_BACTERIA IN THE SOIL_
Surface soils and those rich in organic matter supply a varied field
for the bacteriologist. Indeed, it may be said that the introduction
of the plate method of culture and the improved facilities for growing
anaërobic micro-organisms have opened up possibilities of research into
soil micro-biology unknown to previous generations of workers.
From the nature of bacteria it will be readily understood that their
presence is affected by geological and physical conditions of the soil,
and in all soils only within a few feet of the surface. As we go down
below two feet, bacteria become less, and below a depth of five or six
feet we find only a few anaërobes. At a depth of ten feet, and in the
"ground water region," bacteria are scarce or absent. This is held
to be due to the porosity of the soil acting as a filtering medium.
Regarding the numbers of micro-organisms present in soil, no very
accurate standard can be obtained. Ordinary earth may yield anything
from 10,000 to 5,000,000 per gram, whilst from polluted soil even
100,000,000 per gram have been estimated. These figures are obviously
only approximate, nor is an exact standard of any great value.
Nevertheless, Fränkel, Beumer, Miquel, and Maggiora have, as the result
of experiments, arrived at a number of conclusions respecting bacteria
in soil which are of much more practical use. From these results it
appears that, in addition to the "ground water region" being free, or
nearly so, virgin soils contain much fewer than cultivated lands, and
these latter, again, fewer than made soils and inhabited localities. In
cultivated lands the number of organisms augments with the activity of
cultivation and the strength of the fertilisers used. In all soils the
maximum occurs in July and August.
But the condition which more than all others controls the quantity and
quality of the contained bacteria is the degree and quality of the
organic matter in the soil. The quantity of organic matter present in
soil having a direct effect upon bacteria will be materially increased
by placing in soil the bodies of men and animals after death. Dr.
Buchanan Young two or three years ago performed some experiments to
discover to what degree the soil bacteria were affected by these means.
"The number of micro-organisms present in soil which has been used for
burial purposes," he concludes, "exceeds that present in undisturbed
soil at similar level, and this excess, though apparent at all depths,
is most marked in the lower reaches of the soil."[35] The numbers were
as follows:--
Virgin soil, 4 ft. 6 in. = 53,436 m.o. per gram of soil.
Burial soil (8 years), 4 ft. 6 in. = 363,411 m.o. per gram of soil.
" (3 " ), 6 ft. 6 in. = 722,751 " "
_Methods of Examination of Soil._ Two simple methods are generally
adopted. The first is to obtain a qualitative estimation of the
organisms contained in the soil. It consists simply in adding to
test-tubes of liquefied gelatine or broth a small quantity of the
sample, finely broken up with a sterile rod. The test-tubes are now
incubated at 37° C. and 22° C., and the growth of the contained
bacteria observed in the test-tube, or after a plate culture has been
made. The second plan is adopted in order to secure more accurate
quantitative results. One gram or half-gram of the sample is weighed
on the balance, and then added to 1000 cc. of distilled sterilised
water in a sterilised flask, in which it is thoroughly mixed and
washed. From either of these two different sources it is now possible
to make sub-cultures and plate cultures. The procedure is, of course,
that described under the examination of water (p. 41 _et seq._), and
Petri's dishes, Koch's plates, or Esmarch's roll cultures are used.
Many of the commoner bacteria in soil will thus be detected and
cultivated. But it is obvious that this by no means covers the required
ground. It will be necessary for us here to consider the methods
generally adopted for growing anaërobic bacteria, that is to say those
species which will not grow in the presence of oxygen. This anaërobic
difficulty may be overcome in a variety of ways.
1. The air contained in the culture tube may be removed by _ebullition_
and rapid cooling. And whilst this may accurately produce a vacuum,
it is far from easy to introduce the virus without also reintroducing
oxygen.
2. The oxygen may be displaced by some other gas, and though coal-gas,
nitrogen, and carbon dioxide may all be used for this purpose, it has
become the almost universal practice to grow anaërobes in _hydrogen_.
The production of the hydrogen is readily obtained by Kipp's or some
other suitable apparatus for the generation of hydrogen from zinc and
sulphuric acid. The free gas is passed through various wash-bottles
to purify it of any contaminations. Lead acetate (1-10 per cent.
water) removes any traces of sulphuretted hydrogen, silver nitrate
(1-10) doing the same for arseniated hydrogen; whilst a flask of
pyrogallic acid will remove any oxygen. It is not always necessary to
have these three purifiers if the zinc used in the Kipp's apparatus
is pure. Occasionally a fourth flask is added of distilled water,
and this or a dry cotton wool pledget in the exit tube will ensure
germ-free gas. From the further end of the exit tube of the Kipp's
apparatus an india-rubber tube will carry the hydrogen to its desired
destination. With some it is the custom to place anaërobic cultures
in test-tubes, and the test-tubes in a large flask having a two-way
tube for entrance and exit of the hydrogen; others prefer to pass the
hydrogen immediately into a large test-tube containing the culture
(Fränkel's method). Either method ends practically the same, and the
growth of the culture in hydrogen is readily observed. Yet another plan
is to use a yeast flask, and after having passed the hydrogen through
for about half an hour, the lateral exit tube is dipped into a small
flask containing mercury. The entrance tube is now sealed, and the
whole apparatus placed in the incubator. The interior containing the
culture is filled with an atmosphere of hydrogen. No oxygen can obtain
entrance through the sealed entrance tube, or through the exit tube
immersed in mercury. Yet through this latter channel any gases produced
by the culture could escape if able to produce sufficient pressure.
[Illustration: KIPP'S APPARATUS
For the Production of Hydrogen]
[Illustration: FRÄNKEL'S TUBE
For Cultivation of Anaërobes]
3. _The Absorption Method._ Instead of adding hydrogen to the tube or
flask containing the anaërobic culture, it is feasible to add to the
medium some substances, like glucose or pyrogallic acid, which will
absorb the oxygen which is present, and thus enable the anaërobic
requirement to be fulfilled. To various media--gelatine, agar, or
broth (the latter used for obtaining the toxins of anaërobes)--2 per
cent. of glucose may be added. Pyrogallic acid, or pyrogallic acid one
part and 20 per cent. caustic potash one part, is also readily used
for absorptive purposes. A large glass tube of 25 cc. height, named a
Buchner's cylinder, having a constriction near the bottom, is taken;
and about two drachms of the pyrogallic solution are placed in the
bottom of it. A test-tube containing the culture is now lodged in the
upper part above the constriction. The apparatus is now placed in the
incubator at the desired temperature, and the contained culture grows
under anaërobic conditions. As the pyrogallic solution absorbs the
oxygen it assumes a darker tint.
[Illustration: BUCHNER'S TUBE
For Cultivation of Anaërobes]
4. _Mechanical Methods._ These include various ingenious tricks for
preventing an admittance of oxygen to the culture. An old-fashioned
one was to plate out the culture and protect it from the air by
covering it with a plate of mica. A more serviceable mode is to
inoculate, say, a tube of agar with the anaërobic organism, and then
pour over the culture a small quantity of melted agar, which will
readily set, and so protect the culture itself from the air. Oil may
be used instead of melted agar. Another mechanical method is to make
a deep inoculation and then melt the top of the medium over a bunsen
burner, and thus close the entrance puncture and seal it from the air.
5. _Absorption of Oxygen by an Aërobic Culture._ This method takes
advantage of the power of absorption of certain aërobic bacteria,
which are planted over the culture of the anaërobic species. It is not
practically satisfactory, though occasionally good results have been
obtained.
6. _Lastly, there is the Air-pump Method._ By this means it is
obviously intended to extract air from the culture and seal of it
_in vacuo_. The culture tubes are connected with the air-pump, and
exhausted as much as possible.
Of these various methods it is on the whole best to choose either the
hydrogen method, the vacuum, or the plan of absorption by grape-sugar
or pyrogallic. In anaërobic plate cultures grape-sugar agar plus 0.5
per cent. of formate of soda may be used. The poured inoculated plate
should be placed over pyrogallic solution under a sealed bell-glass
and incubated at 37° C. Pasteur, Roux, Joubert, Chamberland, Esmarch,
Kitasato, and others have introduced special apparatus to facilitate
anaërobic cultivation, but the principles adopted are those which have
been mentioned.
THE QUALITATIVE ESTIMATION OF BACTERIA IN THE SOIL
We may now turn to consider the species of bacteria found in the
interstices of soil. They may be classified in five main groups. The
division is somewhat artificial, but convenient:
1. _The Denitrifying Bacteria._ A group whose function has been
elucidated in recent years (largely by the investigations of Professor
Warington) are held responsible for the breaking down of nitrates. With
these may be associated the _Decomposition or Putrefactive Bacteria_,
which break down complex organic products other than nitrates into
simpler bodies.
[Illustration: A METHOD OF GROWING CULTIVATIONS IN A VACUUM OVER
PYROGALLIC SOLUTION]
2. _The Organisms of Nitrification._ To this group belong the two
chief types of nitrifying bacteria, viz., those which oxidise ammonia
into nitrites, and those which change nitrites into nitrates.
3. _The Nitrogen-fixing Bacteria_, found mainly in the nodules on the
rootlets of certain plants.
4. _The Common Saprophytic Bacteria_, whose function is at present but
imperfectly known. Many are putrefactive germs.
5. _The Pathogenic Bacteria._ This division includes the three types,
tetanus, malignant œdema, and quarter evil. Under this heading
we shall also have to consider in some detail the intimate relation
between the soil and such important bacterial diseases as tubercle and
typhoid.
To enable us to appreciate the work which the "economic bacteria"
perform, it will be necessary to consider shortly the place they occupy
in the economy of nature. This may be perhaps most readily accomplished
by studying the accompanying table (p. 145).
A SCHEME SHOWING THE PLACE AND FUNCTION OF THE ECONOMIC MICRO-ORGANISMS
FOUND IN SOIL
_Water_ _Chemical Substances_ _Gases_
\ [Nitrates, etc.] [CO_{2}, H, N, O]
\ | /
\ | /
_| \|/ |_
PLANT LIFE
|
+------------+----------+---+--------+-------------+----------+
| | | | | |
Carbohydrates Fats Proteids Vegetable Mineral Water
[albumoses, [bodies Acids Salts
sugar, containing
starch, Nitrogen]
etc.] |
|
ANIMAL LIFE
|
+-----------------+------------+---+-----------------------+
| | | |
Gases Water Urea, Albuminoids, Nitrogen in many
[CO_{2}, etc.] Ammonia compounds, etc. forms locked up
in the body
\_______________________________________/
|
PUTREFACTIVE AND DENITRIFYING BACTERIA
|
+--------------+----------+-------------+-------------------+
| | | | |
Free Nitrogen Gases Water Ammonia [Nitrites]
| [CO_{2}] and other elements
| of broken-down
| complex bodies.
|
| \_______________________/
| |
| NITRIFYING BACTERIA
| |
| Nitrites[=Nitrous organism
| | (Nitrosomans)]
NITROGEN-FIXING Nitrates[=Nitric organism
BACTERIA (Nitrosomonas)]
[In soil and in the nodules [In soil and available for
on the rootlets of _Leguminosæ_] plant life]
The threefold function of plant life is nutrition, assimilation, and
reproduction: the food of plants, the digestive and storage power of
plants, and the various means they adopt for multiplying and increasing
their species. With the two latter we have little concern in this
place. Respecting the nutrition of plant life, it is obvious that,
like animals, they must feed and breathe to maintain life. Plant food
is of three kinds, viz., _water_, _chemical substances_, and _gas_.
Water is an actual necessity to the plant not only as a direct food
and food-solvent, but as the vehicle of important inorganic materials.
The hydrogen, too, of the organic compounds is obtained from the
decomposition of the water which permeates every part of the plant,
and is derived by it from the soil and from the aqueous vapour in
the atmosphere. The chief _chemical substances_ of which vegetable
protoplasm is constituted are six, viz, potassium, magnesium, calcium,
iron, phosphorous, and sulphur. These inorganic elements do not enter
the plant as such, but combined with other substances or dissolved in
water. _Potassium_ occurs in salt form combined with various organic
acids (tartaric, oxalic, etc.), _calcium_ and _magnesium_ as salts of
lime and magnesia in combination both with organic and inorganic acids.
_Iron_ contributes largely to the formation of the green colouring
matter of plants, and is also derived from the soil. _Phosphorus_,
one of the chief constituents of seeds, generally occurs as phosphate
of lime. _Sulphur_, which is an important constituent of albumen, is
derived from the sulphates of the soil. In addition to the above, there
are other elements, sometimes described as non-essential constituents
of plants. Amongst these are _silica_ (to give stiffness), _sodium_,
_chlorine_, _iodine_, _bromine_, etc. All these elements contribute to
the formation or quality of the protoplasm of plants.
The _gases_ essential to plants are four: Carbon dioxide (carbonic
acid), Hydrogen, Oxygen, and Nitrogen. By the aid of the green
chlorophyll corpuscles, and under the influence of sunlight, we know
that leaves absorb the carbon dioxide of the atmosphere, and effect
certain changes in it. The hydrogen, as we have seen, is obtained from
the water. Oxygen is absorbed through the root from the interstices
of the soil. Each of these contributes vitally to the existence of
the plant. The fourth gas, _nitrogen_, which constitutes more than
two thirds of the air we breathe (79 per cent. of the total volume
and 77 per cent. of the total weight of the atmosphere), is, perhaps,
the most important food required by plants. Yet, although this is so,
the plant cannot absorb or obtain its nitrogen in the same manner
in which it acquires its carbon--viz., by absorption through the
leaves--nor can the plant take nitrogen into its own substance by any
means as _nitrogen_, with the exception of the flesh-feeding plants
(insectivorus). Hence, although this gas is present in the atmosphere
surrounding the plant, the plant will perish if nitrogen does not
exist in some combined form in the soil. Nitrates and compounds of
ammonia are widely distributed in nature, and it is from these bodies
that the plant obtains, by means of its roots, the necessary nitrogen.
Until comparatively recently it was held that plant life could not
be maintained in a soil devoid of nitrogen or compounds thereof. But
it has been found that certain classes of plants (the _Leguminosæ_,
for example), when they are grown in a soil which is practically free
from nitrogen at the commencement, do take up this gas into their
tissues. One explanation of this fact is that free nitrogen becomes
converted into nitrogen compounds in the soil through the influence
of micro-organisms present there. Another explanation attributes
this fixation of free nitrogen to microorganisms existing in the
rootlets of the plant. These two classes of organisms, known as the
nitrogen-fixing organisms, will require our consideration at a later
stage. Here we merely desire to make it clear that the main supply
of this gas, absolutely necessary to the existence of vegetable life
upon the earth, is drawn not from the nitrogen of the atmosphere,
but from that contained in nitrogen compounds in the soil. The most
important of these are the _nitrates_. Here then we have the necessary
food of plants expressed in a sentence: _water, gases, salts, the most
important and essential gas and some of the salts being combined in
nitrates_.
Plant life seizes upon its required constituents, and by means of the
energy furnished by the sun's rays builds these materials up into
its own complex forms. Its many and varied forms fulfil a place in
beautifying the world. But their contribution to the economy of nature
is, by means of their products, to supply food for animal life. The
products of plant life are chiefly sugar, starch, fat, and proteids.
Animal life is not capable of extracting its nutriment from soil, but
it must take the more complex foods which have already been built up
by vegetable life. Again, the complementary functions of animal and
vegetable life are seen in the absorption by plants of one of the waste
materials of animals, viz., carbonic acid gas. Plants abstract from
this gas carbon for their own use, and return the oxygen to the air,
which in its turn is of service to animal life.
By animal activity some of these foods supplied by the vegetable
kingdom are at once decomposed into carbonic acid gas and water, which
goes back to nature. Much, however, is built up still further into
higher and higher compounds. The proteids are converted by digestion
into albumoses and peptones, ultimately entirely into peptones; these
in their turn are reconverted into proteids, and become assimilated as
part of the living organism. In time they become further changed into
carbonic acid, sulphuric acid, water, and certain not fully oxidised
products,[36] which contain the nitrogen of the original proteid. In
the table these bodies have been represented by one of their chief
members, viz., _urea_.
It is clear that there is in all animal life a double process
continually going on; there is a building up (anabolism, assimilation),
and there is a breaking down (katabolism, dissimilation). These
processes will not balance each other throughout the whole period
of animal life. We have, as possibilities, elaboration, balance,
degeneration; and the products of animal life will differ in degree and
in substance according to which period is in the predominance. These
products we may subdivide simply into excretions during life and final
materials of dissolution after death, both of which may be used more
or less immediately by other forms of animal or vegetable life, or
mediately after having passed to the soil. We may shortly summarise the
final products of animal life as carbonic acid, water, and nitrogenous
remnants. These latter will occur as urea, new albumens, compounds of
ammonia, and nitrogen compounds of great complexity stored up in the
tissues and body of the animal. The carbonic acid, water, and other
simple substances like them will return to nature and be of immediate
use to vegetable life. But otherwise the cycle cannot be completed, for
the more complex bodies are of no service as such to plants or animals.
1. In order that this complex material should be of service in the
economy of nature, and its constituents not lost, it is necessary that
it should be broken down again into simpler conditions. This prodigious
task is accomplished by the agency of two groups of organisms,
_the decomposition and denitrifying_[37] _bacteria_. The organisms
associated with decomposition processes are numerous; some denitrify
as well as break down organic compounds. This group will be referred
to under "Saprophytic Bacteria." The reduction by the denitrifying
bacteria may be simply from nitrate to nitrite, or from nitrate to
nitric or nitrous oxide gas, or indeed to nitrogen itself. In all these
processes of reduction the rule is that a loss of nitrogen is involved.
How that free nitrogen is brought back again and made subservient to
plants and animals we shall understand at a later stage.
Professor Warington has again recently set forth the chief facts known
of this decomposition process.[38] That the action in question only
occurs in the presence of living organisms was first established by
Mensel in 1875 in natural waters, and by Macquenne in 1882 in soils.
If all living organisms are destroyed by sterilisation of the soil,
denitrification cannot take place, nor can vegetable life exist.
"Bacteria reduce nitrates," says Professor Warington, "by bringing
about the combustion of organic matter by the oxygen of the nitrate,
the temperature distinctly rising during the operation." The reduction
to a nitrite is a common property of bacteria. But only a few species
have the power of reducing a nitrate to gas. These few species are,
however, widely distributed. In 1886 Gayon and Dupetit first isolated
the bacteria capable of reducing nitrates to the simplest element,
nitrogen. They obtained their species from sewage, but ten years later
denitrifying bacteria were isolated from manure. That soil contains a
number of these reducing organisms is known by introducing a particle
of surface soil into some broth, to which has been added one per cent.
of nitre. During incubation of such a tube gas is produced, and the
nitrate entirely disappears.
Whenever decomposition occurs in organic substances there is a
reduction of compound bodies, and in such cases the putrefying
substances obtain their decomposing and denitrifying bacteria from
the air. The chief conditions requisite for bringing about a loss of
nitrogen by denitrification are enumerated by Professor Warington as
follows: (1) the specific micro-organism; (2) the presence of a nitrate
and suitable organic matter; (3) such a condition as to aëration that
the supply of atmospheric oxygen shall not be in excess relatively
to the supply of organic matter; (4) the usual essential conditions
of bacterial growth. "Of these," he says, "the supply of organic
matter is by far the most important in determining the extent to which
denitrification will take place." The necessarily somewhat unstable
condition facilitates its being split up by means of bacteria. The
bacteria in their turn are ready to seize upon any products of animal
life which will serve as their food. Thus, by reducing complex bodies
to simple ones, these denitrifying organisms act as the necessary link
to connect again the excretions of the animal body, or after death the
animal body itself, with the soil.
In a book of this nature it has been deemed advisable not to enter into
minute description of all the species of bacteria mentioned. Some of
the chief are described more or less fully. We cannot, however, do
more than name several of the chief organisms concerned in reducing
and breaking down compounds. As we shall find in the bacteria of
nitrification, so also here, the _entire_ process is rarely, if ever,
performed by one species. There is indeed a remarkable division of
labour, not only between decomposition bacteria and denitrification
bacteria, but between different species of the same group. _Bacillus
fluorescens non-liquefaciens_, _Mycoderma ureæ_, and some of the
staphylococci break down nitrates (denitrification), and also decompose
other compound bodies. Amongst the group of putrefactive bacteria found
in soil may be named _B. coli_, _B. mycoides_, _B. mesentericus_, _B.
liquidus_, _B. prodigiosus_, _B. ramosus_, _B. vermicularis_, _B.
liquefaciens_, and many members in the great family of _Proteus_. Some
perform their function in soil, others in water, and others, again, in
dead animal bodies. Dr. Buchanan Young, to whose researches in soil
we have referred, has pointed out that in the upper reaches of burial
soil, where these bacteria are most largely present, there is as a
result no excess of organic carbon and nitrogen. Even in the lower
layers of such soil it is rapidly broken down.
[Illustration: MICROCOCCUS FROM SOIL]
It will be observed, from a glance at the table, that the chief results
of decomposition and denitrification are as follows: free nitrogen,
carbonic acid, gas and water, ammonia bodies, and sometimes nitrites.
The nitrogen passes into the atmosphere, and is "lost"; the carbonic
acid and water return to nature and are at once used by vegetation.
The ammonia and nitrites await further changes. These further changes
become necessary on account of the fact, already discussed, that plants
require their nitrogen to be in the form of nitrates in order to use
it. Nitrates obviously contain a considerable amount of oxygen, but
ammonia contains no oxygen, and nitrites very much less than nitrates.
Hence a process of oxidation is required to change the ammonia into
nitrites and the nitrites into nitrates.
2. This oxidation is performed by the _nitrifying microorganisms_,
and the process is known as _nitrification_. It should be clearly
understood that the process of nitrifaction may, so to speak, dovetail
with the process of denitrification. No exact dividing line can be
drawn between the two, although they are definite and different
processes. In a carcass, for example, both processes may be going on
concomitantly; so also in manure. There is no hard and fast line to be
drawn in the present state of our knowledge. Other organisms beside the
true nitrification bacteria may be playing a part, and it is impossible
exactly to measure the action of the latter, where they began and where
the preliminary attack upon the nitrogenous compounds terminated. In
all cases, however, according to Professor Warington, the formation of
ammonia has been found to precede the formation of nitrous or nitric
acid.
It was Pasteur who (in 1862) first suggested that the production of
nitric acid in soil might be due to the agency of germs, and it is to
Schlösing and Müntz that the credit belongs for first demonstrating (in
1877) that the true nature of nitrification depended upon the activity
of a living micro-organism. Partly by Schlösing and Müntz and partly
by Warington (who was then engaged in similar work at Rothamsted),
it was later established (1) that the power of nitrification could be
communicated to substances which did not hitherto nitrify by simply
seeding them with a nitrified substance, and (2) that the process of
nitrification in garden soil was entirely suspended by the vapour of
chloroform or carbon disulphide. The conditions for nitrification, the
limit of temperature, and the necessity of plant food, have furnished
additional proof that the process is due to a living organism. These
conditions are briefly as follows:
1. Food (of which phosphates are essential constituents). "The
nitrifying organism can apparently feed upon organic matter, but it
can also, apparently with equal ease, develop and exercise all its
functions with purely inorganic food" (Warington).
Winogradsky prepared vessels and solutions carefully purified from
organic matter, and these solutions he sowed with the nitrifying
organism, and found that they flourished. Professor Warington has
employed the acid carbonates of sodium and calcium with distinct
success as ingredients of an ammoniacal solution undergoing
nitrification.
2. The next condition of nitrification is the presence of oxygen.
Without it the reverse process, denitrification, occurs, and instead
of a building up we get a breaking down, with an evolution of nitrogen
gas. The amount of oxygen present has an intimate proportion to the
amount of nitrification, and with 16 to 21 per cent. of oxygen present
the nitrates are more than four times as much as when the smallest
quantity of oxygen is supplied. The use of tillage in promoting
nitrification is doubtless in part due to the aëration of the soil thus
obtained.
3. A third condition is the presence of a base with which nitric acid
when formed may combine. Nitrification can take place only in a feebly
alkaline medium, but an excess of alkalinity will retard the process.
4. The last essential requirement is a favourable temperature. The
nitrifying organism can act at a temperature as low as 37° or 39° F.
(3-4° C.), but at a higher temperature it becomes much more active.
According to Schlösing and Müntz, at 54° F. (12° C.) nitrification
becomes really active, and it increases as the temperature rises to 99°
F. (37° C.), after which it falls. A high temperature or a strong light
are prejudicial to the process.
We are now in a position to consider shortly some of the characters
of these nitrification bacteria. They may readily be divided into
two chief groups, not in consideration of their form or biological
characteristics, but on account of the duties which they perform. Just
as we observed that there were few denitrifying organisms which could
break down ammonia compounds to nitrogen gas, so is it also true that
there are few nitrifying bacteria which can build up from ammonia to
the nitrates. Nature has provided that this shall be accomplished in
two stages, viz., a first stage from ammonia bodies to nitrites, and
a second stage from nitrites to nitrates. The agent of the former is
termed the _nitrous_ organism, the latter the _nitric_ organism. Both
are contributing to the final production of nitrates which can be used
by plant life.[39]
_The Nitrous Organism_ (Nitrosomonas). Prior to Koch's gelatine method
the isolation of this bacterium proved an exceedingly difficult task.
But even the adoption of this isolating method seemed to give no better
results, and for an excellent reason: the nitrifying organisms will
not grow on gelatine. To Winogradsky and Percy Frankland belongs the
credit of separately isolating the nitrous organism on the surface of
gelatinous silica containing the necessary inorganic food. Professor
Warington, in his lectures under the Lawes Agricultural Trust, has
described this important germ as follows:
"The organism as found in suspension in a freshly nitrified
solution consists largely of nearly spherical corpuscles,
varying extremely in size. The largest of these corpuscles
barely reaches a diameter of one-thousandth of a millimetre,
and some are so minute as to be hardly discernible in
photographs. The larger ones are frequently not strictly
circular, and are sometimes seen in the act of dividing.
"Besides the form just described, there is another, not
universally present in solutions, in which the length is
considerably greater than its breadth. The shape varies, being
occasionally a regular oval, but sometimes largest at one end,
and sometimes with the ends truncated. The circular organisms
are probably the youngest.
"This organism grows in broth, diluted milk, and other
solutions without producing turbidity. When acting on ammonia
it produces only nitrites. It is without action on potassium
nitrite. It is, in fact, the nitrous organism which, as we
have previously seen, may be separated from soil by successive
cultivations in ammonium carbonate solution."
The elongated forms appear to be a sign of arrested growth. Normally
the organ is about 1.8 µ long, or nearly three times as long as the
nitric organism. It possesses a gelatinous capsule. "The motile cells,
stained by Löffler's method, are seen to have a flagellum in the form
of a spiral." When grown on silica the nitrous organism appears in
the same two forms--zooglea and free cells--as when cultivated in a
fluid. It commences to show growth in about four days, and is at its
maximum on about the tenth day. Winogradsky found that there were
considerable differences in the morphology of the organism according to
the soil from which it was taken. One of the Java soils he investigated
contained a nitrous organism having a spiral flagellum of thirty
micromillimetres; but its movement was slow.
As we have already seen, the most astonishing property of this organism
is its ability to grow and perform its specific function in solutions
absolutely devoid of organic matter. Some authorities hold that it
acquires its necessary carbon from carbonic acid. The mode of culturing
it is as follows:
To sterilised flasks add 100 cc. of a solution made of one gram of
ammonium sulphate, one gram of potassium sulphate, and 1000 cc. of
pure water. To this add one gram of basic magnesium carbonate which
has been previously sterilised by boiling. Now inoculate the flask
with a small portion of the soil under investigation, and after four
or five days sub-culture on the same medium in fresh flasks, and let
this be repeated half a dozen times. Now, as this inorganic medium
is unfavourable to ordinary bacteria of soil, it is clear that after
several sub-cultures the nitrous organism will be isolated in pure
culture.
Winogradsky employs for culturing upon solid media a mineral gelatine.
A solution of from 3 to 4 per cent. of silicic acid in distilled water
is placed in flasks. By the addition of the following salts to such a
solution gelatinisation occurs:
{ Ammonium sulphate 0.4 gram
(_a_) { Magnesium sulphate 0.05 "
{ Calcium chloride A trace
{ Potassium phosphate 0.1 gram
(_b_) { Sodium carbonate 0.6, 0.9 "
{ Distilled water 100 cc.
The sulphates and chloride are mixed in 50 cc. of distilled water,
and the latter substance in the remaining 50 cc. in separate flasks.
After sterilisation and cooling these are all mixed and added in small
quantities to the silicic acid.
Upon this medium it is possible to sub-culture a pure growth from the
film at the bottom of the flasks in which the nitrous organism is first
isolated.
_The Nitric Organism._ It was soon learned that the nitrous organism,
even when obtainable in large quantities and in pure culture, was not
able entirely to complete the nitrifying process. As early as 1881
Professor Warington had observed that some of his cultures, though
capable of changing nitrites into nitrates, had no power of oxidising
ammonia. These he had obtained from advanced sub-cultures of the
nitrous organism, and somewhat later Winogradsky isolated and described
this companion of the nitrous organism. It develops freely in solutions
to which no organic matter has been added; indeed, much organic matter
will prevent its growing. He isolated it from soils from various parts
of the world on the following media:
Water 1000.0
Potassium phosphate 1.0
Magnesium sulphate 0.5
Calcium chloride A trace
Sodium chloride 2.0
About 20 cc. of this solution is placed in a flat-bottom flask, and a
little freshly washed magnesium carbonate is added. The flask is closed
with cotton wool, and the whole is sterilised. To each flask 2 cc. of
a 2 per cent. solution of ammonium sulphate is subsequently added.
The temperature for incubation is 30° C. Winogradsky concluded that
the oxidation of nitrites to nitrates was brought about by a specific
organism independently of the nitrous organism. He successfully
isolated it in silica jelly. He believes the organism, like its
companion, derives its nutriment solely from inorganic matter, but this
is not finally established.
The form of the nitric organism (or _nitromonas_, as it was once
termed) is allied to the nitrous organism. The cells are elongated,
rarely oval, but sometimes pear-shaped. They are more than half a
micromillimetre in length, and somewhat less in thickness. The cells
have a gelatinous membrane. Like the other nitrifying bacteria, its
development and action are favoured by the presence of the acid
carbonates of calcium and sodium. Of the latter, six grams per litre
or even a smaller quantity gives good results. The sulphate of calcium
can be used, but the organism prefers the carbonates. The differences
between these two bacteria are small, with the exception of their
chemical action. The nitric organism has no action upon ammonia, and
the presence of any considerable amount of ammonium carbonate hinders
its development and prevents its action on a nitrite.[40]
We may here summarise the general facts respecting nitrification.
Winogradsky proposes to term the group _nitro-bacteria_, and to
classify thus:
{ Nitrosomonas, containing at least two species,
Nitrous organisms = { viz., the European and the Java.
{ Nitrosococcus.
Nitric organism = Nitrobacter.
Nitrification occurs in two stages, each stage performed by a distinct
organism. By one (_nitrosomonas_) ammonia is converted into nitrite; by
the other (_nitrobacter_) the nitrite is converted into nitrate.[41]
Both organisms are widely and abundantly distributed in the
superficial soils. They act together and in conjunction, and for one
common purpose. They are separable by employing favourable media.
"If we employ a suitable inorganic solution containing
potassium nitrite, but no ammonia, we shall presently obtain
the nitric organism alone, the nitrous organism feeding on
ammonia being excluded. If, on the other hand, we employ an
ammonium carbonate solution of sufficient strength, we have
selected conditions very unfavourable to the growth of the
nitric organism, and a few cultivations leave the nitrous
organism alone in possession of the field" (Warington).
[Illustration: NITROUS ORGANISM
× 1000]
[Illustration: NITRIC ORGANISM
× 1000]
[Illustration: NITROGEN FIXING ORGANISM FROM SECRETION OF ROOT-NODULES
× 1000]
A word upon the natural distribution of these nitrifying bacteria
before we leave them. They belong to the soil, river water, and sewage.
They are also said to be frequently present in well water. From some
experiments at Rothamsted it appears that the organisms occur mostly
in the first twelve inches, and in subsoils of clay down to three or
four feet. In sandy soils nitrification may probably occur at a greater
depth. These facts should be borne in mind when arranging for the
purification of sewage by intermittent filtration.
We have now given some consideration to the chief events in the
life-cycle of nature depicted in the table. There is but one further
process in which bacteria play a part, and which requires some mention.
It will have been noticed that at certain stages in the cycle there
is more or less appreciable "loss" of free nitrogen. In the process
of decomposition brought about by the denitrifying bacteria, a very
considerable portion of the nitrogen is dissipated into the air
in the form of a free gas. This is the last stage of all proteid
decomposition, so that wherever putrefaction is going on there is a
continual "loss" of an element essential to life. Thus it would appear
at first sight that the sum-total of nitrogen food must be diminishing.
But there are other ways also in which nitrogen is being set free.
In the ordinary processes of vegetation there is a gradual draining
of the soil and a passing of nitrogen into the sea; the products of
decomposition pass from the soil by this drainage, and are "lost" as
far as the soil is concerned. Many of the methods of sewage disposal
are in reality depriving the land of the return of nitrogen which is
its necessity. Again, nitrogen is freed in explosions of gunpowder,
nitroglycerine, and dynamite, for whatever purpose they are used. Hence
the great putrefactive "loss" of nitrogen, with its subsidiary losses,
contributes to reduce this essential element of all life, and if there
were no method of bringing it back again to the soil, it would seem
that plant life, and therefore animal life, would speedily terminate.
It is at this juncture, and to perform this vital function, that the
_nitrogen-fixing bacteria_ play their wonderful part: they bring back
the free nitrogen and fix it in the soil. Excepting a small quantity
of combined nitrogen coming down in rain and in minor aqueous deposits
from the atmosphere, the great source of the nitrogen of vegetation
is the store in the soil and subsoil, whether derived from previous
accumulations or from recent supplies by manure.
Sir William Crookes has recently[42] pointed out the vast importance of
using all the available nitrogen in the service of wheat production.
The distillation of coal in the process of gas-making yields a certain
amount of its nitrogen in the form of sulphate of ammonia, and this,
like other nitrogenous manures, might be used to give back to the soil
some of the nitrogen drained from it. But such manuring cannot keep
pace, according to Sir W. Crookes, with the present loss of fixed
nitrogen from the soil. We have already referred to several ways
in which "loss" of nitrogen occurs. To these may well be added the
enormous loss occurring in the waste of sewage when it is passed into
the sea. As the President of the British Association pointed out,[43]
the more widely this wasteful system is extended, recklessly returning
to the sea what we have taken from the land, the more surely and
quickly will the finite stocks of nitrogen, locked up in the soils of
the world, become exhausted. Let us remember that the plant creates
nothing in this direction; there is nothing in wheat which is not
absorbed from the soil, and unless the abstracted nitrogen is returned
to the soil, its fertility must be ultimately exhausted. When we apply
to the land sodium nitrate, sulphate of ammonia, guano, and similar
manurial substances, we are drawing on the earth's capital, and our
drafts will not be perpetually responded to.[44] We know that a virgin
soil cropped for several years loses its productive powers, and without
artificial aid becomes unfertile. For example, through this exhaustion
forty bushels of wheat per acre have dwindled to seven. Rotation of
crops is an attempt to meet the problem, and the four-course rotation
of turnips, barley, clover, and wheat witnesses to the fact that
practice has been ahead of science in this matter.
The store of nitrogen in the atmosphere is practically unlimited,
but it is fixed and rendered assimilable only by cosmic processes of
extreme slowness. We may shortly glance at these, for it is upon these
processes, plus a return to the soil of sewage, that we must depend in
the future for storing nitrogen as nitrates.
1. Some combined nitrogen is absorbed by the soil or plant from the
air, for example, fungi, lichens, and some algæ, and the absorption
is in the form of ammonia and nitric acid. This is admittedly a small
quantity.
2. Some free nitrogen is fixed within the soil by the agency of porous
and alkaline bodies.
3. Some, again, may be assimilated by the higher chlorophyllous plants
themselves, independently of bacteria (Frank).
4. Electricity fixes, and may in the future be made to fix more,
nitrogen. If a strong inductive current be passed between terminals,
the nitrogen from the air enters into combination with the oxygen,
producing nitrous and nitric acids.
5. Abundant evidence has now been produced in support of the fact that
there is considerable fixation by means of bacteria.
Bacterial life in several ways is able to reclaim from the atmosphere
this free nitrogen, which would otherwise be lost. The first method
to which reference may be made is that involving _symbiosis_. This
term signifies "a living together" of two different forms of life,
generally for a specific purpose. It may be to mutual advantage, a
living for one another, or it may be, by means of an interchange of
metabolism or products, finally to produce or obtain some remote
chemical result. It is convenient to restrict the term symbiosis to
complementary partnerships such as exist between algoid and fungoid
elements in lichens, or between unicellular algæ and Radiolarians,[45]
or between bacteria and higher plants. The partnerships between
hermit crabs and sea-anemones and the like are sometimes defined by
the term _commensalism_ (joint diet). Symbiosis and commensalism must
be distinguished from _parasitism_, which indicates that all the
advantage is on the side of the parasite, and nothing but loss on the
side of the host. The distinction between symbiosis and commensalism
cannot be rigid, but between these conditions which are advantageous
to the partners and parasitism, there is an obvious and radical
difference. Association of organisms together for increase of virulence
and function should be distinguished from symbiosis, and mere existence
of two or more species of bacteria in one medium is not, of course,
symbiosis. Most frequently such a condition would result in injury
and the subsequent death of the weaker partner, an effect precisely
opposite to that defined by this term.
The example of bacteriological symbiosis with which we are concerned
here is that partnership between bacteria and some of the higher plants
(_Leguminosæ_) for the purpose of fixing nitrogen in the plant and in
the surrounding soil.
[Illustration: ROOTLET OF PEA WITH NODULES]
_The Nitrogen-fixing Bacteria_, the third group of micro-organisms
connected with the soil, exist in groups and colonies situated inside
the nodules appearing, under certain circumstances, on the rootlets of
the pea, bean, and other _Leguminosæ_. It was Hellriegel and Wilfarth
who first pointed out that, although the higher chlorophyllous plants
could not directly obtain or utilise free nitrogen, some of them
at any rate could acquire nitrogen brought into combination under
the influence of bacteria. Hellriegel found that the gramineous,
polygonaceous, cruciferous, and other orders depended upon combined
nitrogen supplied within the soil, but that the _Leguminosæ_ did not
depend entirely upon such supplies.
It was observed that in a series of pots of peas to which no nitrogen
was added most of the plants were apparently limited in their growth by
the amount of nitrogen locked up in the seed. Here and there, however,
a plant, under apparently the same circumstances, grew luxuriantly
and possessed on its rootlets abundant nodules. The experiments of
Sir John Lawes and Sir Henry Gilbert at Rothamsted[46] demonstrated
further that under the influence of suitable microbe-seeding of the
soil in which _Leguminosæ_ were planted there is nodule formation on
the roots, and coincidentally increased growth and gain of nitrogen
beyond that supplied either in the soil or in the seed as combined
nitrogen. Presumably this is due to the fixation, in some way, of free
nitrogen. Nobbe proved the gain of nitrogen by non-leguminous plants
(Elœagnus, etc.) when these grow root nodules containing bacteria,
but to all appearances, bacteria differing morphologically from the
_Bacillus radicicola_ of the leguminous plants.
[Illustration: NITROGEN FIXING BACTERIA _IN SITU_ IN NODULE ON ROOTLET
OF PEA
× 400
--Cellular sheath of Rootlet forming capsule of nodule.
--Colonies of bacteria _in situ_.]
[Illustration: NITROGEN FIXING BACTERIA _IN SITU_ IN ROOT-NODULE OF PEA
(Section of Nodule)
× 500]
[Illustration: NITROGEN FIXING BACTERIA _IN SITU_ IN ROOT-NODULE OF PEA
(Section of Nodule)
× 600]
These facts being established, the question naturally arises, How
is the fixation of nitrogen to be explained, and by what species
of bacteria is it performed? In the first place, these matters are
simplified by the fact that there is very little fixation indeed by
bacteria in the soil apart from symbiosis with higher plants. Hence we
have to deal mainly with the work of bacteria in the higher plant. Sir
Henry Gilbert concludes[47] that the alternative explanations of the
fixation of free nitrogen in the growth of _Leguminosæ_ seem to be:
"1. That under the conditions of symbiosis the plant is
enabled to fix the free nitrogen of the atmosphere by its
leaves;
"2. That the nodule organisms become distributed within the
soil and there fix free nitrogen, the resulting nitrogenous
compounds becoming available as a source of nitrogen to the
roots of the higher plant;
"3. That free nitrogen is fixed in the course of the
development of the organisms within the nodules, and that the
resulting nitrogenous compounds are absorbed and utilized by
the host." "Certainly," he adds, "the balance of evidence at
present at command is much in favour of the third mode of
explanation."
If this is finally proved to be the case, it will furnish another
excellent example of the power existing in bacteria of assimilating an
elementary substance.
Most authorities would agree that all absorption of free nitrogen, if
by means of bacteria, must be through the roots. As a matter of fact,
legumes, especially when young, use nitrogen, like all other plants,
derived from the soil. It has been pointed out that, unless the soil is
somewhat poor in nitrogen, there appears to be but little assimilation
of free nitrogen and but a poor development of root nodules.[48] The
free nitrogen made use of by the micro-organism is in the air contained
in the interstices of the soil. For in all soils, but especially
in well-drained and light soils, there is a large quantity of air.
Although it is not known how the micro-organisms in legumes utilise
free nitrogen and convert it into organic compounds in the tissues
of the rootlet or plant, it is known that such nitrogen compounds
migrate into the stem and leaves, and so make the roots really poorer
in nitrogen than the foliage. But the ratio is a fluctuating one,
depending chiefly on the stage of growth or maturity of the plant.
If the nodules from the rootlets of _Leguminosæ_ be examined, the
nitrogen-fixing bacteria can be readily seen. The writer has isolated
these and grown them in pure culture as follows: The nodules are
removed, if possible at an early stage in their growth, and placed for
a few minutes in a steam steriliser. This is advisable in order to
remove the various extraneous organisms attached to the outer covering
of the nodule. They may then be washed in antiseptic solution, and
their capsules softened by soaking. When opened with a sterile knife,
thick creamy matter exudes. On microscopic examination this is found
to be densely crowded with small round-ended bacilli or oval bodies,
known as _bacteroids_. By a simple process of hardening and using the
microtome, excellent sections of the nodules can be obtained which
show these bacteria _in situ_. In the central parts of the section may
be seen densely crowded colonies of the bacteria, which in some cases
invade the cellular capsule of the nodule derived from the rootlet.
Aërobic and anaërobic pure cultures of these bacteria were made. In
some cases these cultures very closely resembled the feathery growth of
the bacillus of anthrax.
4. _The Saprophytic Bacteria in Soil._ This group of micro-organisms
is by far the most abundant as regards number. They live on the dead
organic matter of the soil, and their function appears to be to
break it down into simpler constitution. Specialisation is probably
progressing among them, for their name is legion, and the struggle for
existence keen. After we have eliminated the economic bacteria, most
of which are obviously saprophytes, the group is greatly reduced. It
is also needless to add that of the remnant little beyond morphology
is known, for as their function is learned they are classified
otherwise. It is probable, as suggested, that many of the species of
common saprophytes normally existent in the soil act as _auxiliary
agents_ to denitrification and putrefaction. At present we fear they
are disregarded in equal measure, and for the same reasons, as the
common water bacteria. An excess of either, in soil or water, is not
of itself injurious as far as we know; indeed, it is probably just
the reverse. It is, however, frequently an index of value as to the
amount and sometimes condition of the contained organic matter. The
remarks made when considering water bacteria apply here also, viz.,
that an excess of saprophytes acts not only as index of increase of
organic matter, but as at first auxiliary, and then detrimental, to
pathogenic organisms. It will require accurate knowledge of soil
bacteria generally to be able to say which saprophytic germs, if
any, have no definite function beyond their own existence. It may be
doubted whether the stern behests of nature permit of such organisms.
However that may be, we may feel confident, though at present there
are many common bacteria in soil, as also in water, the life object of
which is not ascertained, that as knowledge increases and becomes more
accurate this special provisional group will become gradually absorbed
into other groups having a part in the economy of nature, or in the
production of disease. At present the decomposition, denitrifying,
nitrifying,[49] and nitrogen-fixing organisms are the only saprophytes
which have been rescued from the oblivion of ages, and brought more or
less into daylight. It is but our lack of knowledge which requires the
present division of saprophytes whose business and place in the world
is unknown.
5. _The Pathogenic Organisms found in Soil._ In addition to these
saprophytes and the economic bacteria, there are, as is now well known,
some disease-producing bacteria finding their nidus in ordinary soil.
The three chief members of this group are the bacillus of Tetanus
(lockjaw), the bacillus of Quarter Evil, and the bacillus of Malignant
Œdema.
_Tetanus._ The pathology of this terrible disease has during recent
years been considerably elucidated. It was the custom to look upon it
as "spontaneous," and arising no one knew how; now, however, after the
experiments of Sternberg and Nicolaier, the disease is known to be due
to a micro-organism common in the soil of certain localities, existing
there either as a bacillus or in a resting stage of spores. Fortunately
Tetanus is comparatively rare, and one of the peculiar biological
characteristics of the bacillus is that it grows only in the absence of
oxygen. This fact contributed not a little to the difficulties which
were met with in securing its isolation.
Tetanus occurs in man and horses most commonly, though it may affect
other animals. There is usually a wound, often an insignificant one,
which may occur in any part of the body. The popular idea that a severe
cut between the thumb and the index finger leads to tetanus is without
scientific foundation. As a matter of fact, the wound is nearly always
on one or other of the limbs, and is infected simply because they come
more into contact with soil and dust than does the trunk. It is not the
locality of the wound nor its size that affects the disease. A cut with
a dirty knife, a gash in the foot from the prong of a gardener's fork,
the bite of an insect, or even the prick of a thorn have before now
set up tetanus. Wounds which are jagged, and occurring in absorptive
tissues, are those most fitted to allow the entrance of the bacillus.
The wound forms a local manufactory, so to speak, of the bacillus and
its secreted poisons; the bacillus always remains in the wound, but
the toxins may pass throughout the body, and are especially absorbed
by the cells of the central nervous system, and thus give rise to the
spasms which characterise the disease. Suppuration generally occurs in
the wound, and in the pus thus produced may be found a great variety
of bacteria, as well as the specific agent itself. After a few days
or, it may be, as much as a fortnight, when the primary wound may be
almost forgotten, general symptoms occur. Their appearance is often the
first sign of the disease. Stiffness of the neck and facial muscles,
including the muscles of the jaw, is the most prominent sign. This is
rapidly followed by spasms and local convulsions, which, when affecting
the respiratory or alimentary tract, may cause a fatal result. Fever
and increased rate of pulse and respiration are further signs of the
disease becoming general. After death, which results in the majority of
cases, there is very little to show the cause of fatality. The wound
is observable, and patches of congestion may be found on different
parts of the nervous system, particularly the medulla (grey matter),
pons, and even cerebellum. Evidence has recently been forthcoming
at the Pasteur Institute to support the theory that tetanus is a
nervous disease, more or less allied to rabies, and is best treated by
intra-cerebral injection of antitoxin, which then has an opportunity of
opposing the toxins at their favourite site. (Roux and Borrel.)
In the wound the bacillus is present in large numbers, but mixed
up with a great variety of suppurative bacteria and extraneous
organisms. It is in the form of a straight short rod with rounded
ends, occurring singly or in pairs of threads, and slightly motile.
It has been pointed out that by special methods of staining, flagella
may be demonstrated.[50] These are both lateral and terminal, thin
and thick, and are shed previously to sporulation. Branching also has
been described. Indeed, it would appear that, like the bacillus of
tubercle, this organism has various pleomorphic forms. Next to the
ordinary bacillus, filamentous forms predominate, particularly so in
old cultures. Clubbed forms, not unlike the bacillus of diphtheria,
may often be seen from agar cultures. Without doubt the most peculiar
characteristic of this bacillus is its sporulation. The well-formed
round spores occur readily at incubation temperature. They occupy a
position at one or other pole of the bacillus, and have a diameter
considerably greater than the rod. Thus the well-known "drumstick" form
is produced. In practice the spores occur freely in the medium and
in microscopical preparation. Like other spores, they are extremely
resistant to heat, desiccation, and antiseptics. They can resist
boiling for several minutes.
[Illustration: BACILLUS OF TETANUS]
As we have seen, this bacillus is a strict anaërobe, growing only in
the absence of oxygen. The favourable temperature is 37° C., and it
will only grow very slowly at or below room temperature.
An excellent culture is generally obtainable in glucose gelatine. The
growth occurs, of course, only in the depth of the medium, and appears
as fine threads passing horizontally outwards from the track of the
needle. At the top and bottom of the growth these fibrils are shorter
than at the middle or somewhat below the middle. For extraction of the
soluble products of the bacillus glucose broth may be used.
In some countries, and in certain localities, the bacillus of tetanus
is a very common habitant of the soil, and when one thinks how
frequently wounds must be more or less contaminated with such soil, the
question naturally arises, How is it that the disease is, fortunately,
so rare? Probably we must look to the advance of bacteriological
science to answer this and similar questions at all adequately. Much
has recently been done in Paris and elsewhere to emphasise the relation
which other organisms have to such bacteria as those of typhoid and
tetanus. When considering typhoid, we saw that in addition to the
presence of the specific germ other conditions were requisite before
the disease actually occurred. So in tetanus, Kitasato and others have
pointed out that the presence of certain other bacteria, or of some
foreign body, is necessary to the production of the disease. The common
organisms of suppuration are particularly accused of increasing the
virulence of the bacillus of tetanus. How these auxiliary organisms
perform this function has not been fully elucidated. Probably, however,
it is by damaging the tissues and weakening their resistance to such a
degree as to afford a favourable multiplying ground for the tetanus. It
is right to state that some authorities hold that they act by using up
the surrounding oxygen, and so favouring the growth of tetanus.
_Quarter Evil_ (or symptomatic anthrax) is a disease of animals,
produced in a manner analogous to tetanus. It is characterised by a
rapidly increasing swelling of the upper parts of the thigh, sacrum,
etc., which, beginning locally, may attain to extraordinary size and
extent. It assumes a dark colour, and crackles on being touched. There
is high temperature, and secondary motor and functional disturbances.
The disease ends fatally in two or three days.
Slight injuries to the surface of the skin or mucous membrane are
sufficient for the introduction of the causal bacillus. This organism
is, like tetanus, an anaërobe, existing in the superficial layers
of the soil. From its habitat it readily gains entrance to animal
tissues. It has spores, but though they are of greater diameter than
the bacillus itself they are not absolutely terminal. Hence they merely
swell out the capsule of the bacillus, and produce a club-shaped rod.
They form gas while growing in the tissues and in artificial culture.
External physical conditions have little effect upon this bacillus,
and the dried and even buried flesh retains infection for a very long
period of time.
[Illustration: B. OF SYMPTOMATIC ANTHRAX]
[Illustration: B. OF MALIGNANT ŒDEMA]
The third disease-producing microbe found naturally in soil is that
which produces the disease known as Malignant Œdema. Pasteur called
this _gangrenous septicœmia_. Unlike quarter evil, malignant
œdema may occur in man in cases where wounds have become septic.
Animals become inoculated with this bacillus from the surface of soil,
straw-dust, upper layers of garden-earth, or decomposing animal and
vegetable matter.
The bacillus occurs in the blood and tissues as a long thread, composed
of slender segments of irregular length. It is motile and anaërobic.
The spores are larger than the diameter of the bacillus, and the
organism produces gas; so much is this the case in artificial culture,
that the medium itself is frequently split up.
Both malignant œdema and symptomatic anthrax are similar in some
respects to anthrax itself. There are, however, a number of points for
differential diagnosis. The enlargement of the spleen, the non-motility
of the bacillus, the enormous numbers of bacilli throughout the body,
the square ends, equal inter-bacillary spaces, aërobic growth, and
characteristic staining afford ample evidence of anthrax.
_The Relation of Soil generally to certain Bacterial Diseases._ Recent
investigations have, in effect, considerably added to our knowledge of
pathogenic germs in soil; and whilst the three species enumerated above
are still considered as types normally present in soil, it must not be
forgotten that other virulent disease producers either live in the soil
or are greatly influenced by its conditions.
Fränkel and Pasteur have both demonstrated the possible presence of
anthrax. Fränkel maintained that it could not live there long, and at
ten feet below the surface no growth occurred. This may have been due
to the low temperature of such a depth. Pasteur held that earthworms
are responsible for conveying the spores of anthrax from buried
carcasses to the surface, and thus bringing about reinfection. Cholera,
too, has been successfully grown in soil, except during winter. The
presence of common saprophytes in the soil is prejudicial to the
development of the cholera spirillum, and under ordinary circumstances
it succumbs in the struggle for existence. From experiments recently
conducted for the Local Government Board by Dr. Sidney Martin, evidence
is forthcoming in support of the view that the bacillus of typhoid can
live in certain soils. Samples of soil polluted with organic matter
formed a favourable environment for living bacilli of typhoid for
456 days, whereas in sterilised soil, without organic matter, these
organisms lived only twenty-three days. Tubercle also has been kept
alive for several weeks in soil.
In passing, a single remark may be made in relation to the long
periods during which bacteria can retain vitality in soil. Farm soils
have, as is well known, been contaminated with anthrax in the late
summer or autumn, and have retained the infectious virus till the
following spring, and it has even then cropped up again in the hay of
the next season. In 1881 Miquel took some samples of soil at a depth of
ten inches, containing six and a half million bacteria per gram. After
drying for two days at 30° C., the dust was placed in hermetically
sealed tubes, which were put aside in a dark corner of the laboratory
for sixteen years. Upon re-examination it is reported that more than
three million germs per gram were still found, amongst them the
specific bacillus of tetanus. Whether or not there is any fallacy in
these actual figures, there is abundant evidence in support of the fact
that bacteria, non-pathogenic and pathogenic, can and do retain their
vitality, and sometimes even their virulence, for almost incredibly
long periods of time.
It is now some years since Sir George Buchanan, for the English Local
Government Board, and Dr. Bowditch, for the United States, formulated
the view that there is an intimate relationship between dampness
of soil and the bacterial disease of Consumption (tuberculosis of
the lungs). The matter was left at that time _sub judice_, but the
conclusion has been drawn, and surely a legitimate one, that the
dampness of the soil acted injuriously in one of two ways. It either
lowered the vitality of the tissues of the individual, and so increased
his susceptibility to the disease, or in some way unknown favoured
the life and virulence of the bacillus. That is one fact. Secondly,
Pettenkofer traced a definite relationship between the rise and fall
of the ground water with pollution of the soil and enteric (typhoid)
fever.[51] A third series of investigations concluded in the same
direction, viz., the researches of Dr. Ballard respecting summer
diarrhœa. This, it is generally held, is a bacterial disease,
although no single specific germ has been isolated as its cause.
Ballard demonstrated that the summer rise of diarrhœa mortality does
not commence until the mean temperature of the soil, recorded by the
four-foot thermometer, has attained 56.4° F., and the decline of such
diarrhœa coincides more or less precisely with the fall in soil
temperature. This temperature (56.4° F.) is, therefore, considered
as the "critical" four-foot earth temperature, that is to say, the
temperature at which certain changes (putrefactive, bacterial, etc.)
take place in the pores of the earth, with the consequent development
of the diarrhœal poison.
After a very elaborate and prolonged investigation on behalf of
the Local Government Board, Dr. Ballard formulates the causes of
diarrhœa in the following conclusions:[52]
(_a_) The essential cause of diarrhœa resides ordinarily in the
superficial layers of the earth, where it is intimately associated with
the life processes of some micro-organism not yet detected or isolated.
(_b_) That the vital manifestations of such organism are dependent,
among other things, perhaps principally upon conditions of season and
the presence of dead organic matter, which is its pabulum.
(_c_) That on occasion such micro-organism is capable of getting abroad
from its primary habitat, the earth, and having become air-borne,
obtains opportunity for fastening on non-living organic material,
and of using such organic matter both as nidus and as pabulum in
undergoing various phases of its life history.
(_d_) That from food, as also from contained organic matter of
particular soils, such micro-organism can manufacture, by the chemical
changes wrought therein through certain of its life processes, a
substance which is a virulent chemical poison.
Here, then, we have a large mass of evidence from the data collected
by Buchanan, Bowditch, Pettenkofer, and Ballard. But much of this work
was done anterior to the time of the application of bacteriology to
soil constitution. Recently the matter has received increased attention
from various workers abroad, and in England from Dr. Sidney Martin,
Professor Hunter Stewart, Dr. Robertson, and others. The greater part
of this work we cannot here consider. But some reference must be
made to Dr. Robertson's admirable researches into the growth of the
bacillus of typhoid in soil. By experimental inoculation of soil with
broth cultures, he was able to isolate the bacillus twelve months
after, alive and virulent. He concludes that the typhoid organism is
capable of growing very rapidly in certain soils, and under certain
circumstances can survive from one summer to another. The rains of
spring and autumn or the frosts and snows of winter do not kill them
off so long as there is sufficient organic pabulum. Sunlight, the
bactericidal power of which is well known, had, as would be expected,
no effect except upon the bacteria directly exposed to its rays. The
_bacillus typhosus_ quickly dies out in the soil of grass-covered
areas. Dr. Robertson holds that the chief channel of infection between
typhoid-infected soil and man is dust. As in tubercle and anthrax,
so in typhoid, dried dust or excreta containing the bacillus is the
vehicle of disease.
Hitherto we have addressed ourselves to those diseases the known
causal organisms of which reside, normally or abnormally, in the
soil. But closely allied to these matters connected with the rôle of
pathogenic bacteria in soil is the question of what has been termed the
_miasmatic_ influence of soil. The term "miasm" has had an extensive
and somewhat diffuse application in medical science. It may happen in
the future that typhoid will be classified strictly as a miasmatic
disease. But at present, in the transition state of the science, it
would hardly be justifiable to classify typhoid with a typically
miasmatic disease like malaria. Yet it is clear that mention should
here be made of a group of diseases of which malaria is the type, and
of which the tropics generally are the native land. The bacterial
etiology of the group is by no means worked out. The cause of malaria
alone is not yet a closed subject. However the details of the etiology
of this group finally arrange themselves, there is little doubt of two
facts, viz., the diseases are probably produced by bacteria or allied
protozoa, and soil plays an important part in their production.
From what has been said, it will be seen that though a considerable
amount of knowledge has been obtained respecting bacteria in the soil,
it may be conjectured that actually there is still a great deal to
ascertain before the micro-biology of soil is in any measure complete
or even intelligent. The mere mention of tetanus and typhoid in the
soil, and their habits, nutriment, and products therein, not to mention
the work of the economic bacteria, is to open up to the scientific
mind a vast realm of possibility. It is scarcely too much to say that
a fuller knowledge of the part which soil plays in the culture and
propagation of bacteria may suffice to revolutionise the practice of
preventive medicine. Truly, our knowledge at the moment is rather a
heterogeneous collection of isolated facts and theories, some of which,
at all events, require ample confirmation; still, there is a basis for
the future which promises much constructive work.