was diluted to one one-thousandth, one ten-thousandth, one
one-hundred-thousandth, and one one-millionth. Each of these dilutions
was used for seeding with five sterilized solutions of ammonium
carbonate, using the method of seeding above described. At the end of
190 days not one of these solutions had nitrified.
Warington supposed that the cause of failure in the method just
mentioned might be due to the alkalinity of the ammonium carbonate.
While this solution could be seeded in the ordinary way with fresh earth
it might be that the faint alkalinity which it presented might prevent
it altogether from action when the nitrifying agent was reduced to a few
organisms.
He therefore changed the solution to one of the following composition:
Water 1,000 parts.
Ammonium chlorid 0.02 part.
Potassium phosphate 0.06 „
Magnesium sulfate 0.03 „
Calcium sulfate 0.03 „
The solution was divided in twenty stoppered bottles which were half
filled. The bottles were divided into four series, A, B, C, D, each one
consisting of five bottles, and these were respectively seeded with one
drop from dilutions to one one-thousandth, one ten-thousandth, one
one-hundred-thousandth, and one one-millionth of a second subculture of
No. 3 in the first dilution series.
After 115 days, nitrification had occurred in ten of the bottles. The
other ten did not nitrify at all. Each of the nitrifying solutions was
spread on gelatin, P and U being employed. Growth took place far more
easily on gelatin U than on gelatin P. Of the ten nitrified solutions
there were three which gave no growth on gelatin U, either when spread
on the surface or introduced into the substance of the jelly. There were
therefore secured nitrifying solutions which did not contain organisms
capable of growing on gelatin. The supposition is therefore fair that
they were pure nitrifying organisms. These fresh, pure organisms had the
faculty of converting ammonia into nitrous acid only and not into nitric
acid.
With the organisms thus prepared a number of solutions of potassium
nitrite containing phosphates and other mineral ingredients were seeded.
In no case was any loss of nitrite found, which is proof that the
solution contained no organisms capable of oxidizing nitrous acid. The
organisms prepared as above, have the power of nitrifying organic
substances containing nitrogenous bodies.
The organism isolated as described and examined under the microscope is
seen to contain two forms. The first one is nearly spherical in shape,
the corpuscles varying in size from mere points to a diameter of one
micromillimeter. The form is very striking and easily stained. The
second form is oval-shaped and attains a length distinctly exceeding one
micromillimeter. Sometimes it is a regular oval and sometimes it is
egg-shaped. This form is stained less easily than the preceding or
spherical form.
=442. Method of Staining.=—The method of staining employed is as
follows:
A drop of the culture-liquid is placed on a glass slide and mixed with
the filtered stain by means of a wire. A cover glass is placed on the
drop and allowed to stand for half an hour. It is then pressed down on
the slide and the liquid which exudes wiped off and hollis glue run
around the cover glass. In this way the organism is stained in its own
culture-fluid and can be seen in its true form without any possibility
of the destruction of its shape by drying. The plate is bright and clear
though colored.
If the preparation is to be mounted in balsam a drop of the culture is
dried in the center of a cover glass. It is then placed for some minutes
in dilute acetic acid to remove matter which would cause turbidity. The
cover glass with its contents is then washed, dried, and stained for
some hours in methyl violet.
=443. Classification of Nitrifying Organisms.=—The names proposed by
Winogradsky for the various organisms are the following:
For the general group of microbes transforming ammonia into nitric acid,
_Nitro-bacteria_.
For the nitrous ferments of the Old World
Genus, _Nitrosomonas_:
Species, _Nitrosomonas europaea_.
_Nitrosomonas javanensis._
For the nitrous microbes of the New World:
Genus, _Nitrosococcus_.
Species, not determined.
For the nitric ferment:
Genus, _Nitrobacter_.
=444. Nitrification in Sterilized Soil.=—The process of nitrification in
sterilized soil, when seeded with pure cultures, is determined as
follows:
_Preparation of Sample._—The fresh sample of arable soil is freed from
pebbles and vegetable débris and reduced to as fine a state of
subdivision as is possible in the fresh state. It is placed in
quantities of about 800 grams in large crystallizing dishes.
One dish is set aside for use in the natural state, and the other,
hermetically closed, is placed in a sterilizing apparatus and subjected
to the action of steam for two and a half hours. This treatment is
repeated three times on as many successive days.
_Seeding of Sample._—Each of the two dishes is moistened with fifty
cubic centimeters of pure water containing 500 milligrams of ammonium
sulfate. The sterilized portion is then seeded with a preparation of the
pure nitrous ferment, produced as before described. The seed is prepared
by filtering a few cubic centimeters of the nitrous culture liquid
through asbestos. The asbestos is well washed and then thrown into a
flask containing a few cubic centimeters of sterilized water and well
shaken. The water carrying the filaments of asbestos is poured drop by
drop on the surface of the soil in as many places as possible. The two
dishes of soil are kept at an even temperature of 20° in a dark place.
Winogradsky found that, treated in this way, the unsterilized soil
produced only nitrates, while the sterilized portions produced only
nitrites.[292]
=445. Variation of the Determinations.=—To vary the conditions of the
experiment Winogradsky uses twelve flasks of the erlenmeyer shape, four
having bottoms twelve centimeters in diameter, and eight of them five
centimeters in diameter. In each of the four large flasks are placed 100
grams of fresh soil, and in each of the eight small flasks twenty-five
grams. The eight small flasks are designated a, b, c, d, and a′, b′, c′,
d′, and the four large flasks A, B, C, D.
The flasks a, b, c, d, and a′, b′, c′, d′, are placed in a stove at 30°
for several days before use, while A, B, C, and D, are kept at 22°–23°
for the same length of time. The soil in the small flasks is, therefore,
somewhat drier than that in the large ones.
The flasks are treated as follows:
a, a′, A, contain the soil as prepared above for control.
b, b′, B, are sterilized at 135° and seeded with a drop of the pure
nitrous culture.
c, c′, C, sterilized as above and seeded with a little of the
unsterilized earth.
d, d′, D, sterilized as above and seeded with pure nitrous and pure
nitric cultures.
After sterilization there was added to the small flasks two cubic
centimeters of a twenty per cent sterilized ammonium sulfate solution,
and to the large ones six cubic centimeters. At the end of a month or
six weeks the contents of the flasks are thrown on a filter and washed
with cold water until a drop of the filtrate gives no blue color with
diphenylamin. The respective quantities of nitrite and nitrate are then
determined in the filtrates by the usual processes, which will be fully
described further along.
=446. Sterilization.=—One of the chief requisites for success in the
bacteriological investigation of soils is found in the thoroughness of
the sterilizing processes. The value of cultures depends chiefly on the
care with which the introduction of foreign germs is prevented. In the
following description a mere outline of the method of sterilization is
presented, while those who wish to study more carefully the details of
the process are referred to the standard works on bacteriology.
=447. Sterilization of the Hands.=—It is important that the hands of the
operator handling apparatus and materials for bacteriological work
should be sterilized. The sterilization may be accomplished in the
following way:
The nails should be cut short and thoroughly cleaned with soap and
brush. The hands are thoroughly washed in hot water with soap. After
washing in hot water the hands should be washed in alcohol and ether.
They are then dipped in the sterilizing solution.
This liquid may consist of a three per cent solution of carbolic acid,
which is the one most commonly employed. A solution of corrosive
sublimate, however, is perhaps the best disinfectant. It should contain
from one to two parts of the crystallized salt to 1,000 parts of water.
It has lately been advised to use the sublimate in an acid solution.
Acetic acid or citric acid may be employed, but hydrochloric acid is
recommended as the best, in a preparation of one-half part per 1,000.
For stronger solutions of sublimate containing more than a half per
cent, equal quantities of common salt should be added. The solution
should be made with sterilized water.
After dipping the hands in the sterilizing solution they should be dried
with a napkin taken directly from a sterilizing oven, where it has been
kept for some time at the temperature of boiling water. Where only
ordinary work in bacteriology is contemplated this sterilization of the
hands is not necessary. It is practiced chiefly in antiseptic surgery.
=448. Sterilizing Apparatus.=—With platinum instruments the most
effective and easiest way for sterilizing is to hold them in the flame
of a bunsen until they are red hot. Steel and copper instruments can not
be treated in this way without injury. They are best sterilized by
submitting them to dry heat in a drying oven at a temperature of
150°–160° for two hours. Glass and porcelain apparatus can be sterilized
best in the same way.
All apparatus and materials employed should be used in a space as free
as possible from dust, so that any germs which might be carried in the
dust can be excluded from the apparatus in transferring it from one
place to another.
=449. Methods of Applying Heat.=—Sterilization by means of heat may take
place in several ways.
_First. Submitting the Materials to Dry Heat Without Pressure._—The
temperature in sterilization of this kind may vary from the temperature
of boiling water at sea-level to 160° obtained by an oil-bath or by an
air-oven.
_Second. Sterilization in a Liquid Under Pressure._—This form of
sterilization may be effected by sealing the liquid in a strong vessel
and submitting it to the required temperature. If the temperature
required be greater than that of boiling water the vessel can be
immersed in a solution of some mineral salt which will raise the
boiling-point.
_Third. Sterilization in Steam Under Pressure._—This method of
sterilization consists in placing the body in a proper receptacle in
vessels to which the steam can have access and then admitting steam from
a boiler at any required pressure. In the case of small apparatus, such
as the autoclave, the steam can be generated in the apparatus itself.
The variety of apparatus used in the above method of sterilization is
very great, but all the forms of apparatus employed depend upon the
principles indicated.
=450. The Sterilizing Oven.=—The apparatus for sterilization by means of
hot, dry air usually consists of a double-walled vessel made of
sheet-iron, usually with a copper bottom. The apparatus is shown in Fig.
71.
The temperature is controlled by means of a thermometer, T, and the
gas-regulator, _R_. This is one of the ordinary gas-regulators by means
of which the amount of gas supplied to the lamp is increased if the
temperature should fall, and diminished if it should rise above the
required degree. The best form of the sterilizing ovens is provided with
a means for circulating the hot air so that the temperature may be made
uniform throughout the mass. This can be accomplished by introducing a
mechanical stirrer, or by the movement of the air itself.
[Illustration:
FIGURE 71. STERILIZING OVEN.
]
Between the walls of the vessel may be placed water, provided the
temperature of sterilization be that of boiling water. If it should
require a higher temperature than boiling water, a solution of salt can
be added until the required temperature is reached, or the space between
the two walls may be left vacant and hot air made to circulate around
the oven.
The exterior of the oven, except at the bottom where the lamp strikes
the copper surface, should be protected by thick layers of asbestos or
other non-conducting material. To avoid danger of flying filaments, this
covering should be coated with some smooth paint which will leave an
even surface not easily abraded.
=451. Sterilization with Steam at High Pressure.=—The apparatus used for
this is commonly called an autoclave and is shown in Fig. 72.
The top is movable and held in place by the clamp, _a_, which is fixed
by the screw worked by the lever, _b_. The vessel itself is
double-jacketed and the pressure is obtained from water in the vessel
heated by means of the lamp, _c_. The actual steam pressure is indicated
by the index _d_. The safety-valve, _e_, allows any excess of steam to
escape above the amount required for the maintenance of the pressure.
This, however, is best regulated by the lamp. The outer jacket permits
the heat from the lamp to circulate around the inner pressure vessel,
and the holes near the top, _oo_, are for the escape of the heated
gases. Enough water is placed in the bottom of the inner pressure vessel
to supply all the aqueous vapor necessary to produce the required
pressure and still leave some water in excess.
[Illustration:
FIGURE 72. AUTOCLAVE STERILIZER.
]
The materials to be sterilized are held on the shelves of the stand and
the vessels may be of various kinds according to the nature of the
material to be sterilized. The vessels containing the material being
covered, the steam does not come in actual contact with it. At the end
of the operation the safety-valve must not be opened to allow the escape
of the steam, otherwise the remaining water would be rapidly converted
into vapor and would be projected over the materials on the shelves. The
lamp should be extinguished and the apparatus allowed to cool. The
autoclave is not only useful for sterilizing purposes but can be made of
general use in the laboratory where heat under pressure, as in the
estimation of starch, etc., is required.
These two forms of apparatus are sufficient to illustrate the general
principles of sterilization by hot air and steam. There are, however,
many variations of these forms designed for special use in certain kinds
of work. For full descriptions of these, reference is made to catalogues
of bacteriological apparatus.
=452. Arnold’s Sterilizing Apparatus.=—A very simple and cheap steam
sterilizer has been devised by Arnold.
[Illustration:
FIGURE 73. ARNOLD’S STERILIZER.
]
Water is poured into the pan or reservoir, B, Fig. 73, whence it passes
through three small apertures into the shallow copper vessel, A. It is
there converted into steam by being heated with any convenient lamp, and
rises through the funnel in the center to the sterilizing chamber. Here
it accumulates under moderate pressure at a temperature of 100°. The
excess of steam escapes about the cover, becomes imprisoned under the
hood, E, and serves to form a steam-jacket between the wall of the
sterilizing chamber and the hood. As the steam is forced down from above
and meets the air it condenses and drips back into the reservoir. Such
an apparatus as this is better suited to commercial purposes, as the
sterilizing of milk, than for scientific uses.
=453. Thermostats for Culture Apparatus.=—It is important in the culture
of micro-organisms that the temperature should be kept constant during
the entire time of growth. Inasmuch as some operations continue for as
much as three months it is necessary to have special forms of apparatus
by means of which a given temperature, during the time specified, can be
maintained. This is secured by means of an oven with an automatic
temperature regulator, practically built on the principle of the hot air
sterilizing oven already described.
The essential principles of construction are, however, that the
regulator for the temperature should be delicate and that the
non-conducting medium surrounding the apparatus should be as perfect as
possible, so that the variations in temperature from changes in the
exterior temperature, are reduced to a minimum. This delicacy is secured
by introducing a drop of chloroform-ether into a confined space over the
mercury of the regulating apparatus. The doors of the chamber are
double, the interior one being of glass so that the exterior door can be
opened for inspection of the progress of the bacterial growth without
materially interfering with the interior temperature. A convenient form
is shown in Fig. 74.
[Illustration:
FIGURE 74. LAUTENSCHLÄGER’S THERMOSTAT.
]
The apparatus figured, is oval in shape, although circular or other
forms are equally as effective. The arrangement of the lamp, _a_,
thermometers, _t t t_, and gas-regulator, _g_, and the double doors,
_d d_, is shown in the engraving and does not require further
description.
The usual temperatures for cultures range from 22° to 35°, and the
apparatus once set at any temperature will remain fixed with extremely
minute variations for an indefinite time. The apparatus possesses a heat
zone which, by the arrangement of the regulator, is kept absolutely
constant. The space between the walls of the apparatus being filled with
water, the temperature is maintained even in every part. The apparatus,
as constructed, is independent not only of the surrounding temperature
within ordinary variations, but also of the pressure of the barometer.
Three thermometers are employed to determine the temperature of the
heating zone, the water space and the inner space. The arrangement of
the gas-regulator is of an especial kind, as mentioned above, by means
of which the consumption of gas is reduced to a minimum. This apparatus
can be regulated to suit the character of the work.
=454. Microscopic Apparatus Required.=—Any good microscope, capable of
accurate observation, of high power, may be used for the bacteriological
observations necessary to soil analysis.
Preference should be given to the patterns adapted to receive any
additional accessories which may be subsequently required for advanced
work. The stage, in addition to being fitted with a sliding bar, should
have a large circular or horseshoe opening to facilitate focusing
operations. A mechanical stage is a desirable acquisition if really well
made, but a plain stage is preferable for many purposes. A rackwork,
centering sub-stage is essential for advanced work, and in the absence
of the more complete form, there should at least be a fitting beneath
the stage to take the diaphragm and condenser. An iris diaphragm will be
found more useful than any other kind in practice, since the size of the
opening can be increased very gradually at will.
One of the best lamps is known as the paraffin lamp and is fitted with a
half-inch wick. This will give even more light than is actually
required, and a steady flame, perfectly under control, may be obtained.
For the minute details to be observed in high-grade microscopic work,
such as is required in the bacteriological examination of soils,
reference must be had to the standard works on bacteriology and
microscopy.
=455. General Conclusions.=—The nitrogenous food of plants is provided
in several ways; _viz._, (1) By the nitrogen brought to soil in rain and
snow. This nitrogen is chiefly in the form of ammonia and nitric acid.
The nitrogen gas in solution in rain water has no significance as a
plant food. (2) By the action of certain anaerobic organisms herding in
the rootlets of leguminous plants, free nitrogen may be oxidized and put
into form for assimilation. (3) By the action of certain organisms on
nitrogenous compounds pre-existing in the soil, ammonia, nitrous acid,
and finally, nitric acid, are produced. It is believed that the plant
organism, unaided by the activity of a micro-organism, is unable to
assimilate nitrogen unless it be fully oxidized to nitric acid. (4)
There exist micro-organisms capable of acting directly on free nitrogen
independent of other plant growth, but the significance of this possible
source of plant food is, at the present time, unknown. (5) The
micro-organisms of importance to agriculture may be isolated and
developed to the exclusion of other organisms of a similar character.
This isolation is best accomplished in culture-media consisting
essentially of a mineral gelatin to which is added only pure
carbohydrates and the necessary mineral nourishment. (6) The nitrifying
ferments consist probably of several species, of different geographic
distribution. Different types of soils probably have nitrifying
organisms of different properties. This is illustrated by the fact that
nitrification is accomplished in dry alkaline soils under conditions in
which the ordinary nitrifying organisms would fail to develop. (7) The
study of typical soils in respect of the kind, activity, and vigor of
their nitrifying organisms has become as important a factor in soil
analysis as the usual determination of physical and chemical
composition.
DETERMINATION OF NITRIC AND NITROUS ACIDS IN SOILS.
=456. Classification of Methods.=—The minute quantities in which highly
oxidized nitrogen exists in soils render the operations of its
quantitative estimation extremely delicate. On the other hand, the easy
solubility of these forms of combination and the absence of absorptive
powers therefor, in the soil, render the separation of them from the
soil a matter of great ease. It is possible, therefore, to secure all
the nitrates and nitrites present in a large quantity of earth in a
solution which can be concentrated under proper precautions to a volume
convenient for manipulation. The method of this extraction is the same
for all the processes of determination. The methods of analysis suited
to soil extracts, as a rule, may also be used in the determination of
the same compounds in rain, drainage, and sewage waters, and for the
qualitative and quantitative control of the progress of nitrification.
The various processes employed may be classified as follows:
1. The conversion of the nitrogen into the gaseous state and the
determination of its volume directly. This is accomplished by combustion
with copper oxid and metallic copper.
2. The conversion of the nitrogen into nitric oxid and the volumetric
determination thereof. The decomposition of a nitrate with ferrous
chlorid in the presence of free hydrochloric acid is an instance of this
type of methods.
3. The oxidation of colored organic solutions and the consequent
disappearance of the characteristic color, or its change into a
different tint. The indigo and indigotin processes are examples of this
method.
4. The production of color, in a colorless or practically colorless
solution, by the treatment thereof with the nitrate in presence of an
acid which decomposes it with the liberation of oxidizing compounds. The
depth of color produced is compared with that formed by a known quantity
of a pure nitrate solution until the two colorations are alike. The
methods depending on the use of carbazol or acid phenol sulfate
illustrate this class of reactions.
5. The conversion of the nitrogen into ammonia by moist combustion with
sulfuric acid in the presence of certain organic compounds, _e. g._,
salicylic acid, and the collection of the ammonia in standard acid, the
excess of which, is determined by titration.
6. The reduction of nitrates to ammonia by nascent hydrogen and the
recovery of the ammonia produced by distillation and collection in
standard acid.
7. The reduction of nitrates by electrolytic action and the collection
of the ammonia as above.