silica recommend dialyzing the soluble glass after treatment with
hydrochloric acid in a parchment tube.[286] The proportions of silicate
and acid are 100 cubic centimeters of the silicate solution (1.06
specific gravity) and 100 cubic centimeters of hydrochloric acid (1.1
specific gravity). With a dialyzing tube placed two days in running
water and one day in distilled water frequently changed it will be found
that the acid is completely removed. One hundred cubic centimeters of
the residual liquor giving no reaction for hydrochloric acid are
concentrated to twenty cubic centimeters. When cold there is added one
cubic centimeter each of a solution of ammonium sulfate and of sodium
carbonate, together with corresponding quantities of the other nutrient
salts commonly employed. The ammonium sulfate should never exceed two to
two and a half, and the sodium carbonate four parts per thousand. To the
flask containing the above substances is added one drop of the
seed-liquor, which may be a soil water or a drop from some previous
culture. The flask is shaken and the mixture poured into a low circular
glass dish which is covered by one slightly larger in diameter (Petri
double dish). To the liquid in the dish is added a drop of a cold
saturated solution of common salt, and it is then stirred with a
platinum spatula. The addition of the salt greatly favors the setting of
the jelly. The jelly may set in from two to three hours, but a longer
time secures better results in the end.
In employing these preparations as seed, after the organisms have grown,
it is absolutely necessary to use the isolated cellules and not the
aggregated masses (zoöglœæ). The latter are rarely free of foreign germs
which adhere to their gelatinous envelope. Since the zoöglœæ can not be
broken up by any artificial means it is necessary to await their
spontaneous disintegration in order to separate the mobile monads. The
opalescence of the culture-liquid is a sure index of this separation.
The particles of mineral gelatin to be used as seed for nitrifying are
best taken as follows:
A glass tube is drawn out immediately preceding the operation, until the
end is as fine as a hair. The surface of the mineral gelatin is
magnified by means of a dissecting microscope magnifying 80 to 100, to
the proper degree and the preparation table is so arranged as to give a
perfect support to the right hand which should hold the filament of
glass. The smallest colony is then pricked with the needle and the end
of the glass is broken and dropped into the flask which is to be seeded.
The seed is thus selected in as small a particle as may be desired, only
a few cells, but it can always be ascertained with certainty that some
of the particles have been obtained by this operation.
The method of cultivation on mineral jelly is considered by Winogradsky
an important resource in the study of the nitrifying organisms. It
removes the chief difficulties heretofore existing in discovering and
characterizing these organisms among the innumerable micro-organisms of
the soil. The long series of cultures necessary to separate the
organisms are rendered nugatory. By directly introducing a little of the
earth into the silicic jelly the active organisms in nitrification can
be at once discovered. It is preferable, however, as indicated below, to
previously produce a nitrification in an aqueous solution by a trace of
earth and to take from it the seed for impregnating the solid medium. In
order to show at once a proof of its nitrifying character, it is only
necessary to take a small bit of the mineral jelly, the size of a grain
of rye, and to throw it into a little sulfuric acid which has been
treated with diphenylamin. There is at once formed a blue spot equal in
intensity to a saturated solution of anilin blue.
In regard to the growths which nitro-organisms make in a medium of the
kind described, they are far from being so marked as are those produced
by ordinary micro-organisms.
A nitro-bacterium is not capable of the energy of growth which is
recognized for the greater number of microbes. The colonies contained in
the gelatin always remain small. The largest among them are just visible
to the naked eye like white points. Along the striae, on the contrary,
there is formed quite a thick white crust. To the naked eye, in general,
there is nothing very characteristic in the formation of colonies in a
medium of this nature. But this impression changes altogether when the
placques are examined with a low magnifying power. The colonies,
especially those of the interior surface, reveal then an aspect so
curious as to be well remembered when once seen.
This mineral gelatin, as has already been noticed, is very unfavorable
to the growth of microbes other than nitro-bacteria and becomes altered
only under the action of the air. If the placques be carefully preserved
from desiccation the culture of these organisms can be continued for
several weeks. Although they do not seem to increase, the colonies, as
well as the jelly, are still in a good condition at the end of that
time. Nevertheless the expectation that this medium would prevent the
formation of any foreign organism has not been realized. Some of the
organisms which accompany the nitro-bacteria in soil, also grow upon the
silicic jelly; but they do not form colonies, properly so-called, and
their growth is extremely slow. They generally make their appearance
before the nitro-bacteria and spread exclusively upon the surface in
form of white spots, so transparent that without careful examination
they would not be discovered. Having reached a certain size the spots do
not change during entire weeks. This circumstance renders the operations
of isolation somewhat delicate, but does not prevent them.
=431. Preparation and Treatment of the Solution to be Nitrified.=—The
organisms having been grown on the siliceous gelatin in the manner
described they are tested for their nitrifying power as follows:
The mineral solution which is to be nitrified with the above preparation
is composed of ammonium sulfate, four-tenths gram; magnesium sulfate,
half a gram; potassium phosphate, one-tenth gram; calcium chlorid,
trace; sodium carbonate, six-tenths to nine-tenths gram; and distilled
water, 100 cubic centimeters. The sulfates with the calcium chlorid on
the one hand, and the phosphate and carbonate on the other, are
dissolved separately and the two solutions sterilized separately and
mixed after cooling. The seeding is then done as described above.
=432. Isolation of the Nitrous and Nitric Organisms.=—Instead of
proceeding immediately to the isolation of special organisms in the
soil, the preliminary period of purification is prolonged by Winogradsky
by allowing the free growth to take place of all the organisms which can
be maintained in the ordinary medium.[287]
The composition of the culture solution employed is as follows:
Distilled water, 1,000 parts; potassium phosphate, one part; magnesium
sulfate, half a part; calcium chlorid, trace. Each flask receives
besides this some magnesium carbonate, freshly washed with boiling water
and added in slight excess.
The flasks thus charged are sterilized, and after sterilization there
are added two cubic centimeters of a solution of two per cent of
ammonium sulfate, which, when added to fifteen or twenty cubic
centimeters of liquid give from two to two and a half parts per
thousand.
They are then seeded with soil. The reasons for this preliminary
treatment are as follows: First, all the observations upon the
enfeeblement of the oxidizing power of these organisms have been made
upon cultures seeded simply by the fresh soil, and in cultures derived
therefrom. In the second place, the existence of the two forms, one
nitrous and the other nitric, prevents at once the isolation of a single
organism.
Samples of soil from Europe, Africa, Asia, Australia, and America, were
used for seed for the experiments. First, the cultures were made by
seeding with a small quantity of each of these samples of soil, and each
one of these cultures served as a point of departure for a series of
subcultures. The temperature of the cultures should be kept constantly
at 30°.
The method of following the nitrification adopted by Winogradsky is
essentially that of Warington, the percentage of ammonia remaining at
any time being determined by nesslerizing. To detect the presence of
nitric acid the nitrous acid is decomposed by boiling with ammonium
chlorid in excess, or with urea, and then diphenylamin is used as a
reagent. By treatment with ammonium chlorid and boiling, the ammonium
nitrite is resolved into free nitrogen and water as indicated by the
equation NH₄NO₂ = N₂ + 2H₂O. Or the total oxidized nitrogen may be
estimated by the Schloesing method or by any of the standard methods
hereafter given. The nitrous acid is then determined by potassium
permanganate and the nitric acid by difference.
A great difference is to be noted between freshly taken earth and that
which has been kept for a long while, especially when sealed. With fresh
earth taken near the surface a mere trace is sufficient to produce
nitrification. With samples of earth which have been kept for a long
while and thoroughly dried, several grams must be added in order to
secure perfect nitrification. The period of incubation with the samples
of earth ranges from three to twenty days. The beginning of the
phenomenon is revealed by the appearance of nitrous acid, of which the
quantity is increased very rapidly, but in the end it disappears and is
transformed into nitric acid.
=433. Statement of the Results.=—The method of stating the results of
examination of soils for nitrifying organisms is illustrated by the
following example:
Soil from Zurich. The culture was seeded on the 11th of October, one
gram of soil being taken. On the 20th of October the nitrous acid had
reached its maximum of intensity and there was no ammonia left. On the
29th of October the nitrous acid remained almost stationary and there
was hardly any nitric acid present. On the 1st of November the reaction
for nitrous acid began to decrease. On the 5th of November the reaction
for nitric acid was very intense. On the 11th of November the nitrous
acid had all disappeared except a mere trace.
The above order of phenomena was observed with all the samples of soil
tried, from which it is concluded with certainty that nitrifying
organisms transplanted directly from their natural medium in the soil
into a liquid easily nitrifiable produce at once nitrous acid in
abundance. The phenomenon of nitrification is divided into two periods
therefore, of which the first is devoted to the production of nitrites,
and the second consists in the oxidation of the nitrites, and this does
not commence until the total disappearance of the ammonia. Occasionally
the formation and oxidation of the nitrites practically go on together,
but never equally, the oxidation of the nitrites being always sensibly
behind their formation.
=434. Method for Subcultures.=—From the mother cultures described above,
Winogradsky makes subcultures as follows:
The solution to be nitrified is prepared as in the mother cultures. The
seeding is accomplished by adding a small quantity of the liquor of the
mother culture after shaking. Subcultures can be made in this way to the
seventh generation.
In respect of the oxidation of the nitrites the results may be entered
as negative if they have not disappeared at the end of two months.
To determine whether the process of oxidizing the nitrites is in
progress or not the total nitrous acid is estimated, and the process
repeated at the end of eight or ten days. Should there be no diminution
of the nitrous acid within this time it may be considered that the
further oxidizing action is not taking place.
=435. Use of a Solid Medium.=—It may be justly claimed that the action
of nitrifying organisms in a liquid is not to be compared with their
action in a solid medium, such as a soil which is their natural habitat.
It might be, therefore, that the inability of the nitrous organism to
produce nitrates is due to the nature of the medium in which it is
cultivated. Winogradsky in order to determine this question cultivated
the organism in a solid medium of two kinds, first a silicate gelatin
impregnated with an ammonium salt and second in sterilized earth. The
silicate jelly is prepared as follows:
Mix a jelly of silica containing some ammonium sulfate with sterilized
soil. The seeding is done with one of the subcultures which no longer
has the power of producing nitrates.
In the case of the jelly the seeding is accomplished as follows:
A minute drop of a culture liquid is taken with a capillary glass tube
and applied in striae to different parts of the solid jelly; or a minute
drop of the culture liquid may be mixed with the jelly before
solidification. The Petri dishes in which these cultures are made can be
preserved in a moist atmosphere and thus the desiccation be easily
prevented for a long time. From time to time small pieces of the jelly
as large as a pea can be taken and tested for the progress of
nitrification.
_Results._—The nitrous reaction, both in the prepared jelly and in
sterilized soil, will appear in a few days. At the end of from seven to
twelve days it will have attained its maximum intensity and will then
remain stationary indefinitely. Sterilized soil has no power to generate
the nitric from the nitrous ferment. The two organisms are, therefore,
of different species.
After a few generations the power of producing nitrates seems to be lost
although the nitrous ferment may still be active. This suppression of
the power to oxidize the nitrites is not due to any pernicious influence
of the culture-medium but to the condition of the successive solutions
at the time of taking the seeding samples.
=436. Microscopic Examination.=—A small particle of the deposit in
the culture-liquid is spread on a glass slide and dried. There is
then added a drop of very dilute perfectly transparent malachite
green solution. Malachite green is Bittermandelölgrün, or
tetramethyldiamidotriphenylcarbinol. Use the zinc chlorid double
salt or oxalate. In about half a minute it is washed and colored by
a very dilute solution of gentian violet which is left to act for
some time. The cells then appear distinctly colored on a colorless
background.
In examining in this way nitrous cultures under a moderate enlargement
there are seen particles of material covered with scattered groups and
massive zoöglœæ composed of cells which are, doubtless, identical.
By their round or roundish forms, by their relative size and especially
by their numbers and uniformity they are at once distinguished from the
other vegetations which are generally of a purely bacillus shape.
With the exception of some shreds of mycelium coming from some oidium in
the soil the microscope reveals nothing but the organisms described. The
microscopic appearance[288] of the nitrous ferment is shown in Fig. 69.
[Illustration:
Figure 69. (Upper figure.) Nitrous ferment prepared by Winogradsky
from soil from Cito.
Figure 70. (Lower figure.) Nitric ferment prepared by Winogradsky from
soil from Cito.
]
The general conclusions of Winogradsky are:
1. Each soil possesses but one organism capable of oxidizing ammonia.
2. Soils from one locality have always the same kind of nitrifying
ferment.
3. Soils from different and distant countries contain nitrifying
organisms which differ from one another in some respects so much so that
it may be necessary to distinguish a few species or even genera in these
bodies.
=437. Isolation of the Nitric Ferment in Soils.=—The principle of the
separation of this ferment as described by Winogradsky rests upon the
fact that in culture solutions of a mineral nature free from ammonia the
nitrous ferment will not grow, whereas if nitrite or nitrous acid be
present the nitric ferment will grow.[289] In a few generations,
therefore, the nitrous ferment will be entirely eliminated.
Solution employed:
Distilled water 1,000 grams.
Potassium phosphate 1 gram.
Magnesium sulfate 0.5 „
Calcium chlorid trace.
Potassium nitrite 0.22 gram.
To culture-flasks containing 100 cubic centimeters of the above mixture
after sterilization about one-tenth gram of fresh soil is added. In
favorable conditions the nitrous acid will disappear in about fifteen
days.
Subcultures are made by seeding fresh portions of the sterilized
solution with one or two cubic centimeters of the mother culture. The
operation is continued until the nitrous ferment is eliminated.
The organisms in the deposit in the culture-flasks are then subjected to
microscopic examination in the manner already described for the nitrous
ferment; or proceed as follows:
=438. Culture on Solid Media.=—Take a liquid which has been employed in
the culture of a nitrous ferment and evaporate to one third of its bulk.
Gelatinize the residue by adding double its volume of the silicic acid
solution prepared as already directed.
The jelly is placed in the glass vessels usually employed. The seeding
may be done with a few drops of a culture-liquid containing the nitric
ferment as obtained above. The first reaction will appear in from eight
to ten days. In about forty-five days the nitrous acid in the jelly will
have entirely disappeared. Two classes of colonies are noticed under the
microscope. The first to appear are small colonies which never extend
beneath the surface of the jelly. In cultures seeded with these colonies
there is no oxidation of nitrous acid. The second class of colonies
extends into the interior of the jelly. They are much larger than the
first, of a yellowish-gray color and not spherical but rather lenticular
in shape. Cultures seeded with these colonies will lose their nitrous
acid in about ten days or two weeks.
The growth of these organisms in a liquid scarcely merit the name of
cultures. The naked eye can usually distinguish no form of vegetation.
The liquid remains clear, the surface is free from any film, no flocks
are deposited. Colored and examined in the microscope the organisms
found are so puny as to make doubtful their oxidizing power. There is an
apparent contradiction between the powerful chemical action that these
organisms can produce and their apparent deficiency in physical
properties.
These organisms are best found by cultivating them in a very limpid
solution. The bottoms of the culture boxes will be found covered with an
extremely tenuous gelatinous deposit communicating to the glass a feeble
grayish-blue tint. The culture bottle is inclined and the bottom
scratched with a recently drawn-out capillary tube. The colonies rise in
the tube together with a little of the liquid. The colonies are dried,
mounted, and colored as already described and when examined with the
microscope are found to be composed exclusively of masses of an organism
of extreme minuteness.
The organism remains attached so firmly to the bottom of the culture
bottle that it can be washed several times with pure water without
danger of detachment and thus rendered more pure.
In old cultures which are sustained by new additions of nitrite an
extremely transparent pellicle on the bottom of the flask can be
distinguished. By shaking the liquid some fragments may be detached and
made to float through the fluid. With a little care and patience these
flocks can be captured, mounted, and colored. Since they show the nitric
organism in its natural state their preparations are of the greatest
interest.
The best preparations are made by coloring with malachite green and
gentian violet and then coloring again hot with magenta. Afterwards the
preparation is washed with warm water at 50°–60° which takes almost the
whole of the color from the gelatinous matter. The cells are then
clearly presented colored a reddish violet on a rose background. These
organisms[290] are shown in figure 70.
The figure shows the cells united by a gelatinous membrane and grouped
in small dense masses composed often of a single layer of organisms. The
cells are generally elongated, rarely regularly spherical or oval. Their
mean length does not exceed half a micromillimeter and their thickness
is from two to three times less.
The difference in form of the nitrous and nitric ferments is very marked
and leaves no doubt of the existence of these two forms which are as
distinct as could be desired in microbic discrimination.
=439. Dilution Method of Warington.=—The method pursued by Warington in
preparing pure cultures of the nitrifying ferment is based on the
well-known principle of dilution which may be expressed as follows:[291]
In a liquid containing bacterial ferments dilution may be practiced
until a drop of the liquid may be taken which will contain no more than
a single organism of any one kind. If now proper solutions be seeded
with single drops of this solution, some of them may give colonies of
pure cultures of any given organism. The solution to be nitrified
employed by Warington had the following composition:
Water 1000 parts.
Ammonium carbonate 0.25 „
Ammonium chlorid 0.50 „
Potassium phosphate 0.04 „
Magnesium sulphate 0.02 „
Calcium sulphate 0.02 „
The ammonium chlorid is added to prevent the precipitation of magnesium
and calcium phosphates. The solution is kept in wide-mouthed, stoppered
bottles to prevent the loss of ammonium carbonate, the bottles being
only half full. About 100 cubic centimeters are taken for each
experiment. These bottles are sterilized and seeded with fresh soil in
the ordinary way. They are then covered with paper caps and placed in a
dark cupboard at a constant temperature of 22°.
_Special Media._—A quantity of arable soil is exhausted of nitrates by
washing with cold water under pressure. The soil is then boiled with
water and filtered. The clear amber-colored solution obtained may be
used instead of water in the above formula.
_Solid Media._—(1) Ordinary ten per cent gelatin made with beef broth
and peptone. (P)
(2) A ten per cent urine solution solidified with six per cent of
gelatin. (U)
(3) A solution of one gram of asparagin, one-half gram sodium acetate,
one-half gram potassium phosphate, two-tenths gram magnesium sulfate,
two-tenths gram calcium sulfate, and one liter of water solidified with
six per cent gelatin. (As)
Other solid media may also be employed for the purpose of favoring, as
much as possible, the growth of the nitrifying organisms.
The first culture in the ammonium carbonate solution given above, is
always made by seeding with a little unmanured arable soil. Subcultures
are seeded from this mother culture by seeding new solutions with a few
drops of the original. In all cases tried by Warington the subcultures
produced only nitrous fermentation while the original cultures produced
the nitric fermentation.
=440. Microscopic Examination.=—The microscopic examination of the
organisms formed is conducted as follows:
The cover glasses for microscopic objects are placed at the bottom of
the culture-flask, the cover glasses being previously sterilized. At the
end of the nitrification the liquid is removed with a pipette and the
flask containing the cover glasses dried at 35°. The cover glasses are
then removed and stained. The microscopic appearance of the organisms
obtained by the previous cultures showed masses of corpuscles usually of
oval shape and having a length generally exceeding one micromillimeter.
An immersion objective giving a magnification of 800 diameters is
suitable for this work.
Other forms of organisms are also met, the whole series being
characterized as follows:
(1) The corpuscles already mentioned. Larger ones are frequently rough
in outline resembling masses of siliceous sea-sand. The smaller oval
corpuscles are regular in form.
(2) Some very small circular organisms often appearing as mere points
and staining much more plainly than the preceding.
(3) A few slender bacilli, staining faintly.
All the cultures obtained by the above method give abundant growth on
gelatin.
=441. Trials with the Dilution Method.=—One part of the third subculture
in the ammonium carbonate solution described above, is mixed with 500
parts of thoroughly boiled water and one drop from a sterilized
capillary tube is added to each of five bottles containing the
sterilized ammonium carbonate solution. In Warington’s experiments one
of the five bottles was found to have nitrified after forty-one days.
After ninety-one days two more were nitrified. Two bottles did not
nitrify at all. All three solutions which nitrified gave growths on
gelatin. The growths took place more speedily on gelatin U and As than
on P.
The organisms obtained on gelatin were seeded in appropriate liquid
media but no nitrification was obtained.