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Edward Payson Roe - "Opening a Chestnut Burr"

Jerome K. Jerome - "Idle Ideas in 1905"

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Books: The Harvard Classics Volume 38

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The following days the organisms multiplied, the deposit of
tartrate gradually disappeared, and a sensible ferment action was
manifest on the surface, and throughout the bulk of the liquid.
The deposit seemed lifted up in places, and was covered with a
layer of dark-grey colour, puffed up, and having an organic and
gelatinous appearance. For several days, in spite of this action
in the deposit, we detected no disengagement of gas, except when
the flask was slightly shaken, in which case rather large bubbles
adhering to the deposit rose, carrying with them some solid
particles, which quickly fell back again, whilst the bubbles
diminished in size as they rose, from being partially taken into
solution, in consequence of the liquid not being saturated. The
smallest bubbles had even time to dissolve completely before they
could reach the surface of the liquid. In course of time the
liquid was saturated, and the tartrate was gradually displaced by
mammillated crusts, or clear, transparent crystals of carbonate
of lime at the bottom and on the sides of the vessel.

The impregnation took place on February 10th, and on March 15th
the liquid was nearly saturated. The bubbles then began to lodge
in the bent part of the exit-tube, at the top of the flask. A
glass measuring-tube containing mercury was now placed with its
open end over the point of the exit-tube under the mercury in the
trough, so that no bubble might escape. A steady evolution of gas
went on from the 17th to the 18th, 17.4 cc. (1.06 cubic inches)
having been collected. This was proved to be nearly absolutely
pure carbonic acid, as indeed might have been suspected from the
fact that the evolution did not begin before a distinct
saturation of the liquid was observed. [Footnote: Carbonic add
being considerably more soluble than other gases possible under
the circumstances.--ED.]

The liquid, which was turbid on the day after its impregnation,
had, in spite of the liberation of gas, again become so
transparent that we could read our handwriting through the body
of the flask. Notwithstanding this, there was still a very active
operation going on in the deposit, but it was confined to that
spot. Indeed, the swarming vibrios were bound to remain there,
the tartrate of lime being still more insoluble in water
saturated with carbonate of lime than it is in pure water. A
supply of carbonaceous food, at all events, was absolutely
wanting in the bulk of the liquid. Every day we continued to
collect and analyze the total amount of gas disengaged. To the
very last it was composed of pure carbonic acid gas. Only during
the first few days did the absorption by the concentrated potash
leave a very minute residue. By April 26th all liberation of gas
had ceased, the last bubbles having risen in the course of April
23rd. The flask had been all the time in the oven, at a
temperature between 25 degrees C. and 28 degrees C. (77 degrees
F. and 83 degrees F.). The total volume of gas collected was
2.135 litres (130.2 cubic inches). To obtain the whole volume of
gas formed we had to add to this what was held in the liquid in
the state of acid carbonate of lime. To determine this we poured
a portion of the liquid from the flask into another flask of
similar shape, but smaller, up to the gaugemark on the neck.
[Footnote: We had to avoid filling the small flask completely,
for fear of causing some of the liquid to pass on to the surface
of the mercury in the measuring tube. The liquid condensed by
boiling forms pure water, the solvent affinity of which for
carbonic acid, at the temperature we employ, is well known. This
smaller flask had been previously filled with carbonic acid. The
carbonic acid of the fermented liquid was then expelled by means
of heat, and collected over mercury. In this way we found a
volume of 8.322 litres (508 cubic inches) of gas in solution,
which, added to the 2.135 litres, gave a total of 10.457 litres
(638.2 cubic inches) at 20 degrees and 760 mm., which, calculated
to 0 degrees, C. and 760 mm. atmospheric pressure (32 degrees F.
and 30 inches) gave a weight of 19.700 grammes (302.2 grains) of
carbonic acid.

Exactly half of the lime in the tartrate employed got used up in
the soluble salts formed during fermentation; the other half was
partly precipitated in the form of carbonate of lime, partly
dissolved in the liquid by the carbonic acid. The soluble salts
seemed to us to be a mixture or combination of 1 equivalent of
metacetate of lime, with 2 equivalents of the acetate, for every
10 equivalents of carbonic acid produced, the whole corresponding
to the fermentation of 3 equivalents of neutral tartrate of lime.
[Footnote: The following is a curious consequence of these
numbers and of the nature of the products of this fermentation.
The carbonic acid liberated being quite pure, especially when the
liquid has been boiled to expel all air from the flask, and
capable of perfect solution, it follows that the volume of liquid
being sufficient and the weight of tartrate suitably chosen--we
may set aside tartrate of lime in an insoluble, crystalline
powder, alone with phosphates at the bottom of a closed vessel
full of water, and find soon afterwards in their place carbonate
of lime, and in the liquid soluble salts of lime, with a mass of
organic matter at the bottom, without any liberation of gas or
appearance of fermentation ever taking place, except as far as
the vital action and transformation in the tartrate are
concerned. It is easy to calculate that a vessel or flask of five
litres (rather more than a gallon) would be large enough for the
accomplishment of this remarkable and singularly quiet
transformation, in the case of 50 grammes (767 grains) of
tartrate of lime.]. This point, however, is worthy of being
studied with greater care: the present statement of the nature of
the products formed is given with all reserve. For our point,
indeed, the matter is of little importance, since the equation of
the fermentation does not concern us.

After the completion of fermentation there was not a trace of
tartrate of lime remaining at the bottom of the vessel: it had
disappeared gradually as it got broken up into the different
products of fermentation, and its place was taken by some
crystallized carbonate of lime--the excess, namely, which had
been unable to dissolve by the action of the carbonic acid.
Associated, moreover, with this carbonate of lime there was a
quantity of some kind of animal matter, which, under the
microscope, appeared to be composed of masses of granules mixed
with very fine filaments of varying lengths, studded with minute
dots, and presenting all the characteristics of a nitrogenous
organic substance. [Footnote: We treated the whole deposit with
dilute hydrochloric acid, which dissolved the carbonate of lime
and the insoluble phosphates of calcium and magnesium; afterwards
filtering the liquid through a weighed filter paper. Dried at 100
degrees C. (212 degrees F.), the weight of the organic matter
thus obtained was 0.54 gramme (8.3 grains), which was rather more
than 1/200 of the weight of fermentable matter.] That this was
really the ferment is evident enough from all that we have
already said. To convince ourselves more thoroughly of the fact,
and at the same time to enable us to observe the mode of activity
of the organism, we instituted the following supplementary
observation. Side by side with the experiment just described, we
conducted a similar one, which we intermitted after the
fermentation was somewhat advanced, and about half of the
tartrate dissolved. Breaking off with a file the exit-tube at the
point where the neck began to narrow off, we took some of the
deposit from the bottom by means of a long straight piece of
tubing, in order to bring it under microscopical examination. We
found it to consist of a host of long filaments of extreme
tenuity, their diameter being about 1/1000th of a millimetre
(0.000039 in.); their length varied, in some cases being as much
as 1/20th of a millimetre (0.0019 in.). A crowd of these long
vibrios were to be seen creeping slowly along, with a sinuous
movement, showing three, four, or even five flexures. The
filaments that were at rest had the same aspect as these last,
with the exception that they appeared punctuate, as though
composed of a series of granules arranged in irregular order. No
doubt these were vibrios in which vital action had ceased,
exhausted specimens which we may compare with the old granular
ferment of beer, whilst those in motion may be compared with
young and vigorous yeast. The absence of movement in the former
seems to prove that this view is correct. Both kinds showed a
tendency to form clusters, the compactness of which impeded the
movements of those which were in motion. Moreover, it was
noticeable that the masses of these latter rested on tartrate not
yet dissolved, whilst the granular clusters of the others rested
directly on the glass, at the bottom of the flask, as if, having
decomposed the tartrate, the only carbonaceous food at their
disposal, they had then died on the spot where we captured them,
from inability to escape, precisely in consequence of that state
of entanglement which they combined to form, during the period of
their active development. Besides these we observed vibrios of
the same diameter, but of much smaller length, whirling round
with great rapidity, and darting backwards and forwards; these
were probably identical with the longer ones, and possessed
greater freedom of movement, no doubt in consequence of their
shortness. Not one of these vibrios could be found throughout the
mass of the liquid.

[Illustration with caption: Figure 10.]

We may remark that as there was a somewhat putrid odour from the
deposit in which the vibrios swarmed, the action must have been
one of reduction, and no doubt to this fact was due the greyish
coloration of the deposit. We suppose that the substances
employed, however pure, always contain some trace of iron, which
becomes converted into the sulphide, the black colour of which
would modify the originally white deposit of insoluble tartrate
and phosphate.

But what is the nature of these vibrios? We have already said
that we believe that they are nothing but the ordinary vibrios of
putrefaction, reduced to a state of extreme tenuity by the
special conditions of nutrition involved in the fermentable
medium used; in a word, we think that the fermentation in
question might be called putrefaction of tartrate of lime. It
would be easy enough to determine this point by growing the
vibrios of such fermentation in media adapted to the production
of the ordinary forms of vibrio; but this is an experiment which
we have not ourselves tried.

One word more on the subject of these curious beings. In a great
many of them there appears to be something like a clear spot, a
kind of bead, at one of their extremities. This is an illusion
arising from the fact that the extremity of these vibrios is
curved, hanging downwards, thus causing a greater refraction at
that particular point, and leading us to think that the diameter
is greater at that extremity. We may easily undeceive ourselves
if we watch the movements of the vibrio, when we will readily
recognize the bend, especially as it is brought into the vertical
plane passing over the rest of the filament. In this way we will
see the bright spot, THE HEAD, disappear, and then reappear.

The chief inference that it concerns us to draw from the
preceding facts is one which cannot admit of doubt, and which we
need not insist on any further--namely that vibrios, as met with
in the fermentation of neutral tartrate of lime, are able to live
and multiply when entirely deprived of air.




V.--ANOTHER EXAMPLE OF LIFE WITHOUT AIR--FERMENTATION OF LACTATE
OF LIME


As another example of life without air, accompanied by
fermentation properly so called, we may lastly cite the
fermentation of lactate of lime in a mineral medium.

In the experiment described in the last paragraph, it will be
remembered that the ferment liquid and the germs employed in its
impregnation came in contact with air, although only for a very
brief time. Now, notwithstanding that we possess exact
observations which prove that the diffusion of oxygen and
nitrogen in a liquid absolutely deprived of air, so far from
taking place rapidly, is, on the contrary, a very slow process
indeed; yet we were anxious to guard the experiment that we are
about to describe from the slightest possible trace of oxygen at
the moment of impregnation.

We employed a liquid prepared as follows: Into from 9 to 10
litres (somewhat over 2 gallons) of pure water the following
salts [Footnote: Should the solution of lactate of lime be
turbid, it may be clarified by filtration, after previously
adding a small quantity of phosphate of ammonia, which throws
down phosphate of lime. It is only after this process of
clarification and filtration that the phosphates of the formula
are added. The solution soon becomes turbid if left in contact
with air, in consequence of the spontaneous formation of
bacteria.] were introduced successively, viz:

Pure lactate of lime. ... . ... . ... . ... . .. 225 grammes
Phosphate of ammonia. ... . ... . ... . ... . .. 0.75 grammes
Phosphate of potassium. ... . ... . ... . ... .. 0.4 grammes
Sulphate of magnesium. ... . ... . ... . ... ... 0.4 grammes
Sulphate of ammonia. ... . ... . ... . ... . ... 0.2 grammes
(1 gramme = 15.43 grains.)

[Illustration with caption: Fig. 11]

On March 23rd, 1875, we filled a 6 litre (about 11 pints) flask,
of the shape represented in FIG. 11, and placed it over a heater.
Another flame was placed below a vessel containing the same
liquid, into which the curved tube of the flask plunged. The
liquids in the flask and in the basin were raised to boiling
together, and kept in this condition for more than half-an-hour,
so as to expel all the air held in solution. The liquid was
several times forced out of the flask by the steam, and sucked
back again; but the portion which re-entered the flask was always
boiling. On the following day when the flask had cooled, we
transferred the end of the delivery tube to a vessel full of
mercury and placed the whole apparatus in an oven at a
temperature varying between 25 degrees C. and 30 degrees C. (77
degrees F. and 86 degrees F.) then, after having refilled the
small cylindrical tap-funnel with carbonic acid, we passed into
it with all necessary precautions 10 cc. (0.35 fl. oz) of a
liquid similar to that described, which had been already in
active fermentation for several days out of contact with air and
now swarmed with vibrios. We then turned the tap of the funnel,
until only a small quantity of liquid was left, just enough to
prevent the access of air. In this way the impregnation was
accomplished without either the ferment-liquid or the ferment-
germs having been brought in contact, even for the shortest
space, with the external air. The fermentation, the occurrence of
which at an earlier or later period depends for the most part on
the condition of the impregnating germs, and the number
introduced in the act, in this case began to manifest itself by
the appearance of minute bubbles from March 29th. But not until
April 9th did we observe bubbles of larger size rise to the
surface. From that date onward they continued to come in
increasing number, from certain points at the bottom of the
flask, where a deposit of earthy phosphates existed; and at the
same time the liquid, which for the first few days remained
perfectly clear, began to grow turbid in consequence of the
development of vibrios. It was on the same day that we first
observed a deposit on the sides of carbonate of lime in crystals.

It is a matter of some interest to notice here that, in the mode
of procedure adopted, everything combined to prevent the
interference of air. A portion of the liquid expelled at the
beginning of the experiment, partly because of the increased
temperature in the oven and partly also by the force of the gas,
as it began to be evolved from the fermentative action, reached
the surface of the mercury, where, being the most suitable medium
we know for the growth of bacteria, it speedily swarmed with
these organisms. [Footnote: The naturalist Cohn, of Breslau, who
published an excellent work on bacteria in 1872, described, after
Mayer, the composition of a liquid peculiarly adapted to the
propagation of these organisms, which it would be well to compare
for its utility in studies of this kind with our solution of
lactate and phosphates. The following is Cohn's formula:

Distilled water. ... . ... . ... . ..20 cc. (0.7 fl. oz.)
Phosphate of potassium. ... . ... ...0.1 gramme (1.5 grains)
Sulphate of magnesium. ... . ... . 0.1 gramme (1.5 grains)
Tribasic phosphate of lime. ... ... 0.01 gramme (0.15
grain)
Tartrate of ammonia. ... . ... . ... 0.2 gramme (3 grains)

This liquid, the author says, has a feeble acid reaction and
forms a perfectly clear solution.] In this way any passage of
air, if such a thing were possible, between the mercury and the
sides of the delivery-tube was altogether prevented, since the
bacteria would consume every trace of oxygen which might be
dissolved in the liquid lying on the surface of the mercury.
Hence it is impossible to imagine that the slightest trace of
oxygen could have got into the liquid in the flask.

Before passing on we may remark that in this ready absorption of
oxygen by bacteria we have a means of depriving fermentable
liquids of every trace of that gas with a facility and success
equal or even greater than by the preliminary method of boiling.
Such a solution as we have described, if kept at summer heat,
without any previous boiling, becomes turbid in the course of
twenty-four hours from a SPONTANEOUS development of bacteria; and
it is easy to prove that they absorb all the oxygen held in
solution. [Footnote: On the rapid absorption of oxygen by
bacteria, see also our Memoire of 1872, sur les Generations dites
Spontanees, especially the note on page 78.] If we completely
fill a flask of a few litres capacity (about a gallon) (Fig. 9)
with the liquid described, taking care to have the delivery-tube
also filled, and its opening plunged under mercury, and, forty-
eight hours afterwards by means of a chloride of calcium bath,
expel from the liquid on the surface of the mercury all the gas
which it holds in solution, this gas, when analyzed, will be
found to be composed of a mixture of nitrogen and carbonic acid
gas, WITHOUT THE LEAST TRACE OF OXYGEN. Here, then, we have an
excellent means of depriving the fermentable liquid of air; we
simply have completely to fill a flask with the liquid, and place
it in the oven, merely avoiding any addition of butyric vibrios,
before the lapse of two or three days. We may wait even longer;
and then, if the liquid does become impregnated spontaneously
with vibrio germs, the liquid, which at first was turbid from the
presence of bacteria, will become bright again, since the
bacteria, when deprived of life, or, at least, of the power of
moving, after they have exhausted all the oxygen in solution,
will fall inert to the bottom of the vessel. On several occasions
we have determined this interesting fact, which tends to prove
that the butyric vibrios cannot be regarded as another form of
bacteria, inasmuch as, on the hypothesis of an original relation
between the two productions, butyric fermentation ought in every
case to follow the growth of bacteria.

We may also call attention to another striking experiment, well
suited to show the effect of differences in the composition of
the medium upon the propagation of microscopic beings. The
fermentation which we last described commenced on March 27th and
continued until May 10th; that to which we are now to refer,
however, was completed in four days, the liquid employed being
similar in composition and quantity to that employed in the
former experiment. On April 23, 1875, we filled a flask of the
same shape as that represented in Fig. 11, and of similar
capacity, viz., 6 litres, with a liquid composed as described at
page 69. This liquid had been previously left to itself for five
days in large open flasks, in consequence of which it had
developed an abundant growth of bacteria. On the fifth day a few
bubbles, rising from the bottom of the vessels, at long
intervals, betokened the commencement of butyric fermentation, a
fact, moreover, confirmed by the microscope, in the appearance of
the vibrios of this fermentation in specimens of the liquid taken
from the bottom of the vessels, the middle of its mass, and even
in the layer on the surface that was swarming with bacteria. We
transferred the liquid so prepared to the 6 litre flask arranged
over the mercury. By evening a tolerably active fermentation had
begun to manifest itself. On the 24th this fermentation was
proceeding with astonishing rapidity, which continued during the
25th and 26th. During the evening of the 26th it slackened, and
on the 27th all signs of fermentation had ceased. This was not,
as might be supposed, a sudden stoppage due to some unknown
cause; the fermentation was actually completed, for when we
examined the fermented liquid on the 28th we could not find the
smallest quantity of lactate of lime. If the needs of industry
should ever require the production of large quantities of butyric
acid, there would, beyond doubt, be found in the preceding fact
valuable information in devising an easy method of preparing that
product in abundance. [Footnote: In what way are we to account
for so great a difference between the two fermentations that we
have just described? Probably it was owing to some modification
effected in the medium by the previous life of the bacteria, or
to the special character of the vibrios used in impregnation. Or,
again, it might have been due to the action of the air, which,
under the conditions of our second experiment, was not absolutely
eliminated, since we took no precaution against its introduction
at the moment of filling our flask, and this would tend to
facilitate the multiplication of anaerobian vibrios, just as,
under similar conditions, would have been the case if we had been
dealing with a fermentation by ordinary yeast.]

Before we go any further, let us devote some attention to the
vibrios of the preceding fermentations.

On May 27th, 1862, we completely filled a flask capable of
holding 2.780 litres (about five pints) with the solution of
lactate and phosphates. [Footnote: In this case the liquid was
composed as follows: A saturated solution of lactate of lime, at
a temperature of 25 degrees C. (77 degrees F.), was prepared,
containing for every 1OO cc. (3 1/2 fl. oz.) 25.65 grammes (394
grains) of the lactate, C6 H5 O5 Ca O (NEW NOTATION, C6 H10 Ca
O6) This solution was rendered very clear by the addition of 1
gramme of phosphate of ammonia and subsequent filtration. For a
volume of 8 litres (14 pints) of this clear saturated solution we
used (1 gramme = 15.43 grains):

Phosphate of ammonia. ... . ... . ... . ... 2 grammes
Phosphate of potassium. ... . ... . ... . ... 1 gramme
Phosphate of magnesium. ... . ... . ... . ... 1 gramme
Sulphate of ammonia. ... . ... . ... . ... 0.5 gramme]

We refrained from impregnating it with any germs. The liquid
became turbid from a development of bacteria and then underwent
butyric fermentation. By June 9th the fermentation had become
sufficiently active to enable us to collect in the course of
twenty-four hours, over mercury, as in all our experiments, about
100 cc. (about 6 cubic inches) of gas. By June 11th, judging from
the volume of gas liberated in the course of twenty-four hours,
the activity of the fermentation had doubled. We examined a drop
of the turbid liquid. Here are the notes accompanying the sketch
(Fig. 12) as they stand in our note-book: "A swarm of vibrios, so
active in their movements that the eye has great difficulty in
following them. They may be seen in pairs throughout the field,
apparently making efforts to separate from each other. The
connection would seem to be by some invisible, gelatinous thread,
which yields so far to their efforts that they succeed in
breaking away from actual contact, but yet are, for a while, so
far restrained that the movements of one have a visible effect on
those of the other. By and by, however, we see a complete
separation effected, and each moves on its separate way with an
activity greater than it ever had before."

[Illustration with caption: Fig. 12]

One of the best methods that can be employed for the

microscopical examination of these vibrios, quite out of contact
with air, is the following. After butyric fermentation has been
going on for several days in a flask, (Fig. 13), we connect this
flask by an india-rubber tube with one of the flattened bulbs
previously described, which we then place on the stage of the
microscope (Fig. 13). When we wish to make an observation we
close, under the mercury, at the point B, the end of the drawn-
out and bent delivery-tube. The continued evolution of gas soon
exerts such a pressure within the flask, that when we open the
tap R, the liquid is driven into the bulb LL, until it becomes
quite full and the liquid flows over into the glass V. In this
manner we may bring the vibrios under observation without their
coming into contact with the least trace of air, and with as much
success as if the bulb, which takes the place of an object glass,
had been plunged into the very centre of the flask. The movements
and fissiparous multiplication of the vibrios may thus be seen in
all their beauty, and it is indeed a most interesting sight. The
movements do not immediately cease when the temperature is
suddenly lowered, even to a considerable extent, 15 degrees C.
(59 degrees F.) for example; they are only slackened.
Nevertheless, it is better to observe them at the temperatures
most favourable to fermentation, even in the oven where the
vessels employed in the experiment are kept at a temperature
between 25 degrees C. and 30 degrees C. (77 degrees F. and 86
degrees F.).