grounds of its being more difficult of digestion, and hence less
wholesome than the raw article. I may only point out that in this, as
in most other matters where opinions may be made or unmade, and in
consequence of the facts available being scanty must be more or less
arbitrary in character, Dr. Duclaux, the successor to Pasteur as
Director of the Pasteur Institute in Paris, has expressed himself as
follows in an article on "La digestibilité du lait stérilisé." After
reviewing the various special researches which have been made on the
subject, he says:--
"Ceci nous amène à une conclusion qu'il faut bien avoir le courage
de tirer, c'est que ces études chimiques sur la digestibilité du
lait ne sont pas adéquates à la question à résoudre.... En
attendant, tenons-nous-en à cette conclusion générale, que le lait
pasteurisé, chauffé ou stérilisé, est encore du lait, devant la
science comme devant la pratique, et que si son emploi présente
parfois des inconvénients, ceux-ci sont légers et amplement
compensés par les avantages."
BACTERIA AND ICE
The fate of bacteria when frozen excited the curiosity of
investigators already in the early years of bacteriology, for in 1871
we find Burdon Sanderson recording the fact that water which he had
obtained from the purest ice contained microzymes, or, as we now
prefer to call them, micro-organisms.
It is quite possible that at the time this announcement was made it
may have been received with some scepticism, for it was undoubtedly
difficult to believe that such minute and primitive forms of vegetable
life, seemingly so scantily equipped for the struggle for existence,
should be able to withstand conditions to which vegetable life in more
exalted circles so frequently and lamentably succumbs.
The tormented agriculturist realises only too well what havoc is
followed by a return in May to that season
"When icicles hang by the wall,
And Dick the shepherd blows his nail
And Tom bears logs into the hall,
And milk comes frozen home in pail."
Again, with what solicitude those of us who have gardens wait to see
what will have survived the iron grip of winter in our favourite
flower borders, and how frequently we have to face blanks in the ranks
of some of its most cherished occupants! Numerous bacteriologists,
however, have now confirmed this fact, the fields of ice and snow have
been repeatedly explored for micro-organisms, and it has been shown
how even the ice on the summit of Mont Blanc has its complement of
bacterial flora, that hailstones as they descend upon the earth
contain bacteria, that snow, the emblem of purity, is but a whited
sepulchre, and will on demand deliver up its bacterial hosts. Quite
apart from its general scientific interest, the bacterial occupation
of ice is of importance from a hygienic point of view, and a large
number of examinations of ice as supplied for consumption have been
made. Thus, Professor Fraenkl and also Dr. Heyroth have submitted the
ice-supply of the city of Berlin to an exhaustive bacteriological
examination. These investigations showed that the bacterial population
of ice as supplied to Berlin is a very variable one, and fluctuates
between great extremes, rising to as many as 25,000 bacteria in a
cubic centimetre (about twenty drops) of ice-water, and falling to as
few as two in the same measure.
There are numerous circumstances which come into play in determining
the density of the bacterial population in ice. First, of course, the
initial quality of the water from which the ice is derived is a factor
of great importance, for the purer the water the fewer will be the
bacteria found in the resulting ice.
Again, if the ice field is wind-swept by air bearing an unduly rich
complement of bacteria, as may be expected in the vicinity of populous
cities, for example, then the ice will reflect in its bacterial
contents the undesirable neighbourhood in which it was produced. Water
in repose, again, yields purer ice than water in movement during
freezing, for during rest opportunity is given for the bacteria
present in suspension to subside, the process of sedimentation or
deposition of bacteria which takes place under these conditions
playing an important part in water-purification; when, however, the
water is disturbed by swift currents, or agitated by storms, this
process is interrupted, and the bacteria become entangled in the ice
and frozen _in situ_.
The importance attaching to the physical conditions under which ice is
produced in enabling an estimate to be formed of the safety or
otherwise of the same for consumption may be gathered from the
following extract from an American report on the subject:--
"On the whole it is evident that the conditions surrounding water
when it freezes are very important factors in determining the
purity of the ice formed. If there is a considerable depth of
water in portions of a somewhat polluted pond or river, and the
ice is formed in these portions in comparatively quiet water with
but little matter in suspension, this ice will probably be
entirely satisfactory for domestic use. On the other hand, ice
formed in shallow portions of such ponds or rivers, even during
still weather, or in any portion if there is a considerable
movement of the water by currents or wind while it is forming, may
be rendered by these conditions entirely unfit for domestic use."
We have learnt that ice contains bacteria, that its bacterial contents
are to a certain extent dependent upon the bacterial quality of the
water before crystallisation, and that an important factor in
determining its purity is afforded by the physical conditions
prevailing at the time of freezing.
It will be of interest to ascertain in more detail what effect the
process of freezing has upon the number of bacteria present in the
water--what is the degree of bacterial purification effected during
the conversion of water into ice.
Now Professor Uffreduzzi, in his investigations on the ice-supply of
Turin, part of which is derived from a much-polluted portion of the
River Dora, found that about 90 per cent. less bacteria were present
in the ice than were present in the water from which it was produced.
In the making of ice, therefore, a remarkable removal of bacteria may
be effected which approaches very nearly the degree of bacterial
purification which is achieved during the best-conducted
sand-filtration of water.
Uffreduzzi's results have been repeatedly confirmed by other
researches. Thus, in regard to ice obtained from the River Merrimac,
water which contained originally about 38,600 bacteria per cubic
centimetre, on its conversion into ice had only from three to six.
Sewage, again, containing about a million and a half bacteria per
cubic centimetre after being frozen only contained under 74,000. It
should be mentioned that this last figure represented the number of
bacteria obtained by thawing the _outside_ of the sewage ice-cake;
_inside_ the cake there were more found--about 121,000. The difference
in these figures is due to the fact that, whereas the outer layers of
ice looked quite clear, towards the centre the ice contained sewage
sludge and hence more bacteria had become arrested; but in spite of
this the bacterial purification effected is very striking, although
not sufficient to render the use of ice from such a polluted source
either palatable or desirable.
It is, of course, a well-known fact that water possesses the power of
purifying itself during its transformation into ice, and that the
process of crystallisation not only prevents a considerable proportion
of the matters in suspension from becoming embodied in the ice, but
also eliminates a large percentage of the matters in solution, the
latter being driven from the water which is being frozen into the
water beneath. If, therefore, ice in the act of forming can get rid of
matters in solution, it is not difficult to understand how it can
eject bacteria, which though so minute are yet bodies of appreciable
dimension and in suspension. But that there are limits to this power
of excluding bacteria, and that, as in the case of other mechanical
processes, an overtaxing of the available resources is at once
reflected in the inferiority of the product, is shown by the frozen
sewage experiment, in which the ice, having had too large a supply of
bacteria in the first instance to deal with, was unable to get rid of
more than a certain proportion, and was obliged to retain a very
considerable number. Hence great as is the degree of purification
achieved by ice in forming, yet it must be recognised that its powers
in this direction are limited, and that the fact of water being frozen
does not necessarily convert a bad water into immaculate ice.
It is worthy of note that the city of Lawrence, in Massachusetts,
obtains the greater portion of its ice from a river which in its raw,
unpurified condition was rejected for purposes of water-supply in
consequence of the numerous and severe epidemics of typhoid fever
which accompanied its use. Since the application of sand-filtration to
this water, however, the death-rate from typhoid in this city, instead
of being abnormally high, has fallen abnormally low, and this
improvement is attributed to the excellent quality of the water
supplied to the city, and has taken place despite the use which still
continues of ice from the polluted river. The authorities consider the
city's immunity from typhoid amply justifies their sanctioning the
distribution of this river-ice, the freezing of the water having
rendered it sufficiently pure to remove all danger to health from its
consumption.
So far we have been considering the effect on bacteria of freezing
carried on under more or less natural conditions; but much interesting
work of a more detailed character has been carried out with reference
to the behaviour of particular varieties of micro-organisms when
frozen under more or less artificial conditions.
Thus Dr. Prudden froze various bacteria in water at temperatures
ranging from -1° C. to -10° C., and he found that different varieties
were very differently affected by this treatment; that, for example, a
bacillus originally obtained from water, and introduced in such
numbers as represented by 800,000 individuals being present in every
twenty drops, after four days' freezing had entirely disappeared, not
one having survived. On the other hand, similar experiments in which
the typhoid bacillus was used resulted in the latter not only enduring
a freezing of four days' duration, but emerging triumphant after it
had been carried on for more than 103 days!
In these experiments it should be borne in mind that, as the ice was
frozen to a solid block or lump, there was no opportunity for the
mechanical committal of the bacteria during freezing to the water
beneath; all the bacteria present were imprisoned in the ice, and the
fact that the typhoid bacteria were not destroyed by being frozen
shows that they can withstand exposure to such low temperatures,
although, as we have seen, the other variety of bacillus employed was
destroyed.
Dr. Prudden, however, discovered an ingenious method by which even
typhoid bacilli were compelled to succumb when frozen. In the course
of his investigations he found that bacteria which had offered the
stoutest resistance under freezing were extremely sensitive to this
treatment if the process was carried on intermittently, or, in order
words, if the temperature surrounding them was alternately lowered and
raised.
In this manner the bacteria may be said to be subjected to a
succession of cold shocks, instead of being permitted to remain in a
continuously benumbed condition. The vitality of typhoid bacilli was
put to the test under these circumstances, the freezing process being
carried on over twenty-four hours, during which time, however, it was
three times interrupted by the ice being thawed. The effect on the
typhoid bacteria was striking in the extreme; from there being about
40,000 present in every twenty drops, representing the number
originally put into the water, there were only ninety at the end of
the twenty-four hours; and after a further period of three days,
during which this treatment was repeated, not a single bacillus could
be found. This signal surrender to scientific tactics forms a marked
contrast to the stout resistance maintained for over 103 days under
the ordinary methods of attack.
But, although the typhoid bacillus appears to submit and meekly
succumb to this plan of campaign, yet the conclusion must not be
rashly drawn that all descriptions of bacteria will be equally feeble
and helpless in these circumstances.
Doctors Percy Frankland and Templeman have shown that the spore form
of the anthrax bacillus is able to successfully challenge all such
attempts upon its vitality. Thus when put into water and frozen at a
temperature of -20° C., the process being extended over a period of
three months and interrupted no fewer than twenty-nine times by
thawings, when examined even after this severe series of shocks, it
showed no signs of submission and clung to life as tenaciously as
ever.
The more sensitive form of anthrax, however, the bacillus, was readily
destroyed; for after one freezing its numbers were already so much
reduced that it was only with difficulty that even one or two could be
found, and after the second freezing every one out of the large number
originally present had died.
Renewed interest has been of late revived in the question of the
behaviour of bacteria at low temperatures, in consequence of the
possibility of obtaining, by means of liquid air and liquid hydrogen,
degrees of cold which were undreamt of by the scientific philosophers
of fifty years ago. Public interest has also been quickened in such
inquiries on account of the important part which low temperatures play
in many great commercial developments, their application rendering
possible the transport from and to all parts of the world of valuable
but perishable foodstuffs, thus encouraging local industries by
opening up markets, and bringing prosperity to countries and
communities which before were seeking in vain an outlet for their
surplus produce.
The application of cold storage for preservation purposes is, however,
no novelty; for nature ages ago set us the example, and of this we
have been lately reminded afresh by the discovery announced by Dr.
Herz of a mammoth in Siberia, which, despite the thousands of years
which have elapsed since it was originally overwhelmed and frozen, is
described as being in a marvellous state of preservation.
Thus we are told that "most of the hair on the body had been scraped
away by ice, but its mane and near foreleg were in perfect
preservation and covered with long hair. The hair of the mane was from
four to five inches long, and of a yellowish brown colour, while its
left leg was covered with black hair. In its stomach was found a
quantity of undigested food, and on its tongue was the herbage which
it had been eating when it died. This was quite green."
Considering that certainly more than eight thousand years had elapsed
since this creature was peacefully consuming what proved to be its
last meal, nature's method of cold storage must indeed be regarded as
unsurpassable in the excellence of its results.
I believe it was in the year 1884 that the first attempts were made to
follow more closely and in greater detail the precise effect upon
different bacteria of submitting them to temperatures of such a low
degree as -130° C., obtained by means of solid carbonic acid. These
experiments were carried out by Pictet and Young, and are recorded in
the _Comptes Rendus_ of the Paris Academy of Sciences.
They differ from those which we have so far been considering, inasmuch
as the bacteria were not frozen in water, but in culture-material, or,
in other words, like the mammoth, whilst enjoying a midday meal!
One of the micro-organisms experimented with was a bacillus known at
that time as the rinderpest bacillus, capable of producing disease in
animals when inoculated into them and existing both in the spore and
bacillar form. Pictet and Young specially state that the spore form
was present in the specimens employed by them, and hence the fact that
this micro-organism was alive after being frozen and exposed to this
low temperature of -130° C. for the space of twenty hours is not,
perhaps, so surprising when we bear in mind the remarkable feats of
endurance exhibited by spores which have with justification obtained
for them a prominent place amongst the so-called curiosities of
bacteriology. But of more interest than their mere survival in these
circumstances is the fact that, on being restored to animation--or, in
other words, released from their ice-prison--these bacteria were
discovered to have retained all their pathogenic properties, this
period of enforced rigidity having in no way affected their
disease-producing powers.
Such results naturally only served to whet the scientific appetite for
more, and the liquefaction of air and of hydrogen placing much lower
temperatures at the disposal of investigators, those bacteriologists
who were fortunate enough to command a supply were not long in
availing themselves of the opportunity thus given them of further
testing the vitality of micro-organisms.
Botanists had already shown that exposure to liquid air, which means a
temperature of about -190° C., and to liquid hydrogen, which means a
temperature of about -250° C., did not impair the germination powers
of various descriptions of seeds, such as those of musk, wheat,
barley, peas, vegetable marrow, and mustard, and that their actual
immersion in liquid hydrogen for the space of six hours did not
prevent them coming up when sown just as well as ordinary seeds which
had not undergone this unique experience; hence the opportunity of
submitting other members of the vegetable kingdom to these low
temperatures was eagerly sought for by bacteriologists. Dr. Macfadyen
found this opportunity in the laboratories of the Royal Institution,
and, Professor Dewar having placed a generous supply of liquid air and
liquid hydrogen at his disposal, he submitted specimens growing in
various culture-materials, such as gelatin, broth, potatoes, etc., of
typhoid, diphtheria, cholera, anthrax with spores, and other bacteria,
for twenty hours and seven days respectively, to a temperature of
about -190° C. In no instance, however, whether exposed when growing
in fluid or solid media, could any impairment of their vitality or the
slightest alteration in their structure be observed. Similar results
were obtained when liquid hydrogen, or a temperature of about -250°
C., was applied. The question of the retention or otherwise of the
disease-producing powers of these bacteria was not investigated, and
in this connection much interest attaches to Mr. Swithinbank's
investigations on the vitality and virulent properties of that
notorious malefactor amongst micro-organisms, the _bacillus
tuberculosis_, when exposed to the temperature of liquid air. The
specimens of the consumption bacillus employed were originally
obtained from the human subject, and they were exposed for periods
varying from six hours to six weeks to -190° C. In each case the
malignant properties of the tubercle bacillus after exposure were
tested by their direct inoculation into animals, and the results
compared with those which followed similar inoculations made with
bacilli which had not been frozen in this manner, but had been grown
in ordinary circumstances. In no single case, Mr. Swithinbank tells
us, were these frozen tubercle bacilli deprived of their virulence,
and the length of exposure, at any rate as far as could be judged
after six weeks, appeared to make no difference in this respect. It is
true that the pathogenic action of the frozen bacilli appeared to be
somewhat retarded--that is, they took rather longer to kill animals
than the ordinary unfrozen bacilli--but in every case their
inoculation produced the typical tuberculous lesions associated with
them.
Of particular interest, however, in view of what has been already
discovered about the lethal effect upon bacteria of violent
alternations of temperature, are Mr. Swithinbank's observations on the
vitality of the tubercle bacillus when exposed to such extreme
variations of temperature as are represented by a passage from -190°
C. to that of 15° C.
The _bacillus tuberculosis_ is admittedly a tough antagonist to deal
with, and enjoys an unenviable notoriety for its robust constitution
amongst the pathogenic members of the microbial world; hence a
knowledge of its behaviour in these trying circumstances, as we now
know them to be to bacterial life, becomes of special interest. Great
must have been the investigator's satisfaction, then, when he
discovered that the vitality of the consumption bacillus had been so
seriously impaired by this treatment that its pathogenic properties
collapsed, and the animals which were inoculated with these specimens,
instead of with the continuously frozen bacilli, suffered no
inconvenience, and remained in good health.
But although no appreciable change either in the structure, vitality,
or malignant properties of the particular bacteria investigated have
been noted as resulting from their exposure to extremely low
temperatures, yet there is no doubt that a certain proportion of the
individual micro-organisms present--those probably whose constitution
is less robust than their more fortunate associates--do succumb under
these trying conditions.
This fact has been well brought out by Dr. Belli, of the University of
Padua, in the experiments which he made with the fowl-cholera bacillus
and the anthrax bacillus in the presence of very low temperatures.
Thus he exposed a large number of fowl-cholera bacilli in broth to the
temperature of liquid air, as many as 396,000 bacilli being present in
every twenty drops of the liquid. After exposing them continuously for
nine hours to -190° C., he had the curiosity, after thawing them, to
count how many were left alive, and he found that an enormous
mortality had taken place amongst them; for, instead of nearly 400,000
bacilli being present in one cubic centimetre, there were only about
9,000. On the other hand, in the broth tubes kept during that time in
ordinary surroundings, the bacilli had flourished remarkably, and had
greatly increased in numbers. Thus not only had no multiplication
amongst these bacilli taken place, which circumstance is always
regarded as indicative of their vital condition--not only, then, had
their vitality been arrested--but a very large number of them had been
actually destroyed in consequence of this severe treatment; but that
the residue were not only alive, but unimpaired in their energies on
being restored to animation, was proved by their being able to destroy
animals, not having parted with any of their malicious propensities.
Dr. Belli carried out similar experiments with the bacilli of anthrax
and obtained very similar results. With regard to both these varieties
of pathogenic bacteria, he mentions that their action upon animals was
not quite so rapid as is characteristic of normal specimens of these
micro-organisms, thus confirming the experiments in this direction
made with frozen tubercle bacilli.
Not content with the exhibition of their powers of endurance, Dr.
Belli determined to make yet another demand upon the vitality of these
bacilli. For this purpose he immersed them in the liquid air itself,
thus bringing them into direct contact with it, effecting this by
lowering into the liquid strips of filter-paper soaked in broth
containing these bacilli. But, in spite of remaining for the space of
eight hours in these surroundings, they emerged triumphant, exhibiting
no modification whatever either in their structure or pathogenic
properties.
There are doubtless many other trials yet awaiting bacteria, to which
they will most certainly be submitted before the limits of their
powers of endurance have been adequately tested, but it is difficult
to conceive of a severer strain upon their vital resources than the
imposition of the conditions of which the above is but a brief sketch.