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results depended upon a defective mode of conducting the experiments. The pro-
cess employed in some of them was identical with that employed by M. Pasteur
himself, and in others, with that made use of by M. Milne Edwards in his one exper-
iment (see his Lefona sur la PhyaiologU et VAnatomie Oomparh, t. viii, pp. 260,
269.) When flasks, holding organic fluids and air, are hermetically sealed and
boiled for firom fifteen minutes to three or four hours, and infusoria make their ap>
pearance nevertheless, the experiment cannot be aillcd a faulty one, so long as only
these results are claimed. The recent experiments of Dr. G. W. Child of Oxfora,
£ogland, Mid those reported in this communication are a sufficient answer to the
criticisms of M. Edwards. The experiment of Pouchet recorded in his Nauoeltes
BxperimcB^, Paris, 1864, p. 224, is conclusive as to possibility of Infusoria appearing
m boiled aolotions exposed to pare air.

Digitized by


158 /. Wyman on living organisms in heated water.

Series A. The flasks used in this series had a capacity of
about 800 c. c. and had % long and slender neck ; this was drawn
outy about an inch from the end to a size which could easily be
melted in a flame and in some instances the stem of a clay pipe,
with a calibre of 0*08 inch, and in others, a copper tube 018
inch in diameter and filled with fine wires, was cemented into
the mouth. The fluid in the flasks was boiled and thus the con-
tained air expelled and replaced by steam. After the boiling
had been continued sufficiently long, the whole was then allowed
to cool slowly, the air to reenter through the tube, which last
was kept meanwhile at a red heat. After the flask was cold it
was hermetically sealed, the heat of the tube being carefully
kept up until this was accomplished. Thus a boiled organic
solution was obtained, in contact only with air which had been
purified by heat.

Mcp. I. — The contents of the flask were a few grains of meat
and sugar, and 20 c. c. of water boiled 25 minutes. An infuso-
rial film was formed on the 5th day ; the flask was opened on
the 81st. Vibrios were found in large numbers.

EScp. n. — 50 c. c. of beef-juice and water were boiled twenty
minutes. On the 4th day a thick infusorial film was formed
over the whole surfiwe of the fluid ; the flask was opened on the
6th day and found to contain Vibrios and Bacteriums.

JEJxp. III. — The flask contained a small piece of beef weighing
about three grammes, and 60 c. c. of water, which were boiled
80 minutes. An infusorial film was formed on the 8d day,
which sank to the bottom and was afterwards replaced by

The flask was opened on the 27th day ; the contents had a
nauseous odor but were not putrid. The muscular fasciculi had
fallen to pieces and the fluid had become slimy and viscid.
Immense numbers of Monads filled the solution. Some feathery-
crystals were noticed, but the most striking circumstance in this
as well as in some other instances in wluch muscle was used,
was the £atty degeneration of the fibres which had taken place
subsequently to the boiling. The fibres were found in many
stages of change, some of them having their contents finely
granular, strisB still seen, while others were filled with oil-glob-
ules, and had no traces of striae left. One specimen was exam-
ined by Dr. Calvin Ellis who was unable to recognize any dif-
ference between the appearances which it presented and those
of ordinary fatty degeneration of the muscles.

JEJrp. IV . — The flask contained very thin wheat paste mixed
with saliva and water ; the boiling was continued 25 minutes.
No film was noticed until the 80th day, the flask was opened on
the 68th day, and the fluid found filled with Monads.

Digitized by


/. Wyman on living organisms in heated water. 159

Exp. y. — A few grains each of meat and sugar in 20 a c. of
water were boiled 26 minutes. An infusorial film appeared on
the 4th day. The flask was opened on the 60th day by Prof.
Henry James Clarke by whom the accompanying figures were
carefully drawn from nature.

Fig. 1. Vibrto baccillusj enlarged two thousand diameters;
these are very numerous and move 2

quickly ; the joints appear to be uni- \ r^

ted by a gelatinous substance which r^ V^V

is quite transparent and move on each ^^ \ ifiNi ^v^^W
other by a series of flexions and ex- 1 \\ ^^1 \ w^

tensions ; the number of segments is >^ ^ >^J

variable. jl

Fig. 2. bacterium; enlarged four 3 - ..\ U,.. - - p ^^^^
thousand diameters. Prof. Clarke has \ ^""^{'^ /^^

given especial attention to these bod- ^^S) (f^j

ies, and has shown that they are some- r'^^/^ir^^^^^^^^*

what more complex than generally ^ A (f^^^^^^ §
supposed. They consist of a dark %flx^

colored oval nucleus, around which is 5 ^^ V>X j

a transparent space, and around this ^ id
a gelatmous envelope of extreme deli- ^

cacy. They are sometimes seen single but generally double;
more than two are rarely united togeuier.

Fig. 3. A linear series of globules enlarged five hundred
diameters ; these have the power of locomotion.

Fig. 4, Excessively faint and colorless bodies of great del-
icacy, and changing their form under the slightest pressure.
They closely resemble the substance described by Virohow as
myetine which not only forms the medullary sheath of the nerves,
but is also found abundantly elsewhere. He says " there scarce-
ly exists a tissue rich in cells in which this suostance does not
exist in large quantities."* ^

Fig. 5. This figure, also drawn by Prof Clarke, represents an
organism commonly seen in nearly all of the experiments. It
is enlarged thirty-five hundred diameters.

Fig. 6, represents the form of the bodies we have called
Monads, and each has a ciliated appendage and is locomotive.

Series B. — The flasks in which the experiments described
below were made, all had the same capacity, viz. about 70 c. c,
and the quantity of solution used in each was from 5 to 8 c. c.
After the fluid to be experimented with had been introduced
they were sealed at the temperature of the room, placed in a
covered vessel containing water, and the whole heated to 212*^
F., and boiled for various periods.

This method corresponds substantially with that of the Need-

* Oellalar Pathology, translated bj Dr. Chance, p. 284. London, 1860.

Digitized by VjOOQIC

160 /. Wyman on limng organisms in heated water.

ham and Spallanzanni. It is in all respects more simple and
easier than- that in which the apparatus is yComplicated with a
heated tube. This last beyond a question destroys all organ-
isms contained in the air which enter the flask through it, but is
without effect on such as ma^ be contained in the solution, or
adhere to the inner surface of the glass. These come in contact
only with boiling water or steam, and unless destroyed by one
or the other of these, would be sufficient to vitiate any exper-
iment, however careful the adjustment and heating, of the tube
may have been. We therefore believe that the tube is an unne-
cessary and useless complication of the apparatus.

Exp. VI. — Six flasks prepared as above, and containing from
5 to 8 c. c. of beef juice and water were boiled 80 minutes. The
albuminous matter was coagulated, but the fluid portion became
perfectly transparent. The contents became turbid and an infu-
sorial film was formed in four of the flasks on the 7th day, and
a few days afterwards in the others.

Exp. V II. — Six flasks, each containing about 1 gram of beef
and 5 c. c. of water were boiled 30 minutes. An infusorial film
was formed on the surface of all of them on or before the 7th day.

Exp. Vni. — A single flask containing a few grains of finely
ground bean flour and 5 c. c. of water was boiled 48 minutes.
The mixture became filled with Vibrios on the Ith day.

Exp. IX. — Six flasks containing beef juice and water were
boiled 80 minutes. A thick infusorial film waa formed in all of
them on or before the 7th day.

Eocp. 'X. — Six flasks containing each a few milligrams of beef
and 5 c. c. of water were boiled 80 minutes. An infusorial film
was formed on all on or before the 17th day.

In the preceding experiments of this series, the boiling was
continued thirty minutes in four, and forty-eight in the fifth.
The following experiments were made for the purpose of ascer-
taining whether prolonged boiling would be attended with a dif-
ferent result.

Exp. XI. — Five flasks, each containing a few grains of beef,
and about 5 c. c. of water were prepared as in the preceding ex-
periments ; one was boiled 82 minutes, one 50 minutes, and the
other three 1 hour and 20 minutes each.

An infusorial film formed in the first on the 2d day, in the
second on the 6th day, in one of those boiled 1 hour and 20
minutes on the 8th, and in the others on the 10th. All were ex-
amined and found to contain Vibrios and Bacieriums; and in ad-
dition the first and second contained the " myeloid substance"
already described, fig 4. Those which were boiled longest were
the latest in developing infusoria ; but of those boiled 1 hour
and 20 minutes, they appeared two days later than in the other.

JSqj. Xn. — Twenty-four flasks each containing about 5 c. c.

Digitized by


/. Wyman on living wgani$m» in heated water. 161

of beef juice were divided into six series of four each, and boiled
for different periods as indicated in the following table ; this
table also gives the day of the appearance of the infusoria in
each of the flasks belonging to a given seriel.


Time boiled.

nay of appearaoce of Infusoria.


0^ 80">

3 4 6


0^ 46"^

4 4 4 4


1^ 00

4 4 4


1^ 16»

4 4


jh 3om



2^ 00

Seven of the flasks produced no infusoria, and the number in
which they did appear becomes less the longer the boiling was

Exp, Xm. — Of seventeen flasks containing beef-juice eleven
were boiled 45 minutes, and 6 were boiled 2 hours. Infusoria
appeared in all of the first series except one, at the end of the
second day, and in the remaining one of this series and in all of
the second on the 8d day.

It will be seen at a glance that Expts. XII and XTTI differ
in their results, and that of four flasks in Ex. XII boiled 1 h.
and 80 m., three produced no infusoria, while in Ex. XIII the
infusoria appearea in all, six of which were boiled two hours.
We have several times met with similar differences in successive
experiments. Sometimes flasks boiled for a single hour became
wholly inert, even when the number of them was quite large.
We can only insist that in the above experiments the flasks were
properly sealed, and that there was no error as to the time for
which the boiling was kept up.

Bxp, XrV. — Twenty flasks containing extract of beef were
treated as follows :

No. of flaaka Id


Daj OD which Inraaoria

eacti •erii'a.


Series I.

5 flasks.


6 6 6 6 6

" II.

6 "


6 6 6 6 7

" in.

6 "



•* IV.

6 "


No flask was opened until the sixth day, though there were
slight indications of infusoria three days previously ; actively
moving Vibrios were found in large numbers. The experiment
was discontinued on the 10th day, as there was no indication of
further change.

JSxp. XV. — Thirty-two flasks containing a boiled solution of
" extract of beef'* were arranged in six series, and boiled for
different times as seen in the following table :

* This is BordsD's oonoeotrated juice of beef, eTaporatsd to a nsarlj solid sob-
■ianot, is fros from tissues, and is entirely soluble.
Ajc Jovs. Scl— taooKD SuuM, Vol. XLIV, Ko. 181.— Sept., 1807.

Digitized by VjOOQ IC


/. Wyman on limng orgoMums in. heated water.




each scries.



Series L



112 2 2

** IL

• 6

1 00

2 2 2 2 2

" ra.


1 30

2 2 2 2 2

" IV.


2 20

12 2 2 2

" V.


S 30

2 2 3 3 9

" VI.


4 00

2 2 2 4 4

(4 4

An infaaorial film formed in all the flasks on the days indica-
ted, except in the one marked doabtfal in series Y, and those of
series YI, in which infosoria appeared on the 4th day. None of
these last had a film, but, as in the one marked doubtful in series
V, had an immense number of minute bodies, which formed a
cloud when the flask was shaken, and were not there when the
experiment was begun. These bodies were spherical, had an
outer wall, and a nucleus which was also a hollow body. The^
were of different sizes, and seemed to be undergoing multiph-
cation by division. Their real nature must be considered doubt-
ful. The films in the other flasks consisted chiefly of Bacteriums.

The preceding experiments show that if the boiling of the
flasks be continued for four hours, as in Exp. XY, the infusoria
may appear nevertheless — though in other cases it has hap-
pened, as in Exp. XII, that life ceased to be manifested if it was
continued only for two hours.

In pushing the experiments still fiirther, we have not found
that mfusona appeared in any instance if the boiling was pro-
longed to five or six houra Several experiments, in which
many flasks were used, were tried, but the result was uniformly
the same. Thus a limit to the development of infusoria in boil-
ing water was reached.

bSCTiON III. JExperimenls to show the effect of boiling water on
living infusoria, — In view of the results given above, viz., the
development of infusoria in sealed flasks, notwithstanding the
precautions taken, the question naturally arises whether the
infusoria which appeared were already in the flask and resisted
the action of the water at 212^ F. If not, then the invasion of
the contents of the flasks by them is easily accounted for, since
experiment shows that even a very few individuals if present
may multiply so rapidly as to fill and make turbid a given solu-
tion in a very short time. But on the other hand, if the infu-
soria perish during the boiling, then any subsequent appearance
of them under the circumstances, must be explained in some
other way than by the common process of reproduction.

Since large numbers of infusoria can be proved at all times to
be floating in the air, we must assume that some are included
in every flask, and since certain organisms live in thermal

Digitized by


/. Wyman on litring arganisnu in heated wattr. 163

waters at a very high temperature, within a few degrees even of
boiling water, the experimenter is called upon to snow whether
water at the boiling point is or is not destructive of life, before
he can venture to offer a theory of the origin of the infusoria in
the flasks. It should not be overlooked that a marked differ-
ence exists between the conditions of life in the flasks and the
thermal springs; in the former, the temperature is suddenly
raised from that of the air to the boiling point, while in the lat-
ter, the organisms inhabiting them have oecome adapted to their
surroundings throuch long periods of time. Furthermore it
must be remembered that in the two cases, we have to deal with
widely different species. It therefore becomes necessary to de*
termine by direct experiment on the species of infusoria found
in the flasks what their powers of resistance are.

Before proceeding to give the result of the experiments we
have made, bearing upon this question, we will notice some of
the statements which are constantly urged in support of the
opinion that infusoria are capable of resisting water of a very
high temperature. Among tnese are the ones relating to the
well known experiments of Doydre and others, in which Tar-
digrades and Kotifers are asserted to have resisted a heat of
248° F. In these cases the important condition that the orffan*
ism was in a dry and not in a moist state is often overlooked.
In truth Doydre himself expressly mentions that in a moist con-
dition they perished at 122° F.*

In the alleged instances of seeds resisting the action of boiling
water, it may be reasonably doubted whether in these the results
have not been misunderstood. Spallanzanni clearly proved that
if the seeds experimented upon by him were soaked previouslv to
the boiling, they did not germinate.f So long as the water does
not penetrate to the germ, this is no more influenced by it than
if the ^rm were exposed to dry air heated to the same degree,
and this it easily resists. Water penetrates the seeds of many
plants and especially of some of the Leguminosce very slowly ;
m the case of those of Okidtichia and Laburnum^ we have found
several days and even weeks necessary for the complete pen-
etration of cold water, though when the water is hot it pen-
etrates much more readily. If therefore the seeds are dry
when immersed, and are boiled for a few minutes only, they
may still germinate. If they are moistened beforehand, the
action of boiling water has been found uniformly fatal. In one
of our experiments twenty-eight seeds of Oleiditchia were soaked

* Annales des Sciencefl NaturelleR, t. iriii, 1842, p. 29. The vhole mibject as
regards the redstance of dried Roiifera to heat, war inTestigated io an exhaustira
manner by a commission consisting of Balbiani, Berthelot, Brown Scquard, Dareste,
Ouillermen and Robin. See Comptes Rendus et Memoirei de la Society de Bi-
ologie, 1860.

\ Opo«x>li, Milaoo, 1836, p. 68.

Digitized by


164 J. Wyman on Htnng arganiims in heated waUr.

until their ooverings became soft and swollen ; one half were
planted at onoe, and the others after haying been boiled five
minutes. None of the boiled ones germinated while all the
others did. Similar experiments with beans and with several
other kinds of seeds ended in a similar manner.

Pasteur has given the result of his own observations on the
effects of high temperature on the spores of some of the differ-
ent kinds of Cryptogams, and states that thej resist when dry
248° to 267'' F., but perish at 266''.* He gives no experiments
in which the spores were exposed to heatra water, or to steam.
He excuses himself, however, on the ground of bein^ unable to
devise a sufficiently rigorous method of experimentation.! We
believe the alleged difficulty can be met, and shall endeavor to
show by a series of comparative experiments that definite re-
sults may be obtained. From the manner in which Pasteur
states his own results, he gives the impression that in view of
the &ct that the spores resist when dry a temperature of 257°
F., it is reasonable to suppose that they will resist 212° when
moist. He does not assert this, but leaves his reader to infer it.
We have tried many experiments upon different kinds of moulds
and yeast plants and have found, as nearly all observers have,
that they perish at 212° F. Every one is familiar with the pro-
cess of " scalding " in domestic economy, which destroys the
kinds of cryptogams ordinarily concemea in fermentation, and
on the efficacy of which the preservation of canned meats and
other articles depends. As regards moulds the following exper-
iment is decisive. Take any number of cups of paste and sow
them all freely with spores ; put one half m a covered vessel
containing a small quantity of water ; boil this for a few min-
utes, thus exposing the paste and spores to steam, and then set
all the cups in similar conditions for light and heat ; none of the
flpores in the series which has been steamed will germinatei
while all others will.

Payen is quoted by many writers as asserting that the q>ores
of Oidium aurantiacum germinated after having been exposed
to 248° F. Pasteur himself expresses the belief that in this
case the temperature has been erroneously determined.^ Pay-
en's own statements are somewhat conflicting. In the one most
commonly quoted he says that spores heated in ordinary dough
to 120° C. (248° F.), may still germinate, but are so altered at
140° C. (284° F.) that they do not germinate.§ In another
and later statement he presents the matter quite differently.
"The spores of Oiitum," he says, "preserve their vegetative
faculty m the soft part of the bread, (la mie du pain) the tem-

* Add. dM Se. Nai« t xvi. 1861, p. 81. f Ibid^ p. 86.

1 Add. des Sc Nat, t xW, 1861, p. 81.

I Ado. de Chim. «t d« Fkyn., 8me aerie, t zxi?, 1848, p. 264.

Digitized by


/• Wyman on living organisms in hsated water. 165

perature of which does not exceed 100^ G. during the baking ;*
while in the crust, the temperature which exceeds 200^ C. (892'^
F.) destroyed the vitality of the Oiciium."f There are here two
conditions of the bread recognized ; the moist interior which
destro;98 the spores at 212° F., and the dry exterior which does
80 at 892° F. Mention has also been made of the germination
of seeds taken from the raspberry jam, as proof that these had
resisted the action of boiling water. But in none of these cases
have we seen any evidence adduced to show that they had
really been boiled. We have not been able to find a single
instance in which seeds or spores or infusoria, excepting only
Vibrios, Bacteriums and Monads, thoroughly moistened before
the experiment, have resisted the prolonged action of boiling

The organisms which we have most commonly met with in
our experiments in flasks, in fact almost the only ones, when the
boiling was prolonged, are Vtbrios, Bacteriums and Monads. The
first and second are without doubt plants allied to the Alga; the
nature of Monads is more obscure. They are all among the
iowest of living organisms. Leaving out of consideration cil-
iated infusoria which perish more easily, our inquiry now is,
at what temperaiurej or after how long an txposwre to the action of
boiling vmter are the Vibrios^ Bacteriume and Monads killed^ avid
by what signs can we know that they are deadf — This question is
a difficult one to answer.

The usual signs of life manifested by the infusoria found in
the flasks are the following: 1st, locomotion in nearly all of the
species ; 2d, growth and reproduction ; 8d, their reaction on the
surrounding fluid, producing fermentation or putrefaction. The
second and third are so intimately associated that the presence
of one is almost a certain indicotion of the other. It a clear
organic solution ferments, or becomes turbid, it may be safely
inferred that living infusoria are present; nevertheless the cor-
rectness of this inference must be tested with the microscope.
The absence of either of the above signs alone cannot be consid-
ered as a proof of death, and under certain circumstances all
signs of life may cease, but the infursoria may still be alive. If
for example, they are developed in a sealed flask, as soon as the
organic matter convertible into infusoria is exhausted their ac-
tivity ceases, and they remain dormant for months; we have
kept them in this way for a year ; but if fresh material is sup-
plied to them, they at once resume their activity. Inactivity in
the presence of organic material suitable for nourishment, and of

* The baking in this case was for the purpoaa of dettrojing the sporet in loaves
in store, and not for those going into the oren for the firat time.

k Precis de Ohim^Indust Paris, 1869, t u, i>. 160.

The assertion of M. Ooste, that encysted infnsoria reaiet the action of boilii^
Trater, has been shown to be incorrect bj the cenclnsive ezperimeota of M. Yictor

Digitized by


IM /. Wyman on living organisms in heated water.

air at the ordinary temperatare, added to the absence of the
other signs of life, must be considered as the best indication ot

1. Arresi of motion. — The temperature at which motion ceases
was determined as follows: a wide-mouthed bottle oontainins
infusoria was set in a water-bath, and a thermometer suspended
with its bulb in the infusorial fluid. The whole was gradually
heated, and drops of the fluid were examined from time to time
with the microscope, until the motions of the infusoria ceased.
The following table gives the result of the examination of fluids
taken from several different sources. The movements of the
Vibrios lasted longer than those of all other kinds, and the tem-
perature given below is that at which the motions of these were
arrested. The motion of all the ciliated infusoria stopped at less
than 180° F.

Motion ceased

In inftitoria from a maoerating tab at
In infufioria from another tub at
In infusoria from beef juice at
In infusoria from a source not stated at

Online LibraryJohn AlmonThe American journal of science and arts → online text (page 70 of 102)