9 and 10, plus 0.5 per cent. K2HPO4.

Note. — The dibasic potassium phosphate is readily added from a solution.
For this purpose prepare a stock solution.

4. Incubate at room temperature for four days.

5. Determine the amount of ammonia.

TABLE 16. — Effect of Dibasic Potassium Phosphate on Rate of Ammonification

Ammonia nitrogen in 100 gm. of soil.

No.

Treatment.

Nitrogen
ammonified.

Total.

Blank
subtracted.

Average.

Per cent.

Mgm.

Per cent.

None.

do.

o.i K2HPO4

do.

0.2 K2HP04

do.

0.5 K2HP04

do.

SOIL BACTERIOLOGY

Exercise 12
Ammonification by Pure Cultures of Bacteria

1. Prepare ten portions, 50 grams each, of field soil.

2. Add i gram of clover and bring the moisture to two-
thirds saturation.

3. Cover the tumblers with Petri dishes and sterilize
in the autoclave at 15 pounds' pressure for two hours on
two consecutive days.

4. When cool, inoculate each tumbler of soil with a
i-c.c. suspension of the bacterial culture to be tested.

Note. — Prepare a suspension of bacteria by shaking a forty-eight-hour-old
culture of the organism in sterile water.

(a) i and 2, uninoculated.

(6) 3 and 4, unknown ammonifier (i).

(d) 7 and 8, Bacillus subtilis.

(e) o and 10, Bacillus tumescens.

5. Numbers i and 2 should be analyzed at once.

6. Incubate for ten days at 28° C.

7. At the end of this time analyze.

TABLE 17. — Ammonification by Pure Cultures of Bacteria

Ammonia nitrogen in 100 gm. of soil.

No.

Culture.

Nitrogen
ammonified.

Total.

Blank
subtracted.

Average.

Mgm.

Per cent.

No. i

do.

No. 2

do.

B. subtilis

do.

B. tumescens

do.

NITRIFICATION IN SOLUTION 47

Exercise 13
Nitrification in Solution

A. Nitrite Qualitative:

1. Prepare five 150-0.0. Erlenmeyer flasks with 2O-c.c.
portions each of nitrite solution (m. 26).

2. Inoculate two of the flasks.

(a) Add approximately o.i gram of field soil.

(b) Add approximately o.i gram of garden soil.

3. Incubate at 28° C.

4. At regular intervals of four to six days remove, with
a sterilized platinum needle, i drop of the solution from
each flask and test as follows :

(a) Presence of nitrites — Trommsdorf's reagent (see page 136).

(b) Absence of ammonia — Nessler's reagent (see page 135).

Use the spot plate for this test. Record the date and
results of test in the table on p. 48.

5. As soon as the culture shows the presence of nitrites
and absence of ammonia, make loop subinoculations into
a sterile flask of the same medium.

6. If it is desirable to study the nitrite bacteria in enrich-
ment cultures, repeated subinoculations may be made.

B . Nitrate Qualitative :

1. Prepare five 150-0.0. Erlenmeyer flasks with 2O-c.c.
portions each of nitrate solution (m. 28).

2. Inoculate two of the flasks.

(a) Add approximately o.i gram of field soil.

(b) Add approximately o.i gram of garden soil.

SOIL BACTERIOLOGY

Control.

Garden soil.

2
.£

Nitrites.

Control.

Garden soil.

Field soil.

NITRIFICATION IN SOLUTION 49

3. Incubate at 28° C.

4. At regular intervals of four and six weeks remove,
with a sterilized platinum needle, i drop of the solution
from each flask and test as follows:

(a) Absence of nitrites — Trommsdorf's reagent (see page 136).
(6) Presence of nitrates — Diphenylamin reagent (see page 137).

Use the spot plate for this .test. Record the date and
results of tests in the table below.

5. As soon as the culture shows the presence of nitrates
and absence of nitrites, make loop subinoculations into a
sterile flask of the same medium.

6. If it is desirable to study the nitrate bacteria in en-
richment cultures, repeated subinoculations may be made.

C. Nitrification Quantitative:

1. Place loo-c.c. portions of medium 29 into eight i -liter
Erlenmeyer flasks.

2. Inoculate each flask with i-c.c. portions of water
extract of different soils.

Note. — Shake 50 grams of soil with 100 c.c. of sterile water and allow
to settle.

(a) i, 2, 3, and 4, field soil.
(6) 5, 6, 7, and 8, garden soil.

3. Immediately after inoculation add 5 c.c. of con-
centrated sulphuric acid to flasks numbered i, 2, 5, and 6.

4. Incubate all of the flasks at 28° C. for three to four
weeks.

5. At the end of this time determine the amount of
nitrate nitrogen (see page 143).

6. Tabulate results as shown on p. 50.

50 SOIL BACTERIOLOGY

TABLE 19. — Nitrification in Solution (Quantitative).

Nitrate nitrogen in 100 c.c. of solution.

, *

Total.

Blank
subtracted.

Average.

Mgm.

Per cent.

Field soil.

do.

Garden soil.

do.

•

Exercise 14
Isolation of Nitrifying Organisms

1. Prepare eight tubes of acid sodium potassium silicate
(m. 30).

2. Dilute the second enrichment cultures, nitrite, and
nitrate organisms until i c.c. represents i : 1000, i : 10,000,
i : 100,000, and i : 1,000,000 of the original culture.

3. Pour "the acid silicate into a sterile Petri dish with
the culture dilutions; add the nutrient salts and enough
sodium carbonate to harden the silicate.

4. When hard, invert the plates and incubate under a.
moist bell jar for three to six weeks.

5. Examine at weekly intervals, using the low-power
objective. As soon as small colonies appear, make trans-
fers to sterile nitrite or nitrate solution.

6. The nitrifying organisms may be grown on washed
agar (m. 31).

Exercise 15

Nitrification of Various Substances

i. Prepare six portions of field soil, 100 grams each, in
tumblers or flasks. Mix and sieve the soil well before using.

NITRIFICATION OF VARIOUS SUBSTANCES 5!

2. Treat as follows:

(a) i and 2, untreated.

(b) 3 and 4, 30 mgm. of nitrogen from (NH4)2SO4.

The proper amount of nitrogen is most conveniently
added from solution. Prepare a stock solution in such a
way that 5 c.c. equals 30 milligrams of nitrogen in the form
of casein or ammonium sulphate.

3. Mix these substances thoroughly with the soil.

4. Add sterile water to make half -saturation.

5. Cover with Petri dishes and incubate at 28° C.

6. Weigh each week and restore loss of water by evap-
oration.

7. Analyze for nitrate nitrogen after ten and twenty
days.

8. Express the results in terms of milligrams of nitrate
nitrogen in 100 grams of soil; also as percentages of the
original substance nitrified.

9. Tabulate results as follows:

TABLE 20. — Nitrification of Various Substances

No.

Treatment.

Nitrate nitrogen in 100 gm. of soil.

Nitrified.

After ten days.

After twenty days.

Total.

Blank
subtracted.

Total.

Blank
subtracted.

Mgm.

None.

Mgm.

Per cent.

30 N. from
(NH4)2S04

30 N. from

casein.

SOIL BACTERIOLOGY

Exercise 16
Effect of Soil Type on Nitrification

1. Weigh out four loo-gram portions of garden soil,
field soil, and acid soil into clean, dry tumblers.

2. In one-half of the tumblers mix thoroughly with the
soil i per cent, of clover tissue.

3. Arrange as follows:

(a) i and 2, garden soil untreated.

(6) 3 and 4, garden soil plus i per cent, clover.

(d) 7 and 8, field soil plus i per cent, clover.

(e) 9 and 10, acid soil untreated.

(/) ii and 12, acid soil plus i per cent, clover.

4. Add moisture until the soil is two-thirds saturated,
allow it to stand for one hour, and remix.

5. Cover with Petri dishes and incubate at 28° C.

6. After three weeks determine the nitrate nitrogen
(see p. 143) in numbers i, 3, 5, 7, 9, and n; after 6 weeks
in numbers 2, 4, 6, 8, 10, and 12.

TABLE 21.— Effect of Soil Type on Nitrification

Nitrate nitrogen in 100 gm. of soil.

No.

Soil and treatment.

After twenty-one days.

After forty-two days.

Nitrified.

Total.

Blank
subtracted.

Total.

Blank
subtracted.

Per cent.

Mgm.

Mgm

Mgm.

Per cent.

Garden.

Garden i clover.

Field.

Field i clover.

Acid.

Acid i clover.

EFFECT OF MOISTURE ON NITRIFICATION

Exercise 17
Effect of Moisture on Nitrification

1. Prepare eight loo-gram portions of field soil in clean
tumblers.

2. Add to each, 30 milligrams of nitrogen in the form of
ammonium sulphate.

3. Arrange thus:

(a) i and 2, air dry.

(6) 3 and 4, 15 per cent, moisture.

(d) 7 and 8, 45 per cent, moisture.

4. After standing for one hour, mix thoroughly the con-
tents of the tumblers.

5. Cover with Petri dishes and incubate for fourteen
days at 28° C.

6. At the end of the period determine the nitrate nitro-
gen.

7. Tabulate results.

TABLE 22.— Effect of Moisture on Nitrification

Nitrate nitrogen in 100 gm. of soil.

No.

"M" " f

Nitrified.

Total.

Blank
subtracted.

Average.

Per cent.

Mgm.

Per cent.

Air dry.

do.

SOIL BACTERIOLOGY

Exercise 18
Effect of Limestone on Nitrification

1. Prepare ten tumblers of soil, 100 grams in each.
Two types of soil may be used, neutral and acid.

2. Arrange each soil type as follows:

(a) i and 2, untreated.

(&) 3 and 4, 30 mgm. of nitrogen as ammonium sulphate.

CaC03.

(d) 7 and 8, 30 mgm. of nitrogen as gelatin.

(e) 9 and 10, 30 mgm. of nitrogen as gelatin plus i gram CaCOs.

3. Stir in the chemicals thoroughly by means of a sterile
spatula.

4. Add water and incubate for ten to twenty days at
room temperature. From time to time replace the water
lost by evaporation.

5. At the end of the period of incubation analyze for
nitrates.

TABLE 23. — Effect of Limestone on Nitrification

No.

Treatment.

Nitrate nitrogen in 100 gm. of soil.

Nitrified.

Total.

Blank
subtracted.

Average.

Per cent.
None.

Mgm

Mgm.

Per cent.

do.

3
4
5

Ammonium sulphate,
do.
Ammonium sulphate
plus i limestone,
do.

7
8

Gelatin,
do.

Gelatin plus i lime-

stone.

do.

REDUCTION OF NITRATES TO NITRITES 55

Exercise 19
Isolation of Denitrifying Organisms

1 . Fill five test-tubes about two-thirds full of denitrifying
solution (m. 33).

2. Inoculate as follows:

(a) Uninoculated.

(&) Inoculated with approximately o.i gram of garden soil.

3. Incubate at 28° C. until all nitrates have disappeared.
The destruction of nitrates is generally indicated by foam-
ing.

4. At regular intervals, daily if possible, make qualitative
tests (spot plate) for the presence of nitrates, nitrites, and
ammonia.

.5. As soon as the nitrates are destroyed transfer a loopful
of the old culture to a new tube of denitrifying solution.
This may be repeated several times, although a pure
culture is readily isolated from the second transfer.

6. Follow the same method of isolation as given in the
previous exercises. Pour plates of denitrifying agar, and
incubate them until the plates show a good growth.

7. Now pick off several isolated colonies, making trans-
fers into tubes of sterile denitrifying solution.

8. From* the pure culture showing the most vigorous
denitrification make a transfer to denitrifying agar. Pre-
serve this pure culture for later study.

Exercise 20
Reduction of Nitrates to Nitrites

1. Prepare four tubes of starch nitrate agar (m. 35).

2. Dilute two soil types with sterile water until i c.c.
represents from 10 to 50 organisms.

56 SOIL BACTERIOLOGY

3. From these suspensions of soil bacteria prepare dupli-
cate plates with starch-nitrate agar.

4. When the colonies are well developed, pour over the
surface of one of the duplicate plates a very dilute solution
of potassium iodid in dilute hydrochloric acid. Allow to
react for a moment or two, then pour off. The production
of a blue zone around colonies indicates a reduction of
nitrates to nitrites.

KI + HC1 = KC1 + HI
2HI + 2HNO2 = 2H2O + 2NO + I2

5. Note the relative proportion of organisms capable of
reducing nitrates to nitrites.

6. Note the general characteristics of such colonies, and
from similar colonies upon the untreated plates make
transfers to nitrate agar slopes (m. 33).

Exercise 21
Reduction of Stains by Denitrifying Organisms

1. Inoculate duplicate tubes of nitrate solution (m. 33)
with pure cultures of denitrifying organisms.

2. Add to each tube 0.5 c.c. of a sterile i : 1000 (highest
purity) methylene-blue solution and mix thoroughly.

3. In order to exclude partially the oxygen, pour paraffin
oil to a depth of about 2 cm. in one-half of the tubes.

4. Incubate at 28° C.

5. Note each day the change in color. This change
furnishes a method for detecting nitrites. As long as
nitrites are present the solution remains blue. A colorless
solution indicates that all of the nitrite nitrogen is destroyed.

DENITRIFICATION -BY PURE CULTURES OF BACTERIA 57

This should be confirmed by qualitative tests with Tromms-
dorf's and diphenylamin reagents.

Methylene-blue. Leuco-base.

CeHs— N— (CHs)2 CeH,— N— (CHs)2

\ / \

^>S + H2 = HN^ ^>S + HC1

CeHa = N— (CH3)2 C6H3— N— (CH3)2

Exercise 22
Denitrification by Pure Cultures of Bacteria

1. Prepare ten 2oo-c.c. portions of denitrifying solution
(m. 33) in 3oo-c.c. Erlenmeyer flasks.

2. Inoculate as follows:

(a) i and 2, uninoculated.

(b) 3 and 4, pure culture of unknown denitrifier from Exercise 19.

(d) 7 and 8, Bacillus Hartlebii.

(e) 9 and 10, Bacillus coli.

3. Incubate all cultures for two weeks at 28° C.

4. At the end of the incubation period make qualitative
tests of each culture for ammonia, nitrites, and nitrates.
If present, determine the amount according to quantitative
methods.

5. In all of the cultures, inoculated and uninoculated,
determine the total nitrogen. Use the modified Kjeldahl
method (see page 147). For total nitrogen analysis take
duplicate portions of 50 c.c. each of the cultures.

6. For determining the nitrate nitrogen take lo-c.c.
portions of the control, dilute with 500 c.c. of distilled
water, and of this evaporate lo-c.c. portions to dryness.

SOIL BACTERIOLOGY

In the case of the inoculated cultures with nitrates present
proceed as follows: (a) Evaporate 10 c.c. to dryness, and
(b) dilute 10 c.c. to 100 c.c., and evaporate 10 c.c. of this
to dryness (see page 143).

7. From the results of the quantitative analyses fill in
the following table:

TABLE 24. — Denitrification by Pure Cultures of Bacteria

No.

Treatment.

Nitrogen in 100 c.c. of solution.

As nitrate.

Total nitrogen.

Begin.

End.

Loss.

Begin.

End.

Loss.

Control,
do.

Mgm.

3
4

Unknown denitrifier.
do.

5
6

Bacillus pyocyaneus.
do.

7
8

Bacillus Hartlebii.
do.

•

9
10

Bacillus coli.
do.

Exercise 23

Denitrification with the Formation of Nitrous Oxid
(Optional)

i. Prepare in glass-stoppered bottles medium 34 plus
80 grams potassium nitrate in each liter.

DENITRIFICATION IN SOIL 59

2. Inoculate two bottles with 10 to 20 grams of garden
soil; two with a pure culture of a denitriner.

3. Incubate at 37° C.

4. Place bottles in plates, so that the overflow is collected.

5. After forty-eight to seventy-two hours remove the
stopper and insert a glowing splinter. The nitrous oxid
should behave much like pure oxygen.

Exercise 24
Denitrification in Soil

1 . Prepare eight loo-gram samples of field soil in tumblers.

2. Add to each 100 grams of soil 60 milligrams of nitrogen
in the form of potassium nitrate.

3. Treat the series as follows:

(a) i and 2, control untreated.

(6) 3 and 4, add 2.5 grams of dextrose.

(d) 7 and 8, add 2.5 grams of dextrose.

4. Mix these materials thoroughly by means of a spatula.

5. To soil portions i to 4 add sterile water to bring the
moisture content to about one-half saturation.

6. To soil portions 5 to 8 add sterile water to bring
moisture up to total saturation.

7. Incubate for two weeks at 28° C.

8. At the end of this time remove a sample for nitrate
determination and dry the remainder for total nitrogen
analysis. Use the modified Kjeldahl method to include
nitrates (see page 147).

9. From these results calculate the percentage of the
nitrogen denitrified, and note the effect of excessive
moisture and excessive organic matter on the loss of ni-
trogen.

60 SOIL BACTERIOLOGY

10. Tabulate results.

TABLE 25. — Denitrification in Soil

No.

Treatment.

Nitrogen in 100 gra. of soil.

As nitrate.

Total nitrogen.

Begin.

End.

Loss.

Begin.

End.

Loss.

\ water control,
do.

Mgm.

3
4

\ water 2.5 dextrose,
do.

5
6

Total water control,
do.

7
8

Total water 2.5 dextrose
do.

Exercise 25
Autotrophic Denitrifying Bacteria

1. Prepare six tall tubes of Lieske's culture-medium
(m. 38). The tubes for this exercise should be at least
10 to 15 inches long and filled four-fifths full of the
culture-medium .

2. Inoculate:

(a) i and 2, uninoculated.

(&) 3 and 4, i gram of garden soil.

3. Incubate the cultures for six to eight weeks at 280:t3^

4. Determine the total nitrate content of the different
cultures.

ISOLATION OF AZOTOBACTER 6 1

Exercise 26
Nitrogen Fixation in Solution

1. Measure into four 750-0.0. Erlenmeyer flasks loo-c.c.
portions of mannit solution (m. 39).

2. Weigh accurately just 10 grams of soil into each
flask.

3. To two of the flasks add concentrated sulphuric
acid at once, or sterilize.

4. Incubate the cultures for three weeks at 28° C.

5. At the end of this time analyze for total nitrogen
according to the Kjeldahl method (see page 145).

6. Subtract the nitrogen in the soil and culture at the
beginning, from that in the culture after three weeks'
growth. The difference represents the amount of nitrogen
fixed by micro-organisms.

Exercise $27\
Isolation of Azotobacter

1. Prepare four flasks (loo-c.c. Erlenmeyer) of mannit
liquid medium, 20 c.c. in each.

2. Inoculate with i or 2 grams of soil.

3. Incubate at 28° C. and note changes occurring in
cultures.

4. Every two days examine the films in hanging-drop
preparations and note the predominating type of organism.
Also examine some of the surface film in a drop of water
mixed with a drop of Meissner's or Gram's iodin solution
(see pages 127, 128).

5. Dilute two loops of surface film in a loo-c.c. sterile
water blank containing 50 grams of clean sand.

Note. — The presence of sand in the water blank aids in breaking up the
gelatinous clumps of the Azotobacter.

62 SOIL BACTERIOLOGY

6. Shake vigorously, and transfer i c.c. to a- second
loo-c.c. blank, and so on to a third.

7. From the third dilution pour plates, using i c.c. for
each. It is frequently difficult to separate Azotobacter
from a small organism known as Bacillus radiobacter.

Fig. 3. — Azotobacter stained with methylene-blue; X 1200.

Exercise 28
Nitrogen Fixation in Soil

i. Weigh two 5oo-gram portions of field soil into soup

plates.

NITROGEN FIXATION IN SOIL

2. Treat as follows:

(a) Control untreated.

(b) Add 2 per cent, of mannit.

3. Mix the mannit thoroughly with the soil by means of
a spatula.

4. Raise the moisture content of the soil to optimum,
and at regular intervals of two days add water to replace
the loss by evaporation.

5. Incubate these at 28° C. for from fourteen to twenty-
one days.

6. Now prepare the soil for analysis. When dry, pass
it through a 20-mesh sieve, mix thoroughly, and draw a
small sample for analysis; about 100 to 150 grams is enough.
This smaller sample should be pounded in a mortar until
the entire mass passes through a loo-mesh sieve. Weigh
out from three to six portions of 10 grams each into 8oo-c.c.
Kjeldahl flasks.

7. Analyze according to the Kjeldahl method (see p. 145).

8. Run moisture determinations on the soil at the time
samples are taken for nitrogen analysis.

9. Tabulate results.

TABLE 26. — Nitrogen Fixation in Soil

Nitrogen in 100 gm. of dry soil.

Total.

Average.

Gain due to
treatment.

Per cent.

Mgm.

None.

do.

2 mannit.

do.

SOIL BACTERIOLOGY

Exercise 29
Nitrogen Fixation by Pure Cultures of Azotobacter

1. Prepare six i-liter Erlenmeyer flasks with 100 c.c.
each of mannit agar. In place of the flasks large pans or
moist chambers may be used. The object is to use a vessel
that will give a large surface exposure.

2. After sterilization, inoculate the agar films with a
pure culture of Azotobacter. This may be accomplished
by using i-c.c. transfers from a suspension in sterile water.

3. Immediately after inoculation remove half of the
cultures for analysis. These may be treated with sulphuric
acid or sterilized.

4. A few days after inoculation add 10 c.c. of sterile
water to each culture.

5. Incubate the cultures in such a position that only a
portion of the surface will be covered with water, and
from day to day rotate. In this way it is possible to get
an even film over the entire surface.

6. About 28° C. is a favorable temperature for growth.

7. After twenty-one days analyze all of the cultures for
total nitrogen.

TABLE 27. — Nitrogen Fixation by Pure Cultures of Azotobacter

Nitrogen in 100 c.c. of agar.

Total.

Average.

Gain.

Per cent.

Mgm.

None.

do.

Azotobacter.

do.

RELATION OF AZOTOBACTER TO OXYGEN 65

Exercise 30

Effect of Variation in Culture-media on the Growth of
Azotobacter (Optional)

1. Prepare eight tubes of agar slopes.

2. Arrange the culture-media as follows:

(a) i and 2, mannit agar.

(b) 3 and 4, agar without mannit.

(d) 7 and 8, mannit agar without phosphate.

3. Inoculate all cultures from a suspension of Azoto-
bacter.

4. Incubate at 28° C. Examine every two days for
twelve days or longer.

5. Record the growth of Azotobacter on the various
culture-media.

Exercise 31

Relation of Azotobacter to Oxygen

1. Prepare six mannit agar slope cultures of Azotobacter
chroococcum.

2. Treat as follows:

(a) i and 2, untreated.

(6) 3 and 4, place in an atmosphere free of oxygen (see page 133).
(c) 5 and 6, seal the tubes by melting the glass and drawing out the
ends.

3. Incubate at room temperature.

4. Examine weekly for growth and pigment formation.
If the sealed tubes fail to show a brown to black pigment
after two weeks, open and note the change in color after
three or four days.

66 SOIL BACTERIOLOGY

Exercise 32
Anaerobic Nitrogen Fixation (Clostridiae)

1. Prepare four small flasks of Winogradsky's solution
(m. 41). Arrange to have the liquid high in the necks of
the flasks.

2. After sterilization inoculate the liquid with a pasteur-
ized soil extract.

Note. — Heat 50 grams of soil with 200 c.c. of water for fifteen minutes
at 80° C.

3. Allow the coarse particles to settle and pipet 5 -c.c.
portions into the flasks.

4. Arrange as follows:

(a) i and 2, sterilize immediately, or add 5 c.c. of sulphuric acid.
(6) 3 and 4, incubate at 28° C.

5. After twenty-one . days analyze for total nitrogen.
Transfer the entire contents to a Kjeldahl flask. In order
to avoid too rapid evolution of carbon dioxid, the sulphuric
acid should be added slowly.

Exercise 33
Isolation of Anaerobic Nitrogen-fixing Organisms

1. From the cultures obtained in the previous exercise
make transfers into tubes of sterile Winogradsky's solution.

2. Incubate under anaerobic conditions for ten to four-
teen days at 28° C.

3. Inoculate from these cultures into tubes of sterile
water, and from these dilutions into two tubes (in series) of
Winogradsky's agar liquefied and cooled to 40° C.

4. Pour plates in the usual way.

BACILLUS RADICICOLA FROM DIFFERENT LEGUMES 67

5. Incubate under anaerobic conditions for seven to
ten days at 20° C., and make transfers from several well-
isolated colonies into tubes of Winogradsky's solution,
and incubate as before. If the cultures are not pure,
they should be replated.

Exercise ffif
Isolation of Bacillus Radicicola from Different Legumes

i. Thoroughly wash the roots of several legumes (e. g.,
red clover, alfalfa, sweet clover, vetch, and soy beans)
under the tap.

Fig. 4. — Plate colonies of Bacillus radicicola from clover, after ten days at

28° C.; X 2.

2. Compare the number, size, and position of the nodules
on the roots of these different legumes.

SOIL BACTERIOLOGY

3. Select a large and firm nodule, cut off, and immerse
for three to five minutes in mercuric chlorid solution
(i : 500), and finally in alcohol.

4. Remove alcohol by flaming and place the nodule on a
sterile surface (flamed slide).

' Fig. 5. — Bacillus radicicola from a root nodule of alfalfa; X 1200.

5. Cut open with a sterile knife and take out some of the
inner contents.

6. Inoculate this bacterial mass from the nodule into a
few drops of water in a Petri dish.

7. Make two or more loop transfers from the first Petri

FORMATION OF NODULES BY BACILLUS RADICICOLA 69

dish to a second containing a few drops of water. Repeat
these dilutions to a third and fourth Petri dish.

8. Pour mannit agar (m. 42) into each dish, agitate until
the organisms are equally distributed, and incubate at 28° C.

9. After six to eight days examine plates. The legume
colonies should be characterized by a raised moist surface
and round entire form, at first glistening, later changing
to an opaque white. In size these colonies vary from ij
to 4 mm. in diameter.

Exercise 35
Formation of Nodules by Bacillus Radicicola

1. Wash thoroughly the seeds of several legumes (alfalfa,
crimson clover, red clover, etc.) and immerse in mercuric
chlorid (HgCfe) solution (i : 500) for three to five minutes.
For careful work, treat the seeds with mercuric chlorid
in a partial vacuum.

2. Remove from mercuric chlorid solution and rinse
in-sterile water.

3. Next drop one or two seeds of each legume into a
large tube containing soft mannit agar or a loose mass of
filter-paper pulp.

4. Arrange as follows:

(a) i and 2, alfalfa uninoculated.

(6) 3 and 4, alfalfa inoculated with culture iso^Ri by student.

In the case of large legumes — ef g., soy beans — it is
necessary to use vessels larger than ordinary test-tubes.

5. To inoculate, use a forty-eight-hour-old culture of
the legume bacteria. Prepare a water suspension and from
this take i c.c. for each tube.

SOIL BACTERIOLOGY

6. Under favorable conditions nodules will begin to form
in ten to fifteen days.

Fig. 6. — Showing inoculated alfalfa plant.

7. Keep the cultures in a warm place — greenhouse or
near a window.

8. After four and six weeks examine carefully for nodules,
noting the number, size, shape, and location.

EFFECT .OF BACILLUS RADICICOLA ON ALFALFA 71

kLFALFA ALFALFA

'NOCULATEO

Fig. 7. — Alfalfa in sterilized sand to which plant food minus nitrogen has

been added.

Exercise 36

Effect of Bacillus Radicicola on the Growth and the
Nitrogen Content of Alfalfa (Optional)

i. Prepare four J-gallon jars of clean sand and four of
coal-ashes.

72 SOIL BACTERIOLOGY

2. Plant to alfalfa as follows:

(a) i and 2, sand uninoculated.
(&) 3 and 4, sand inoculated.

(d) 7 and 8, coal-ashes inoculated.

3. One week after seedlings begin to germinate add
100 c.c. per jar of plant food minus nitrogen.

Note. — Plant food: Add 10 grams of CaSO4 -\- 2HzO, 4.6 grams of
KH2PO4, and 2.3 grams of MgSO4 + 7H2O to 1000 c.c. of water.

4. This nutrient solution should be added at intervals
of every two weeks or whenever needed. A few drops of
iron chlorid or iron phosphate will be found beneficial.

5. After four to six weeks examine for nodules.

6. When mature, cut and analyze the tissue for total
nitrogen.

Exercise 37

Effect of Caffein on the Formation of Bacteroids (Optional)

1. Inoculate in duplicate tubes of agarwith and without
cafTein (m. 46).

2. Prepare caffein agar slant cultures of the following
organisms: Bacillus radicicola, from alfalfa, pea, and vetch.

3. At regular two-day intervals study the morphology
of the organisms from the different cultures.

Exercise 38
Nitrogen Fixation by Bacillus Radicicola in Solution

1. Prepare four 5oo-c.c. Erlenmeyer flasks with 100 c.c.
each of mannit soil extract (m. 43).

2. Arrange as follows:

(a) i and 2, uninoculated.

(6) 3 and 4, inoculated with a pure culture of alfalfa bacteria.

ARTIFICIAL CULTURES FOR INOCULATION OF LEGUMES 73

3. Incubate at 28° C. for three weeks.

4. Then analyze for total nitrogen.

Exercise 39
Production of Gum by Bacillus Radicicola

1. Inoculate two tubes of different media (42 and 47)
with Bacillus radicicola, and at the same time leave two
tubes uninoculated.

2. Incubate for four to five weeks at 28° C., and test for
gum.

3. Add 10 c.c. of alcohol (95 per cent.) or 5 c.c. of acetone
to each tube.

4. Note the precipitation of gum.

Exercise 40
Artificial Cultures for the Inoculation of Legumes

A. i. Inoculate one bottle of mannit agar (m. 42) and
one of mannit solution with a pure culture of legume
bacteria. Use 5oo-c.c. bottles with flat sides. In the
liquid culture take 300 c.c. of the medium; in the agar
culture, 100 c.c. Dilute a young legume culture of the
desired organism with 5 c.c. of sterile water. Shake
thoroughly and pipet i-c.c. portions into the bottles of
liquid and solid media. Be sure that the inoculum covers
the entire surface of the agar.

2. Incubate the cultures at 28° C. for four to ten days.

3. Determine the number of bacteria in each bottle. The
liquid culture may be treated as follows: Shake thoroughly,
remove i c.c. to a gg-c.c. water blank, and continue the
dilution to i : 1,000,000 and i : 10,000,000. Pour mannit
agar plates.

74 SOIL BACTERIOLOGY

Follow the same procedure with the solid culture, adding
200 c.c. of sterile water to the agar. Shake thoroughly,
dilute, and plate as given in the preceding directions.

B. i. Place about ten bacteria-free seeds in each culture
bottle. Shake and pour off the liquid.

2. With sterile forceps remove about five seeds to a
sterile Petri dish.

3. Allow seeds to dry in the Petri dish.

4. After twenty-four to forty-eight hours count the
number of legume bacteria on each seed.

5. Place the seeds in a zoo-c.c. water blank, shake,
and plate i-c.c. portions.

Exercise 41
Nitrogen Content of Bacteria (Optional)

1. Grow a mass culture of bacteria in a large Petri dish
or pan. Any vigorous growing organism may be used,
e. g., Azotobacter.

Note. — Use 2 to 2.5 per cent. agar.

2. When cool, inoculate the surface of the agar with
5 c.c. of a rich suspension of Azotobacter in sterile water.

3. Incubate ten to fifteen days.

4. At the end of this time remove growth by carefully
scraping off with a clean glass slide.

5. Dry the mass culture at 100° C. and pulverize by
grinding in a mortar.

6. Analyze for total nitrogen. If desirable, a portion
of the material may be saved for further analysis — e. g.,
potassium and phosphorus.

SECTION III
RELATION OF MICROORGANISMS TO THE CARBON CYCLE

Exercise i
Fermentation of Cellulose in Impure Cultures (Liquid)

1. FILL six large test-tubes about half -full of Omelianski's
solution (m. 48).

2. Add four strips of filter-paper to each tube.

3. Treat as follows:

(a) i and 2, uninoculated.

(b) 3 and 4, stable manure.

4. Incubate at 28° C.

5. Cover the solution in tubes i, 3, and 5 with f-inch
layer of paraffin oil.

6. Examine the cultures at regular intervals, taking note
of the changes in the filter-paper.

7. When the filter-paper shows the first evidences of
disintegration, make transfers to new tubes of Omelianski's
medium.

Exercise 2
Fermentation of Cellulose in Impure Cultures (Soil)

i. Place 2oo-gram portions of garden soil in three i -liter
Erlenmeyer flasks.

76 SOIL BACTERIOLOGY

2. Treat as follows:

(a) Control.

(&) Add i per cent, of sugar.

3. Bring the moisture content of the soil up to two- thirds
saturation.

4. Insert one round filter-paper, diameter smaller than
that of the flask, into each culture. Partially cover the
filter-paper with soil.

5. Incubate at 28° C.

6. At weekly intervals examine. Note the change in the
filter-paper.

Exercise 3
Fermentation of Cellulose in Soil

1. Prepare four large soup plates with 500 grams each of
soil. Use two plates of field and two plates of garden
soil.

2. Add to each plate 5 grams of dry filter-paper cut into
strips about 2 inches long and J inch wide. These should
be thoroughly mixed with the soil and a little more water
than necessary for half -saturation added.

3. In order to avoid rapid evaporation cover with inverted
plates and keep the moisture constant.

4. After six to eight weeks remove the remaining paper
from the soil by passing the soil through a fine mesh sieve.
Now wash the paper, dry, and weigh. Although this
procedure does not always give uniform results, it will
show the relative cellulose-destroying power of the soil.

ISOLATION OF CELLULOSE BACTERIA 77

Exercise 4

— — .

Fermentation of Cellulose by Denitrifying Bacteria

1. Fill completely two Erlenmeyer flasks of 2oo-c.c.
capacity each with medium 37.

2. Inoculate as follows:

(a) i c.c. of a rich sewage suspension.
(&) i gram of garden soil.

3. Incubate at 35° C. in a pan or plate so arranged as to
catch the overflow from the flasks. After one week the
cultures should begin to show fermentation, and by the
end of the second or third week all nitrates should be de-
stroyed.

4. When the solution no longer reacts for nitrates pour
the turbid liquid off without removing the paper. Refill
the flask with the same medium minus the paper. Now
the process should proceed very much faster than in the
case of the first inoculation.

5. The addition of new culture-media may be repeated
several times.

Exercise 5

Isolation of Cellulose Bacteria

A. Without Enrichment Cultures:

1. Dilute samples of soil in such a way that plates may
be made representing various dilutions, about i : 10,000
and i : 100,000 of a gram of the original material.

2. Garden, field, and marsh soil should be used.

3. Plate all of these inocula with medium 52.

B. With Enrichment Cultures:

i. At the same time plates are poured for A make
transfers from the enrichment culture, Exercise i, in sterile
99-c.c. water blanks.

78 SOIL BACTERIOLOGY

2. From the first gg-c.c. water blank make i-c.c. transfer
to a second and a third.

3. Pour cellulose agar plates from the second and third
dilution.

4. Incubate all of the plates under a bell jar at 28° C.
Very often the cellulose organisms do not appear for several
weeks.

5. Look for the colonies with clear zones.

Exercise 6
Formation of Carbon Dioxid from Organic Substances

1. For this exercise use a field soil and adjust the moisture
content to about half -saturation.

2. Mix thoroughly so as to have the entire sample
uniform.

3. Weigh into suction flasks (2-liter capacity) four equal
quantities of the soil, i kilo each, and treat as follows:

(a) Untreated.

(6) Add 2 per cent, of finely ground vegetable matter, clover, corn,
beet leaves, or something similar.

(d) Add i per cent, of cane-sugar.

4. Connect to a glass cylinder so arranged with glass
beads and alkali that the carbon dioxid (C02) will be
caught as it is drawn through the solution.

5. Every forty-eight hours determine the amount of
carbon dioxid by drawing a current of air through the
apparatus for twenty minutes with a water-pump (see page
150). Free the current of air from carbon dioxid by pass-
ing through strong N/i potassium hydroxid. In order to
regulate the air, count the number of air bubbles.

FORMATION OF HUMUS IN SOIL (OPTIONAL) 79

6. Allow the experiment to run for twelve days.

7. Arrange the results in a table.

TABLE 28. — The Influence of Organic Substances on the Evolution of Carbon
Dioxid from Soil

Carbon dioxid in too gm. of soil.

Two-day periods.

Control.

Clover
2 per cent.

Barnyard
manure
2 per cent.

Cane-sugar
2 per cent.

Mgm.

Mg-m.

Total

Exercise 7
Formation of Humus in Soil (Optional)

1. Prepare a uniform sample of soil (air-dry) by passing
through a sieve.

2. Weigh out 5oo-gram portions and treat as follows:

(a) Control.

(b) Add 2 per cent, finely ground wheat straw.

(d) Add 2 per cent, finely ground dry manure.

(e) Sterile, no additional treatment.

(/) Add 2 per cent, finely ground wheat straw; sterilize.

(g) Add 2 per cent, finely ground alfalfa; sterilize.

(h) Add 2 per cent, finely ground dry manure; sterilize.

3. Pint or quart Mason jars can be used as receptacles
for the various soils.

80 SOIL BACTERIOLOGY

4. Add sufficient sterile water to bring moisture content
up to two- thirds saturation.

5. Cover loosely with a Petri dish and incubate three to
four months, restoring moisture from time to time.

6. At the end of this time prepare the cultures for analysis.

7. Dry and determine the amount of humus in lo-gram
samples (see page 148).

SECTION IV

RELATION OF MICROORGANISMS TO THE SULPHUR
CYCLE

Exercise i

Reduction of Sulphates with the Formation of Hydrogen

Sulphid

1. PREPARE three small bottles of sulphate solution

(m. 55)-

2. Inoculate as follows:

(a) Uninoculated.

(&) i gram of rich soil.

3. Stopper tightly with paraffined corks.

4. Incubate at 28° C. for two or four weeks.

5. At the end of this time remove bottles from incubator;
note the change in color and odor.

6. Hold over the open mouth of the bottle a small piece
of filter-paper saturated with a solution of lead acetate.
A blackening of the paper shows the presence of hydrogen
sulphid.

7. Remove a few cubic centimeters with a pipet to a
test-tube or small Erlenmeyer flask.

8. Add a few drops of BaCl2 solution.

9. Compare the amount of white precipitate in the
inoculated cultures with that in the uninoculated control.

6 81

8 2 SOIL BACTERIOLOGY

10. The amount of hydrogen sulphid may be determined
quantitatively by titrating with iodin and sodium thio-
sulphate (see page 154).

Exercise 2
Isolation of Hydrogen Sulphid Organisms

1. Prepare four tubes of sulphate-reducing gelatin
(m. 56).

2. Inoculate shake cultures of the gelatin with various
dilutions of the impure cultures from the previous exercise.

3. Harden the gelatin cultures in cold water and incubate
at room temperature four to seven days in an anaerobic
jar (seepage 133).

4. In case black colonies appear in the tubes, attempt to
isolate the organisms by making subinoculations into the
sulphate gelatin.

Exercise 3
Hydrogen Sulphid from Protein and Sulphur (Optional)

1. Prepare two small bottles of culture solutions (i)
and (2) (m. 59).

2. Treat as follows:

(a) Solution i, uninoculated.

(b) Solution i, i gram garden soil.

(d) Solution 2, i gram garden soil.

3. Compare the changes that take place in solutions
(!) and (2).

OXIDATION OF THIOSULPHATES 83

Exercise 4
Oxidation of Thiosulphates

1. Place in four flasks (300-0.0. Erlenmeyer) about 20
c.c. each of Nathansohn's solution (m. 60).

2. Inoculate two flasks with o.i gram of soil each.
Leave two flasks uninoculated.

3. Incubate for four weeks at 28° C.

4. Remove the flasks from incubator and test for sul-
phates.

SECTION V

RELATION OF MICROORGANISMS TO THE IRON CYCLE

Exercise i
A Method for Growing Crenothrix and Spirophyllum

1. CLEAN and sterilize a Berkefeld filter.

2. Connect the filter to the city water-supply and allow
the water to run slowly for twenty-four hours.

3. Remove the metal cap from the filter and place in a
large beaker of iron solution (m. 61).

4. Incubate in the ice-box or at 15° to 20° C.

5. At regular two-day intervals examine the deposit
on the sides of the filter.

6. If bacteria are found, test for iron. Add a few drops
of a 5 per cent, hydrochloric acid solution and a 4 per cent,
potassium ferrocyanid solution. In the presence of ferric
salts an intense blue color is formed.

7. In order to stain the higher forms of iron bacteria
it is well to remove the deposit of iron by treating with a
5 per cent, hydrochloric acid solution.

Exercise 2
Iron Precipitating Bacteria

1. Shake 20 grams of field soil with 200 c.c. of water.
Dilute until i c.c. equals i : 100,000.

2. Pour plates with medium 64.

3. Incubate the plates for several weeks at 28° C.

4. Note the precipitation of iron compounds around
certain colonies.

jrig> g.— Iron bacteria: A, Crenothrix thread showing germination of
spores within sheath; X 850. B, Chlamydothrix showing simple and
curved threads; X 850.

Fig. 9. — Iron bacteria: A, Gallionella, Chlamydothrix, and Spirophyllum ;

X 850. B, Spirophyllum; X 850.
86

SECTION VI

RELATION OF MICROORGANISMS TO THE PHYSICAL
PROPERTIES OF SOIL

Exercise i
Movements of Soil Water

1. PREPARE four glass cylinders or large test-tubes as
follows: Fill two three-fourths full of quartz sand and
two three-fourths full of soil.

2. Treat as follows:

(a) Sand plus i per cent, of sugar bouillon previously inoculated with

5 c.c. of a rich soil suspension.
(6) Sand plus i per cent, of sugar bouillon previously inoculated with

5 c.c. of a rich soil suspension.

(d) Soil plus i per cent, sugar.

3. Add enough bouillon to the sand and enough water to
the soil to completely saturate the columns.

4. In order to prevent bacterial growth add 5 c.c. of
a i : 5 mercuric chlorid solution to cylinders (a) and (c),
and the same amount of water to cylinders (b) and (d).

5. Mark on the cylinders the height of the column of
water.

6. Incubate at 28° C., and examine each day.

FORMULAE AND METHODS

CLEANING GLASSWARE

1. ALL glassware must be thoroughly cleaned before it
is ready to use. Test-tubes, Petri dishes, flasks, and similar
glassware should be boiled in a 5 per cent, soda solution
or washed in hot soapsuds until free from organic matter.
When it is desirable to use very clean glassware, immerse
for ten minutes or longer if possible in the dichromate
solution.

Potassium (K2Cr2O7) or sodium dichromate (Na2Cr2O7) ... 80 gm.

Water 300 c.c.

Sulphuric acid (H2SO4) 460 c.c.

Note. — Dissolve the dichromate in warm water and, when cool, add
slowly concentrated sulphuric acid. If properly prepared, the liquid should
be thick, with small crystals. It may be used repeatedly, provided the
crystals 'are present.

2. After removing from the cleaning solution rinse
thoroughly in distilled water.

3. Dirty cover-glasses and slides may be treated in the
same manner. Drop these, one at a time, into the dichro-
mate mixture and allow to remain for several hours. Re-
move from this solution, wash, and wipe with a soft, clean
cloth.

4. A simple and more rapid method, suitable for general
work, is to rub the slides with moist Bon Ami, and when
dry to polish them with a clean cloth.

5. In order to remove fat pass the cover-slips through a
flame. Where it is desirable to have very clean slides and

BOUILLON OR NUTRIENT BROTH 89

cover-slips it is well to heat them in water and then in 50
per cent, sulphuric acid. After rinsing in distilled water,
wash in alcohol and wipe with a clean cloth. These should
be kept in a clean, covered dish.

SECTION VII

PREPARATION OF CULTURE-MEDIA

The culture-media are arranged according to the groups
of soil microorganisms. Since it is not possible to grow all
of the strains of bacteria in one group on the same medium,
several formulae are given.

MEDIA FOR THE DETERMINATION OF THE NUMBER AND FOR
THE SEPARATION OF SOIL BACTERIA

Medium i

Bouillon or Nutrient Broth

Peptone 10 gm.

Liebig's meat extract 3 gm.

Distilled water 1000 c.c.

1. Add to i liter of distilled water 3 grams of meat
extract and 10 grams of peptone.

2. Record the weight of vessel and contents.

3. Heat not above 50° C. in steamer or double boiler until
extract and peptone are dissolved.

4. Titrate and adjust reaction to i per cent, acid, with
phenolphthalein as an indicator (+i).

5. Boil over the free flame for fifteen minutes.

6. Restore the loss in weight with distilled water.
' 7. Titrate again.

QO SOIL BACTERIOLOGY

8. Sterilize broth in large flask and allow to stand until
next laboratory period.

9. Refilter through fine paper and tube.

10. Fill tubes about one-third full.

11. Plug the tubes and sterilize in autoclave at 120° C.
for fifteen minutes.

Note. — To titrate, remove 5 c.c. of the medium to a casserole or small
flask containing about 45 c.c. of distilled water. Boil one minute with
constant stirring, add 3 drops phenolphthalein, and neutralize excess acid
with N/20 NaOH. If i c.c. N/20 NaOH is required to neutralize 5 c.c.
of the medium, the reaction is correct. In this way calculate the amount
of normal alkali or acid necessary to adjust the reaction of the entire bulk
of culture-medium to i per cent. (+i).

All reactions must be expressed with reference to the phenolphthalein
neutral point. They are stated in percentages of normal acid or alkaline
solutions required to neutralize them (Fuller's scale). Alkaline media
should be recorded with the minus sign ( — ) before the percentage of normal
acid needed for their neutralization; acid media should be written with the
plus sign (+) before the percentage of normal alkaline solution necessary
for their neutralization.

The example below will illustrate the method. If the required reaction
is (+1) and the buret reading shows that i.S c.c. of N/20 NaOH has been
used in neutralizing the 5 c.c. of broth, then the problem may be stated as
follows:

5 c.c. of broth require the addition of 1.8 c.c. N/20 NaOH to neutralize
it.

100 c.c. of broth require the addition of 36 c.c. N/2O NaOH or 1.8 c.c.

N/i NaOH to neutralize it.

looo c.c. of broth require the addition of 18 c.c. N/i NaOH to neutralize
it.

The figures above show that the broth as titrated is 0.8 per cent, too
acid, and that 8 c.c. of normal NaOH per liter must be added to obtain the
proper reaction.

Do not neutralize medium first and then readjust by the addition of acid.
This tends to precipitate certain substances which are favorable to bac-
terial development.

The broth prepared in this way should be of a golden color and
should not develop a precipitate upon subsequent sterilization in the
autoclave. Adjust reaction by adding normal hydrochloric acid or
sodium hydroxid.

NUTRIENT GELATIN 9 1

The hydrogen electrode may be used to determine the reaction of culture-
media.

Fuller, G. W., Jour. Pub. Health Assoc., vol. xx, pp. 381-399, 1895.
Standard Methods of Water Analysis, 1915.
Clark, W. M., Jour. Inf. Diseases, vol. xvii, pp. 109-136, 1915.
Anthony and Ekroth, Jour. Bact., vol. i, pp. 230-232, 1916.
Itano, A., Bui. 167, Mass. Agr. Exp. Sta., 1916.

12. Titration of Broth. — Titrate the two samples of
broth prepared by the instructor. Determine the amount
of N/i alkali or acid required to make i liter of these
solutions (+i). Do this for each sample. Titrate sample
No. 2, hot and cold, using phenolphthalein and litmus as
indicators. Record results.

Medium 2
Nutrient Gelatin

Gelatin 100 to 150 gm.

Liebig's meat extract 3 gm.

Peptone 10 gm.

Distilled water 1000 c.c.

1. In a convenient vessel measure 1000 c.c. of nutrient
broth.

2. Add 10 per cent., on the dry basis, of gold label sheet
gelatin. Let the gelatin soak five to ten minutes.

3. Heat over water-bath until dissolved.

4. Adjust the reaction as directed in the preparation of
nutrient broth. Gelatin is decidedly acid and will require
more NaOH to neutralize it than bouillon or agar.

5. Cool this mass to about 60° C. Add the whites of
two eggs or 3 grams of powdered egg-albumen to 25 c.c.
of water. Stir into the gelatin and heat in a double boiler.
The egg-albumen will coagulate and inclose most of the

92 SOIL BACTERIOLOGY

impurities. When this coagulum has settled to the bottom,
pour the cleared gelatin through the filter.

6. If properly prepared, gelatin may be filtered through
filter-paper. Otherwise it will be necessary to use an ab-
sorbent cotton filter.

Note. — A cotton filter is prepared as follows: In the base of a large funnel
place a small amount of clean excelsior. In place of the excelsior a small
spiral of copper wire may be used. On top of this put two or three layers of
absorbent cotton. Split a piece of absorbent cotton, somewhat larger
than the top of the funnel, horizontally into two layers of equal thickness.
Place one layer of cotton above the other, so that the fibers are at right
angles. Pour the medium, slowly at first, on to the filter. (In order to
avoid breaking the filter use a glass rod to direct the fluid to the center of
the filter.) When the filtrate begins to come through the cotton, fill the
funnel. If the first filtrate is not clear, the turbid liquid should be refiltered
through the same cotton.

7. Sterilize in the autoclave for ten minutes at 120° C.

8. As soon as removed from the autoclave stand in cold
or ice-water. Gelatin is easily decomposed, and if heated,
too high or too long will not solidify.

Medium 3

Nutrient Agar

Agar 15 gm.

Liebig's meat extract 3 gm.

Peptone 10 gm.

Distilled water 1000 c.c.

1. In a vessel containing 1000 c.c. of water add 15 grams
of thread agar.

2. Heat in the steamer or double boiler until the agar is
dissolved. This requires at least one hour.

3. Add 3 grams extract of meat and 10 grams of peptone.

HEYDEN-NAHRSTOFF AGAR 93

4. When completely dissolved adjust the reaction to

5. Clear with egg-albumen and filter as directed under
the preparation of gelatin.

6. Tube, and sterilize in the autoclave for fifteen minutes.
The amount of agar to place in tubes will depend on the
purpose for which the agar is to be used. For slants,
about 5 c.c. is enough; for plates, about 10 c.c.

Note. — For making especially clear agar, adjust the reaction to (+1.5)
with N/i HC1 before adding the agg-albumen. Heat in the steamer until
the albumen is coagulated and settled to the bottom of the dish. If it will
not settle, stir the agar vigorously and continue heating. It may be three
or four hours before it is ready to be filtered. Filtering then consists only
in decanting the cleared agar through either a cotton filter or filter-paper.
Do not pour the dirt and albumen on to the filter. Titrate and adjust the
reaction to (+i).

Medium 4
Heyden-Nahrstoff Agar

Agar ........................................ 1 2.0 gin.

Heyden-Nahrstoff ............................. 7.5 gm.

Distilled water ........... .................... 1000.0 c.c.

1. To 500 c.c. of cold distilled water in a flask add 7.5
grams of Heyden-Nahrstoff. Shake until a good suspension
is obtained and allow the mixture to stand for thirty minutes
or more.

2. Heat in steamer or double boiler for one hour, or
until the upper portion of the solution is clear.

3. While hot filter through paper.

4. Dissolve 1 2 grams of agar in 500 c.c. of water. Filter
and mix the Heyden-Nahrstoff and agar solutions.

5. It is not necessary to adjust the reaction of this medium.

Heyden-Nahrstoff, The Heyden Chemical Works, 135 Williams St.,
New York City, N. Yt

94 SOIL BACTERIOLOGY

Medium 5
Casein Agar

Agar 10 gm.

Casein 10 gm.

Sodium hydroxid N/i (NaOH) 7 c.c.

Distilled water 1000 c.c.

1. Measure into an Erlenmeyer flask 100 c.c. of distilled
water and 10 grams of casein (Hammarsten) .

2. Add to this 7 c.c. of normal NaOH.

3. Heat in a double boiler or steamer to get a perfect
solution.

4. Dissolve 10 grams of agar in 900 c.c. of distilled water.

5. Mix and filter the casein-agar solution.

6. Adjust the reaction between (+0.1) and (+0.2) per
cent. If the casein is weighed accurately and the normal
solution is correct, the reaction will be about (+0.2).

7. Tube, and sterilize in autoclave for fifteen minutes.
Cool quickly in cold or iced water.

Note. — The final reaction of the medium will be about (+0.1), Fuller's
scale. If the medium is alkaline, the bacterial growth will be restricted.
If the medium is more than (+0.1) some of the casein may be precipitated
during sterilization. The casein agar should be clear and almost colorless
when poured into a Petri dish. Sometimes the casein will be slightly pre-
cipitated during the sterilization or the cooling. This is of no consequence,
since the precipitate, when poured into plates, is so finely divided that it
becomes invisible. Casein agar should be incubated for six days at 30° C.

Ayres, S. H., United States Dept. Agr. Bur. Animal Indus., 28th Ann.
Report, pp. 225-235, 1911.

Medium 6

Soil- extract Agar

Agar 15 gm.

Dextrose (C6Hi2O6) i gm.

Soil extract 100 c.c.

Water 900 c.c.

ASPARAGIN-DEXTROSE AGAR FOR SOIL BACTERIA 95

1. Dissolve the agar in. 900 c.c. of water by heating in
the steamer for one hour or longer. Add 100 c.c. of the
stock soil-extract solution.

2. Add the dextrose just prior to tubing.

3. The reaction should be (+0.5) or nearly neutral.

Note. — Stock Solution of Soil Extract. — This is prepared by heating 1000
grams of garden soil with 1000 c.c. of tap- water in the autoclave at 5 to 10
pounds' pressure for thirty minutes. A small amount of calcium carbonate
is added and the whole is filtered through a double paper filter. The turbid
filtrate should be poured back on to the filter until it comes through clear.

Medium 7
Soil-extract Gelatin

Gelatin 100 to 150 gm.

Dextrose (C6Hi2O6) i gm.

Soil extract 100 c.c.

Water 900 c.c.

1. Dissolve the gelatin in the diluted soil-extract solution
by heating slowly in the steamer.

2. Clarify the medium with egg-albumen.

3. Add i gram of dextrose and adjust the reaction to

(+0.5).

Conn, H. J., Bui. 38, N. Y. Agr. Exp. Sta., 1914.

Medium 8
Asparagin-dextrose Agar for Soil Bacteria

Agar 15.0 gm.

Dibasic potassium phosphate (K2HPO4) 0.5 gm.

Magnesium sulphate (MgSO4 + yH2O) 0.2 gm.

Asparagin (C4H8N2O3 + H2O) i.o gm.

Dextrose (C6H12O6) i.o gm.

Distilled water . 1000.0 c.c.

96 SOIL BACTERIOLOGY

1. After dissolving the agar by steaming for one hour or
more, add the dibasic potassium phosphate and magnesium
sulphate.

2. The asparagin and dextrose should be added just
before sterilizing.

3. Adjust the reaction to (+1.0).

Medium 9
Urea-ammonium Nitrate Agar for Soil Bacteria

Agar 15.0 gm.

Dibasic potassium phosphate (K2HPO4) 0.5 gm.

Magnesium sulphate (MgSO4 + 7H2O) 0.2 gm.

Dextrose (C6Hi2O6) 10.0 gm.

Urea (CO(NH2)2) 0.05 gm.

Ammonium nitrate (NHUNOs) o.i gm.

Ferric sulphate (Fe2(SO4)3) • trace

Distilled water 1000.0 c.c.

The urea, ammonium nitrate, and dextrose should not be
added until the medium is ready for sterilization. The
reaction should be about (+0.25).

Cook, R. C., Soil Science, vol. i, No. 2, pp. 153-161, 1916.

Medium 10
Sodium Asparaginate Agar for Soil Bacteria

Agar 1 2.0 gm.

Ammonium biphosphate (NH4)H2PO4 1.5 gm.

Magnesium sulphate (MgSO4 + yH2O) 0.2 gm.

Sodium asparaginate (NaC4H6NO4 -f- H2O) i.o gm.

Dextrose (CeH^Oe) i.o gm.

Calcium chlorid (CaCl2) o.i gm.

Potassium chlorid (KC1) o.i gm.

Ferric chlorid (FeCla + 6H2O) trace

Distilled water 1000.0 c.c.

The sodium asparaginate and dextrose should not be added
until the medium is ready for sterilization. The reaction

SOIL EXTRACT (FLAGELLATES AND CILIATES) 97

should be between (+0.8 or +1.0). It requires about
10 c.c. of N/i NaOH per liter. In place of clarifying with
the white of egg, the author recommends heating for half
an hour at 15 pounds' pressure without disturbing the sedi-
ment and decanting through a cotton filter.

Conn, H. J., Bui. 38, New York Agr. Exp. Sta., 1914.

COUNTING SOIL PROTOZOA

Medium n
Hay-soil Extract

Soil extract 100 c.c.

Hay extract (o.i per cent, of dry hay) 100 c.c.

Calcium carbonate (CaCO3) 5 gm.

Tap-water 800 c.c.

Medium 12
Hay Infusion

Hay 10 gm.

Tap-water 1000 c.c.

Medium 13
Hay Egg-albumen (Ciliates)

Hay 100 gm.

Egg-albumen 50 gm.

Tap-water 1000 c.c.

Medium 14
Soil Extract (Flagellates and Ciliates)

Soil extract 100.0 c.c.

Dibasic potassium phosphate (K^HPC^) 0.5 gm.

Tap-water 900.0 c.c.

98 SOIL BACTERIOLOGY

Medium 15
Mannit Solution
See Culture-medium No. 39, page 108.

AMMONIFICATION

Medium 16

Peptone Solution

Peptone 10 gm.

Distilled water 1000 c.c.

Heat in the autoclave for thirty minutes and filter.

Medium 17
Gelatin Solution

Gelatin 5 gm.

Distilled water. 1000 c.c.

Bring the reaction to (+1.0).

Medium 18

Casein Solution

Casein 10 gm.

Normal sodium hydroxid (NaOH) 7 c.c.

Distilled water 1000 c.c.

Prepare according to directions on page 94.

Medium 19
Urea Solution

Urea (CO(NH2)2) 20 gm.

Bouillon ( — i.o) looo c.c.

UREA GELATIN 99

1. Heat in the steamer or over a free flame until the
precipitate settles.

2. Filter and sterilize.

3. In order to diminish the loss of ammonia from urea
the culture-media should be sterilized in the steamer
twenty minutes on three successive days.

Medium 20
Urea Solution

Certain forms of urea fermenters prefer a medium richer
in urea. This may be prepared by adding 10 per cent, of
urea to the bouillon.

Medium 21

Urea Solution

Urea (CO(NH2)2) 30.0 gm.

Dibasic potassium phosphate (K2HPO4). 0.5 gm.

Calcium citrate (Ca3(C6H5O7)2 + 4H2O) 10.0 gm.

Tap-water 1000.0 c.c.

Sohngen, N. L., Centbl. Bakt. (etc.), Abt. 2, Bd. 23, p. 94, 1909.

Medium 22

Urea Solution

Urea (CO(NH2)2) 50.0 gm.

Dibasic potassium phosphate (K2HPO4) 0.5 gm.

Soil extract 100.0 c.c.

Tap-water 900.0 c.c.

Lohnis, F., Centbl. Bakt. (etc.), Abt. 2, Bd. 14, p. 714, 1905.

Medium 23
Urea Gelatin

Gelatin 120 to 150 gm.

Urea (CO(NH2)2) 20 c.c.

Bouillon ( — i.o) 1000 c.c.

100 SOIL BACTERIOLOGY

The urea decreases the solidifying properties of gelatin.
If agar medium is wanted, take 15 grams to i liter.

Medium 24
Hippuric Acid Solution

Sodium hippurate (NaC9H8NO3) 3.0 gm.

Dibasic potassium phosphate (K2HPO4) 0.5 gm.

Tap-water 1000.0 c.c.

Medium 25
Uric Acid Solution

Uric acid (C5H4N4O3) 3.0 gm.

Dibasic potassium phosphate (K2HPO4) 0.5 gm.

Tap-water 1000.0 c.c.

Lohnis, F., Landwirtschaftliche-bakteriologisches Praktikum, Berlin, pp.
112, 113, 1911.

NITRIFICATION

Medium 26
Solution for Nitrite Formation

Ammonium sulphate (NH4)2SO4 i.o gm.

Dibasic potassium phosphate (K2HPO4) i.o gm.

Magnesium sulphate (MgSO4 + yH^O) 0.5 gm.

Sodium chlorid (NaCl) 2.0 gm.

Ferrous sulphate (FeSO4 + 7H2O) 0.4 gm.

Magnesium carbonate (MgCOs) in excess about 5.0 gm.

Distilled water 1000.0 c.c.

In order to prevent any loss of ammonia, it is well to sterilize
the ammonium sulphate separately. A 10 per cent, solu-
tion will be found very convenient. When cool, the proper
amount of ammonium sulphate may be added with a
sterile pipet.

Winogradsky, Lafar, Technische Mykologie, Bd. 3, pp. 132-181, 1904.

SOLUTION FOR NITRITE

Medium 27
Solution for Nitrite Formation

Magnesium ammonium phosphate (Mg(NH4)PO4

+ 6H2O) 2.0 gm.

Dibasic potassium phosphate (KaHPO^ 0.5 gm.

Magnesium sulphate (MgSO4 + 7H2O) 0.5 gm.

Sodium chlorid (NaCl) i.o gm.

Ferrous sulphate (FeSO4 + 7H2O) 0.4 gm.

Magnesium carbonate (MgCO3) 5-° gm-

Distilled water 1000.0 c.c.

It is not necessary to sterilize the magnesium ammonium
phosphate separately.

Medium 28
Solution for Nitrate Formation

Sodium nitrite (NaNO2) i.o gm.

Dibasic potassium phosphate (K2HPO4) 0.5 gm.

Magnesium sulphate (MgSC>4 + 7H2O) 0.3 gm.

Sodium chlorid (NaCl) 0.5 gm.

Ferrous sulphate (FeSO4 + yH2O) 0.4 gm.

Sodium carbonate (Na2CO3) (anhydrous) 0.3 gm.

Distilled water : IOOG.O c.c.

The formation of nitrates takes place rapidly, provided
the cultures are grown under conditions that supply an
abundance of oxygen.

Medium 29
Solution for Nitrite and Nitrate Formation

Ammonium sulphate (NH4)2SO4 2.0 gm.

Dibasic potassium phosphate (K2HPC>4) i.o gm.

Magnesium sulphate (MgSC>4 + ?H2O) 0.5 gm.

Sodium chlorid (NaCl) 2.0 gm.

Ferrous sulphate (FeSO4 + yH2O) 0.4 gm.

Calcium carbonate (CaCOs) 5.0 gm.

Distilled water. . . 1000.0 c.c.

102 SVOIL BACTERIOLOGY

The ammonium sulphate or the calcium carbonate should
be sterilized separately and added after cooling. In place
of 2 grams of ammonium sulphate, 7.4 grams of magnesium
ammonium phosphate may be used. This solution is
suited to a quantitative study of nitrification.

Medium 30
Silicate Jelly for the Nitrifying Bacteria

A. Undialyzed

1. Prepare a solution of sodium silicate (Na2Si03) of
approximately 8 per cent. Weigh out the sodium silicate
and boil in water for thirty minutes, filter through cotton
and coarse grained filter-paper. It is more convenient to
use Merck's concentrated solution of sodium silicate and
dilute to the desired strength. This must be kept tightly
stoppered.

2. Prepare a solution of hydrochloric acid (HC1) of such
a strength that i c.c. of the acid neutralizes i c.c. of the
sodium silicate, using methyl-orange as an indicator; or
use normal hydrochloric acid and determine the amount of
sodium silicate required to neutralize the acid.

3. To 1 20 c.c. of the hydrochloric acid solution add, with
stirring, 100 c.c. of the sodium silicate solution. If normal
acid is used, be sure there is 20 c.c. excess of acid in each
220 c.c. of the mixture.

4. Tube i2-c.c. portions of the mixture and sterilize in
the autoclave for ten minutes at 15 pounds' pressure. If
the tubes are sealed tightly this mixture may be kept for
several weeks. In case the mixture becomes a milky color
or solid when taken from the autoclave, it indicates an

SILICATE JELLY FOR THE NITRIFYING BACTERIA 103

insufficient quantity of hydrochloric acid was added.
Repeat, using more acid.

5. Prepare a solution containing the nutrient salts
suitable for the growth of the desired organism. This
solution should contain the salts in from 2.5 to 5 times the
desired strength.

6. Tube and sterilize the nutrient solution. If 5 times
normal strength is taken, use about 3 c.c. per tube, or 5 c.c.
if 2.5 times normal strength.

7. Pour the silicic acid mixture into a sterile Petri dish.
Inoculate the sterile nutrient solution and add a sufficient
amount of a sodium carbonate solution to neutralize the
excess silicic acid in the mixture and a few drops, in excess.
Now pour this into the dish with the silicic acid, rotate,
and allow the plate to harden on an even surface. After
a few moments this mixture should harden. The plates
may be handled similar to agar plates. The concentration
of silicic acid mixtures determines the strength of the
nutrient solution to use.

A modification of the Stevens and Temple method, Centbl. Bakt. (etc.),
Abt. 2, Bd. 21, pp. 84-87, 1908.

B. Undialyzed

1. Dissolve 8.40 grams of sodium silicate (Na2SiO3) and
24 grams of potassium silicate (K2Si03) in 500 c.c. of
distilled water. A mixture of sodium and potassium
silicate decreases the sodium salt in the final medium.

2. Prepare dilute hydrochloric acid in such a way that it
requires slightly more than i c.c. of the sodium potassium
silicate solution to neutralize i c.c. of the HC1.

3. Add to the hydrochloric acid solution the nutrient
salts suitable for the growth of the nitrifying bacteria.

4. With methyl-orange as an indicator, standardize the

104 SOIL BACTERIOLOGY

hydrochloric acid mixture against the silicate so that i c.c.
equals i c.c.

5. In a similar manner standardize a solution of sulphuric
acid and phosphoric acid without the salts.

6. The three acids should then be mixed. Approxi-
mately, i c.c. of the acid mixture will neutralize i c.c. of
the silicate mixture.

Doryland, C. J. T., Jour, of Bact., vol. i, No. 2, pp. 143-148, 1916.

C. Partially Dialyzed

1. Make a solution of sodium silicate as in the undialyzed
procedure.

2. Mix with approximately normal hydrochloric acid,
making the mixture decidedly acid.

3. Dialyze in running water, using parchment, animal
membrane, or collodion sacs until nearly all the chlorids
have disappeared. Make sure all the chlorids do not
dialyze out, or the mass will solidify.

Note. — Collodion is conveniently prepared by dissolving soluble gun-
cotton in a mixture of equal parts of 95 per cent, alcohol and sulphuric ether.
Take about 5 grams of clean, white guncotton per 100 c.c. of fluid. It
requires at least twenty-four hours to completely dissolve the guncotton.

Pour the collodion slowly into clean test-tubes and rotate. Try to
moisten the interior of the tube without forming air bubbles. The ex-
cess of collodion should be poured back into the bottle and the tube slowly
rotated in order to keep the interior of the tube covered with a uniform
layer. After pouring off the excess, stand the tube upright, mouth down,
on a sheet of clean paper to drain. Wipe off the excess of collodion from
about the mouth of the tube. Now rotate the tube for five minutes or
more with the mouth in a draft. When dry, remove the sac by cutting
around mouth of tube and filling with water. Allow the collodion sacs to
stand in water until ready to use.

Fill the sacs with the acid sodium silicate and tie the mouth with rubber
bands. When dialyzed, pour the silicate jelly out of the collodion sacs
into a clean beaker and boil for one or two minutes over an open flame.
This should remove the absorbed air.

MAGNESIUM-GYPSUM BLOCKS 105

4. Make the mixture 10 per cent, acid with strong
hydrochloric acid.

5. Tube and sterilize as before, and proceed as with the
undialyzed media.

6. In case the silicic acid does not solidify, the dialyzed
solution may be concentrated.

Medium 31
Washed Agar for the Nitrifying Bacteria

1. Heat ordinary agar with distilled water until in solu-
tion.

2. Pour into Erlenmeyer flasks and allow to solidify.

3. After standing for one or two weeks with several
changes of water, all the soluble organic substances will
have been removed.

4. Add the inorganic substances and sterilize. It is well
to use precipitated calcium carbonate and hydrogen am-
monium sodium phosphate (NH4NaHP04 + 4H20).

Medium 32

Magnesium-gypsum Blocks

1. Add i per cent, of magnesium carbonate (MgC03) to
dried gypsum (CaS04 + H2O) and mix thoroughly.

2. To this mass add water until the mixture has the
consistency of sour cream.

3. Pour upon plate glass and cut into circular blocks for
Petri dishes. It is best to cut with a Petri dish of a size
smaller than the dish for which it is intended. If the
glass plate or Petri dish is previously washed in soapy
water, the gypsum block is readily removed.

4. When hard, remove blocks from glass, place bottom

106 SOIL BACTERIOLOGY

up in dishes, and add enough culture-media to half cover
blocks.

5. Inoculate the surface of blocks and incubate at 25°
to 30° C.

DENITRIFICATION

Medium 33
Solution for Nitrate Reduction

(a) Potassium nitrate (KNO3) i gm.

Asparagin (C^s^Os + H2O) i gm.

Water 250 c.c.

(&) Citric acid (C6H8O7 + H2O) 5.0 gm.

or Neutral sodium citrate 8.5 gm.

Monobasic potassium phosphate (KH2PO4).. . . i.o gm.

Magnesium sulphate (MgSO4 + 7H2O) i.o gm.

Calcium chlorid (CaCl2 + 6 H2O) . 0.2 gm.

Ferric chlorid (FeCl3 + 6H2O) trace

Distilled water 250.0 c.c.

Neutralize the citric acid solution with a 10 per cent,
solution of sodium or potassium hydroxid, using phenol-
phthalein as an indicator. Mix the two solutions, cool to
15° C., and add sufficient water to make i liter. If the
asparagin and potassium nitrate are dissolved along with
the other salts, a decomposition may occur. This is marked
by a browning of the liquid due to the presence of nitrous
acid.
For a solid medium add 15 grams of agar to i liter.

Medium 34
Nitrate Bouillon

Potassium nitrate (KNO3) 5 gm.

Bouillon . 1000 c.c.

SOLUTION FOR NITRATE REDUCTION 107

Medium 35
Starch Nitrate Agar

Agar ....................................... ... 10 gm.

Potassum nitrate (KNO3) ........................ i gm.

Starch (C6HioO5)n .............................. 5 gm.

Bouillon ....................................... 1000 c.c.

After colonies develop, treat one series of plates with a
weak solution of potassium iodid (KI) in dilute HC1. The
treatment should result in the development of a character-
istic blue halo about the colonies that reduce nitrate to
nitrite.

Hoffmann, C., Centbl. Bakt. (etc.), Abt. 2, Bd. 34, p. 386, 1912.

Medium 36
Solution for Nitrate Reduction

Dibasic potassium phosphate (K2HPO4) ......... 0.5 gm.

Potassium nitrate (KNO3) ..................... 10.0 gm.

Ethyl alcohol (C2H5OH) ....................... 5.0 c.c.

Tap-water ................................... IOOG.O c.c.

Beijerinck, M. W., Centbl. Bakt. (etc.), Abt. 2, Bd. 25, p. 35, 1910.

Medium 37
Solution for Nitrate Reduction

Dibasic potassium phosphate (KaHPC^) ......... 0.5 gm.

Potassium nitrate (KNOs) ..................... 2.5 gm.

Filter-paper in strips .......................... 20.0 gm.

Tap-water ................................... 1000.0 c.c.

Iterson, C. V., Centbl. Bakt. (etc.), Abt. 2, Bd. n, p. 689, 1904.

108 SOIL BACTERIOLOGY

Medium 38
Inorganic Solution for Nitrate Reduction

Sodium thiosulphite (Na2S2O3 + 5H2O) 5.0 gm.

Potassium nitrate (KNO3) 5.0 gm.

Sodium bicarbonate (NaHCO3) i.o gm.

Dibasic potassium phosphate (K2HPO4) 0.2 gm.

Magnesium chlorid (MgCl2 + 6H2O) o.i gm.

Calcium chlorid (CaCl2 + 6H2O) trace

Ferric chlorid (FeCl3 + 6H2O) trace

Distilled water , 1000.0 c.c.

Lieske, R., Ber. d. deutsch. bot Gesell., Bd. 30, 1912.

NITROGEN ASSIMILATING ORGANISMS

A. Free Nitrogen-fixing Bacteria (Aerobic)

Medium 39

Mannit Solution

Mannit (C6H8(OH)6) 15.0 gm.

Magnesium sulphate (MgSO4 + yH2O) 0.2 gm.

Monobasic potassium phosphate (KH2PO4) 0.2 gm.

Sodium chlorid (Nad) 0.2 gm.

Calcium sulphate (CaSO4 + 2H2O) o.i gm.

Calcium carbonate (CaCO3) 5.0 gm.

Distilled water. 1000.0 c.c.

Dissolve the phosphate separately in a little water and make
the solution neutral to phenolphthalein with N/i NaOH;
then add to the other ingredients. For a solid medium
add 15 grams of agar to each liter.

Ashby, S. F., Jour. Agr. Sci., vol. 2, p. 38, 1907.

MANNIT SOLUTION IOQ

Medium 40

— __

Dextrose Solution

Dibasic potassium phosphate (K^HPCU) 0.2 gm.

Dextrose (CeHiaOe) 10.0 gm.

Tap-water 1000.0 c.c. *

For a solid medium add 1.5 per cent, of agar.

(Anaerobic)

Medium 41
Solution for Anaerobic Organisms

Dibasic potassium phosphate (K2HPO4) i.o gm.

Magnesium sulphate (MgSO4 + 7H2O) 0.2 gm.

Sodium chlorid (NaCl) o.oi gm.

Ferrous sulphate (FeSO4 + yH^O) o.oi gm.

Manganese sulphate (MnSO4 + 4H2O) o.oi gm.

Dextrose (CeEfoQu) 20.0 gm.

Calcium carbonate (CaCO3) 30.0 gm.

Distilled water 1000.0 c.c.

Sterilize at 15 pounds' pressure for fifteen minutes; or, bet-
ter, steam twenty minutes for three consecutive days.

Winogradsky, S., Centbl. Bakt. (etc.), Abt. 2, Bd. 9, p. 49, 1902.

B. Symbiotic Nitrogen-fixing Bacteria

Medium 42
Mannit Solution

Mannit solution same as No. 39 with an excess of cal-
cium carbonate removed.

/•'I

HO SOIL BACTERIOLOGY

Medium 43
Soil Extract

Soil extract. 100.0 c.c.

Dibasic potassium phosphate (K2HPO4) 0.5 gm.

Mannit (C6H8(OH)6) 10.0 gm.

Distilled water 900.0 c.c.

See note, page 95.

Medium 44

Saccharose Solution

Saccharose (CuHaOu) 10.0 gm.

Monobasic potassium phosphate (KH2PO4) i.o gm.

Magnesium sulphate (MgSO4 + 7H2O) 0.2 gm.

Distilled water 1000.0 c.c.

Medium 45
Maltose Solution

Maltose (CiaHaOii + H2O) 10.0 gm.

Monobasic potassium phosphate (KH2PO4) i.o gm.

Magnesium sulphate (MgSO4 + yH2O) o.i gm.

Sodium chlorid (NaCl) : trace

Ferrous sulphate (FeSO4 + 7H2O) trace

Calcium chlorid (CaCl2) fused trace

Distilled water 1000.0 c.c.

In order to prepare a solid medium, add 1 5 grams of agar to
each liter of the above solutions.

Medium 46
Bean-extract Caffein Agar

Agar 15 gm.

Caffein (C8H10N4O2 + H2O) 2 gm.

Dextrose (C6Hi2O6) 20 gm.

Bean extract. . . 1000 c.c.

SOLUTION FOR CELLULOSE FERMENTATION III

Note. — Bean extract: Add to 100 grams of powdered bean seed in a
mortar 100 c.c. of N/i KOH. Allow this to stand a few minutes, then add
water sufficient to make 5 liters. This should stand twenty-four hours.
Siphon off the clear liquid, neutralize with phosphoric acid (H3PO4 + aq.),
and make the volume up to 5 liters.

Zipfel, H., Centbl. Bakt. (etc.), Abt. 2, Bd. 32, pp. 107-131, 1911.

Medium 47
Peptone Saccharose Solution

Monobasic potassium phosphate (KH2PO4) 2.0 gm.

Magnesium sulphate (MgSO4 + yEkO) o.i gm.

Peptone i.o gm.

Saccharose (Ci2H22On) 20.0 gm.

Distilled water 1000.0 c.c.

Buchanan, R. E., Centbl. Bakt. (etc.), Abt. 2, Bd. 22, p. 392, 1909.

CELLULOSE DESTROYING ORGANISMS

(Anaerobic)

Medium 48
Solution for Cellulose Fermentation

Dibasic potassium phosphate (K2HPO4) i.o gm.

Ammonium sulphate ((NH4)2SO4) i.o gm.

Magnesium sulphate (MgSO4 + 7H2O) 0.5 gm.

Calcium carbonate (CaCO3) 2.0 gm.

Sodium chlorid (NaCl) trace

Distilled water 1000.0 c.c.

Fill large test-tubes about half -full. Add strips of filter-
paper.

Omelianski, W., Centbl. Bakt. (etc.), Abt. 2, Bd. 8, p. 226, 1902.

112 SOIL BACTERIOLOGY

(Aerobic)

Medium 49

Solution for Cellulose Fermentation

Dibasic potassium phosphate (K2HPO4) ......... 0.5 gm.

Ammonium chlorid (NH^Cl) ................... i.o gm.

Calcium carbonate (CaCO3) .................... 10.0 gm.

Tap-water ................................... 1000.0 c.c.

Prepare in shallow layers (loo-c.c. portions in 750-0.0.
Erlenmeyer flasks) and add one sheet of filter-paper about
10 cm. in diameter to each culture.

Iterson, C. V., Centbl. Bakt. (etc.), Abt. 2, Bd. n, p. 693, 1904.

Medium 50
Solution for Cellulose Fermentation

Dibasic potassium phosphate (KaHPC^) ........... i gm.

Magnesium sulphate (MgSC>4 + 7H2O) ............ i gm.

Sodium carbonate (Na2CO3), anhydrous ........... i gm.

Ammonium sulphate ((NH^SC^) ....... . ........ 2 gm.

Calcium carbonate (CaCO3) ...................... 2 gm.

Tap-water ...................................... 1000 c.c.

Fill large test-tubes about half-full of the above medium,
or put i5o-c.c. portions into 300-0.0. Erlenmeyer flasks.
Immerse in the liquid of the test-tubes one or two strips of
filter-paper. In the Erlenmeyer flasks use a single sheet
of filter-paper of such a size that when dropped into the
liquid it will nearly cover the bottom of the flask.

McBeth and Scales, Bui. 266, United States Dept. Agr. Bur. Plant
Indus., p. 26, 1913.

CELLULOSE AGAR 113

Medium 51
Solution for Cellulose-fermenting Molds

Rye bread or bran 10.0 gm.

Dibasic potassium phosphate (K2HPO4) 0.5 gm.

Dextrose (C6Hi2O6) 2.0 gm.

Distilled water • 1000.0 c.c.

The rye bread or bran and water are boiled together for
thirty minutes. The mixture is filtered, the loss in weight
restored, and the phosphate and dextrose added.

Medium 52
Cellulose Agar

Agar (washed) • 15.0 gm.

Cellulose 2.5 gm.

Dibasic potassium phosphate (K2HPO4) 0.2 gm.

Magnesium sulphate (MgSC>4 + 7H2O) 0.2 gm.

Potassium carbonate (K^COs) 0.4 gm.

Calcium chlorid (CaCl2) fused 0.02 gm.

Ferric sulphate (Fe2(SO4)3) 0.02 gm.

Sodium chlorid (NaCl) 0.02 gm.

Peptone 0.50 gm.

Distilled water 1000.0 c.c.

Dissolve the peptone in 40 c.c. of distilled water and filter.
Dissolve the salts in the filtrate and make volume equal to
100 c.c. Add 400 c.c. of the aqueous cellulose suspension
and 3 per cent, of aqueous washed agar. Tube in i5-c.c.
portions.

Note. — Hydra ted cellulose may be prepared as follows: To 100 c.c. of
concentrated sulphuric acid in a 2-liter flask add 60 c.c. of water. When
cooled to 60° C. add 5 grams of moist filter-paper. Shake this mass violently
until the cellulose is dissolved. Now fill the flask with water containing

114 SOIL BACTERIOLOGY

crushed ice. Transfer to a filter and wash until all traces of the acid are
removed. When the volume of the nitrate is reduced to about 200 c.c.,
punch a hole in the filter; wash filtrate into a flask. Make volume up to
800 c.c.

Scales, F. M., Centbl. Bakt. (etc.), Abt. 2, Bd. 44, p. 661, 1915.

Medium 53
Cellulose Agar

(a) Agar 10 gm.

Dibasic potassium phosphate (K2HPO4) i gm.

Magnesium sulphate (MgSC>4 + 7H2O) i gm.

Sodium chlorid (NaCl) i gm.

Ammonium sulphate ((NH4)2SO4) 2 gm.

Calcium carbonate (CaCOs) 2 gm.

Tap-water 500 c.c.

(6) Cellulose solution 500 c.c.

1. Pour 1000 c.c. of ammonium hydroxid, sp. gr. 0.90,
into a glass-stoppered bottle; add 250 c.c. of distilled water
and 75 grams of pure copper carbonate; shake the solution
vigorously until all the copper is dissolved. (About ten
to fifteen minutes are ordinarily required.)

2. To the copper-ammonium solution add 15 grams of
high-grade sheet filter-paper; shake vigorously at intervals
of ten minutes for one-half hour. Examine the solution
carefully to see that the paper is completely dissolved.
If any particles of paper remain in the solution, the shaking
must be continued until the solution is perfectly clear.
Dilute 250 c.c. of the amonium-copper-cellulose solution
to 10 liters with tap-water; add slowly, with frequent
shaking, a weak hydrochloric acid solution prepared by
adding 500 c.c. of concentrated acid to 10 liters of tap-
water. Continue the addition of the acid until the blue

STARCH AGAR I 15

color disappears; add a slight excess of acid, shake thor-
oughly, and allow to stand a few minutes. The finely
precipitated cellulose will rise to the top, due to the large
quantity of free hydrogen liberated in the precipitation
process. Shake the solution vigorously at intervals of a
few minutes to dislodge the hydrogen. As soon as the
free hydrogen has escaped the cellulose will settle rapidly.

3. Wash through repeated changes of water untill free
from copper and chlorin. After the washing is complete,
bring the cellulose in the solution up to 0.5 per cent, by
allowing to settle a few days, and siphoning off the clear
solution or by evaporating. Add the nutrient salts desired,
together with i per cent, of thoroughly washed agar; heat
in autoclave or boil until the agar is dissolved; tube and
sterilize in the usual way.

McBeth, I. G., Soil Science, vol. i, No. 5, pp. 438, 439, 1916.

Medium 54
Starch Agar

(a) Agar 10 gm.

(Salts the same as for Cellulose Agar, Medium 52.)
Tap-water 500 c.c.

(6) Starch solution 500 c.c.

To 10 grams of potato starch suspended in a little cold
water add 800 c.c. of boiling water. Concentrate by
boiling to 500 c.c. This breaks up the starch grains and
should give a nearly transparent starch solution.

Il6 SOIL BACTERIOLOGY

SULPHUR ORGANISMS

Reduction and Oxidation

Medium 55
Solution for Sulphate Reduction

Dibasic potassium phosphate (K2HPO4) 0.5 gm.

Magnesium sulphate (MgSO4 + 7H2O) i.o gm.

Ferrous sulphate (FeSO4 + 7H2O) trace

Asparagin (C4H8N2O3 + H2O) i.o gm.

Sodium lactate (NaCsHsOa) 5.0 gm.

Tap-water 1000.0 c.c.

Van Delden, Centbl. Bakt. (etc.), Abt. 2, Bd. n, p. 88, 1904.

Medium 56
Gelatin for Sulphate Reduction

Gelatin 120.0 to 150.0 gm.

Dibasic potassium phosphate (K2HPO4) 0.5 gm.

Magnesium sulphate (MgSO4 + yH2O) i.o gm.

Iron-ammonium sulphate (FeSO4(NH4)2(SO4) +

6H2O) trace

Asparagin (C4HsN2O3 + H2O) i.o gm.

Sodium lactate (NaCsHaOs) 5.0 gm.

Distilled water- 1000.0 c.c.

Sterilize in the autoclave at 10 pounds' pressure for fifteen
minutes. Cool in ice-water.

Medium 57
Solution for Sulphate Reduction

Dibasic potassium phosphate (K2HPO4) 0.5 gm.

Sodium lactate (NaCaHsOs) 5.0 gm.

Ammonium sulphate ((NH4)2SO4) 2.0 gm.

Ferrous sulphate (FeSO4 + 7H2O) trace

Tap-water 1000.0 c.c.

SOLUTION FOR HYDROGEN SULPHID FORMATION 1 17

Medium 58

Sulphate Reduction

Iron lactate (Fe(C3H5O3)2 + 3H2O) 5.0 gm.

Ammonium sulphate ((NH4)2SO4) 2.0 gm.

Dibasic potassium phosphate (K2HPO4) 0.5 gm.

Water 1000.0 c.c.

To prepare a solid medium add the above ingredients to
15 per cent, gelatin. Heat the medium in a steamer until
the precipitate has settled, and filter. Sterilize at a low
temperature, about 10 pounds' pressure for fifteen minutes,
or in the steamer for twenty minutes for three consecutive
days.

Medium 59

Solution for Hydrogen Sulphid Formation from Protein
and from Sulphur

(a) Iron ammonium-sulphate (FeSO4(NH4)2(SO4) +

6H2O) i gm.

Bouillon. . . . 1000 c.c.

(b) Iron-ammonium sulphate (FeSO4(NH4)2(SO4) +

6H2O) i gm.

Sulphur flowers (S) i gm.

Bouillon. . . . 1000 c.c.

In the presence of the proper organisms, hydrogen sulphid
formation will be noted in solution (b) long after solution
(a) has been reduced.

Beijerinck, W. M., Centbl. Bakt. (etc.), Abt. 2, Bd. i, p. 5, 1895.

Il8 SOIL BACTERIOLOGY

Medium 60
Solution for the Oxidation of Thio sulphates

Sodium thiosulphate (Na2S2O3 + sH2O) 5.0 gm.

Sodium hydrogen carbonate (NaHCO3) i.o gm.

Dibasic potassium phosphate (K2HPO4) 0.2 gm.

Ammonium chlorid (NEUCl) o.i gm.

Magnesium chlorid (MgCl2) + 6H2O) o.i gm.

Tap-water 1000.0 c.c.

Nathansohn, Mitt. a. d. zoolog. Station Neapel, Bd. 15, p. 655, 1902.
Beijerinck, W. M., Centbl. Bakt. (etc.), Abt. 2, Bd. n, pp. 594-597, 1904.

IRON ORGANISMS

Medium 61

Solution for Thread Bacteria

Potassium acetate (KC2H3O2) 0.5 gm.

Ferrous carbonate (FeCOs) . : 0.5 gm.

Tap-water 1000.0 c.c.

Medium 62
Solution for Thread Bacteria

Ferrous ammonium citrate (about 16 per cent. Fe) 0.5 gm.
Tap-water 1000.0 c.c.

Medium 63

Solution for Isolating Chlamydothrix

(a) Agar 10.0 gm.

Manganese peptone (4 per cent. Mn2O3) 0.5 gm.

Tap-water 1000.0 c.c.

(b) Gelatin 100.0 gm.

Manganese peptone (4 per cent. Mn2O3) 0.25 gm.

Peat extract 1000.0 c.c.

Make the reaction slightly alkaline with normal potassium
hydroxid.

SOLUTION FOR YEASTS IIQ

Medium 64

Agar for Isolating Iron-precipitating Bacteria

Ferrous ammonium citrate (about 16 per cent. Fe) 0.5 gm.
Heyden-Nahrstoff agar (see page 93) 1000.0 c.c.

Medium 65

Solution for Testing the Effect of Bacteria on Insoluble
Phosphates

Asparagin (C4H8N2O3 + H2O) / 5.0 gm.

Sodium chlorid (NaCl) i.o gm.

Potassium sulphate (K2SO4) i.o gm.

Ferrous sulphate (FeSO4 + ?H2O) o.oi gm.

Bone-meal 4-° gm.

Distilled water 1000.0 c.c.

The bone-meal should be passed through a fine sieve and
thoroughly washed. In order to secure the largest amount
of soluble phosphoric acid the cultures should be incubated
for sixty to ninety days.

Sackett, Patton and Brown, Bui. 43, Mich. Agr. Exp. Sta., 1908.
YEASTS

Medium 66
Solution for Yeasts

Dibasic potassium phosphate (K2HPO4) 0.75 gm.

Ammonium sulphate ((NH^SC^) 5.0 gm.

Magnesium sulphate (MgSO4 + 7H2O) o.io gm.

Tartaric acid (C4HeO6) i.o gm.

Dextrose (CeH^Oe) 100.0 gm.

Distilled water. . . 1000.0 c.c.

120 SOIL BACTERIOLOGY

Medium 67
Raisin Extract

Raisins 375 gm.

Ammonium chlorid (NH4C1) 2 gm.

Distilled water 1000 c.c.

Allow the raisins to stand in i liter of water for one to two
days. Mash, add the ammonium chlorid, cook in the
steamer for thirty minutes, and filter.

Medium 68
Solution of Yeast-water

Yeast cells 250 gm.

Distilled water 1000 c.c.

Take 250 grams of pressed yeast or 500 c.c. of washed
yeast; steam in i liter of water for one hour. Filter while
warm and steam again for thirty minutes. Make the
reaction neutral to phenolphthalein, filter, and sterilize in
the steamer for three successive days.

Dextrose-yeast-water may be prepared by dissolving
10 per cent, of dextrose in the yeast- water.

FUNGI

Medium 69
Solution for Fungi

Ammonium nitrate (NH4NO3) 10.0 gm.

Dibasic potassium phosphate (K2HPO4) 5.0 gm.

Magnesium sulphate (MgSO4 + 7H2O) 2.5 gm.

Ferrous chlorid (FeCl2 + aq.) trace

Saccharose (CwHaOn) 50.0 gm.

Distilled water. . . . 1000.0 c.c.

POTATO AGAR 121

Medium 70

Agar for Soil Fungi

Agar 15.0 gm.

Dibasic potassium phosphate (K2HPO4) 0.5 gm.

Magnesium sulphate (MgSCU + 7H2O) 0.2 gm.

Dextrose (C6Hi2O6) 10.0 gm.

Soil extract 200.0 c.c.

Water 800.0 c.c.

Jensen, C. N., Bui. 315, Cornell Agr. Exp. Sta., p. 430, 1912.

Medium 71
Solution for Fungi

Dibasic potassium phosphate (K2HPO4) i.o gm.

Magnesium sulphate (MgSO4 + 7H2O) 0.5 gm.

Potassium chlorid (KC1) 0.5 gm.

Ferrous sulphate (FeSC>4 + 7H2O) o.oi gm.

Sodium nitrate (NaNOs) 2.0 gm.

Cane-sugar (Ci2HaOu) 30.0 gm.

Distilled water. . . 1000.0 c.c.

Medium 72
Potato Agar

Potato 200 gm.

Agar 30 gm.

Dextrose (C6Hi2O6) 20 gm.

Distilled water. . . 1000 c.c.

Peel and slice 200 grams of potatoes. Cook in 1000 c.c.
water for one hour in the steamer. Strain or decant the
clear liquid and restore it to original volume. Add 20
grams glucose and 30 grams agar. Heat in steamer until
the agar is dissolved. Filter through cotton filter.

122 SOIL BACTERIOLOGY

Medium 73

Clover Agar

Clover (green) 500.0 gm.

Agar 25.0 gm.

Saccharose (CwHaOu) 2.0 gm.

Potassium nitrate (KNO3) 0.5 gm.

Tap-water 1000.0 c.c.

Extract the clover tissue by heating for one hour in the
steamer; filter and add the other ingredients. Add i to 2

drops of N/io hydrochloric acid to each tube of the medium
just before pouring plates.

ACTINOMYCETES

Medium 74
Solution for Actinomycetes

Dibasic potassium phosphate (K2HPO4) 0.5 gm.

Potassium nitrate (KNO3) 3.0 gm.

Calcium malate (CaC4H4O5)2 + 6H2O) 10.0 gm.

Tap-water 1000.0 c.c.

Krainsky, A., Centbl. Bakt. (etc.), Abt. 2, Bd. 41, pp. 649-688, 1914.

Medium 75

Agar for Actinomycetes

Agar 15.0 gm.

Glycerin 10.0 gm.

Sodium asparaginate (NaC4H6NO4 + H2O) i.o gm.

Glucose (C6Hi2O6) i.o gm.

Ammonium hydrogen phosphate (NH4H2PO4). ... 1.5 gm.

Magnesium sulphate (MgSO4.+ 7H2O) 0.2 gm.

Calcium chlorid (CaCl2) fused o.i gm.

Potassium chlorid (KC1) o.i gm.

Ferric chlorid (FeCl3 + 6H2O) trace

Distilled water , 1000.0 c.c.

Conn, H. J., Jour. Bact., vol. i, p. 198, 1916.

SOLUTION FOR ALG.E 123

Medium 76

Solution for Actinomycetes
Same as No. 71.

ALG.E

Medium 77
Solution for Algae

Ammonium nitrate (NH^NOs) 0.5 gm.

Magnesium sulphate (MgSO4 + 7H2O) 0.2 gm.

Calcium chlorid (CaCl2) fused o.i gm.

Ferrous sulphate (FeSO4 + 7H2O) o.oi gm.

Dibasic potassium phosphate (K2HPO4) 0.2 gm.

Distilled water 1000.0 c.c.

For a solid medium add 2 per cent, of washed agar. Let
the agar stand in a weak solution of alkali for several days,
then wash.

Beijerinck, W. M., Centbl. Bakt. (etc.), Abt. 2, Bd. 4, p. 785, 1898.

Medium 78

Solution for Algae

Calcium nitrate (Ca(NO3)2 + 4H2O) 1.65 gm.

Potassium chlorid (KC1) 0.50 gm.

Magnesium sulphate (MgSO4 + 7H2O) 0.50 gm.

Monobasic potassium phosphate (KH2PO4) 0.50 gm.

Ferric chlorid (FeCl3 + 6H2O) trace

Distilled water 1000.0 c.c.

For a solid medium add 2 per cent, of washed agar.

124 SOIL BACTERIOLOGY

HIGHER PLANTS

Medium 79
Solution for Growing Higher Plants

(a) Calcium nitrate (Ca(N03)2 + 4H2O) .......... 100 gm.

Potassium nitrate (KNOs) ................... 25 gm.

Sodium chlorid (NaCl) ....................... 15 gm.

Distilled water .............................. 1000 c.c.

(6) Monopotassium phosphate (KH2PO4) .......... 25 gm.

Distilled water .................... . ......... 1000 c.c.

Distilled water ............................... 1000 c.c.

(d) Ferric chlorid (FeCls + 6H2O) ................ 5 gm.

Distilled water .......... . .................. 250 c.c.

Take lo-c.c. portions of solutions (a), (£>), and (c) to 1000
c.c. of water. Add i to 2 drops of solution (d).
Tollens, B., Jour. f. Landw., Bd. 30, pp. 537-540, 1882.

Medium 80
Solution for Growing Higher Plants

(a) Ammonium nitrate (NH^NOs) .............. 32.0 gm.

Distilled water ............................ 1000.0 c.c.

(b) Monocalcium phosphate (CaH4(PO4)2) ....... 10.0 gm.

Distilled water ............................. 1000.0 c.c.

Distilled water ............................ 1000.0 c.c.

(d) Magnesium sulphate (MgSO4 + 7H2O) ...... 8.0 gm.

Distilled water ............................ 1000.0 c.c.

(e) Ferric chlorid (FeCl3 + 6H2O) .............. o.i gm.

Distilled water ............................. 250.0 c.c.

SOLUTION FOR HIGHER PLANTS 125

Solutions should be prepared with ammonia-free water
and chemically pure salts.

Dilute lo-c.c. portions of (a), (6), (c), and (d) and i c.c.
of (e) in 1000 c.c. of water. If a nitrogen-free medium is
desired, omit (a). Plant food solutions should be renewed
at regular intervals of about one week each.

Hopkins and Pettit, Soil Fertility Laboratory Manual, p. 22, 1910.

Medium 81
Solution for Higher Plants

Potassium nitrate (KNO3) i.oo gm.

Magnesium sulphate (MgSO4 4- 7H2O) 0.25 gm.

Calcium sulphate (CaSO4 + 2H2O) 0.25 gm.

Tricalcium phosphate (Ca3(PO4)2) 0.25 gm.

Ferrous phosphate (Fe3(PO4)2) 0.25 gm.

Distilled water. . . 1000.00 c.c.

This solution is supposed to prevent the growth of algae.

Crone, G., Stizungsber-Niederrhein. Gesell. Nat. und Heilkunde, Bonn.,
pp. 167-173, 1902.

Medium 82
Soft Agar for Plants

For growing plants 0.75 per cent, of agar in mannit solu-
tion (see page 108) is very satisfactory.

Medium 83
Solution for Higher Plants

(a) Monobasic potassium phosphate (KHaPC^).. , i22.5gm..
Distilled water ............... ....... ....... 1000.0 c.c.

126 SOIL BACTERIOLOGY

(6) Calcium nitrate (Ca(NO3)2) '42.7 gm.

Distilled water 1000.0 c.c.

(d) Ferric phosphate (FePO4 + 4H2O) . . . 2.2 gm.

Distilled water. 1000.0 c.c.

To prepare the complete nutrient solution, take 2o-c.c.
portions of (a), (b), and (c) and dilute to i liter; now add
0.5 c.c. of solution (d).

Shive, J. W., Physiological Researches, Vol. I, No. 7, Johns Hopkins
University, pp. 327-397, 1915.

Medium 84
Solution Similar to Sea-water

Sodium chlorid (Nad) . 26.0 gm.

Magnesium chlorid (MgCl2 + 6H2O) 3.7 gm.

Potassium chlorid (KC1) i.o gm.

Magnesium sulphate (MgSC>4 + 7H2O) 1.7 gm.

Calcium sulphate (CaSO* + 2H2O) i.o gm.

Distilled water 1000.0 c.c.

This solution is recommended for the cultivation of organ-
isms accustomed to sea-water.

Medium 85

Agar for Preserving Plate Cultures

Washed agar 20 gm.

Glycerin (C3H6(OH)3) 500 c.c.

Distilled water 500 c.c.

Dissolve the agar in the water by heating in a steamer, add
the glycerin, and filter through glass wool.

SECTION VIII

PREPARATION OF STAINS

PREPARE saturated alcoholic solutions of methylene-blue,
gentian- violet, and fuchsin.

1. Place the dye in a large test-tube (about one-fourth
full) and fill with 95 per cent, alcohol.

2. Let stand twenty-four hours and shake from time to
time.

(1) Loffler's Methylene-blue:

Saturated alcoholic solution of methylene-blue 30 c.c.

Potassium hydroxid in distilled water .(i : 10,000) ... 70 c.c.

(2) Gentian-violet (Aqueous):

Saturated alcoholic solution of gentian- violet 5 c.c.

Distilled water 95 c.c.

(3) Carbol-fuchsin:

Saturated alcoholic solution of fuchsin 10 c.c.

Aqueous solution of carbolic acid (5 per cent.) 90 c.c.

(4) Gram's lodin Solution:

Metallic iodin i gm.

Potassium iodid 2 gm.

Water 300 c.c.

Dissolve in a few cubic centimeters of water. When in
solution bring volume to 300 c.c.

137

128 SOIL BACTERIOLOGY

(5) Meissner's Solution:

Metallic iodin 7 gm.

Potassium iodid 20 gm.

Water.. . 100 c.c.

(6) Aniline Oil Gentian-violet:

Saturated alcoholic solution of gentian-violet 10 c.c.

Aniline oil in water 30 c.c.

Note. — Prepare aniline water by shaking 2 c.c. of aniline oil with 100 c.c.
of water and filter until clear.

Directions for the Use of Stains

1. Take a clean cover-glass or a slide, flame, and add a
loopful of sterile water.

2. With a platinum needle remove a small quantity of
the bacterial colony. Do not take too much growth on
the needle. Mix this thoroughly with the water and spread
over the cover-glass. If the cover-glass is not clean, the
water will collect in small drops.

3. Allow to dry by passing back and forth high above the
flame, and finally pass rapidly two or three times through
the flame. This should fix the bacteria to the cover-glass
and precipitate albuminous substances.

4. Flood the cover-glass with stain and let stand for
ten to thirty seconds. In order to secure a deeper stain,
warm gently.

5. Wash in water until the mount is clear.

6. For a temporary mount, invert the wet cover-glass
on a slide, blot with filter-paper, and examine under micro-
scope. For a permanent mount, dry the cover-glass above
the flame and mount in Canada balsam or euparal.

DIRECTIONS FOR THE USE OF STAINS I2Q

7. The mount should be labeled as follows: Name of
organism, stain, and date :

& o

1. Gram's Stain:

(a) Spread an even, thin film.

(b) Fix in the flame.

utes.

(d) Wash in water.

(e) Apply Gram's iodin for one minute, or until the

preparation has a coffee color.
(/") Wash in water.
(g) Decolorize with 95 per cent, alcohol until no more

violet color streams out.
(ti) Wash in water.

2. Endospore Stain (Double):

(a) Spread thin film.

(b) Fix by passing through flame three times.

boil).

(d) Clean under side of slide with 2\ per cent, acetic acid.

(e) Decolorize the smear with i\ per cent, acetic acid

until the pink color is nearly removed from film.

130 SOIL BACTERIOLOGY

(/) Wash thoroughly with distilled water.

(g) Dry and blot.

(h) Counterstain with Loffler's methylene-blue for ten

seconds,
(i) Wash in water, mount, and examine.

3. Capsule Stain:

(a) Spread film without the use of water.

(b) Air dry.

(d) Apply glacial acetic acid, drain immediately (clo not

wash in water).

(e) Wash off acid with carbol-fuchsin three times, as

rapidly as possible.
(/") Wash in i per cent, salt solution.
(g) Mount in the salt solution.

4. Flagella Stain (Preparation of Culture):

(a) Transfer cultures twice each day for five days; in

the morning into bouillon, and in the evening on
to fresh slopes of bouillon agar.

(b) Carefully inoculate a tube of sterile city water.

(Slant the tube and make the inoculation at the
base. Then gently raise tube to upright position.)
Incubate for one hour at 37° C.

using the greatest care in each step to prevent the
breaking of the flagella.

(d) Air dry.

(e) Flame once only.

DIRECTIONS FOR THE USE OF STAINS 13!

5. Loffler's Flagella Stain:

(a) Preparation of the Mordant. — Dissolve 2 grams of
desiccated tarmic acid in 15 c.c. of distilled water
(heat gently), and add 5 c.c. of a saturated solution
of ferrous sulphate, i c.c. of an alcoholic solution
of fuchsin (i gram of fuchsin, 25 c.c. warm ab-
solute alcohol), i c.c. of i per cent, sodium hydrate
solution. Filter.

(a) Preparation of Stain. — One part of a saturated alco-
holic solution of fuchsin to 4^ parts of a 5 per cent,
solution of carbolic acid.

0) Filter.

(a) Method of Staining. — Mordant one minute.

(b) Wash in distilled water. (Have water in wide-

mouthed beaker.)

gently).

(d) Wash in water.

(e) Dry thoroughly.

(f) Treat with xylol.

(g) Mount in balsam.

6. Zettnow's Flagella Stain:

(a) Solution (i): Dissolve 2 grams of tartar emetic

(2K(SbO)C4H4O6 + H2O) in 40 c.c. of water.

(b) Solution (2) : Dissolve 10 grams of tannic acid

(Ci4HioO9) in 200 c.c. of water.

tartar emetic solution (i). The turbidity of the
mordant should entirely clear up on heating.

(d) Next dissolve i gram silver sulphate in 250 c.c.

distilled water.

132 SOIL BACTERIOLOGY

(e) Of this solution take 50 c.c., and add to it, drop by
drop, ethylamin (33 per cent, solution) until the
yellowish-brown precipitate which forms at first
is entirely dissolved and the fluid is clear. Only
a few drops are required.

(/") Float the cover-slips in a little mordant contained
in a Petri dish which is heated over a water-bath
for five minutes.

(g) Take the dish off the water-bath, and as soon as the
preparation becomes slightly opalescent, wash
thoroughly in distilled water.

(ti) Then heat a few drops of the ethylamin-silver
sulphate solution upon the mordanted cover
preparation until it just steams and the margin
appears black.

(i) Wash and mount in balsam.

SECTION IX

PREPARATION OF REAGENTS AND QUALITATIVE METHODS
OF ANALYSIS

Pyrogallic Acid for Absorbing Oxygen. — For every 100
c.c. of air space take i gram of pyrogallic acid and 10
c.c. of a 10 per cent, solution of sodium or potassium
hydroxid.

Note. — To Prepare an Anaerobic Jar. — Cover the bottom of an anaerobic
jar with ^-inch layer of pyrogallic acid. Fit the cover tightly to the jar
with vaselin and draw out the air with a suction-pump, and when there is
a good vacuum, run in 75 to 100 c.c. alkali.

(1) Phenolphthalein :

Phenolphthalein 10 gm.

Alcohol (86 per cent.) 1000 c.c.

Dissolve the phenolphthalein in the alcohol and neu-
tralize with sodium hydroxid until faintly pink.

(2) Methyl-orange:

Methyl-orange 0.2 gm.

Distilled water 1000.0 c.c.

Dissolve the solid methyl-orange in hot water, allow to
cool, and if a deposit forms, filter. If the sodium salt is
used instead of the acid, take 0.22 gram to i liter of water.
Add 0.67 c.c. of normal hydrochloric acid, let stand, and
filter.

133

134 SOIL BACTERIOLOGY

(3) Methyl-red:

Methyl-red 2 gm.

Alcohol (95 per cent.) 1000 c.c.

Dissolve the methyl-red in alcohol and filter.

(4) Cochineal:

Cochineal (pulverized) 6 gm.

Alcohol (95 per cent.) 50 c.c.

Distilled water. . . . 200 c.c.

Shake the cochineal in the mixture of water and alcohol.
Allow to stand for two days at room temperature. Filter
until clear. The color of this solution should be a deep
ruby red ; in the presence of alkali a violet color, and in the
presence of acid a yellowish-red color.

(5) Preparation of Standard Solution of Sulphuric Acid. —
A normal solution of sulphuric acid is one-half the molecular
weight of H2SO4 in grams, diluted to i liter with distilled
water. Since the molecular weight of sulphuric acid is
(2+32+64) 98, then 49 grams, one-half of 98, is the amount
necessary for each liter.

1. In order to secure 49 grams of H2SO4, it requires 49
divided by 1.80, or 27.2 c.c. of chemically pure acid. To
be sure that sufficient acid has been used, measure out
about 27.5 c.c. of acid.

2. Place in looo-c.c. graduated flask, make up to 1000
c.c., and mix carefully.

3. From this mixture remove lo-c.c. portions, accurately
measured in a ic-c.c. pipet, and place in weighing bottles
which have been thoroughly cleaned, dried in an oven,
cooled, and weighed.

REAGENTS AND QUALITATIVE METHODS 135

4. One c.c. of chemically pure ammonia is added to each
weighing bottle to neutralize the sulphuric acid.

5. The water and excess of ammonia is then evaporated
in an oven at 100° C. and the ammonium sulphate remains
behind. If the chemicals are pure, 1000 c.c. of the solution
should give 49 grams of sulphuric acid. In 10 c.c. of the
solution there should be 0.49 gram of H2SO4.

H2S04 : (NH4)2SO4 :: 98 : 132
49 : x :: 98 : 132
x = 0.66

If the solution is exactly normal, there should be 0.6600
gram of (NH^SC^ formed from 10 c.c. In case the amount
of (NH4)2SO4 formed is too great, its factor is determined
by dividing 0.6600 into the weight of ammonium sulphate
found. If, for instance, the weight of ammonium sulphate
is 0.6675, the factor of the solution is 1.0113 + . This
means that 10 c.c. of the solution is equivalent to 10.113 c-c-
of normal solution.

(6) Nessler's Reagent for Ammonia:

1. Dissolve 50 grams of potassium iodid in a small
quantity of cold distilled water.

2. Add a saturated solution of mercuric chlorid until a
slight precipitate persists.

3. Now add 400 c.c. of a 50 per cent, solution of potassium
hydroxid made by dissolving the potassium hydroxid and
allowing it to clarify by sedimentation before using.

4. Dilute to 1000 c.c., allow to settle for one week, and
decant. This solution gives the required color with am-
monia within five minutes after addition.

5. Keep the Nessler's solution in a well-stoppered bottle
away from the light.

136 SOIL BACTERIOLOGY

Test for Ammonia. — Add to a drop of Nessler's solution
in a test plate a loopful of the solution to be tested. A
deep golden-yellow color indicates the presence of ammonia.

(7) Trommsdorfs Reagent for Nitrites:

1. Add slowly, with constant stirring, a boiling solution
of 20 grams of zinc chlorid in 100 c.c. of distilled water to
a mixture of 4 grams of starch in water. Continue heating
until the starch is dissolved as much as possible, and the
solution is nearly clear.

2. Then dilute with water and add 2 grams zinc iodid.

3. Dilute to i liter and filter.

4. Store in well-stoppered bottles in the dark.

Test for Nitrites. — Place 3 drops of Trommsdorfs reagent
in depression on test plate. Add i drop of dilute sulphuric
acid (i 13). Remove a loopful of the solution to be tested
and touch to surface of reagent. A blue color indicates the
presence of nitrites.

(8) Sulphanilic Reagent for Nitrites:

(a) Sulphanilic acid 0.5 gm.

Acetic acid (33 per cent.) 15°-° c.c.

(&) Alpha-naphthylamin o.i gm.

Distilled water 20.0 c.c.

Acetic acid (33 per cent.) 150.0 c.c.

Dissolve the alpha-naphthylamin by heating in 20 c.c. of
water, filter, then add the acetic acid. Combine solutions
(a) and (6), and keep in a tightly stoppered bottle. This
solution is sensitive to 2 parts of nitrite in 10,000,000,
giving a reddish-pink color.

Griess, Ber. d. deutsch. chem. Gesell., 12, p. 426, 1870.

REAGENTS AND QUALITATIVE METHODS 137

(9) Diphenylamin Reagent :

1. Dissolve 0.7 gram of diphenylamin in a mixture of
60 c.c. of concentrated sulphuric acid and 28.8 c.c. of
distilled water.

2. Cool this mixture and add slowly 11.3 c.c. of con-
centrated hydrochloric acid (sp. gr. 1.19). After standing
overnight some of the base separates, showing that the
reagent is saturated.

Withers and Ray, Jour. Amer. Chem. Soc., vol. xxxiii, pp. 708-711, 1911.

Test for -Nitrates. — Place i drop of the substance to be
tested in a^epression on the test plate. Add i drop of
diphenylamn^solution and allow the solutions to mix
thoroughly. ^ien add 2 drops of concentrated sulphuric
acid. A deep blue color indicates nitrates. This test
cannot be made in the presence of nitrites, chloric and
selenic acids, ferric chlorid, and many other oxidizing
agents.

In order to detect nitrates in the presence of nitrites,
add a concentrated solution of urea to a small amount of
the liquid in a test-tube. Now add in the bottom of the
tube (by means of a pipet) a dilute solution of sulphuric
acid. This should destroy a greater part of the nitrous
acid.

CO(NH2)2 + 2HNO2 = CO2 + 3H20 + 2N2

(10) Brucin Reagent. — Dissolve i.o gram of brucin in
10 c.c. of 50 per cent, pure concentrated sulphuric acid and
make up to 100 c.c. with distilled water.

Test for Nitrates. — Place i drop of the substance to be
tested in a depression on the test plate and add 3 drops of
concentrated sulphuric acid. Now add i drop of brucin
solution. If nitrates are present, a red color develops

138 SOIL . BACTERIOLOGY

quickly, which changes to orange, then slowly to lemon or
yellow, and finally becomes a greenish yellow.

(n) Phenolsulphonic Acid. — Dissolve 25 grams of pure
white phenol crystals in 150 c.c. of pure concentrated
sulphuric acid, add 75 c.c. of fuming sulphuric acid (13 per
cent. SOs), stir well, and heat for two hours at about
100° C. The reagent prepared in this way should not
contain any mono-acids or any tri-acids. Two c.c. of
this reagent give reliable results with not more than 5
milligrams of nitrate nitrogen.

Chamot, Pratt, and Redfield, Jour. Amer. Chem. Soc., vol. xxxiii, pp.
381-384, 1911.

(12) Fehling' s Reagents :

(a) Copper sulphate (CuSO4 + sH2O) 34-639 gm.

Distilled water 500.0 c.c.

(b) Sodium potassium tartrate (KNaC4H4O6 +

4H2O) 178.0 gm.

Sodium hydroxid 50.0 gm.

Distilled water 500.0 c.c.

Pulverize the crystalline substances before attempting
to dissolve.

Qualitative Test. — Mix equal quantities of (a) and (ft),
about 5 c.c. of each. Add an equal amount of the solution
to be tested and boil. A red precipitate indicates reducing
sugar.

(13) Citric Acid Reagent:

Mercuric sulphate solution 50 gm.

Sulphuric acid (cone.) 200 c.c.

Distilled water 1000 c.c.

Test for Citric Acid. — To a water solution of the citric
acid add 2 c.c. of the mercury reagent and boil. Now add 5

REAGENTS AND QUALITATIVE METHODS 139

to 10 drops of a potassium permanganate solution (2 grams
to 1000 c.c.). In the presence of citric acid the solution
becomes colorless and a white precipitate is formed.

Abderhalden, E., Handbuch der Biochemischen Arbeitsmethoden, Bd. 5,
p. 409, 1913.

Tests for Indol and Skatol :

(A) i. Add a few drops of a 5 per cent, solution of

vanillin in 95 per cent, alcohol to 5 or 6 c.c. of
the indol solution.

2. Make the mixture strongly acid with 3 to 4 c.c.

of concentrated hydrochloric acid. A beautiful
orange color denotes indol.

3. If skatol is present, the same reagents produce a

deep violet color upon heating. These tests
are very sensitive.

4. The color formed with indol is only very slightly

soluble in chloroform, while the color with
skatol is soluble in chloroform.

Nelson, V. E., Jour. Biol. Chem., vol. xxiv, pp. 527-532, 1916.

(B) i. Add i c.c. of a o.oi per cent, solution of potassium

nitrite.

2. Now add a few drops of sulphuric acid and warm

in a water-bath. In the presence of indol a
pink color appears.

3. If a solution containing skatol is treated with a

few drops of nitric acid and a dilute solution of
potassium nitrite, a white turbidity is noted.

(14) Dillon's Reagent:

Mercury metallic 50 gm.

Nitric acid (sp. gr. 1.42) 100 gm.

Dissolve the mercury in its weight of concentrated
nitric acid and dilute with an equal volume of water. Only

140 SOIL BACTERIOLOGY

freshly prepared solution should be used. Proteins when
heated with Millon's reagent turn a brick red.

Biuret Reaction. — Add sodium or potassium hydroxid
to a dilute sulphuric acid solution containing protein until
alkaline, and a few drops of a very dilute solution of copper
sulphate. The presence of protein will be marked by the
gradual spreading of a reddish-violet color through the
solution.

Mercuric Chlorid. — A stock solution is prepared and
diluted to the desired strength.

Stock Solution: Add i part of mercuric chlorid to 2.5
parts of commercial hydrochloric acid (40 per cent. HgCl2
inHCl).

In order to prepare a i : 1000 solution, the concentration
commonly used for disinfecting purposes, take 2.5 c.c. of
the stock solution and dilute to 1000 c.c.

Solution for Sealing Bottles. — Melt together equal parts
of gutta-percha and paraffin.

Seal for Museum Jars. — A transparent, seal for museum
jars may be made by wetting celluloid with acetone. Cut
strips or rings of celluloid a little wider than the ground-
glass surface, dip in acetone, and place upon the edge of
the jar. Cover before the acetone evaporates and press
slightly.

Preserving Plants in Natural Colors. — Saturate with
copper acetate a 50 per cent, glacial acetic acid solution in
water.

Take 4 parts of water to i part of the stock solution.
Boil the plant tissue to be preserved for five to ten minutes,
or until the color becomes yellowish and then green. Wash
in water and preserve in a 4 or 5 per cent, solution of
formalin.

SECTION X

QUANTITATIVE METHODS OF ANALYSIS

(i) Moisture. — Weigh from 5 to 10 grams of soil into a
glass or aluminum dish and dry at 100° C. until there is no
further change in weight. About six to twelve hours are
generally sufficient. Cool in a desiccator and weigh.
Determine all percentages of moisture on the dry basis.

Record data as follows:

Weight of dish and moist soil

Weight of dish and dried soil

Loss .

(A D S — W F S)
Percentage of moisture = — - X 100

A. D. S. = air-dry soil.
W. F. S. = water-free soil.

(2) Ammonia (Distillation) :

Sulphuric acid solution N/I4

Sodium hydroxid solution N/I4

Indicator, cochineal or methyl-red.
Magnesium oxide.

1. Transfer the culture to be analyzed to an 8oo-c.c.
Kjeldahl or a copper flask, using about 200 c.c. of distilled
water.

2. Add 5 grams of magnesium oxid and some shavings of
paraffin to prevent foaming.

3. Connect to a condenser, the lower end of which is in
N/i4 acid.

141

142 SOIL BACTERIOLOGY

4. Erlenmeyer flasks of good quality should be used to
collect the distillate.

5. The apparatus for this work differs from the usual
Kjeldahl stand in that a jet of steam is passed directly
into the distilling flask. It is so arranged that the rubber
stopper for the Kjeldahl flask has two holes, one for the
condensing bulb and one for the steam tube. Steam is
allowed to bubble slowly through the solution in the bottom
of the Kjeldahl flasks. In order to hasten the analysis a
very low flame should be kept under the distilling flask.
The period of distillation will vary with the amount of
ammonia present. As a rule, one hour is long enough to
drive off all ammonia nitrogen.

6. If methyl-red is used as an indicator, the distillate
should be boiled for a few minutes, cooled to 15° or 20° C.,
about 5 drops of methyl-red added, and the solution titrated.

7. The distillate is titrated with standard alkali, and
from the cubic centimeters of standard acid neutralized by
the distillate the weight of nitrogen liberated as ammonia
is calculated.

(3) Ammonia (Nesslerization). — Ammonia-free Water. —
This is readily prepared by adding sodium hydroxid and
potassium permanganate to laboratory water and redistil-
ling. Discard the first portion of the distillate. After
about one-fourth of the water has been evaporated, the
subsequent distillate will be free of ammonia. .

Standard Ammonium Chlorid Solution. — Dissolve 3.82
grams of ammonium chlorid in 1000 c.c. of distilled water;
dilute 10 c.c. of this to 1000 c.c. with ammonia-free water.
One c.c. equals o.oi mg. of nitrogen.

i. Prepare a series of sixteen Nessler's tubes which con-
tain the following number of cubic centimeters of the
standard ammonium chlorid solution, dilute to 50 c.c.

QUANTITATIVE METHODS OF ANALYSIS 143

with ammonia-free water, namely, o.o, o.i, 0.3, 0.5, 0.7,
i.o, 1.4, 1.7, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 6.0.

2. These will contain o.oi mg. of ammonia nitrogen for
each cubic centimeter of the standard solution used.

3. Nesslerize the standards and also the distillates by
adding approximately 2 c.c. of Nessler's reagent to each
tube.

4. Do not stir the contents of the tubes.

5. After Nesslerizing, allow the tubes to stand for ten
minutes.

6. Compare the color produced in these tubes with that
in the standards by looking vertically downward through
them at a white surface placed at an angle in front of a
window, so as to reflect the light upward.

(4) Nitrates (Colorimetric) :

1. Evaporate in a porcelain dish on a water-bath a
convenient quantity of unknown nitrate solution, depending
upon the amount of nitrate present, to dryness.

2. When evaporated, add 2 c.c. of phenoldisulphonic acid
and stir with the rounded end of a glass rod for about ten
minutes so as to loosen the residue.

Note. — Equations for the action of phenoldisulphonic acid on a nitrate:
H2SO4 + 2KNO3 = 2HNO3 + K2SO4
C6H3(OH)(S03H)2 + HN03 = C6H2(OH) (SO3)2(NO2) + H2O

C6H3(OH)(S03H)2(NO2) + 3NH4OH =

C6H2(ONH4)(S02ONH4)2N02 + 3H2O

3. Dilute with water and add ammonia solution (strong
ammonium hydroxid diluted with an equal volume of water)
until alkaline ; a yellow color is formed. This is then diluted
to a known volume and compared with the standard.

For example, take 500 c.c. of water to 100 grams of soil,

144 SOIL BACTERIOLOGY

and in order to clarify add about 2 grams of calcium oxid.
To secure a fair sample, mix by rubbing in a mortar or by
shaking in a wide-mouthed bottle. Filter through folded
filter-paper until clear. Take a convenient volume, for
example, 25 c.c., and determine the nitrate present. This
is equal to 5 grams of soil. Use the colorimeter to compare
the standard solution with the unknown.
Formula for calculating results:

Where X = Number of milligrams of N as NO3 per 100 grams dry soil.
W = Weight of dry soil.
S = Cubic centimeters of water added to W.
A = Aliquot taken for evaporation.

d = Number of cubic centimeters to which A was diluted.
K = Reading on scale of standard solution.
U = Reading on scale of unknown solution.
M = Milligrams of N as NO3 in i c.c. oi standard solution as
diluted for reading.

Standard Nitrate Solution—Dissolve 0.722 gram of pure
dry potassium nitrate in 1000 c.c. of water. Of this strong
solution dilute 10 c.c. to 100 c.c., and from this take 10 c.c.
for a standard. Evaporate to dryness in a porcelain dish
on a water-bath and treat as described above. Make up
volume to 100 c.c. Each cubic centimeter of this standard
is equal to o.ooi milligram of N as nitrate, or 100 c.c. of
this standard is equal to o.i milligram of nitrogen.
• (5) Nitrates (Reduction) :

i. Add to 250 or 500 c.c, of aqueous soil extract in an
8oo-c.c. Kjeldahl flask 5 c.c. of a 50 per cent, sodium
hydroxid solution ; partially close the mouth of the flask with
a small funnel to prevent spattering and boil for half an
hour.

QUANTITATIVE METHODS OF ANALYSIS 145

2. Replace the water driven off in heating.

3. When cool, add 2 grams of finely divided Devarda's
alloy (about 60 mesh) and connect the flask with the
distilling apparatus.

4. The distillation should not be hurried.

5. Allow the solution to boil for thirty to sixty minutes.
(6) Total Nitrogen Without Nitrates:

Sulphuric acid N/I4

Sodium hydroxid N/I4

Sulphuric acid (concentrated).

Potassium or sodium sulphate.

Copper sulphate.

Pumice powder.

Sodium hydroxid (50 per cent.).

1. Place the substance to be analyzed in a Kjeldahl
flask (the amount for analysis will depend on the nitrogen
content) ; if soil is used, about 10 grams.

2. Add 5 grams of powdered potassium sulphate or
sodium sulphate, 0.5 gram copper sulphate, 30 to 40 c.c.
of sulphuric acid, and mix thoroughly. It is important
that the substance be thoroughly moistened by the sulphuric
acid before heating.

3. Place the flask on the digestion shelf under a hood and
heat slowly until frothing ceases. Avoid a very high
flame; do not allow the flame to touch the flask above the
part occupied by the liquid. If sugar is present, for ex-
ample, mannit agar culture, the acid mixture will foam
very badly. In order to prevent any loss it is well to heat
very slowly until all foaming has ceased. Sometimes this
requires one hour or more.

4. Now raise the heat (avoid a very hot flame) until
the acid boils rapidly.

5. Digest for thirty minutes after the acid mixture is

146 SOIL BACTERIOLOGY

colorless. If sugar is absent, about two to three hours is
sufficient for complete digestion.

6. In case the contents of the flask are likely to become
solid before digestion is complete, cool, and add 10 c.c.
more of sulphuric acid.

7. When digestion is complete, cool, and add 200 c.c.
of water. Shake until the mixture is thoroughly in solution.
Be sure that none of the digested material remains caked
to the sides of the Kjeldahl flask.

8. Recool, add a teaspoonful of powdered pumice to
prevent bumping, and shake thoroughly.

9. Add 100 c.c., or more if necessary, of a saturated so-
dium hydroxid solution. (The stock solution of alkali
should be prepared two days or more before it is to be used
in order that the sodium carbonate may precipitate out.
Avoid the deposit in the bottom of the alkali.) Enough
alkali should be added to make the solution react strongly
alkaline. A few strips of litmus-paper may be added in
order to test the reaction. The alkali should be poured
slowly down the sides of the flask. After about half of the
alkali is added, it is well to shake the solution. Now add
the remaining alkali and connect at once to the condenser.

10. See that the rubber stopper fits snugly in the flask.
Now mix the contents thoroughly by shaking.

n. Just before connecting the flask have a very low
flame burning on the distillation shelf. After the alkali
and acid mixture are well mixed, raise the flame.

12. The proper amount of standard acid should be
measured into flasks connected to the distillation shelf
prior to adding the alkali.

13. Distil slowly. After the first fifteen minutes the
flame may be raised, but never so high that the distillate
collects in the condensing bulbs. Generally the first two-

QUANTITATIVE METHODS OF ANALYSIS 147

thirds of the original volume recovered as distillate will
contain all the ammonia.

14. The distillate is now titrated with standard alkali,
and from the cubic centimeters of standard acid neutralized
by the distillate the weight of nitrogen liberated as am-
monia is calculated.

15. This should be reported as percentage of nitrogen
on the dry basis.

(7) Total Nitrogen with Nitrates Present:

Same as for (6); in addition:
Salicylic acid.
Sodium thiosulphate.

1. Add to the substance to be analyzed in a Kjeldahl
flask 35 to 40 c.c. of sulphuric acid with salicylic acid
(i gram in 30 c.c. of sulphuric acid); shake until thoroughly
mixed and allow to stand five or ten minutes, with frequent
shaking.

2. Now add 5 grams of crystallized sodium thiosulphate
and heat the solution gently for five minutes, then bring to
boiling for five minutes; cool; add 0.5 gram of copper
sulphate and boil. This reduces the danger of foaming.

3. Heat very gently until foaming ceases, then heat
strongly until colorless. Continue boiling for thirty
minutes after the substance is colorless. The entire
process requires five to six hours.

4. Cool and add about 200 c.c. of distilled water.

5. Cool again, and add a few pieces of granulated zinc or
pumice powder to keep the contents of the flask from bump-
ing during distillation.

6. Next add 100 c.c. or more of strong soda solution
sufficient to make the reaction strongly alkaline, pouring it
down the sides of the flask so that it does not mix at once
with the acid.

148 SOIL BACTERIOLOGY

7. Connect the flask with the condenser (having prepared
the acid to receive the ammonia). Mix the contents thor-
oughly by shaking, and distil until all the ammonia has
passed over into the standard acid.

(8) Humus:

1. Extract 10 grams of air-dry soil in a Gooch crucible
with i per cent, hydrochloric acid until the nitrate gives
no precipitate with ammonium hydroxid and ammonium
oxalate.

2. Wash until all the acid is removed. In the case of
clay soil, the washing should be done chiefly by decantation
from a cylinder or tall beaker.

3. Wash the contents of the crucible (including the
asbestos filter) into a glass-stoppered cylinder, with 500
c.c. of 4 per cent, ammonium hydroxid. (Mix 300 c.c. of
water with 200 c.c. of ammonia (sp. gr. .90) and add more
water or ammonia until the hydrometer reads .9604, which
is exactly 20 per cent, solution of ammonium hydroxid
(NH4OH). Dilute this to 4 per cent, with water.)

4. Allow to remain, with occasional shaking, for twenty-
four hours. During this time the cylinder is inclined as
much as possible without bringing the contents in contact
with the stopper, thus allowing the soil to settle on the side
of the cylinder and exposing a very large surface to the
action of the ammonium hydroxid.

5. Place the cylinder in a vertical position and leave for
twelve hours to allow the sediment to settle.

6. Draw off 300 c.c. of the supernatant liquid with a
pipet, without stirring up the sediment, place in a stoppered
5oo-c.c. flask, and let stand for forty-eight hours.

7. Carefully pipet off 200 c.c. of the liquid, free of clay
particles, into a 3oo-c.c. beaker, evaporate it on a steam-
bath, and let the residue heat on the bath for two hours.

QUANTITALIVE METHODS OF ANALYSIS 149

8. Dissolve out the humus with 200 c.c. of 4 per cent,
ammonium hydroxid and filter through paper to separate
the flocculated clay.

9. Evaporate 50 c.c. aliquots, dry at 100° C., and weigh.

10. Ignite the residue and reweigh.

11. Calculate the humus from the difference in weights
between the dried and ignited residues.

12. Report as percentage of the dry soil.
(9) Carbon Dioxid :

1. Connect a large Erlenmeyer suction flask of about
2-liter capacity with a long glass tube by means of a 5o-c.c.
pipet bent as shown in Fig. 10.

2. Fill the glass cylinder about two-thirds full of glass
beads and 10 c.c. of normal potassium hydroxid. The
beads prevent the carbon dioxid from passing through the
alkaline solution too rapidly, and afford much greater
surface. The 5o-c.c. pipet prevents suction of the alkali
back into the flask.

3. Place i kilogram of soil in the Erlenmeyer flask.

4. Connect the 5o-c.c. pipet to the flask and close the
ends of the glass tubes by means of clamps.

5. In order to conduct the carbon dioxid into the potas-
sium hydroxid solution draw a current of air slowly through
the apparatus for ten to twenty minutes each day.

6. The current of air should be freed of carbon dioxid by
passing through strong alkali or a series of soda-lime tubes.
The amount of air may be determined by counting the
air bubbles.

7. After the carbon dioxid has been collected in the
potassium hydroxid, disconnect the suction flask and
transfer the contents of the glass tower into a 5oo-c.c.
Erlenmeyer flask by successive washings with small portions
of carbon-dioxid-free water. This is easily accomplished

SOIL BACTERIOLOGY

by inclining the end of the pipet into the flask and pouring
the wash- water on to the beads. Washing should be re-
peated until all traces of alkali have disappeared. Place
a few drops on a watch-glass containing phenolphthalein.
If there is no change in color, the washing is complete.

Fig. 10. — Apparatus for determining carbon dioxid.

Avoid using too large a quantity of water for each washing,
otherwise the volume of liquid to titrate will be very large.

Note. — Carbon-dioxid-free water may be prepared by drawing a current
of carbon-dioxid-free air through distilled water for twenty-four hours or
longer.

QUANTITATIVE METHODS OF ANALYSIS 151

8. Now add about 6 drops of phenolphthalein to the
solution and titrate with N/2 sulphuric acid until the
pink color begins to change. Care should be taken not to
overtitrate. In order to secure the best results titrate with
the tip of the buret in the solution.

9. At this point, when the carbon exists as acid carbonate,
add 5 drops of methyl-orange and titrate with N/io sul-
phuric acid until the color changes.

10. Prepare color standards for each indicator by adding
to carbon-dioxid-free water the same amount of the in-
dicator as used in titrating.

11. The number of cubic centimeters of N/io sulphuric
acid used during the titration with methyl-orange as an
indicator, minus the blank, multiplied by 1.2, represents
the weight in milligrams of carbon produced as carbon
dioxid from i kilogram of soil.

Equation for the double titration of carbon dioxid:

2KOH + CO2 = K2CO3 + H2O

K2CO3 + H2SO4 = KHSO4 + KHCO3

KHCO3 + H2SO4 = KHSO4 + H2CO3 = H2O + C02

(10) Soil Acidity:

Lead acetate paper.

Calcium chlorid solution plus zinc sulphid solution (2 per cent, of
ZnS in 20 per cent. CaCl2 + 2H2O).

1. Place 10 grams of the soil to be analyzed in a 3OO-C.C.
Erlenmeyer flask.

2. Add 50 c.c. of a mixture of 45 c.c. of water and 5 c.c.
of a well-shaken suspension of zinc sulphid in calcium
chlorid solution.

3. In order to remove the zinc sulphid adhering to the
vessel, refill with 50 c.c. of water and add to the flask.

152 SOIL BACTERIOLOGY

4. Place the flask at once over the flame and boil for one
minute after violent bubbling starts.

5. Now place a strip of lead acetate paper moistened with
not more than 3 drops of water over the mouth of the flask.

6. Boil for two minutes, remove, and dry the paper.

7. Compare the paper with standard color chart.

Truog, E., Bui. 249, Wis. Agr. Exp. Sta., 1915.

(n) Reducing Sugars (Defren-O'Sullivan) :

1. Mix 15 c.c. of Fehling's copper solution (see p. 138)
(a) with 15 c.c. of the alkaline tartrate solution (b) in a
300-c.c. Erlenmeyer flask, and add 50 c.c. of distilled
water.

2. Place the flask and its contents in a water-bath
containing boiling water and allow it to remain five minutes.

3. Then run rapidly from a buret into the hot liquor in
the flask 25 c.c. of the sugar solution to be tested, which
should contain not more than | per cent, of reducing sugar.

4. Allow the flask to remain in the boiling water just
fifteen minutes after the addition of the sugar solution,
remove, and, with the aid of a vacuum, filter the contents
rapidly through a porcelain Gooch crucible containing a
layer of prepared asbestos fiber about £ inch thick, the
Gooch, with the asbestos, having been previously ignited,
cooled, and weighed.

5. The cuprous oxid precipitate is washed thoroughly
with boiling distilled water until the water ceases to be
alkaline. The asbestos used should be of the long-fibered
variety and should be especially prepared as follows: Boil
first with nitric acid (sp. gr. 1.05-1.10), washing out with
hot water; then boil with a 25 per cent, solution of sodium
hydroxid; and finally wash out the alkali with hot water.
Keep the asbestos in a wide-mouthed bottle and transfer

QUANTITATIVE METHODS OF ANALYSIS

153

it to the Gooch by shaking it up in the water and pouring
it quickly into the crucible while under suction. The
excess of fine asbestos should be poured off before adding
to crucible.

6. Dry the Gooch with its contents in the oven, and
finally heat to dull redness for fifteen minutes, during which
the red cuprous oxid is converted into the black cupric
oxid. Considerable care must be taken to avoid cracking
the crucible, the heat being increased cautiously, and the
operation conducted preferably in a muffle furnace.

7. After oxidation as above, the crucible is transferred to
a desiccator, cooled, and quickly weighed.

8. From the milligrams of cupric oxid calculate the
milligrams of dextrose according to the following table:

Leech, Food Inspection and Analysis, New York, p. 595, 1913.
Dej "ren's Table for Dextrose, Maltose, and Lactose

Cupric oxid.

Dextrose.

Maltose.

Lactose.

Cupric oxid.

Dextrose.

Maltose.

Lactose.

Mgm.

13.2

21.7

18.8

180

80.4

131.8

114.6

17.6

29.0

25.2

190

84.9

I39.I

I2I.O

22.1

36.2

31-5

2OO

80.5

146.6

127-5

26.5

43-5

37-8

2IO

94.0

I54-I

I34.I

30.9

50.8

44.1

2 2O

98.6

161.5

I4O.6

35-4

58.1

50.5

230

103.1

169.1

147.0

39-9

65.5

56.8

24O

107.7

176.6

153.5

100

444

72.8

63.2

250

112.3

184.1

1 60.0

48.9

80. i

69.5

260

116.9

191.6

166.5

1 20

53-3

87.4

75-9

270

121.4

199.2

173.0

130

57.8

94-8

82.4

280

I26.I

206.8

179.6

140

62.2

IO2.I

88.7

290

130.7

214.3

186.2

150

66.8

109.5

95-2

300

135-3

221.9

192.8

1 60

71-3

Il6.9

101.7

310

139-9

229.6

199-3

170

75-8

124.4

108.2

320

144-5

237-2

205.9

154 SOIL BACTERIOLOGY

(12) Hydrogen Sulphid:

1. Prepare the following reagents : N/ioo iodin and N/ioo
sodium thiosulphate. Standardize the iodin against the
thiosulphate solution. One c.c. of the N/ioo iodin solution
is equivalent to 0.17 milligram of hydrogen sulphid, using
starch as an indicator.

2. Add to a large glass-stoppered bottle 10 c.c, of the
iodin solution and 2 grams of potassium iodid.

H2S + 2! = 2HI + S

3. Take 1000 c.c. of the sample to be tested, pour it into
the large bottle containing the iodin, shake thoroughly, and
allow to stand for some tune.

4. Titrate the excess iodin with N/ioo thiosulphate.

5. In the presence of large amounts of hydrogen sulphid
use tenth-normal iodin instead of one-hundredth normal.

Treadwell, Analytical Chemistry, vol. ii, p. 687, 1912.

SECTION XI
SPECIAL METHODS

FOR a detailed discussion of sterilization see one of the
standard works on bacteriologic technic. Here only special
phases of this subject will be considered.

Seed Sterilization. — Although a great number of methods
employing various agents have been recommended for
removing micro-organisms from seed, only a few of the
more promising ones will be given. Where it is not neces-
sary 'to render the seeds free of bacteria, but merely to
destroy the majority of the flora, alcohol may be used.

Fig. ii. — Apparatus for seed sterilization.

Among the chemicals that have proved satisfactory for
sterilizing seed, mercuric chlorid, hypochlorate of lime, and
silver nitrate are the most commonly used. The effective-
ness of these substances depends on many factors: strength
of solution, time of exposure, temperature, pressure, and
nature of the seed coat.

155

156 SOIL BACTERIOLOGY

Mercuric Chlorid in Vacuum :

1. Set up the apparatus shown in Fig n, using heavy
walled bottles (milk) and heavy steam-proof rubber connec-
tions.

2. Fill flask B with 0.25 per cent, solution of mercuric
chlorid and C with distilled water.

3. After the whole apparatus is connected, the clamps
between bottles B-D and C-D fastened, sterilize in the
autoclave at 10 pounds' pressure for fifteen minutes.

4. Cool to 40° C., connect to a vacuum pump, and place
the seed in flask D. If the seed are small, place a layer of
cheese-cloth over the mouth of bottle before inserting
stopper. The seed should be thoroughly clean before
sterilizing. It is well to wash with 60 to 70 per cent,
alcohol.

5. By means of the vacuum pump draw the mercuric
chlorid from B to D; then close the screw-clamp and
exhaust D for three to five minutes. This should remove
the air particles from around the seed coats and allow
the disinfectant to come in direct contact with the
seed.

6. At the end of this time invert D and withdraw the
mercuric chlorid solution. Now run in a small amount of
sterile water from C, shake vigorously, empty, and repeat
this process three or four times.

7. Remove some of the seed to sterile Petri dishes and
pour over them a layer of bouillon agar.

8. After the agar hardens, invert and place in the in-
cubator at 20° to 25° C. In two or three days the seed
should germinate. If bacteria or molds are present, they
may be readily noted on the agar.

Hutchinson and Miller, Jour. Agr. Sci., vol. iii, p. 185, 1908.

SPECIAL METHODS 157

Calcium Hypochlorite:

1. Add 10 grams of commercial chlorid of lime (titrating
28 per cent, chlorin) to 140 c.c. of water.

2. Allow the mixture to settle for five or ten minutes and
decant the supernatant liquid. This solution should con-
tain about 2 per cent, of chlorin.

3. For seed sterilization the solution may be diluted or
used full strength. The volume of the liquid should be
about five times that of the seed.

4. Place the seed in a sterile test-tube and cover with a
i per cent, chlorin solution (original solution diluted one-
half).

5. The time required for sterilizing varies with the dif-
ferent seed, about six hours for alfalfa, eight hours for
corn, and fifteen hours for wheat.

Wilson, J. K., Amer. Jour. Bot, vol. ii, pp. 420-427, 1915.

Silver Nitrate. — According to Schroeder the Gramineae
are not readily penetrated by silver nitrate, and withstand
treatment with a 5 per cent, solution for twelve to twenty-
four hours. In order to remove the silver nitrate wash
thoroughly in a sodium chlorid solution and allow the seed
to stand in a dilute solution of sodium chlorid for twenty-
four hours.

Schroeder, H., Centbl. Bakt. (etc.), Abt. 2, Bd. 28, pp. 492-505, 1910.

Soil Sterilization. — In order to destroy all forms of
microorganisms in soil a high temperature for a long
period of time is required. It is impossible to sterilize
soil by the methods commonly employed for culture-media.
Unfortunately, the temperature required to kill bacterial
spores in soil brings about other changes, chemical and
physical. In some cases sterilization results in undesirable

158 SOIL BACTERIOLOGY

changes; in others it seems to improve the crop-producing
power of the soil.

For a discussion of this problem see the publications of
Richter, Pickering, Seaver, and others.

Richter, Landw. Vers. Stat, Bd. 47, p. 269, 1896.

Pickering, Jour. Agr. Sci., vol. ii, p. 411, 1908; vol. iii, p. 32, 1908.

Seaver, Mycologia, vol. i, p. 131, 1909.

Seaver and Clark, Biochemical Bui. No. 9, p. 413, 1912.

Lyon and Bizzell, Bui. 275, Cornell Exp. Sta., 1910.

Lathrop, Bui. 89, Bur. of Soils, U. S. Dept. Agr., 1912.

Johnson, J., Science, vol. xliii, pp. 434, 435, 1916.

Method for Sterilizing Soil:

1. Small test-tubes of soil may be sterilized by heating
in the autoclave for two hours on two successive days at
15 pounds' pressure.

2. When it is desirable to sterilize much larger amounts
of soil, the time of heating should be increased.

3. If earthenware jars are used, the cold air in the bottom
is removed very slowly, therefore it is necessary to heat for
several hours.

4. A 4-liter earthenware jar of soil requires at least six
hours or longer at 15 pounds' pressure to kill all forms of
bacteria.

Growing Plants Free of Microorganisms. — The method
to follow in growing plants free of bacteria depends largely
upon two factors: the size of the plant and the time of
growing period. If small plants are used — clover, alfalfa,
etc. — and it is not necessary to grow to maturity, large
test-tubes or glass cylinders may be used.

Kellerman, K. F., Cir. 120, U. S. Dept. Agr. Bur. Plant Ind., 1913.

In order to grow plants for a long period of time in a
medium free from infection a vessel of special design is

SPECIAL METHODS

159

necessary. Although there are a great number of vessels
designed for this purpose, only one will be described. It
is obvious from the start that apparatus of this nature
must be somewhat complicated. A modification of the
Schulze and Schulow methods has been found fairly satis-
factory.

Apparatus:

One large Woulfe's bottle, about 3-liter capacity, with three openings.

One large 2- or 3-liter flask, Erlenmeyer form.

Two U tubes.

One cylinder constructed as shown in Fig. 13.

The apparatus is arranged according to Fig. 12.

Schulow, I., Ber. d. Dent. Bot. Gesell., Bd. 29, p. 504, 1911.
Schulze, C., Landw. Jahrb., Bd. 30, p. 327, 1901.

Fig. 12. — Complete apparatus for growing plants free of bacteria.

All stoppers and connecting tubes should be made of
steam-resistant rubber. The U tube A and the long
hard-glass tube B, enlarged at one end for sterile cotton

l6o SOIL BACTERIOLOGY

and stopper, serve io remove any micro-organisms from the
air. If a liquid medium is used, the aeration tubes A and
B may be omitted. A' and B' are prepared in the same
way as A and B. The tubes A', B', D, and C are used to
carry water over from the flask to Woulfe's bottle.

Prior to filling the Woulfe bottle with soil wrap some
glass-wool around the end of tube B, and cover the bottom
of the bottle with an inch layer of gravel. Now add the
soil and raise the water content to about half -saturation.
The special glass cylinder E which fits loosely around the
middle neck of Woulfe's bottle consists of a large glass
cylinder 4 to 5 cm. in diameter and 15 cm. in length. With-
in this cylinder there is a glass tube about ii to ij cm.
in diameter and 20 cm. long, which reaches to the wire net
(see Fig. 13). The top of the glass tube carries a cotton
plug. Between the two cylinders there is loose cotton
packing and three glass rods about 0.4 cm. in diameter and
15 cm. long (see Fig. 13).

The entire apparatus should be connected as shown in
Fig. 12; the flask filled almost full of distilled water, a
screw-clamp fastened between C and D in order to prevent
the water in the flask from flowing over into the bottle, and
sterilized for two hours at 15 pounds' steam pressure for
two consecutive days. If carefully wrapped in paper,
heated and cooled slowly, there is not much danger of
cracking the glass. When cool, seal all stoppers with a
beeswax-rosin mixture.

In view of the long time required for plant growth and
the danger of infection, it is well to keep the cultures in a
clean room as free from contamination as possible.

To plant, remove the cotton plug from the inner glass
cylinder or tube (see Fig. 13), and by means of sterilized
forceps drop the seed down the inner tube on the wire net.

SPECIAL METHODS

161

As soon as the young shoots are 5 to 10 cm. long, raise the
inner glass tube slightly (Fig. 13, B) and push the cotton
firmly around the base of the young shoot. This should

. ^

A / f B f i C

Fig. 13. — Apparatus for growing seedlings free of bacteria.

be repeated two or three times until there is a cotton
plug of 3 to 5 cm. around the shoot. Now remove the
inner cylinder, excess cotton, and glass rods.

162

SOIL BACTERIOLOGY

Conversion of Degrees of Temperature on One Scale to Another
Degrees C. X 1.8 + 32 = Degrees F.

F.-32

Degrees

1.8

Degrees C.

Comparison of Metric and English Units
Lengths

Millimeters to inches.

Inches to millimeters.

0.03937
0.07874

0.11811
0.15748
0.19685
0.23622
0.27559
0.31496
0-35433

25.4001

50.8001

76.2002

101.6002

127.0003

152.4003

177.8004

203.2004

228.6005

Cubic centimeters to liquid ounces.

Capacities

Liquid ounces to cubic centimeters.

0.03381
0.06763

o.ioi44
0.13526
0.16907

0.20288

0.23670
0.27051

0.30432

1 =

2 =

3 =

4 =

5 =

6 =

7 =

8 =

9 =

29-574

59-147

88.721

118.295

147-869

177.442

207.016

236.590

266.163

Masses

Grams to Avoirdupois ounces.

1 =

2 =

4 =

5 =

6 =

7 =

8 =

9 =

0.03527
0.07055
0.10582
0.14110
0.17637
0.21164
0.24692
0.28219
0.31747

Avoirdupois ounces to grams.

1 =

2 =

3 =

4 =

5 =

6 =

7 =

8 =

9 =

28.3495

56.6991

85.0486

113.3981

141.7476

170.0972

198.4467

226.7962

255-I457

SPECIAL METHODS

163

Avoirdupois pounds to kilograms. Kilograms to Avoirdupois pounds.

0-45359

i =

2.20462

0.90718

2 =

4.40924

1.36078

3 =

6.61387

1.81437

4 =

8.81849

2.26796

5 =

11.02311

2.72155

6 =

13.22773

3.I75I5

7 =

15-43236

3.62874

8 =

17.63698

4.08233

9 =

19.84160

Steam Pressures and Their Corresponding Temperatures

Steam
pressure. . Temperature.

Pounds,
o

I
2

3
4
5
6

7
8

9
10
ii

12
13
14
15
2O

25
30
40

Centigrade (°).
IOO.O
IO2.4
104.1
105.8
107.3

108.8
110.3
111.7
113.0
II4-3
II5-5
116.8
118.4
119.0

I2O.O
121. 2
126.1
130.6
134.6
140.9

164

SOIL BACTERIOLOGY

c> -3- r>- TJ- M N coco qoqioto<NqTttoqwqioq^q q <-oo
c^ to M o ^~ doo j^ co x>. oj M t^. d ^~ c-i oo d-oo ^oo M o ^ ON to o
qoo q toTj-t^-w o <NOO coco IN to o coo M ^oo co to co t^- ex) o o^

.............. '.'. . . . . . '. ,1 } M

CO
If

>•£? co HH ^- ^O CO to cs O^ t^>O "^i" ^d" "^i" O O ^" d CO CN ri" O O O M to O O O
J,» O M c. ON XOOO --

I O .

' 5

co O t~~ <N O M r-~ too

<NGO q\coq q c>^oq q q <N TJ-

w d cs CN o ^ O to

o I-CXD

<N COJ>-rOO

M M<N

q co q\ to H cj

M lOO t^ O\

INDEX

ACIDITY in soil, determination of, 151
Actinomycetes, agar for, 122

solution for, 122
Agar for preserving plates, 126
nutrient, 92

to clear', 93
washed, 105

Alcohol for denitrifying solution, 107
Alfalfa plant with nodules, 70

with and without bacteria, 71
Algae, solution for, 123
Ammonia, determination of, distilla-
tion, 141, 142
Nesslerization, 142, 143
Ammonification by pure cultures, 46
effect of depth on rate, 42
of limestone on rate, 44
of moisture on rate, 43
of potassium phosphate on rate,

of soil type on rate, 41
of blood-meal, 39
of casein, 39
of clover hay, 39
of gelatin, 38
of urea, 35, 36

Ammonifiers, isolation of, 39
Ammonium sulphate, nitrification of,

5i
Anaerobic jar, 133

nitrogen fixation, 66

fixing organisms, isolation of, 66
Aniline oil gentian- violet, 128

Apparatus for determining catalytic
power of soils, 33

for one student, 1 1
Artificial cultures for inoculation of

legumes, 73
Ashby's solution, 108
Asparagin-dextrose agar, 95
Atomic weights, 164
Azotobacter, formation of pigment,

65
influence of various culture-media

on growth, 65
isolation of, 61
nitrogen fixation by pure culture

of, 64

relation to oxygen, 65
stained with methylene-blue, 62

BACILLUS coli, 57
Hartlebii, 57
pyocyaneus, 57

radicicola, effect of, on growth and
nitrogen content of alfalfa, 71,
72

from alfalfa, 68
from clover, 67
gum formation, 73
isolation from different legumes,

nitrogen fixation in solution, 72
radiobacter, 62
subtilis, 46
tumescens, 46

165

1 66

INDEX

Bacteria, counting, 16-20
effect of cultivation on number of,

of depth on number of, 22, 23
of limestone on number of , 27, 28
of manures on number of, 25, 26
of moisture on number of, 24, 25
of partial sterilization on number

of, 28, 29

of plant roots on number of, 29
of season on number of, 30
iron precipitating, 84
nitrogen content of, 74
number, according to dilution

method, 16
to plate method, 18
in artificial cultures for inoculat-
ing legumes, 73
Bean extract, in
Berkefeld filter, 37
Biuret reaction, 140
Blank, 25

Blood-meal, ammonification, 39
Bouillon, 89, 90

nitrate, 106
Brucin reagent, 137

CAFFEIN agar, no

effect on nodules, 72
Calcium carbonate for soil acidity, 27
Capsule stain, 130
Carbol-fuchsin, 127
Carbon bisulphid, effect of, on num-
ber of bacteria, 28

cycle, 75-80

dioxid, determination of, 149-151
formation of, from organic sub-
stances, 78
Casein, agar, 94

ammonification of, 39

nitrification of, 51

solution, 98
Catalase, 32-34

Catalytic power of soils, 32-34
Cellulose, agar, 113

bacteria, isolation of, 77

fermentation by denitrifying bac-
teria, 77

in impure cultures, 75
in soil, 76
solution for, 112

fermenting molds, solution for, 113

hydra ted, 113
Centigrade scale, 162
Chlamydothrix, solution for isolat-
ing, 118

threads, 85
Ciliates, growth, 18
Citric acid reagent, 138
Cleaning glassware, 88
Clostridium pasteurianum, 66
Clover, agar, 122

bacteria, 67

hay, ammonification of, 39
Cochineal 134
Collodion sacs, 104
Colonies, spreading, 20
Control, 25

Counting bacteria, 16-20
Crenothrix, method for growing, 84

threads, 85
Crone's solution, 125
Cultivation, effect of, on number of

bacteria, 31

Culture-media, comparison of num-
ber of bacteria on different, 21

filtration of, 92

neutralization of , 90, 91

preparation of, 89-126

reaction of, 90, 91

titration of, 90, 91

DENITRIFICATION by pure cultures,

57,58

in inorganic solutions, 60
in soil, 59

INDEX

i67

Denitrification with formation of

nitrous oxid, 58
Denitrifying bacteria, fermentation

of cellulose, 77
isolation of, 55
organisms, reduction of stains by,

56
solution, 106

inorganic, 108

Depth, effect of, on number of bac-
teria, 22, 23
Dextrpse and nitrate reduction, 59

solution, 109

Dilution method of counting bac-
teria, 1 6
protozoa, 17
Diphenylamin reagent, 137

EGG-ALBUMEN, 93

English units, 162, 163

Enrichment cultures, Azotobacter, 61

cellulose, 77

denitrifying, 55

nitrifying, 50

FAHRENHEIT scale, 162
Fehling's reagents, 138
Fermentation of cellulose by denit-
rifying bacteria, 77
in impure cultures, 75
in soil, 76

Filter, Berkefeld, 37
for culture-media, 92
paper for denitrifying solution, 107
in fermentation of cellulose, 75
Fixation of nitrogen in soil, 62

in solution, 61
Flagella stain, Loffler's, 130, 131

Zettnow's, 131, 132
Formulae, 88-126
Fuller's scale, 90
Fungi, agar, 121

solution for, 120, 121

GALLIONELLA, 86
Gelatin, 91

ammonification of, 38

for sulphate reduction, 116

soil extract, 95

solution, 98
Gentian- violet, 127
Giltay's solution, 106
Glassware, cleaning, 88
Gram's iodin solution, 127

stain, 129
Gypsum blocks, 105

HANGING-DROP, 61
Hay egg-albumen, 97

infusion, 97
Hay-soil extract, 97
Heyden-Nahrstoff agar, 93
Higher plants, solution for, 124
Hippuric acid, 100
Hopkins and Pettit solution, 124, 125
Humus, determination of, 148

formation of, 79
Hydrogen peroxid, 34

sulphid, determination of, 154
organisms, isolation of, 82
solution for its formation from
protein and sulphur, 117

INCUBATION of plates, 20
Indol and skatol, 139
Iron cycle, 84-86
Iron-precipitating bacteria, 84

agar for isolation of, 119
Isolation of ammonifiers, 39

of Azotobacter, 61

of Bacillus radicicola from different
legumes, 67

of cellulose fermenting bacteria, 77

of chlamydothrix, 118

of clostridiae, 66

of denitrifying bacteria, 55

of hydrogen sulphid organisms, 82

i68

INDEX

Isolation of nitrifying bacteria, 50
of urea fermenting organisms, 36,37

Iterson's solution for cellulose fer-
mentation, 112

LABELING slides, 1 29

Laboratory rules, 14, 15

Lactic acid for casein agar plates, 21

Lactose, effect of, on growth of Azoto-

bacter, 65
Legumes, artificial cultures for the

inoculation of, 73
Lieske's solution, 108
Limestone, effect of, on number of

bacteria, 27, 28
Literature, 12, 13
LofEer's methylene-blue, 127

MALTOSE solution, 1 10

Mannit, effect of, on growth of Azoto-

bacter, 65

for nitrogen fixation, 63
solution, 1 08

Manure, effect of, on number of bac-
teria, 25, 26

Meissner's solution, 128

Mercuric chlorid, 140

Methods, 127-163

Methylene-blue for reduction, 56

Methyl-orange, 133

Methyl-red, 134

Metric units, 162, 163

Microorganisms in soil, 16
relation of, to physical properties
of soil, 87

Millon's reagent, 139

Moisture, determination of, 141
effect of, on number of bacteria,
24, 25

NESSLER'S reagent, 135
Neutralization of culture-media, 90,

Nitrate bouillon, 106
formation, 47-49
reducing bacteria, 55

colonies on starch agar, 55
reduction by pure cultures, 57, 58

solution for, 106
solution, 101
Nitrates, determination of, colori-

metric, 143
reduction, 144
Nitrification of ammonium sulphate,

casein, 51

effect of limestone on, 54
of moisture on, 53
of soil type on, 52
in solution, 47-50
Nitrifying bacteria, isolation of, 50
Nitrite formation, 47

and nitrate solution, 101
solution for, 100
Nitrogen content of bacteria, 74
cycle, 35-74

determination of, total, with ni-
trates, 147, 148
without nitrates, 145-147
fixation, anaerobic (clostridiae) , 66
by Bacillus radicicola, 72
by pure cultures, 64
in soil, 62
in solution, 61
Nodules, formation of, 69
Number of bacteria in artificial

legume cultures, 73, 74
Nutrient broth, 89, 90

OMELIANSKI'S solution for cellulose
fermentation, in

PARTIAL sterilization, effect of, on
number of bacteria, 28, 29

Pasteurized soil, 66

Peptone saccharose solution, 1 1 1
solution, 98

INDEX

169

Phenolphthalein, 133
Phenolsulphonic acid, 138
Phosphate dissolving bacteria, solu-
tion for, 119
Pigment formation with Azotobacter,

Plant food without nitrogen, 72
roots, effect of, on number of bac-
teria, 29

Plants, to grow, free of microorgan-
isms, 158-161

Plate method of counting bacteria,
18

Potato agar, 121

Preserving plants, 140

Protozoa, dilution method of count-
ing, 17

Pure cultures, ammonification by,
46

Pyrogallic acid for absorbing oxygen,
133

RAISIN extract, 120

Reaction of culture-media, 90, 91

Reductase, 56

Reduction of nitrates, 55

to nitrites, 55

to nitrous oxid, 58
of sulphates, 81
Root nodules, 69
Rules, laboratory, 14, 15

SACCHAROSE solution, no

Sampling soil, 16

Seal for bottles, 140
for museum jars, 140

Season, effect of, on number of bac-
teria, 30

Sea- water, 126

Seed sterilization, 155-157

Shive's solution, 125, 126

Silicate jelly, partially dialyzed, 104
undialyzed, 102, 103

Sodium asparaginate agar, 96

silicate, 102
Soil acidity, determination of, 151

apparatus for determining cata-
lytic power of, 33

catalytic power of, 32-34

directions for drawing samples of,
16

microorganisms in, 16

pasteurized, 66

sterilization, 157, 158

suspension of, 38

water, movement of, 87
Soil-extract agar, 94

culture-media, no

for protozoa, 97

gelatin, 95

stock solution, 95
Spore stain, 129
Spirophyllum, 86

method for growing, 84
Spreading colonies, 20
Stains, how to use, 128
Starch agar, 115

nitrate agar, 107
Steam pressure, 163
Sterilization, partial, effect of, on

number of bacteria, 28, 29
Sugars, determination of, 152, 153
Sulphanilic reagent, 136
Sulphate reduction, solution for, 116,

117

Sulphur cycle, 81-83
Sulphuric acid, standard solution,

134, i35

TABLES, 162, 163

Thermophilic bacteria, occurrence of,

3i
Thiosulphates, oxidation of, 83

solution for oxidation of, 118
Thread bacteria, solution for, 118
Titration of culture-media, 90, 91

170

INDEX

Tollen's solution, 1 24
Trommsdorf s reagent, 136
Turmeric paper, 37

UREA, ammonification of, 35, 36
gelatin, 99
solution, 98, 99

Urea-ammonium nitrate agar, 96
Urea-fermenting organisms, crystals

around, 37
isolation of, 36, 37

Urease, preparation of, 37
Uric acid, 100

WASHED agar, 105
Water blanks, 19

Weights, conversion tables, 162, 163
Winogradsky's solution for nitrogen
fixing bacteria, 109

YEASTS, solution for, 119
Yeast-water, 120

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M. D., of Munich. Edited by G. CARL HUBER, M. D., Professor
of Embryology at the Wistar Institute, University of Pennsyl-
vania. Octavo of 528 pages, 377 illustrations. Flexible cloth, $3. 50
net. Second Edition.

This work is conceded to be the most complete text-book on human
histology published. Particularly full on microscopic technic and
staining, it is especially serviceable in the laboratory. Every step in
technic is clearly and precisely detailed. It is a work you can depend
upon always.

New York Medical Journal : " There can be nothing but praise for
this model text-book and laboratory guide."

Military Hygiene and Sanitation. By LIEUT.-COL. FRANK R.
KEEFER, Professor of Military Hygiene, United States Military
Academy, West Point. i2mo of 305 pages, illustrated. Cloth,
$1.50 net.

You get here chapters on the care of troops, recruits and recruiting, per-
sonal hygiene, physical training, preventable diseases, clothing, equip-
ment, water-supply, foods and their preparation, hygiene and sanitation
of posts, barracks, the troopship, marches, camps, and battlefields; dis-
posal of wastes, tropic and arctic service, venereal diseases, alcohol, etc.

Saunders' College Text-Books

KftffiL®

General Bacteriology. By EDWIN O. JORDAN, Ph. D., Professor
of Bacteriology, University of Chicago. Octavo of 650 pages,
illustrated. Just Out— New (sth) Edition.

This work treats fully of the bacteriology of plants, milk and milk
products, dairying, agriculture, water, food preservation; of leather
tanning, vinegar making, tobacco curing; of household administration
and sanitary engineering. A chapter of prime importance to all stu-
dents of botany, horticulture, and agriculture is that on the bacterial
diseases of plants.

Prof. T. J. Burrill, University of Illinois: "I am using Jordan's Bac-
teriology for class work and am convinced that it is the best text in
existence."

Bacteriologic Technic. By J. W. H. EYRE, M. D., Bacteriologist
to Guy's Hospital, London. Octavo of 525 pages, illustrated.
Cloth, $3.00 net. Second Edition.

Dr. Eyre gives clearly the technic for the bacteriologic examination of
water, sewage, air, soil, milk and its products, meats, etc. It is a work
of much value in the laboratory. The illustrations are practical and
serve well to clarify the text. The book has been greatly enlarged.
The London Lancet: " It is a work for all technical students, whether
of brewing, dairying, or agriculture."

Ch«ffiHic&S

Qualitative Chemical Analysis. By A. R. BLISS, JR., Ph. G., M. D.,
Professor of Chemistry and Pharmacy, Birmingham Medical Col-
lege. Octavo of 250 pages. Cloth, $2.00 net.

This work was prepared specially for laboratory workers in the fields
of medicine, dentistry, and pharmacy. It gives you systematic pro-
cedures for the detection and separation of the most common bases and
acids, and in such a manner that, in a short time, you will be enabled
to gain a good practical knowledge of the theory and methods of quali-
tative chemical analysis.

Saunders* College Text-Books

Elements of Nutrition. By GRAHAM LUSK, Ph. D., Professor of
Physiology, Cornell Medical School. Octavo of 402 pages, illus
trated. Cloth, $3.00 net. Second Edition.

The clear and practical presentation of starvation, regulation of tem-
perature, the influence of protein food, the specific dynamic action
of food-stuffs, the influence of fat. and carbohydrate ingestion and of
mechanical work render the work unusually valuable. It will prove
extremely helpful to students of animal dietetics and of metabolism
generally.

Dr. A. P. Brubaker, Jefferson Medical College: " It is undoubtedly the
best presentation of the subject in English. The work is indispensable."

Physiology. By WILLIAM H. HOWELL, M. D., Ph. D., Professor

of Physiology, Johns Hopkins University. Octavo of 1020 pages,

, illustrated. Cloth, $4.00 net. New (6th) Edition.

Dr. Howell's work on human physiology has been aptly termed a
"storehouse of physiologic fact and scientific theory." You will at
once be impressed with the fact that you are in touch with an expe-
rienced teacher and investigator.

Prof. G. H. Caldwell, University of North Dakota: "Of all the text-
books on physiology which I have examined, Howell's is the best."

Hygiene. By D. H. BERGKV, M. D., Assistant Professor of Bac-
teriology, University of Pennsylvania. Octavo of 529 pages, illus-
trated. Cloth, $3.00 net. New (sth) Edition.

Dr. Bergey gives first place to ventilation, water-supply, sewage, indus-
trial and school hygiene, etc. His long experience in teaching this sub-
ject has made him familiar with teaching needs.

J. N. Hurty, M. D., Indiana University: " It is one of the best books
with which I am acquainted."

IO Saunders* College Text-Books

Momrow's

Immediate Care of the Injured. By ALBERT S. MORROW, M. D.,
Adjunct Professor of Surgery, New York Polyclinic. Octavo of
360 pages, 242 illustrations. Cloth, $2.50 net. Second Edition.

Dr. Morrow's book tells you just what to do in any emergency, and it
is illustrated in such a practical way that the idea is caught at once.
There is no book better adapted to first-aid class work.

Health: "Here is a book that should find a place in every workshop
and factory and should be made a text-book in our schools."

American Illustrated Medical Dictionary. By W. A. NEWMAN
BORLAND, M. D., Member of Committee on Nomenclature and
Classification of Diseases, American Medical Association. Octavo
of 1137 pages, with 323 illustrations, 119 in colors. Flexible
leather, $4.50 net; thumb indexed, $5.00 net. New (8th) Edition.

If you want an unabridged medical dictionary, this is the one you
want. It is down to the minute; its definitions are concise, yet accu-
rate and clear; it is extremely easy to consult; it defines all the newest
terms in medicine and the allied subjects; it is profusely illustrated.
John B. Murphy, M. D., Northwestern University: "It is unquestion-
ably the best lexicon on medical topics in the English language, and
with all that, it is so compact for ready reference."

American Pocket Medical Dictionary. Edited by W. A. NEW-
MAN BORLAND, M. B. 693 pages. Flexible leather, $1.00 net;
thumb index, $1.25 net. New (gth) Edition.

A dictionary must be full enough to give the student the information
he seeks, clearly and simply, yet it must not confuse him with detail.
The editor has kept this in mind in compiling this Pocket Dictionary.

I. V. S. Stanislaus, M. D., Medico-Chirurgical College: "We have
been strongly recommending this little book as being the very best."

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