Dujardin, in 1841, in his Natural History of the Zoophytes, accepted the
classification of Eherenberg, except that he unites the spirillum and
spirochœte, calling them all spirillum. Up to this time all bacteria had
been considered animals, but a close study of their life history and
habitat by those who followed declared them to belong to the vegetable
kingdom, and as such they are accepted to-day.
In 1853, M. Chas. Robin pointed out the relationship of bacteria to
Leptothrix, a form of fungi closely allied to that of mildew; and M.
Davaine, in 1868, clearly demonstrated their relationship to the
vegetable world. From this time the progress of bacteriological
investigation has made rapid strides. Prof. Pasteur in the organisms of
fermentation and the role they play therein; Davaine and Hallier in
demonstrating the specific relationship of bacteria with charbon or
anthrax; and the work of Koch, Nageli, Kohn, Bilroth, Miguel, Burdon,
Sanderson, Klein, Weigert, Klebs, Ehrlich, Sternberg, and many others,
are too recent to require special mention.
Few have more than the faintest conception of the minuteness of these
organisms. Prof. Cohn, justifying himself for the unscientific method of
comparison which he uses in class instruction by Prof. Tyndall’s
argument on the scientific use of the imagination, says he compares man
to the cheese mite, as the Strasburg cathedral to a sparrow. Of the
animalcules which Leeuwenhoek discovered, they are to man as the bee is
to the horse. As improvements have been made in microscopes, just so
fast have we penetrated into the world of micro-organisms, until now the
proportion between the smallest we can see and man, is as man is to Mont
Blanc.
Of course, with these exceedingly minute structures, nothing can be made
out except points. Among some of the larger forms, a few have been able
to see cellia, and in some cases the growth of the spores; but in the
present state of microscopical optics the work is slow, and progress in
this direction is waiting an advance in the science of optics.
Like all living organisms, bacteria propagate themselves. The most usual
method is by fission or by partition, though Magnin and Cohn have
recorded their observations on the formation of spores and sporangia,
and I have myself witnessed the last named method. It is of importance
to note that while the bacterium is killed by continued exposure to
temperatures of freezing or 176° F., the spores will germinate after
protracted exposure to temperature as high as 205° F. or as low as °123
F. These spores will also withstand complete desiccation, and it is in
this form, mixed with the air we breathe and move in, that present the
conditions from which all zymotic diseases originate. Miguel has shown
that, while the air contains very few adult bacteria, it contains
myriads of their spores. To the researches of Koch, Pasteur, and others,
we are indebted for the certain information that, while these
omnipresent germs withstand such vicissitudes of temperature, they
require certain food for their maintenance; and though we cannot as yet
tell what that food is, we know that when nutrient material is submitted
to their action they thrive for a time, and when the particular
principle which supports them is exhausted they die. This is
particularly true of pathogenic germs, and the accepted theory of the
bacillus tuberculosis, or the germ of consumption, is a good
illustration. It has been demonstrated by Koch, Klein, Pasteur,
Frankell, Sternberg, and others, that they require some product of
inflammatory action for their support within the body of their victim.
This is also true of cholera, at least so far as their dietary
requirements are concerned. The animal cannot be infected with
tuberculosis by merely introducing the germ-laden material into the
stomach or upon any of the mucous membranes; but if an inflammatory
condition be present, either due to the puncture of the introducing
needle or scalpel, or to extraneous causes, such as a catarrhal
condition of the lungs, tuberculosis is as sure to follow as the sun is
to rise again.
The human mind can scarcely comprehend the enormous numbers of these
omnipresent atoms without a resort again to the legitimate use of the
imagination. A computation of the increase from a parent germ shows as
follows: We know that the parent grows until it reaches double its
original size, when it constricts itself in the middle like a figure
eight and breaks into two individuals. Each of these divides again, and,
on account of the rapidity with which this is done, we find them usually
in chains or squares. The warmer the air, the faster this proceeds, and
at the temperature of the body the entire life history of a germ, from
the time of fission of the parent to the time of his own subdivision
into two new individuals occupies less than one hour. This gives us a
known quantity for our problem. Let us look at the result. From a single
germ increasing by the power of two each hour, we have at the end of
twenty-four hours 16,777,220; at the end of two days the number has
increased to 281 billions, and in three days to the enormous number of
48 trillions, and in one week the number can only be expressed by
figures of fifty-places. In order to make this number comprehensible,
let us figure the mass and weight of this, the result of a single
bacterium. A single Bacterium Termo has an average width of 1/1,000 mm.
A cubic mm. would therefore contain six hundred and thirty-three
millions, and in one day would be one-fortieth full. At the end of the
following day there would be required 444,570 such cubes to contain the
product of the parent, or say half a litre. Suppose the seas of the
earth cover two-thirds of its surface with a mean depth of one mile, the
aqueous product would be 929 million miles. Now, our parent germ and its
product would in five days completely fill this space. More wonderful
still is a gravimetric estimation. Suppose we call the specific weight
of the parent germ the same as water, which cannot be far from right, it
would appear that the parent weighs, or his equal bulk of water weighs,
136 millionths of a gramme; in forty-eight hours, 442 grammes; in three
days, nearly 7½ million kilograms; and, inside of thirty days, the
weight of the earth itself.
Prof. Cohn, in offering these figures, says: “I don’t consider this idle
play; without it we can form no conception of not only the enormous
increase, but the tremendous destruction of these germs which is going
on around us. Food is lacking to support more than a comparatively small
proportion of the product of the parent, and, as it is demonstrated that
they feed from their environment, one can readily understand that
without a constant supply a given infectious germ will with its
followers soon destroy its nidus or perish from starvation.”
Our breweries demonstrate the truth of this hypothesis; for, in
twenty-four hours, a single yeast cell, which is 8/1,000 mm. in
diameter, will yield one hundred-weight of yeast.
I have endeavored to present the subject in a condensed but general way
without burdening you with technical details of species, genera or life
history. The subject is a vast one and to which the best minds of the
scientific world are devoting themselves. To those who are or may become
interested in bacteriology and particularly to those who study the
relation of these germs to disease, is held forth the reward which is
sure to come to those who work persistently and intelligently.
The method which I shall employ to-night is eclectic. Doubtless each
investigator will find fault with some parts of the process and perhaps
suggest a better one. The following, however, has in my hands worked
well and given entire satisfaction, so far as I know, to those who were
and are most interested.
The apparatus necessary is as follows:
One two-inch glass funnel.
One package filter papers to fit same.
Four medium size test tubes.
Two glass or porcelain staining glasses.
One glass or agate mortar and pestle.
One cover holder.
One pair pincetts.
One alcohol lamp.
Package of wooden toothpicks.
The cover holder may be easily made by taking a piece of thin platinum,
two inches long and one-eighth wide, splitting one end for half an inch
up and bending into a Y shape, then lashing to a small handle (I use a
match). This little tool is most convenient for floating cover glasses
in staining fluids.
The reagents necessary are as follows:
A five per cent. solution of nitric acid in alcohol (95 per cent.).
Saturated alcoholic solution of fuchsine.
Saturated alcoholic solution of methyl blue.
Small quantity of alcohol, 80 to 95 per cent.
Pure colorless aniline oil (anilin).
The method is as follows:
First pour enough aniline into a test tube to cover the bottom and half
fill with water, shake violently for two minutes, and filter through
funnel, which has previously had wet filter paper fitted. It is
essential that the filter paper be saturated with water, else the
aniline oil will separate during filtration. Our next step is to deposit
specimen of sputum in mortar (if very viscid, add a few drops of water),
and triturate thoroughly in order to break up encapsulated colonies, and
distribute evenly through the specimen.
Now remove an amount which will just cover end of toothpick, and deposit
it on a previously cleaned cover glass, which should not be over 1/100
inch thick, and thinner if possible; immediately cover with another
cover glass, allowing sputum to spread by capillarity or slight
pressure, and separate by sliding apart, and put aside to dry without
heat. I have found that specimens dried without heat (and consequent
coagulation of albumen) will show a much larger number of bacilli than
when heat is used. I believe this is due to the fact that the fuchsine
penetrates more thoroughly through the albumen when not coagulated, or
that when it is coagulated by heat it to a greater or less extent it
protects them from the action of the stain. While the covers are drying
we will pour out a sufficient quantity of the aniline water, which by
this time has filtered into one of the staining glasses, and add one or
two drops (not more) fuchsine solution. Now, placing one of the cover
glasses on our cover holder, sputum side down, we lower it into the
staining fluid and withdraw holder from the side, and repeat the
operation for the other cover glass. It is my habit to allow the covers
to remain in this solution for at least eight hours or over night. The
time may be reduced to ten or fifteen minutes by heating the red stain
to about 140 or 150 F., but the result is not so brilliant, nor is it
sure, as I have frequently failed to find the bacilli by the short
method, but have been able to demonstrate their presence by the long
one.
At the end of either of the above periods of time, the cover glass is
lifted out of the staining solution and, without washing, immersed in
our five per cent. solution of nitric acid and alcohol. It is this part
of the process, if any, which will give trouble, as the time of
immersion is governed by the thickness and general character of the
sputum. My custom is to hold the first cover immersed until the color
has just disappeared, or say fifteen seconds, and the second five
seconds longer; but a very little experience will remove any difficulty
from over-decolorizing.
From the decolorizing solution they are immediately immersed in water
and thoroughly washed, when they may be again floated in the
contra-stain, which is prepared by filling the other staining glass with
water to which a few drops (three or four) of our methyl blue has been
added. They should remain here for from five to eight minutes, when they
are again removed with the pincetts, and a few drops of alcohol poured
over them to wash off the surplus stain. Again wash in clean water, and
dry by gentle heat (which will now do no harm) over the alcohol lamp,
and place sputum side up on table.
A very small drop of thin benzole balsam is now placed in the centre of
each cover, and a cleansed slide gently lowered over one in such a
position that both covers may be mounted on a single slide. As soon as
the slide has been sufficiently lowered to come in contact with the drop
of balsam, it spreads by capillarity, and draws the cover close to the
slide without the slightest danger from air bubbles being engaged, and
the slide may at once be inspected by a _dry_ objective.
I have found it necessary to use an objective at least as high as
one-fifth or one-sixth, with central illumination without diaphragm, as
cases will frequently occur where the staining is so faint, that with a
lower power they will escape observation, though a good, wide angle,
four-tenths inch, will show them well when strongly stained.
I have endeavored to explain the method with perhaps too strict a regard
to detail, but am sure that one who follows the various steps once or
twice cannot fail to acquire the necessary technique without occupying
more than fifteen minutes of working time; that is to say, five minutes
to the first staining, and then the following morning to prepare and
mount for observation.