_BACTERIA IN THE AIR_
METHODS OF EXAMINING AIR FOR BACTERIA
The basis of the usual methods in practice is to pass air over or
through some nutrient medium. By this means the contained organisms
are waylaid, and finding themselves under favourable conditions of
pabulum, temperature, and moisture, commence active growth, and thus
reveal themselves in characteristic colonies. These are examined, as
directed on page 43, by the microscope and sub-culture. Quantitative
estimation is not generally made, as a fixed standard is even less a
possibility than in milk and soil. Returns of the number of bacteria in
the sample taken may be made for the sake of information, but little
or no conclusion of value can be drawn from such data. The standard
recognised in Europe is the cubic metre, and one may speak, for
example, of the air of a room containing 500, 1000, or 3000 germs per
cubic metre.
The following are the chief methods:
1. _Pouchet's Aëroscope._ This apparatus was in use some time ago in
France, and by its means all the solid matter of a given quantity of
air was drawn through an air-tight glass tube by aspiration and made
to impinge upon a small plate of glycerine. The air escaped to the
aspirator at the sides, leaving upon the glycerine plate only its
particulate matter. This remnant could then be examined.
2. _Koch_ adopted the simplest of all the culture methods, viz.,
exposing a plate of gelatine or agar for a longer or shorter time to
the air of which examination is desired. By gravity the suspended
bacteria fall on the plate and start growth. As a matter of
quantitative exactitude, this method is not to be recommended, but it
frequently proves an excellent method for qualitative estimation.
3. _The Method of Miquel._ Pasteur was the first to analyse air by the
culture method, and he adopted a plan which in principle is _washing_
the air in some fluid culture medium which will retain all the
particulate matter, which may then be cultured directly or sub-cultured
into any favourable medium.
[Illustration: MIQUEL'S FLASK]
Miquel has contrived a simple piece of apparatus for the carrying out
of this principle. It consists of a flask with a central tube through
its own neck for the entrance of the air. On one side of the flask is
a tube to be connected with the aspirator, on the other side of the
flask a tube through which to pour off the contained fluid at the end
of the process. In the flask are placed 30 cc. of sterilised water (or,
indeed, if it be preferred, sterilised broth). The entrance tube is now
unplugged, and the aspirator draws through a fair sample of the air in
the room (say ten litres). This air perforce passes through the water
and by the exit tube to the aspirator, and is thereby washed, leaving
behind in the water all its bacteria. The aspiration is then stopped,
and the entrance tube closed. The water (plus bacteria) is now poured
out into test-tubes of media or plated out on Petri's dishes. Provided
the apparatus has been absolutely sterilised, and that the water was
also sterile, any colonies developing upon the Petri dish are composed
of micro-organisms from the air examined.
4. _The Method of Hesse._ This method is somewhat akin to Pouchet's
aëroscope, but is in addition a culture method. Hesse's tube is
about 2 feet long and 1-1/2 inches bore throughout. At one end is an
india-rubber stopper bored for a glass tube to the aspirator. The other
end is open. Before using, the tube is sterilised, and 40 or 50 cc. of
sterilised gelatine replaced in it. The tube is now rapidly rotated
in a groove on a block of ice or under a cold-water tap, and by this
simple means the gelatine becomes fixed and forms a layer inside the
tube throughout. We have therefore, so to speak, a tube of glass with
a tube of gelatine inside it. The apparatus is now ready for use. It
is fixed on the tripod, and fifteen litres of air are drawn through,
and the tube is properly plugged and incubated at room temperature. In
a day or two days the colonies appear upon the gelatine. They are most
numerous generally in the first part of the tube, and might be roughly
estimated as follows:
15 litres of air, 6 colonies.
⁂ 6/15 × 10,000 = 4000 aërobic bacteria in the cubic metre.
The disadvantages of this process are that dried gelatine does not
catch germs like the broth cultures of Pasteur or Miquel, and that many
organisms are able to go straight through the tube, and failing to be
deposited, pass out at the aspirator exit, and thus are neither caught
nor counted. The Hesse tube is generally used in practice with a pump
consisting of two flasks and a double-way india-rubber tube. The flasks
have a capacity for one litre of water. By a simple adaptation it is
possible to secure siphon action, and hence measure with considerable
exactitude the amount of air passing through the tube.
5. _Methods of Filtration._ To-day most of the above methods have been
discarded, with the exception, perhaps, of Miquel's and modifications
thereof.
[Illustration: SEDGWICK'S SUGAR-TUBE]
Frankland, Petri, Pasteur, Sedgwick, and others have suggested the
adoption of methods of filtration. These depend upon catching the
organisms contained in the air by filtering them through sterilised
sand or sugar, and then examining these media in the ordinary way.
Many different kinds of apparatus have been invented. Petri aspirates
through a glass tube containing sterilised sand, which after use is
distributed in Petri dishes and covered with gelatine. The principal
objection to this method is the presence of the opaque particles of
sand in and under the gelatine. Probably it was this which suggested
the use of soluble filters like sugar. Pasteur introduced the
principle, and Frankland and others have followed it out. The apparatus
most largely used is that known as Sedgwick's Tube. This consists of
a comparatively small glass tube, about a foot long. Half of it has a
bore of 2.5 cm., and the other half a bore of .5 cm. It is sterilised
at 150° C., after which the dry, finely granulated cane-sugar is
inserted in such a way as to occupy an inch or more of the narrow part
of the tube next the wide part. Next to it is placed a wool plug, and
the whole is again sterilised at 130° C. for two hours, care being
taken that the sugar does not melt. After sterilisation an india-rubber
tube is fixed to the end of the narrow portion, and thus it is attached
to the aspirator. The measured quantity (5-20 litres) of air is drawn
through, and any particulate matter is caught in the sugar. Warm,
nutrient gelatine (10-15 cc.) is now poured into the broad end of
the tube, and by means of a sterilised stilette the sugar is pushed
down into the gelatine, where it quickly dissolves. We have now in
the gelatine all the micro-organisms in the air which has been drawn
through the tube. After plugging with wool at both ends, the tube is
_rolled_ on ice or under a cold-water tap in order to fix the gelatine
all round the inner wall of the tube, which is incubated at room
temperature. In a day or two the colonies appear, and may be examined.
[Illustration: SEDGWICK'S TUBE
Fixed upon Tripod for Air Examination]
_Micro-organisms in the Air._ Schwann was one of the first to point
out that when a decoction of meat is effectually screened from the
air, or supplied solely with calcined air, putrefaction does not set
in. Helmholtz and Pasteur confirmed this, but it may be said with some
truth that Schwann originated the germ theory, and Lister applied it
in the treatment of wounds. Lister believed that if he could surround
wounds with filtered air the results would be as good as if they were
shut off from the air altogether.
It was Tyndall[21] who first laid down the general principles upon
which our knowledge of organisms in the air is based. That the dust in
the air was mainly organic matter, living or dead, was a comparatively
new truth; that epidemic disease was not due to "bad air" and "foul
drains," but to germs conveyed in the air, was a prophecy as daring
as it was correct. From these and other like investigations it came
to be recognised that putrefaction begins as soon as bacteria gain an
entrance to the putrefiable substance, that it progresses in direct
proportion to the multiplication of bacteria, and that it is retarded
when they diminish or lose vitality.
Tyndall made it clear that both as regards quantity and quality of
micro-organisms in the air there neither is nor can be any uniformity.
They may be conducted on particles of dust--"the raft theory"--but
being themselves endowed with a power of flotation commensurate
with their extreme smallness and the specific lightness of their
composition, dust as a vehicle is not really requisite. Nevertheless
the estimation of the amount of dust present in a sample of air is a
very good index of danger. It is to Dr. Aitken that we are indebted for
devising a method by which we can measure dust particles in the air,
even though they be invisible. His ingenious experiments, reported
in the _Transactions of the Royal Society of Edinburgh_ (vol. xxxv.),
have demonstrated that by supersaturation of air the invisible dust
particles may become visible. As is now well known, Dr. Aitken has been
able to prove that fogs, mists, and the like do not occur in dust-free
air, and are due to condensation of moisture upon dust particles. But
it should be remembered that, though dust forms a vehicle for bacteria,
dusty air is often comparatively free from bacteria. Hence, after all,
the necessary conditions for dissemination of bacteria in air are two,
namely, some degree of air-current and dry surfaces.
This latter condition is one of essential importance. Bacteria
cannot leave a moist surface either under evaporation or by means
of air-currents.[22] Only when there is considerable molecular
disturbance, such as splashing, can there possibly be microbes
transmitted to the surrounding air. This fact, coupled with the
influence of gravitation, is the reason why sewer gas and all air
contained within moist perimeters is almost germ-free; whereas from dry
surfaces the least air-current is able to raise countless numbers of
organisms. Quite recently this principle has been admirably illustrated
in two series of investigations made upon expired and inspired air. In
a report to the Smithsonian Institution of Washington (1895) upon the
composition of expired air, it is concluded that "in ordinary quiet
respiration no bacteria, epithelial scabs, or particles of dead tissue
are contained in the expired air. In the act of coughing or sneezing
such organisms or particles may probably be thrown out." The interior
of the cavity of the mouth and external respiratory tract is a moist
perimeter, from the walls of which no organisms can rise except under
molecular disturbance. The position is precisely analogous to the
germ-free sewer air as established by Messrs. Laws and Andrewes for the
London County Council. The popular idea that infection can be "given
off by the breath" is contrary to the laws of organismal pollution
of air. The required conditions are not fulfilled, and such breath
infection must be of extremely rare occurrence. The air can only be
infective when filled with organisms arising from dried surfaces.
The other series of investigations were conducted by Drs. Hewlett and
St. Clair Thompson, and dealt with the fate of micro-organisms in
inspired air and micro-organisms in the healthy nose. They estimated
that from 1500 to 14,000 bacteria were inspired every hour. Yet, as we
have pointed out, expired air contains practically none at all. It is
clear, then, that the inspired bacteria are detained somewhere. Lister
has pointed out, from observation on a pneumo-thorax caused by a wound
of the lung by a fractured rib, that bacteria are arrested before they
reach the air-cells of the lung; hence it is at some intermediate stage
that they are detained. Hewlett and Thomson examined the mucus from
the wall of the trachea, and found it germ-free. It was only when they
reached the mucous membrane and moist vestibules and vibrissæ of the
nose that they found bacteria. Here they were present in abundance. The
ciliated epithelium, the moist mucus, and the bactericidal influence
of the wandering or "phagocyte" cells probably all contribute to their
final removal.[23]
There can be no doubt that the large number of bacteria present in the
moist surfaces of the mouth is the cause of a variety of ailments,
and under certain conditions of ill-health organisms may through
this channel infect the whole body. _Dental caries_ will occur to
everyone's mind as a disease possibly due to bacteria. As a matter of
fact, probably acids (due to acid secretion and acid fermentation) and
micro-organisms are two of the chief causes of decay of teeth. Defects
in the enamel, inherent or due to injury, retention of débris on and
around the teeth, and certain pathological conditions of the secretion
of the mouth are predisposing causes, which afford a suitable nidus for
putrefactive bacteria. The large quantity of bacteria which a decayed
tooth contains is easily demonstrated.
From the two series of experiments which we have now considered we may
gather the following facts:
(_a_) That air may contain great numbers of bacteria which may be
readily inspired.
(_b_) That _in health_ those inspired do not pass beyond the moist
surface of the nasal and buccal cavities.
(_c_) That here there are various influences of a bactericidal nature
at work in defence of the individual.
(_d_) That expired air contains, as a rule, no bacteria whatever.
The practical application of these things is a simple one. To keep air
free from bacteria, the surroundings must be moist. Strong acids and
disinfectants are not required. Moisture alone will be effectual. Two
or three examples at once occur to the mind.
_Anthrax_ spores are conveyed from time to time from dried infected
hides and skins to the hands or bodies of workers in warehouses in
Bradford and other places. If the surroundings were moist, and the
hides moist, anthrax spores and all other bacteria would not remain
free in the air.
The bacilli or spores of _tubercle_ present in sputum in great
abundance cannot, by any chance whatever, infect the air until, and
unless, the sputum dries. So long as the expectorated matter remains on
the pavement or handkerchief _wet_, the surrounding air will contain
no bacilli of tubercle. But when in the course of time the sputum
dries, then the least current of air will at once infect itself with
the dried spores and bacilli.
_Typhoid Fever_, too, occupies the same position. Only when the
excrement dries can the contained bacteria infect the air. It is of
course well known that the common channel of infection in typhoid fever
is not the air, whereas the reverse holds true of tuberculosis. The
writer recently obtained some virulent typhoid excrement, and placed
it in a shallow glass vessel under a bell-jar, with similar vessels
of sterilised milk and of water, all at blood-heat. So long as the
excrement remained moist, even though it soon lost its more or less
fluid consistence, the milk and water remained uninfected. But when the
excrement was completely dried it required but a few hours to reveal
typhoid bacilli in the more absorptive fluid, milk, and at a later
stage the water also showed clear signs of pollution. This evidence
points in the same direction as that which has gone before. If the
excrement of patients suffering from typhoid dries, the air will become
infected; if, on the other hand, it passes in a moist state into the
sewer, even though untreated with disinfectants, all will be well as
regards the surrounding air.
Before passing on to consider other matters concerning organisms
in the air, we may draw attention to some interesting observations
recorded by Mr. S. G. Shattock[24] on the negative action of sewer air
in raising the toxicity of lowly virulent bacilli of diphtheria. Some
direct relationship, it has been surmised, exists between breathing
sewer air and "catching" diphtheria. Clearly it cannot be that the
sewer air contains the bacillus. But some have supposed that the sewer
air has had a detrimental effect by increasing the virulent properties
of bacilli already in the human tissues. Two cultivations of lowly
virulent bacilli were therefore grown by Mr. Shattock in flasks upon a
favourable medium over which was drawn sewer air. This was continued
for two weeks or five weeks respectively. Yet no increased virulence
was secured. Such experiments require ample confirmation, but even
from this it will be seen that sewer air does not necessarily have a
favouring influence upon the virulence of the bacilli of diphtheria.
It should be noted that the bacilli of diphtheria are capable of
lengthened survival outside the body, and are readily disseminated by
very feeble air-currents. The condition necessary for their existence
outside the body for any period above two or three days is moisture.
Dried diphtheria bacilli soon lose their vitality. It is probably owing
to this fact that the disease is not as commonly conveyed by air as,
for example, tubercle.[25]
_The influence of gravity_ upon bacteria in the air may be observed
in various ways, in addition to its action within a limited area
like a sewer or a room. Miquel found in some investigations in Paris
that, whereas on the Rue de Rivoli 750 germs were present in a cubic
metre, yet at the summit of the Pantheon only 28 were found in the
same quantity of air. At the tops of mountains air is germ-free, and
bacteria increase in proportion to descent. As Tyndall has pointed
out, even ultra-microscopic cells obey the law of gravitation. This is
equally true in the limited areas of a laboratory or warehouse and in
the open air.
The conditions which affect the number of bacteria in the air
are various. After a fall of rain or snow they are very markedly
diminished; during a dry wind they are increased. In open fields, free
from habitations, they are fewer, as would be expected, than in the
vicinity of manufactories, houses, or towns. A dry, sandy soil or a dry
surface of any kind will obviously favour the presence of organisms in
the air. Frankland found that fewer germs were present in the air in
winter than in summer, and that when the earth was covered with snow
the number was greatly reduced. Miquel and Freudenreich have declared
that the number of atmospheric bacteria is greater in the morning and
evening between the hours of six and eight than during the rest of the
day. But we venture to express the hope that such coincidental facts
may not be exalted into principles.
There is no numerical standard for bacteria in the air as there is in
water. The open air possibly averages about 250 per cubic metre. On
the seacoast this number would fall to less than half; in houses and
towns it would rise according to circumstances, and frequently in dry
weather reach thousands per cubic metre. When it is remembered _that
air possesses no pabulum_ for bacteria as do water and milk, it will be
understood that bacteria do not live in the air. They are only driven
by air-currents from one dry surface to another. Hence the quality
and quantity of air organisms depend entirely upon environment and
physical conditions. In some researches which the writer made into
the air of workshops in Soho in 1896, it was instructive to observe
that fewer bacteria were isolated by Sedgwick's sugar-tube in premises
which appeared to the naked eye polluted in a large degree than in
other premises apparently less contaminated. In the workroom of a
certain skin-curer the air was densely impregnated with particles
from the skin, yet scarcely a single bacterium was isolated. In the
polishing-room of a well-known hat firm, in which the air appeared to
the naked eye to be pure, and in which there was ample ventilation,
there were found four or five species of saprophytic bacteria. Quite
recently Mr. S. R. Trotman, public analyst for the city of Nottingham,
estimated the bacterial quality of the air of the streets of that town
during "the goose fair" held in the autumn. He used a modification of
Hesse's apparatus in which the gelatine is replaced by glycerine. The
air was slowly drawn through and measured in the usual way. Sterilised
water was then added to bring the glycerine to a known volume, the
liquid thoroughly mixed, and a series of gelatine and agar plates made
with quantities varying from 0.1 to 2 cc. By this method a large number
of bacteria were detected in this particular investigation, including
_Staphylococcus pyogenes aureus et albus_, the common _Bacillus
subtilis_, and _B. coli communis_.[26]
During a six years' investigation the air of the Montsouris Park
yielded, according to Miquel, an average of 455 bacteria per cubic
metre. In the middle of Paris the average per cubic metre was nearly
4000. Flügge accepts 100 bacteria per cubic metre as a fair average.
From this fact he estimates that "a man during a lifetime of seventy
years inspires about 25,000,000 bacteria, the same number contained in
a quarter of a litre of fresh milk."[27] Many authorities would place
the average much below 100 per cubic metre, but even if we accept that
figure it is at once clear how relatively small it is. This is due, as
we have mentioned, to sunlight, rain, desiccation, dilution of air,
moist surfaces, etc. So essentially does the bacterial content of air
depend upon the facility with which certain bacteria withstand drying
that Dr. Eduardo Germano[28] has addressed himself first to drying
various pathogenic species and then to mixing the dried residue with
sterilised dust and observing to what degree the air becomes infected.
Typhoid appears to withstand comparatively little dessication, without
losing its virulence. Nevertheless, it is able to retain vitality in
a semi-dried condition, and it is owing to this circumstance in all
probability that it possesses such power of infection. Diphtheria,
on the other hand, is, as we have pointed out, capable of lengthened
survival outside the body, particularly when surrounded with dust. The
question of their power of resisting long drying is an unsettled point.
The power of surviving a drying process is, according to Germano,
possessed by the streptococcus. This is not the case with cholera or
plague. Dr. Germano classifies bacteria, as a result of his researches,
into three groups: first, those like plague, typhoid, and cholera,
which cannot survive drying for more than a few hours; second, those
like the bacilli of diphtheria, and streptococci, which can withstand
it for a longer period; thirdly, those like tubercle, which can very
readily resist drying for months and yet retain their virulence. It
will be obvious that from these data it is inferred that Groups 1 and
2 are rarely conveyed by the air, whereas Group 3 is frequently so
conveyed. Miquel has recently demonstrated that soil bacteria or their
spores can remain alive in hermetically sealed tubes for as long a time
as sixteen years. Even at the end of that period the soil inoculated
into a guinea-pig produced tetanus.[29]
The presence of pathogenic bacteria in the air is, of course, a much
rarer contamination than the ordinary saprophytes. Tubercle has been
not infrequently isolated from dry dust in consumption hospitals, and
in exit ventilating shafts at Brompton the bacillus has been found.
From dried sputum it has, of course, been many times isolated, even
after months of desiccation. M. Lalesque failed to isolate it from the
dry soil surrounding some garden seats in a locality frequented by
phthisical patients. The writer also failed to isolate it from the same
soil. But a very large mass of experimental evidence attests the fact
that the air in proximity to dried tubercular sputum or discharges may
contain the specific bacillus of the disease. Diphtheria in the same
way, but in a lesser degree, may be isolated from the air, and from the
nasal mucous membrane of nurses, attendants, and patients in a ward
set apart for the treatment of the disease. Delalivesse, examining
the air of wards at Lille, found that the contained bacteria varied
more or less directly with the amount of floating matter, and depended
also upon the vibration set up by persons passing through the ward and
the heavy traffic in granite-paved streets adjoining. _Bacillus coli_,
staphylococci, and streptococci, as well as _B. tuberculosis_, were
isolated by this observer.
Some new light has been thrown upon the subject of pathogenic organisms
in air by Neisser in his investigations concerning the amount and
rate of air-currents necessary to convey certain species through the
atmosphere. He states that the bacteria causing diphtheria, typhoid
fever, plague, cholera, and pneumonia, and possibly the common
_Streptococcus pyogenes_, are incapable of being carried by the
molecules of atmospheric dust which the ordinary insensible currents of
air can support, whilst _Bacillus anthracis_, _B. pyocyaneus_, and the
bacillus of tubercle are capable of being aërially conveyed. This work
will require further confirmation, but if its truth be established,
it proves that attempted aërial disinfection of the first group of
diseases is useless.