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NOTES AND MEMORANDA.
Dr. Koch's New Method of Pure Cultivation of Bacteria At
the recent meeting of the International Medical Congress in
London, during August, Dr. Koch, well known by his
researches on the life-history of Bacillus anthracis (see this
Journal, Vol. XVII, p. 87), gave a series of demonstrations
in the physiological laboratory of King's College, which
were of the greatest interest and importance.
Dr. Koch has recently been appointed to the charge of a
laboratory of experimental research connected with the State
Department of Public Health in Berlin, and aided by his
two assistants, he brought to London material and instruments for the purpose of exhibiting to the members of the
Congress the methods of research into the relation of Bacteria to disease, devised by him. The series of photographs
of various forms of Bacteria shown by Dr. Koch were valuable, as affording convincing evidence of the necessity of
making use of photography as the means of obtaining and
preserving a record of the specific form and character of
Bacterian growths. Of great interest also Avere the cultivations of the Bacteria of blue milk, and of those of blue pus,
exhibited by Dr. Koch, and of the septic Bacterium of
putrid blood, the toxic effects of which were experimentally
demonstrated.
Of most general importance, and in our judgment likely
to mark altogether a new era in the study of the relations
of Bacteria to certain diseases, and to other fermentative
processes, was the demonstration by Dr. Koch of a new
and yet absolutely simple and obvious method of obtaining
pure cultivations of the species of Bacteria.
It is a well-known fact that there are a large number of
species of Bacteria differing from one another in the effects
which they produce in the medium wherein they are cultivated. It is also well known that Bacteria are so ubiquitous that the examination of any natural medium attacked
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by them is almost sure to yield evidence of the presence of
more than one species, the varibus species growing together
in inextricable confusion. On this account it has been
found a matter of extreme difficulty to determine what
effects are due to one species of Bacterium and what to
another. And it has indeed been often impossible to determine in such a mixture of forms those which are genetically
related to one another, and therefore to distinguish one
species from the other forms which are adventitiously associated with it.
To effect the separation of species in a mixture, Mr.
Lister employed a method of dilution and division described in his well-known research on the Lactic ferment
(see this Journal, vol. xviii, p. 191). Making use of a fluid
as the nutrient medium of cultivation (as hitherto has been
the almost universal practice in such cultivations), Mr. Lister
introduced a drop of sour milk containing possibly twenty
kinds of Bacteria, and among them the Bacterium of lactic
fermentation, into a large quantity of pure water, the dilution
and spacing (so to speak) of the Bacteria thus affected being
calculated so to render it probable that a single drop removed
frbin the diluted Bacterian mixture would contain a single
Bacterium. Such drops were then removed and placed each
into a separate culture-tube containing sterilized fluid nutriment, and thus in a certain number of the tubes a pure cultivation consisting of the progeny of a single Bacterium,
andj therefore, unquestionably of but one species, was
obtained.
This method is tedious and liable to failure owing to the
great care necessary to ensure and maintain sterilization of
the cultivation fluid whilst exposed for the purpose of inoculation and again for further examination. Dr. Koch was
led to this new method of cultivation, which essentially consists in the substitution of a solid for a fluid medium of
cultivation, by the use of the method^ known to all mycologists of cultivation, upon slices of potato or beet-root.
It is readily observed when slices of boiled potato are exposed in a damp condition to the atmosphere that the surface of the slice becomes the seat of development of various
Bacteria and of moulds, the spores of which fall from the
atmosphere on to the exposed slice, a fact which struck Dr.
Koch as of importance in reference to the slices of potato
was this—that the various spores falling on to it remain
where they fall, and from the spot where each spore or germ
originally fell it proceeds to multiply, producing around it a
symmetrical hemispherical growth of perfect purity. In fact,
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NOTES AND MEMORANDA.
owing to the solid character of the nourishing support the
germs and spores cannot get mixed as they do in a liquid,
each remains distinct from its neighbour even though in very
close proximity, and without any trouble from the resulting
growth, which proceeds in a day or two from each germ—
new and perfectly pure cultivations may be started in suitable
sterilized fluids.
Dr. Koch's method consists in substituting for the potato
slice a layer of gelatine which is so saturated with water as
just to become solid on cooling. The gelatine liquid is
readily sterilized by boiling, and into it can be introduced
either Pasteur's salts, peptones, blood-serum, or other nutrient material required by one or other species of Bacterium. The gelatine-medium thus prepared may be kept
n a tube and a cultivation thus carried on—on its surface, or (and this is its principal use) it may be spread when
liquid on a microscope object-slide and allowed to cool. Then
such a gelatine plate may be inoculated by touching its
surface with material containing the Bacteria which it is
desired to study. The plate is readily protected from the
access of accidental atmospheric germs, and maintained at
such temperature and degree of moisture (by a glass shade)
as the experimenter may desire. The main point of advantage, however, is this—that the point of inoculation on the
surface of the gelatine can, owing to its transparency, be
readily examined with the highest powers of the microscope
and the growth of the Bacteria followed—whilst further,
owing to the fact that the medium in which the growth
takes place is solid, no mixture of the different kinds which
may be present occurs, but each Bacterium produces around
it a little spherical nest of its own kind. From these nests,
with a sterilized needle-point, individuals can be removed to
start new pure cultivations.
But it is obvious that, if the original point of inoculation
was very minute, there is no danger of any accidental contamination from atmospheric germs, for these are not likely
to fall on the identical spot no bigger than the puncture of
a needle's point, where the experimental culture is going
on. As a matter of fact, where they fall on to the gelatine
there they remain and grow, and fifty such accidental
spores may fall on to the gelatine plate without in the least
interfering with the purity of the experimental culture.
There is yet, further, a very simple device which enables
Dr. Koch to use this gelatine surface as a means of
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spacing " and dividing the various species in a mixture of
Bacteria, JJe dips a stpvilized needle into such a mixture,
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and then makes a long shallow streak with the needle's
point upon the surface of the gelatine. The Bacteria which
were adhering to the needle's point are in this way dropped
at intervals along the streak, some nearer some further
apart, but all (with rare exceptions) in such a way that
their subsequent growth keeps clear of that of a neighbour,
and can, with the aid of a low power or even without any
microscope, be visited by a sterilized needle point, and thus
used to start on another gelatine plate a perfectly pure cultivation.
These pure cultivations, such as Lister aimed at by his
method of dilution and division, may be called, in order to
indicate to what an extent they are known to be pure,
" monosporous cultivations," since the principle which distinguishes them is that all the growth is the offspring of a
single isolated germ or spore.
It is only by such monosporous cultivations that we can
arrive at solid conclusions in reference to the forms and
activities of the Bacteria, e.g. as to whether one form can
give rise to progeny of another form when its food and conditions of growth are changed, and again, as to whether
special fermentative powers can be lost or acquired in the
course of generations derived from one parent germ, but
subjected to different conditions as to food, temperature, and
oxygen.
The method of gelatine cultivation devised by Dr. Koch,
places the means of following out these inquiries in the
hands of every careful microscopist. Such- methods as
Lister's were too troublesome and too difficult for general
and widespread application; but now that monosporous
cultivation of Bacteria has been rendered a comparatively
simple and certain affair, we may expect immediate and
immense advances in our knowledge of the whole series
of phenomena to which the Bacteria are related.
Amongst problems which require immediate investigation
by the new method are the distinctive properties of the various
kinds of Bacteria which may infest the wounds of surgical
practice, and their specific susceptibility to the destructive
influence of carbolic acid and other antiseptics ; further, the
possibility of isolating a specific Bacterium in contagious
diseases not yet investigated: and (of great physiological
interest) the isolation and investigation of the properties
of the specific Bacterium of the ammoniacal fermentation of
urine.
Dr. Koch and his assistants will, no doubt, shortly publish
a detailed account of |;he researches which they haye been.
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engaged in during the past year, and will give particulars
as to the methods of investigation employed by them, which
had not (we believe), previously to the meeting of the
International Medical Congress, been given to the public.
A remarkable negativfe result obtained by Dr. Koch, so
far as his experiments With the new method of monosporous
culture have yet extended, is, that there is no transition of
forms amongst) at any rate, the pathogenous Bacteria—a
Micrococcus produces Micrococei, and no other form; a
Bacillus produces only Bacilli; a biscuit-shaped form (Bacterium proper) only biscuit-Shaped forms; a Spirillum only
Spirilla. Moreover, the fades of the discoid'dl or spherical
mass formed by a growth, as seen with a low power excavating its way in the gelatine is characteristic of species, so that
a practised observer can, in some cases, recognise a particular
Bacillus or Micrococcus by the naked-eye appearance of the
growth alone, or, at any rate, without actually observing the
individual units of the growth.—L.