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[From the Proceedinos op the Royal Society, No. 145, 1873.]

FURTHER ORSERVATIONS

ON THE

TEMPERATURE AT WHICH BACTERIA, VIBRIO NES,

AND THETH

SUPPOSED GERMS ARE KILLED

WHEN EXPOSED TO HEAT IN A MOIST STATE ; AND ON THE
CAUSES OF PUTPtEFACTION AND FERMEMTATION.

BY

H. CHARLTON BASTIAN, M.A., M.D., F.R.S.,

PROFKSSOR OF PATHOLOGICAL ANATOMY IN UNIVERSITY COLLEGE,

LONDON.

Whilst a heat of 140° F. (60° C.) appears to be destructive to Bacteria,
Vibriones, and their supposed germs in a neutral saline solution, a heat of
149° or of 158° F. is often necessary to prevent the occurrence of putre-
faction in the inoculated fluids when specimens of organic infusions are
employed. AVhat is the reason of this difference ? Is it owing to the
fact that li\ing organisms are enabled to withstand the destructive in-
fluence of heat better in such fluids than when immersed in neutral saline
solutions ? At first sight it might seem that this was the conclusion to
be drawn. We must not, however, rest satisfied with mere superficial
considerations.

The problem is an interesting one ; yet it should be clearly understood
that its solution, whatever it may be, cannot in the least affect the vali-
dity of the conclusion arrived at in my last paper, viz. that living matter
is certainly capable of arising de novo. We were enabled to arrive at
the conclusion above mentioned regarding Archebiosis by starting with
the undoubted fact that a heat of 158° F. reduces to a state of potential
death all the Bacteria, Vibriones, and their supposed germs which an or-
ganic infusion may contain. The inquiry upon which I now propose to
enter, therefore, touching the degree of heat beloiv this point which may
sufllce to Idll such organisms and their supposed germs in an organic
infusion, and touching the cause of the delayed putrefaction apt to take
place in inoculated organic infusions which have been heated to tempe-
ratures above 140° and below 158° F., is one lying altogether outside the
chain of fact and inference by which the occurrence of Archebiosis is
proved.

It seems to me that the solution of the problems which form the sub-
ject of the present communication can only be safely attempted by
keeping constantly before our minds two main considerations : —

Thus, in the experiments whose results it is now our object to en-
deavour to explain, the fluids have been inoculated with a compoun
c;onsisting partly {a) of living units, and partly (b) of a drop of a solu-
tion of organic matter in a state of molecular change ; so that in many
cases where putrefaction has been initiated after the inoculating com-
pound has been heated to certain temperatures, there is the possibility
that this process of putrefaction may have been induced (in spite of the
death of the organisms and their germs) owing to the influence of 6, the
dissolved organic matter of the inoculating compound ; that is to say

326

Prof. H. C. Bastian on the Heat

the heat to which the mixture has been exposed may have been adequate
to kill all the living units entering into the inoculating compound,
although it may not have been sufficient to prevent its not-living organic
matter acting as a ferment upon the infusion *.

And there are, I think, the very best reasons for concluding that in all
the cases in which turbidity has occurred after the organic mixtures have
been subjected to a heat of 140° 1\ (60° C.) and upwards, this turbidity
has been due, not to the survival of the living units, but rather to the
fact that the mere dead organic matter of the inoculating compound has
acted upon the more unstable organic infusions in a way which it was
not able to do upon the boiled saline fluids.

In order more fully to explain the grounds upon which this conclusion
is based, it will now be necessary to recast the results of the 102 inocu-
lation experiments recorded in my last communication f. They require
to be thrown into a new tabular form, in order to show how the results
differed amongst themselves when organic infusions of different strengths
were employed. The consideration of this aspect of the question was
purposely delayed, in order to avoid the introduction of unnecessary
complications into my last communication, seeing that the conclusion
which I then sought to establish was in no way affected by these facts.

Neutral Hay-Infusion.

Infusion of sp. gr. 1005.

Infusion of sp. gr. 1002.

Tempe-
rature.

Number

of
Expts.

Date of
Turbidity,
if any.

Results at the
Expiration of
the 8th day.

Number

of
Expts.

Date of
Turbidity,
if any.

Results at the
Expiration of
the 8th day.

122° F.
(50° C.)
131° F.

} >

6

24 hrs.
48 hrs.

Turbid.
All turbid.

1

48 hrs.

Turbid.

140° F.
(60° C.)

149° F.

1 in 48 hrs.
6 in 60 hrs.

1 All turbid.

^ {
^ {

1 in 3 days.

1 in 8 days.

2 in 5 days.
1 in 8 days.

1 All turbid.

Three turbid.
One clear.

158° F.
(70° C.)
167° F.
176° F.
(80° C.)

}

All clear.

15
4

All clear.
All clear.

* See ' The Beginnings of Life,* vol. ii. p. 2.

t See Proceedings of the Royal Society, No. 143, p. 230.

necessary to kill Bacteria, Vibriones, i^c. 327

Acid Turnip-Tnfusion.

Infusion of sp. gr. 1008.

Infusion of sp. gr

1005.

Tempe-
rature.

Number

nf
Expts.

Date of

JL li L LJil.llL> )

if any.

Kesults at tbe
the 8th day.

Number
Expts.

Date of

J. Ul UKllLyj

if any.

Results at the
Expiration of
the 8th day.

122° F.
131° F.

5

24 brs.

All turbid.

2

48 brs.

All turbid.

140° F.
149° F.

6
3

40 brs.
5 days.

All turbid.
All turbid.

• {
'1

4 in 3 days.
2 in 4 days.
1 in 3 da^'s.

1 in 7 days.

2 in 8 days.

All turbid.

Four turbid.
Three clear.

ir)8° F.
1G7°F.
176° F.

17
4

All clear.
All clear.

lieference will be made to these Tables in the setting forth of my
reasons for the conclusion that the more or less dela3'^ed putrefaction which
takes place in inoculated organic infusions raised to the temperature of
140° 1\, and to other degrees of heat above this point, is due to the in-
fluence of the not-living ingredient (b) of the inoculating compound.
These reasons are the follo\^-ing : —

1. Because the turbidity which has occurred in iuoculated organic in-
fusions that have been subjected to a temperature of 140° F. has always
manifested itself appreciably later, and advanced much more slowly than
in similar mixtures which had not been heated above 131° F.; whilst it
has commenced even later, and progressed still more slowly, when occur-
ring in mixtures previously heated to 149° F. Such facts might be
accounted for by the supposition that exposure in these organic fluids
to the slightly higher temperature suffices to retard the rate of growth
and multiplication of the living units of the inoculatiug compound, al-
though the facts are equally explicable upon the supposition that the
later and less energetic putrefactions are due to the sole influence of the
mere organic matter of the inoculating compound.

2. So far as the evidence embodied in the Tables goes, it tends to show
that the more unstable different specimens of siuiilar infusions are (that
is, the stronger they are), the more rapidly and frequently does late tur-
bidity ensue, and the more this late turbidity approaches, both in time
of onset and in rate of increase, to that which occurs when inoculated
infusions are not heated to more than ]31°F.— Mhen both living and
not-living elements of the inoculating compound act conjointly as
ferments. Such facts show quite clearly that where the intrinsic or
predisposing causes of change are strong, there less potent exciting
agencies are more readily capable of coming into play ; but they still do

338

Prof. H. C. Bastiaii on the Heat

not enable us to decide whether the exciting cause of this delayed turbi-
dity is in part the living element whose vitality and rate of reproduction
has been lowered by the heat, or whether the effects are wholly attribu-
table to the mere organic matter of the inoculating compound.

• So far, therefore, we have concomitant variations which are equally
compatible with either h}'pothesis. But it will be found that each of the
three succeeding arguments speaks more and more plainly against the
possible influence of the living element, and in "favour of the action of
the organic matter of the inoculating compound, as an efficient exciting
cause of the delayed putrefactions occurring in the cases in question.

3. As stated in my last communication *, when single drops of slightly
turbid infusions of hay or turnip previously heated to 140° F. are mounted
and securely cemented as microscopical specimens, no increase of turbi-
dity takes place, although drops of similar infusions heated only to
122° F. do notably increase in turbidity (owing to the multiplication of
Bacteria) when mounted in a similar manner. Under such restrictive
conditions as these, in fact, a drop of an inoculated and previously heated
organic infusion behaves in precisely the same manner as a drop of a
similarly treated ammonic-tartrate solution. In each case, when heated
to 140° r., turbidity does not occur, apparently because there are no
living units to multiply, and because in these mere thin films of fluid
dead ferments are as incapable of operating upon the organic fluids as
they are upon the ammonic-tartrate solutions.

4. Because, in the case of the inoculation of fluids which are not easily
amenable to the influence of dead ferments, such as a solution containing
ammonic tartrate and sodic phosphate, this delayed turbidity does not
occur at all. Such inoculated fluids become rapidly turbid when heated
to 131° F., though they remain clear after a brief exposure to a tempera-
ture of 140° F. When the living units in the inoculating compound are

led, there is nothing left to induce turbidity in such solutions. The
mere fact that these fluids do not undergo change when exposed to the
air proves conclusively that they are very slightly amenable to the in-
fluence of the ordinary dead organic particles and fragments ^ith which
the atmosphere abounds. The absence of delayed turbidity in these
fluids serves, therefore, to throw much light upon the cause of its occur-
rence in the organic infusions.

5. And, lastly, I can adduce crucial evidence supplied by the " Method
of Difference," speaking with its accustomed clearness. Two portions of
the same hay- or turnip-infusion can be inoculated in such a manner as
to supply us with the information we require. In the one case we may
employ a drop of a turbid ammonic-tartrate solution previouslv heated
to 140° F., in which, therefore, the living units would certainly be killed ;
whilst in the other we may add an unheated drop of the same turbid
saline solution to the organic fluid, and then heat this mixture also to

* Loc. cif. p, 228.

necessary to kill, Bacteria, Vibrioncs, ^c.

329

the temperature of 140° F. The coinpnTative behaviour of these two
iuocuhxtecl fhiids (phiced, in the ordinary manner, in previously boiled
corked phials) should be capable of showing us whether the living
elements of the inoculating compound, were able to survive when heated
in the organic infusion. If they did survive, the fluids inoculated in this
manner ought to undergo putrefaction earlier and more rapidly than
those inoculated with the drop of turbid fluid, in which we know that the
Bacteria, Vibriones, and theii' supposed germs would have been reduced
to a state of potential death. With the view of settling this question,
therefore, the following experiments were made : —

Description of Experiments.

A. Eoiled aminonic-tai-trate
solution, inoculated with an un-
heated drop of a similar solution
turbid with Bacteria &c.

B. Boiled ammonic-tartrate
solution, inoculated with a drop
of a turbid saline solution pre-
viously heated to 140° F.

C. Boiled turnip- and hay-in
fusions, inoculated with a drop of
a turbid saline solution previous-
ly heated to 140° F.

D. Boiled turnip- and hay-in-
fusions, inoculated with a drop of
an unhealed tiu-bid saline solu
tion, the inoculated fluid bein<
subsequently heated to 140° F.

Results.

Turbid in 40 hrs.

Clear at expiration
of 8th day.

Turnip-infusions

turbid in 2i days
Hay-infusions
clear at exp i ration
of 8th day.
Turnip-infusions

turbid in 2i days
Hay-infu.sions
clear at expiration
of 8th day.

B. Boiled turnip- and hay-in- Turnip-infusions
fusions, inoculated with a drop of turhid in 28 hrs.
an unheated saline .solution, the Hay-infusions
inoculated fluid being subse- turbid in 38 hrs.
quently heated to 131° F.

Inferences.

That boiled ammonic-tartrate so
lution is a fluid inoculable by
living Bacteria &c., and favour-
able for their growth and rapid
multiplication.
That Bacteria, Vibriones, and
their supposed germs are either
killed or deprived of all power
of multiplication when heated
to 140° F. in this fluid.
^The precisely similar behaviour
of the turnip- and hay-infu
sions of series C and series
D respectively shows that
the Bacteria, Vibriones, and
their supposed germs are as
inoperative in series D as
they are known to be in
series C ; whilst the beha'
viour of the hay-infusions
shows that they are little
amenable to the influence of
the drop of the saline fluid
when its living units are
killed.

Siaows that a heat of 131° F. is
not sufTicient to kill Bacteria,
Vibriones, and their supposed
germs in organic infusions, and,
again, that turnip-infusions are
more rapidly influenced by such
an inoculating agent than some
hay-infusions*.

No experiments could speak more decisively. Those of series B show
that Bacteria, Vihriones, and their supposed germs are either actually or
potentially killed when heated to 140° P. in the neutral saline fluid,

* These experiments of series C, D, and E were many times repeated with specimens
of the same turnip- and hay-infusions, the specific gravity of the former being about
1008 and that of the latter 1005. Different specimens of hay especially vary so much
that it becomes absolutely essential to use portions of the same infusion for the compa-
rative experiments of these different series.

330

Prof. H. C. Bastian on the Heat

w liioh the experiments of series A show to be eminently favourable for
their grow th and reproduction. Being certain, therefore, that the ]x\mg
units are killed in the drops with which the fluids of series C were inocu-
lated (because they were drops of the same fluid as was employed in
series B), we may be equally certain that the turbidity and putrefaction
which did ensue in the turnip-solutions of series C were due to the in-
fluence of the mere dead constituents of these drops of the turbid saline
fluid; whilst, seeing that the behaviour of the fluids of series D was
precisely similar to those of series C, we have a perfect right to infer that
this series of fluids (D) was as devoid of living units as those of C are
knov\Ti to be — that is, that Bcccteria, Vibriones, and their supposed germs
are Ivilled by the temperature of 140° F. in organic fluids, just as they are
in saliue fluids, although, as shown by the experiments of series E, they
do not succumb to a heat of 131° F. These experiments of series C and
D further illustrate the different degrees of amenability of different
organic fluids to the same dead ferments ; whilst the comparison of the
results mth the hay-infusions of series C and D mth those previously
cited (in which the inoculating compound was a drop of an organic infusion
heated to the same temperature of 140° F.) will illustrate the different
influence of dissimilar dead ferments upon infusions of the same kind.

The evidence now in our possession shows, therefore, that whilst the
temperature at which living ferments cease to be operative varies wdthin
very narrow limits (131°-140°F.)*, that which destroys the virtues of
not-living ferments A'aries mthiii much ^^ider limits, and depends not
only upon the amount of heat employed, but also upon the nature of the
putrescible or fermentable liquid to which such ferment is added, in con-
junction with the degree of heat and other conditions to Avhich the mix-
ture is subsequently exposed t. Here, therefore, we have evidence as to
the existence of a most important difference between living and not-
living ferments, which has always been either unrecognized or more or
less deliberately ignored by M. Pasteur and his followers J. This differ-

* Licbig has proved that a temperature of 140° P. kills Torulcs, and always suffices to
arrest a process of fermentation taking place under their influence in a sugar solution.
Toruke iieated in water to 140° F. also fail to initiate fermentation in a sugar solution.
I have also found tliat an exposure to a temperature of 131° F. for five minutes always
suffices to destroy the life of Desmids, Euglense, Amoebo;, Monads, Ciliated Infusoria,
Rotifers, Nematoids, and other organisms contained in specimens of pond-water. All
these lower organisms seem to be destroyed at about the same temperature, as might
have been expected from the fundamental I'elatiouship which must exist between these
several varieties of the one substance — living matter.

t See 'The Beginnings of Life,' vol. i. p. 437.

I See, for instance, all M. Pasteur's celebrated experiments in wliich he had recourse
to an "ensemencement des poussieres qui existent en suspension dans I'air," as re-
corded in chaps, iv. & v. of his memoir in 'Ann. de Chimie et dc Physique,' 18G2.
M. Pasteur was engaged in an investigation one of the avowed objects of whicli was to
determine whether fermentation could or could not take place without the intervention of

necessary to kill Bacteria, Vibriones, ^c.

331

eneo is, moreover, tlioroughly in accordance w ith the broad physico-cheuii-
cal theory of fernieutation w hich has been so ably expounded by Baron
Liebig and others, and the truth of which may now be regarded as defniitely
established. According to this theory "livhig" matter, as a ferment,
would take rank merely as a chemical compound having a tolerably defi-
nite constitution ; and this, we might reasonably infer, would, like other
chemical compounds, be endowed with detinite properties — and amongst
others that of being decomposed or radically altered by exposure to a certain
amount of heat. Looked at also from tlus essentially chemical point of
view, it would be only reasonable to expect that the molecular mo^•em(^nts
of living ferments mth a lowered vitality might not be more marked or
energetic than those which many not-living orgaiuc substances are apt to
undergo ; and this being the case, we might expect that there would often
be a gi'eat practical ditficulty in ascertaining whether a fenneut belonging
to the arbitrary and artificial (though, in a sense, justifiable and natural)
category of "living" things had or had not been in operation.

It has, moreover, been most unmistakably proved that the limits of
vital resistance to heat which Bacteria, Vihriones, and theu' supposed
germs are capable of displacing are essentially the same in the three type
fiiiids which I have employed — that is, in a weak saline fluid, in a neu-
tral organic infusion, and in an acid organic infusion. No evidence exists
really tending to show that these organisms or their germs are capable
of withstanding the effects of heat better in one of such fluids than in
another. We may therefore safely affirm that M. Pasteur never had
any v alid evidence in support of his conclusion that the germs of Bacteria
and Vihriones can resist heat better in neutral or slightly alkaline solu-
tions than in slightly acid mixtures. The experimental results which
led him to arrive at such a conclusion were not logically capable of re-
ceiving any such iuterpretation, whilst they can be legitimately accounted
for in accordance with the broader physico-chemical theory of fermenta-
tion, the truth of wliich has now been established*. We may also
safely affirm that M. Pasteur's more specific statement, to the effect that

living organisms, wliich M. Pasteur held (in opposition to many other chemists) to be the
only true ferments. In his inoculating compound (dust filtered fi'om the atmosphere),
there was, as M. Pasteur was fully aware, a large amount of what his scientific opponents
considered not-living ferment, vrhiXst possibly there existed a certain number of living
ferments. In explaining the results of his experiments, however, M. Pasteur and
others tliought he was pursuing a logical and scientific method when he attributed these
results to the action of the possibly existing element of the inoculating compound, wiiilst
he ignored altogether the other element which was certainly present in comparatively
large quantity, and the testing of whose eflicacy was the ostensible object of his research,

* I attempted to show, nearly three years ago (see 'Nature,' July 14, 1870, pp. 224-228),
that the differences which M. Pasteur ascribed to differences of vital resistance of or-
ganisms in particular fluids were just as explicable in accordance with tlie physico-
chemical theory of fermentation, by reference to the different degrees of fcrmentability
of the sevci-al fluids.

332 Prof. H. C. Bastian on the Heat

the germs of some Bacteria and VibHones are Ccapable of resisting the
influence of a heat of 212° F. when in the moist state, though they are
killed by a temperature of 230° F., was a conclusion altogether unwar-
ranted by the evidence which he adduced. Finding that certain fluids
treated after the manner introduced by Schwann always remained quite
devoid of living organisms, M. Pasteur A-ery legitimately concluded that
preexisting organisms and gei-ms had been killed during the boiling of the
liquid ; but finding that when a little powdered chalk was added to fluids
of the same Icind (which in all other respects were treated in a similar
manner) living organisms were after a time invariably found to appear,
although they as invariably failed to appear when the same fluids were
heated to a temperature of 230° F. (110° C), two equally legitimate pro-
visional conclusions were open to M. Pasteur in explanation of these
facts. What did M. Pasteur do ? Following the same method as he
had formerly employed*, he again ignored one of the equally possible
interpretations, and unsuccessfully attempted to prove, by a repe-
tition of similar reasoning t, that the different results in the two
series of experiments w^ere due to the fact that the germs of Bacteria
and Vihriones which had been killed by the temperature of 212° F. in the
first series, were not killed by this temperature in the second series (in
which a slightly alkaline fluid had been employed), although they were
destroyed by the higher temperature of 230° F. Thus results which were
due to the action of not-Hving ferments were ascribed to Living ferments,
and the possible action of not-living ferments was ignored, although, as
I have said 1)eforo, the ostensible object of M. Pasteur's researches was
to inqmre into the relative importance of not-living and living ferments,
or whether, iii fact, " dead " substances (in the ordinary acceptation of
the word) could act as ferments.

"When viewed from the stand-point of the physico-chemical theory of
fermentation, the apparently contradictory results arrived at by the same
experimenter at different times or by different experimenters, in this line
of research, cease to be the inexplicable puzzle which they must always
appear to those who place implicit faith in the narrower and too exclusive
" vital " theory of fermentation advocated by M. Pasteur and his followers.

My investigations have convinced me that, with regard to degree of
fermentability, the various fermentable fluids and mLxtures are divisible
into three distinct subclasses : —

I. There are what may be called self-fermentable fluids or mixtures

that is, fluids or mixtures which, after exposure to a temperature of
212° F. or higher, are still capable of undergoing fermentative changes
without the addition of less-heated matter, either not-living or livino-.
The changes occurring in these self-fermentable fluids (in which preexist-
ing living things have been killed), when strictly protected from contact
v ith adventitious particles, vary in rapidity and in intensity fi'om the
* See note \ on page 330.

t See Ann. de Chim. et de Phys, 1862, pp. GO-65.

necessary to kill Bacteria, Vibrioncs, 6f6\

333

liighest. to the very lowest degrees of fornientability. These gradations
are dependent principally npon the nature of the fluids or mixtures em-
ployed, and upon the degree of heat to which they have been submitted,
though partly also to the temperature, pressure, presence or absence of
filtered air, and degree of light to which the mixtures are subsequently
exposed, l^or the sake of convenience, these gi-adations may be ranged
into several distinct groups, though of course any such divisions as I am
now about to sketch are purely artificial and are connected ^\itl^ one
another in nature by innumerable transitions.

Nature of Fluids.

A. Turnip-infusion with cheese, turnip-
infusion neutraliicecl by liquor potassaj,
ordinary turnip-infusion, strong hay-in-
fusion, &c.

B. Tuniip-infusion neiitralized by liquor
potassif, ordinary turnip-infusion, ordi-
nary hay-infusion, &c.

C. Beer-wort*, &c.

D. Weak liay-infusions, xirine, solu-
tions containing amnionic carbonate and
sodic phosphate with minute organic im-
purities, &c.

E. Weak hay-infusions, urine, solutions
containing ammonio-citrate of iron and
minute organic impurities t, &c.

F. Solutions of ammonie tartrate and
Bodic phosphate with minute organic im-
purities, &c.

Nature of Results.

Within two to four days marked turbidity,
owing to the ap])earance of swarms of
Bacteria and V/hriones. Fluids more
or less foDtid. (Putrefaction.)

No uniform turbidity, but growth of floc-
culi in a more or less clear liquid. After
a time the flocculi (composed of aggre-
gated Bacteria and Vihriones) gradually
subside, and the activity of the process
ceases. Fluids either foetid or having a
mere sour odour.

Fluids which become more or less uni-
formly and rapidly turbid, owing to the
appearance of swarms of Torulce.

Do not become visibly turbid or produce
visible flocculi, although on microsco-
pical examination they may be found to
contain living Bacteria pretty uniformly
distributed, but in comparatively small
quantities. The odour is often not
more appreciably altered than the clear-
ness of such solutions.

Same as in the last group J, though after
weeks or months a dirty-looking sedi
mentary matter slowly accumulates at
the bottom of the flask, which on mi-
croscopical examination is found to be
composed partly of Bacteria with Vi-
hriones and Lejitothrix, and partly of
Torulm or more thick-walled fungus-
germs.

Same as in the last group, only the dirty
sedimentary matter which accumulates
never contains eitlier Bacteria, VitirioneR,
or Lejitothrix. Living Torula and
thick-walled fungus-germs in various
stages of formation are I'requontly met
with, and also, occasionally, a mycelium
resulting from the development of some
of these bodies.

* I have had no experience with such a fluid myself. M. Pouchet's observations
were, however, most striking on this subject (see his 'Nouvelles Experiences,' Paris,
1864,' p. 190).

t In solutions containing iron, green organisms may subsequently be found (see
' Beginnings of Life,' vol. ii. p. 157).

\ This, in fact, is in many cases tbe kind of change which the fluids last described
ultimately undergo.

334

Prof. H. C. Bastian on the Heat

Nat.ure of Fluids. Nature of Results.

G, Weak or strongly acid infusions, and May remain permanently barren, and

also many saline solutions cont<iining or- never show any traces of organisms,

ganic impurities. either dead or living *

■ II. To the second subclass belong fluids which, after exposure to a
temperature of 212° E. or higher, may be kept clear or apparently un-
altered so loiTg as they are shut off from contact with unheated atmo-
spheric or other organic particles, but which do undergo putrefaction, or
more or less marked fermentation, soon after they are brought into con-
tact even with mere not-liAdng organic matter.

The experiments recorded in this commiuiication have most conclu-
sively proved the efficacy of not-living organic matter as a ferment or
inciter of change in previously barren fluids. And combining the know-
ledge derived from these experiments with that which w'e now possess
concerning the absence of living Bacteria, Vihriones, and their germs in
the air, together with the known prevalence of minute organic particles
and fragments of various kinds, the explanation of M. Pasteur's cele-
brated experiments in which he had recourse to an " ensemen cement
des poussieres qui existent en suspension dans lair," becomes quite easy
and legitimate without having recourse to the hypothesis of Pan-
spermismf. Now, also, are we enabled to understand all the apparent
inconsistencies of those experiments in M^iich previously boiled fluids
have been exposed to the ordinary air of different localities, and have then
been resealed. If many specimens of these fluids remained unchanged,
whilst others, after a few days, swarmed with Bacteria and Vihriones, we
may now very safely attribute these previously puzzling results to the
comparative absence or presence of organic fragments in the particular
volumes of air which chanced to get into the flasks, and to the different
nature of the fluids employed by different experimenters J.

* See many negative experiments recorded in ' The Beginnings of Life,' vol. i. cli. xi.
Mr. W. N. Hartley has laboured very industriously to disprove something wliich I
never asserted (see Proceedings of Royal Society, vol. xx. p. 140). In my early paper in
' Nature ' I expressly stated that organic impurities were always present in the saline solu-
tions which I employed ; and, as may be seen by the note appended to the conclusions of
that paper, I never claimed to have established that living organisms could appear in
saline solutions free from traces of organic impurity. Mr. Hartley did attempt to work
with approximately pure saline solutions, and in other respects also the conditions of his
experiments differed so much from mine, that the results which he obtained could not
possibly be considered to disprove what I had previously stated. Some of his flasks were
heated to 180° C, a temperature about which I had said nothing ; and whilst his organic
infusions were too weak, some of his saline solutions were too concentrated, though the
strengths of others were not given at all. Fluids were also employed (such as urine,
heated to 130= C.) which I had not made use of, and which 1 should not have thought
of experimenting with.

t See the experiments before alluded to,which are recorded in chaps, iv. & v. of
his Memoir,

X See M. Pastem-'s Memoir, chap, vii., and also Compt. Rend. Nov. 5, 1860. See

necessai-y to kill Bacteria, Vibrioncs, ^c.

335

Many of the fluids which habitually remaiu clear after a previous ebul-
lition in flasks whose necks have been plugged with cotton-wool, many
times bent, or hermetically sealed after the entry of calcined air, or when
enclosed in vessels which are completely full (ia Gruithuisen's fashion),
belong to this subclass. In other cases, however (as in many of those
instances where urine or hay- or turnip-infusions have been employed),
those who do not content themselves with a mere naked-eye inspection of
the apparently pure fluids would find on microscopical examination of
the sediment that such fluids were to a low degree self-fermentable —
that they correspond, in fact, with group E of the last subclass * ; whilst,
in addition, my researches have shown that many of such fluids are
capable of being rendered self -fermentable to a marked degree, if, instead
of subjecting them to contact with calcined air or variously filtered air,
its reflux after ebulHtiou is altogether prevented by hermetically sealing
the neck of the flask during ebulhtion. Operating in this way, I have
repeatedly found that fluids freed from the pressure of air and from its
influence altogether become to a marked extent self -fermentable, although
the same fluids exposed to filtered or calcined air under ordinary atmo-
spheric pressure remain unaltered and barren, or at most exhibit the
very low degree of f ermeutability referred to as characterizing group E f.
But just as amongst the self-fermentable fluids we find there are some
which only engender Tbrulce or other allied fungus-germs, so now we find
that some previously boiled fluids, even when fully exposed to the air,
swarm only with Torulce. Those exciting agents derived from the atmo-
sphere which, with one set of fluids, initiate changes leading to the evolu-
tion of Baetena, -with another set lead only to the evolution of Torulce.
And whilst telling us that the Bacteria which appear in previously barren
fluids after exposure to air are not due to their contamination with, germs
of Bacteria, some observers would have us conclude that the Torulce
which appear in other previously barren fluids after a similar exposure
are the products of preexisting aerial germs of such organisms. This
conclusion, however, cannot readily be accepted in the face of the evidence
derived from the closed-flask experiments with self-fermentable fluids of
the lowest degree J. Such experiments, in fact, render the hypothesis as

also a record of other experiments'made with the air of alpine regions by MM. Pouchet,
Joly, and Musset, in Compt. Eend. Sept. 21, 1863.

* Other fluids richer in organic matter or otherwise more favourably endowed, in-
stead of presenting this low degree of self-fermentability, are notably prone to undergo
change when we attempt to preserve them in the manner described, although such
modes of preparation do sufBce for preserving so many fluids. This has been fully
admitted by Schrceder and Dusch, Schwann, Pasteur, and others.

f In illustration of this statement see ' The Beginnings of Life,' Appendix C, Exps.
viii., ix., xiv., xv., xviii., xx., xxvi.,xxx., xxxiii., and xxxvi.

J The only evidence in favour of such a conclusion is not one jot more conclusive
than that which was formerly adduced in favour of the universal prevalence of Bacteria-
germs in the air.

336

Prof. H. C. Bastian on the Heat

to the widespread distribution of aerial germs of Torulce wholly unne-
cessary, by shov\iug that certain fliuds, by reason of certain intrinsic
peculiarities, when they undergo fermentation give rise to Torulce only.
We are thus led to conclude that Avhilst some fluids are capable of en-
gendering both kiuds of organisms, others tend only to produce one or other
of them — whether the fluids are contained in closed flasks or in open
vessels exposed to the incidence of atmospheric particles. I have more
than once seen nothing but TorulcB appear in an infusion of turnip ex-
posed to the air after it had been heated in a closed tube to a temperature
of 293° 1\ for t^venty minutes, and I have once seen the same thing occur
in an unheated infusion of turnip exposed to the air, though on all other
occasions such infusions have swarmed only with Bacteria and Vihriones.
On the other hand, a boiled ammonic-tartrate solution exposed to the air,
though protected from an excess of atmospheric particles (for the advent
of a large number of these might in some cases incite putrefaction), is
never found to contain Bacteria ; the fluid continues clear, though a
sediment gradually accumulates at the bottom of the flask, amongst which
Tondce and other fungus-germs are constantly to be found — more nume-
rous though otherwise very similar to those which are to be met with in
flasks closed during ebullition, or in others to vi^hich only filtered air is
admitted. Although Torula; only appear in such fluids, they continue all
the time to be eminently inoculable hj Bacteria] and, again, when the
Torulce begin to decay they are apt to incite a more or less manifest
putrefaction, during which the fluid gradually becomes turbid ^ith Bacteria.
it is, in fact, a general rule that putrefaction is apt to supervene upon
a fermentation of a more smouldering type.

. III. In the third subclass I include fluids which, after exposure to
212° F, or higher temperatures, are unable, either alone or under the in-
fluence of ordinary atmospheric particles or fra,gmeuts, to undergo putre-
faction, although such a process can invariably be initiated by bringing
the fluids iuto contact with living ferments. As examples of such fluids,
I may cite the neutral saline solution to which I have so often referred
and that known as Pasteur's solution. Other fluids of the same kind
have lately been referred to by Professor Iluiziuga*. The fact that
certain fluids cannot be made to undergo putrefaction by the influence of
dead organic particles, although they become at once amenable to the in-
fluence of living units, unmistakably shows the superior potency of living
ferments ; their action has, moreover, invariably proved to be certain
and. inevitable in all the cases in which they were know n to be present.
Even these least fermentable fluids of om- third subclass invariably be-
come turbid wdthin three days after inoculation with living units, if main-
tained at a temperature of about 70° P. ; whilst when other more change-
able fluids are inoculated, putrefaction ensues with equal certainty, though
with much greater rapidity.

* See ' Nature,' March 20th, 1873, p. 380.

necessary to kill Bacteria, Vibriones, S^c. 337

What we have learned, therefore, concerning the invariable unifonnity
of simple inoculation experiments should of itself teach us how diflicult
it would be to account for cases of delayed putrefaction, or for cases in
which a mere smouldering fermentation is set up, by the old though now
well-nigh exploded notion of contamination by preexisting germs. Where
living ferments really exist, the course of events is definite and almost in-
variable in its rapidity ; but where fermentation takes place as a residt of
chemical changes occurring in the fluid itself (either by its own unaided
powers, or under the stimulating influence of a less-heated organic fer-
ment) there is abundant room for all the irregularity and variation actually
encountered. These cases of irregularity and variation have always, on
other grounds, defied all legitimate attempts to bring them individually
within the pale of a narrow and exclusively " vital " theory of fermenta-
tion ; and now a wider experience with living ferments equally tends to
show the impossibility of legitimately explaining a great mass of irregular
phenomena by means of agents whose action is shown to be constant
and almost invariable.

Thus it can now be proved, by evidence of a most unmistakable nature,
that the process of putrefaction which invariably occurs in previously
boiled putrescible infusions contained in flasks with narrow but open
necks is not commonly (is, perhaps, only very rarely) initiated by living
germs or organisms derived from the atmosphere ; it can also be proved
that putrefaction and the appearance of swarms of li\ing organisms may
occur in some boiled fluids \A hen they are simply exposed to air which
has been filtered through a firm plug of cotton-wool or through the nar-
row and bent neck of a flask, to air whose particles have been destroyed
by heat, or even in fluids hermetically sealed in flasks from which all
:iir has been expelled. The evidence in our possession is therefore most
complete on this part of the subject : it shows beyond all doubt, not only
that putrefaction may and does very frequently occur under conditions in
Avhich the advent of atmospheric particles, whether living or dead, is no
longer possible, but also that liviug particles derived from the atmosphere
can only be very rare and altogether exceptional initiators of the putre-
faction which invariably occurs in previously boiled infusions exposed to
the air.

Again, the e\idence which we now possess with reference to the influence
of heat upon Bacteria, Vibriones, and their supposed germs is no less deci-
sive. It has been unmistakably proved that such organisms and their
imaginary germs are either actually or potentially killed by a brief expo-
sure to the temperature of 140° F. when in the moist state ; and it had
also been previously established that they are invariably killed by desic-
cation even at much lower temperatures*.

* See the experiments and conclusions of Dr. Burdon Sanderson in Thirteenth
Eeport of Med. OflBcer of Privy Council, p. 61. This fact of the inability of these or-

338 On the Heat necessary to kill Bacteria, Vibriones, S^c,

But if living germs do not come from the air to contaminate the pre-
viously boiled fluids, and if it is not possible for any of them to have
escaped the destructive iufluence of heat in the boiling fluid or on the
walls of the vessel in which the fluid is contained, what can be the mode
of origin of the swarms of living things which so rapidly and invariably
appear in such infusions when contained in open flasks, and which so
frequently appear when the infusions are contained in flasks whose necks
are closed against atmospheric particles of all lands? They can only
have arisen by the process which I have termed Archebiosis.

Conclusions.

If a previously boiled ammonic-tartrate solution remains free from
Bacteria and Vihriones when exposed to the air, it is because the air does
not contain living organisms of this kind or their supposed germs, and
because mere dead organic particles are not capable of initiating putre-
faction in such a fluid.

And if ordinary organic infusions previously boiled and exposed to the
air do rapidly putrefy, though some of the same infusions when exposed
only to filtered air remain pure, it is because such fluids are, in the ab-
sence of living units, quite amenable to the influence of the dead organic
particles which the air so abundantly contains, although they are not self-
fermentable.

Whilst if other more changeable fluids, after previous boiling, when
exposed to filtered air or cut off altogether from contact with air, do
nevertheless undergo putrefaction or fermentation, it is because these
fluids are seK-fermentable, and need neither living units nor dead organic
particles to initiate those putrefactive or fermentative changes which lead
to the evolution of living organisms.

ganisms and their germs to resist desiccation shows the futility of some objections which
have been from time to time raised by those who thought tliat Bacteria, Vibrioncs, and
their germs might resist the destructive influence of heat by adhesion to the glass above
the level of the fluid, or even in the fluid itself, just as dried and very thick-coated seeds
have been known to do. Dry heat would seem to be even more fatal to such orga-
nisms and their germs than a moist heat of the same degree, owing to their extreme
inability to resist desiccation : if they become dry they are killed at a temperature of
about 104° R, whilst if they remain moist they succumb, as wo have seen, to a tempe-
rature of 140° F.

I