Chapter XXI: Introduction (20)

The method of Warington is the simplest and best to follow.[280] The samples of soil are taken by digging a pit of convenient depth usually from eight to ten feet. A fresh surface is then cut on one of the sides of the pit at the spot selected for sampling. This surface is scraped with a freshly ignited platinum spatula. The spatula should then be washed, re-ignited, and cooled, and a small portion of the soil, at the depth required, detached with the spatula and transferred at once into one of the growth bottles already described.

The growth solution best suited for the purpose contains four cubic centimeters of urine per liter. Each bottle should also contain some freshly precipitated calcium carbonate. In sterilizing urine solutions the calcium carbonate should be added before the heating instead of afterwards. The quantity of soil taken for each seeding should be about one-tenth of a gram.

Inasmuch as the cotton stopper has to be lifted to introduce the soil, opportunity is given for the entrance of any organisms floating in the air. Experience, however, has shown that air free from soil dust very seldom contains nitrifying organisms. The seeded bottles are placed in a dark cupboard of moderate temperature as already described.

=426. Sterilized Urine Solution.=—The sterilized urine solution used for the determination of the distribution of the nitrifying organisms in the soil, is made by taking four cubic centimeters of healthy urine, diluting to one liter, adding some freshly precipitated calcium carbonate, stoppering with cotton wool and heating for several hours at the boiling temperature of water.

As a result of Warington’s experiments it was shown that the nitrifying organism in the soil did not exist, at least in portions of one-tenth of a gram, to a greater depth than eighteen inches. In only one case was nitrification produced from a sample of soil taken at a greater depth and this may have been due to the accidental introduction of organisms from other sources. It may be assumed that any long delay in the commencement of nitrification under favorable conditions, implies the presence of a very limited quantity of organisms in the solution. Thus a comparative study of the period of incubation and the progress of nitrification in solutions seeded with soils taken at different depths or at different places, becomes a fair index of the number and vitality of the nitrifying organisms contained therein.

=427. Depth to Which Micro-Organisms are Found.=—Koch states that at the depth of about one meter, the soil is nearly free from every kind of bacteria.[281] These observations have been corroborated by Pumpelly and Smyth who find that no infection of a bacterial nature is produced in a sterilized solution from samples of clay taken at the depth of nine feet below the surface.[282]

It is evident from the nature of the experiments above described that the nitrifying processes go on almost exclusively in those portions of the soil which are subject to cultivation, while in the subsoil and below the processes of nitrification are either retarded or arrested. Any stores of nitrogenous matter, therefore, in an insoluble state, resting in the subsoil, are preserved from oxidation and consequent waste until such time as they may be removed to near the surface.

=428. Isolation of the Nitrous and Nitric Organisms in the Soil.=—The action of the organisms which produce nitrification either in form of nitrites or nitrates, having been thoroughly established, and the method of testing the soil therefor given, it remains to describe a method by means of which these organisms in the soil may be isolated and obtained in a state of purity. The difficulties attending this process are extremely great on account of the similarity of the two organisms. All earlier attempts to make pure cultures of the two separate organisms were attended with but little success.

According to Winogradsky the method of culture on gelatin so long practiced is not to be relied upon.[283] It is very difficult to eliminate by this process the organisms which grow rapidly in gelatin and which mature their colonies in two or three days, but where they require eight or ten days to produce a colony the method is successful. In fact, by the gelatin process as it was at first practiced, a good deal was owing to chance, but sometimes by a happy accident a pure nitro-bacterium might be isolated.

Formerly it was considered that a liquid could be regarded as sterile if it gave no growth upon gelatin. It has, however, now been demonstrated that a liquid may contain large numbers of nitro-bacteria and still produce no growth upon gelatin. However, for the organisms which accompany the nitro-bacteria in soils, it is regarded as certain that if no growth on gelatin is produced by them they are absent. Therefore in the case of a solution which has been seeded with a soil, if it can be brought to such a state as to produce no growth on gelatin, it may be safely assumed that it contains no bacterial organisms save those which are capable of producing nitrites or nitrates. Therefore if such a solution produce nitrification and at the same time no growth upon gelatin, it may be considered as a proof of the isolation of the nitro-organisms from all others.

This method was also worked out independently by Mr. and Mrs. Frankland.[284]

Winogradsky says further he confesses that he has advanced these views only provisionally and without being convinced of their infallibility. Strictly speaking, the proof of seeding gelatin is not sufficient alone because the absence of growth can not be regarded as the exclusive privilege of the nitro-bacteria. Such might be the case sometimes for an accidental mixture of microbes, introduced with any given sample of soil into the cultures, but the criterion is not absolute. Microbes, for example, of a sulfurous or ferruginous nature may be cited, for which the gelatin layer is not only unfavorable but even fatal. It may thus happen that there may be eliminated from the solution all that will grow upon gelatin without freeing it from some special kinds of cultures, refractory like the nitro-bacteria, but which might reappear if they should be resown in some favorable nutritive solution. On account of this fault in the process, Winogradsky has been impressed with the necessity of bringing out a better method.

In using the gelatin media it is necessary to find the one that is suited to nourish these organisms, which would evidently be the way promising the greatest success. This having been found, and those organisms which produce colonies being easily recognizable, a great step towards the solution of the problem will have been made and the more so as the medium would be at the same time absolutely unfavorable to other forms of microbes. On account of the slow degree of development of the nitro-organisms, all others would probably have opportunity to grow and strengthen to their exclusion, unless these interfering organisms could be completely removed.

=429. The Culture Solution.=—The culture-solution, first proposed by Winogradsky, had the following composition:

To ten grams of gelatin or one part of agar-agar in 100 cubic centimeters of water add potassium phosphate, one-tenth of a gram; magnesium sulfate, five-hundredths of a gram; calcium chlorid, trace; and sodium carbonate, half a gram. The solution being sterilized in the usual way by heating, there are added to it a few cubic centimeters of a sterilized solution containing two-tenths per cent of ammonium sulfate. Such a solution has been proved to be very favorable to nitro-organisms. Nevertheless the experiments with such solutions gave no definite results and they were abandoned.

The non-success of this method led Winogradsky to adopt a nitrifying solution which absolutely excluded all organic substances. Instead of using an animal or vegetable gelatinous substance he used one of a mineral nature, first proposed by Graham and Kühne.[285] Two of these gelatinous mineral substances were considered; _viz._, the aluminum hydroxid and the hydrate of silica. The latter was chosen.

=430. Preparation of the Mineral Gelatinous Solution.=—The soluble glass which is found in commerce is generally of a thick, sirupy consistence. It is first diluted with three times its volume of water. One hundred cubic centimeters of this liquid are poured with constant stirring into fifty cubic centimeters of dilute hydrochloric acid and the mixture placed in a dialyzer. It is useless to employ a standard solution of silica. All that is necessary is to submit to dialysis a liquid with an excess of acid and sufficiently dilute not to be exposed to the danger of being spontaneously gelatinized in the dialyzer. The dialyzer is left for one day in running water and two days in distilled water, often renewed. The solution is then ready for use. This is the case when it is no longer rendered turbid on the addition of silver nitrate, showing that the hydrochloric acid has been entirely extracted. The solution is then to be sterilized by boiling, and preserved in a glass flask closed with a plug of cotton.

More recent instructions by Winogradsky for preparing the gelatinous 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.

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.

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:

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.

FIGURE 71. STERILIZING OVEN.
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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.

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.

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.

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.