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:

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:

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.

A subculture from solution No. 2 of the first dilution mentioned above, 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:

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.

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.

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.

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.

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.

8. The decomposition of nitrates with the quantitative evolution of a different element, and the direct or indirect measurement of the evolved substance. The quantitative evolution of chlorin on treating a nitrate with hydrochloric acid, the collection of the chlorin in potassium iodid, and the determination of the iodin set free, form a process belonging here.

457. Relative Merit of Methods.—The processes mentioned in the classifications embraced under numbers (1) and (5) of the preceding schedule are sufficiently described in the paragraphs devoted thereto, under soil and fertilizers. In practice at the present time it is rare to determine the nitrogen in nitrates by the copper oxid method. The more rapid and equally exact processes of colorimetric comparison or reduction by nascent hydrogen are in all respects to be preferred.

The indigo methods among the colorimetric processes are not so much in use now as those which depend on the development of a color. Lawes and Gilbert considered them far inferior to the Schloesing method. The developed color methods are especially delicate and are to be preferred in all cases where the detection of the merest traces of nitrates is desired. Where nitrates are present in considerable quantities the reduction method with nascent hydrogen is to be preferred over all others. In all these cases the judgment of the analyst must be exercised. The particular method to be employed in any given case can not be determined save by the intelligent discrimination of the operator.

458. The Extraction of Nitric Acid from the Soil.—The easy solubility of nitric acid and of nitrates in water is taken advantage of in the separation of these bodies from the soil. A convenient quantity, usually about 1,000 grams of the fine soil, is taken for the extraction. Instead of freeing the soil entirely from water, it is better to determine the amount of water in the air-dried or prepared sample, and base the calculation on 1,000 grams of the water-free soil.

All samples of soil, when taken for the purpose of examining for nitrates, should be rapidly dried to prevent the process of nitrification from continuing after the sample is taken. For this purpose the soil should be placed in a thin layer in a warm place, 50°–60°, until air-dried. It still contains in this case a little moisture but not enough to permit nitrification to go on.

One thousand grams of soil prepared as above are treated with 2,000 cubic centimeters of distilled water, free of nitric acid, and allowed to stand for forty-eight hours with frequent shaking. One thousand cubic centimeters of the extract are then filtered, corresponding to 500 grams of the dry soil. A small quantity of pure sodium carbonate should be added to the filtrate which is then evaporated on a water-bath to a volume of about 100 cubic centimeters. Should a precipitate be formed during evaporation it should be separated by filtration, the filter washed thoroughly, and the filtrate again evaporated to a volume of 100 cubic centimeters.

In taking a soil for the determination of nitrates, it is well to extend the sampling to a considerable depth. If the sample be taken only to the depth of nine inches, it should be in dry weather when the nitrates are near the surface.

The temperature at which a soil is dried has also an influence on the quality of nitric nitrogen remaining after desiccation.

If a wet soil be dried at 100°, the nitrates present will suffer partial decomposition. This is probably due to deoxidation by organic matter present. On the other hand, ordinary air-drying affords opportunity for continued nitrification, thus increasing the residuum of oxidized nitrogen. The above method is essentially that followed by Warington at Rothamstead.

The method of drying practiced at Rothamstead, in order to secure results as nearly accurate as possible is the following:^[293]

The soil is broken up directly after it is taken from the field, and spread on trays in layers one inch deep. The trays are then placed in a room at 55°. The drying is completed in twenty-four hours. After drying, stones and roots are removed, and the soil is finely powdered and placed in bottles.

For extracting the nitrates, a funnel is prepared by cutting off the bottom from a bottle four and a half inches in diameter. A nicely fitting disk of copper gauze is placed in the bottom of this funnel, and this is covered with two filter papers, the upper one having a slightly greater diameter than the lower. The paper is first moistened, and then from 200 to 500 grams of the dry powdered soil introduced. The funnel is connected with the receiving flask of a filter pump, and pure water poured on the soil until it is thoroughly saturated. The water is then added in small quantities. When the filtrate amounts to 100 cubic centimeters the process may be discontinued, since all the nitrates in the soil will be found in this part of the filtrate.

The extract obtained above is evaporated to convenient bulk for the determination of nitric nitrogen.

THE NITRIC OXID PROCESS.

459. Method of Schloesing.—The processes for estimating nitrogen by combustion with copper oxid and by moist combustion with sulfuric acid have both been used for the determination of the quantity of nitrogen existing in a highly oxidized state. These processes will be fully discussed under the head of fertilizers. In the case of soil extracts, drainage waters, etc., it will be sufficient to discuss, for the present, only those processes adapted especially to a quick and accurate estimation of oxidized nitrogen.

The principle of the method of Schloesing depends on the decomposition of nitrates in the presence of a ferrous salt and a strong mineral acid.^[294] The nitrogen in the process appears as nitric oxid, the volume of which may be directly measured, or it may be converted into nitric acid and titrated by an alkali.

The typical reactions which take place are represented in the following equation:

6FeCl₂ + 2KNO₃ + 8HCl = 3Fe₂Cl₆ + 2KCl + 4H₂O + 2NO.

460. Schloesing’s Modified Method.—The Schloesing method as now practiced by the French chemists is conducted in the apparatus shown in Fig. 75.^[295] The carbon dioxid is generated by the action of the hydrochloric acid in F on the fragments of marble in A. After passing the wash-bottle the gas enters the small tubulated retort, C, which contains the nitrate in solution. For ordinary soils 100 grams are placed in an extraction flask, plugged with cotton, and a layer of the same material is placed over the soil for the purpose of securing an even distribution of the extracting liquid. This liquid is distilled water containing in each liter one gram of calcium chlorid. The purpose of using the calcium chlorid is to prevent the soil from becoming compacted which would render the extraction of the nitrate difficult. The extracting liquid is allowed to fall drop by drop from a mariotte bottle until the filtrate amounts to 500 cubic centimeters. This volume is concentrated on a sand-bath until it is reduced to ten or fifteen cubic centimeters when it is transferred to a flat-bottomed dish and the evaporation finished over steam, care being taken not to allow the temperature to exceed 100°.