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THE INFLUENCE
OF ILLUMINATING GAS AND ITS CONSTITUENTS
ON CERTAIN BACTERIA AND FUNGI
by
Clint on Albert Ludwig
1917
A dissertati on submitted in partial fulfillment
of the requirements for the degree of Doctor of Philosophy
in the University of Michigan.

T A B L E O F C O N T E N T S
I. Introduction * * * * * * * * * * * * * * * * * * * * * e e - e. e. e. e. e. e. e. e. e º s
II. Historical - - - - - - - - - - - - * - - - - - - - - - - - - - - - - - - - - - - - -
III. Investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e - -
A. Organisms used and general methods . . . . . . . . .
B. Experimental
l. Illuminating gas
a. Source and composition of the gas . . . . . . .
b. Effect on the different organisms . . . . . . .
2. Ethylene
a. Production and purification of the gas,
etc. , … -e - - - - - - - - - - - - - - - - - - - - - º
b. Effect on the different organisms . . . . . . .
3. Carbon monoxide
a. Production and purification of the
gas, etc. . . . . . . . . . . . . --------- ---------
b. Effect on the different organisms --- - - - -
4, Methane
a, Production and purification of the
gas, etc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
b. Effect on the different organisms . . . . . . .
5. Methyl iodide vapor . . . . . . . . . . . . . . . . . . . . . . .
6. Tobacco smoke
a. Preparation and composition of the smoke,
etc. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
b. Effect on the different organisms . . . . .
7. General observations and discussion . . . . . . .
IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11
l3
29
31
35
35


THE INFLUENCE OF ILLUMINATING GAS AND IT'S CONSTITUENTS
ON CERTAIN BACTERIA AND FUNGI
I N T R O D U C T I O N
It has been known ever since the observations of
Girardin (6) in 1864 that certain phanerogfams are susceptible
to injury by the presence of illuminating gas in the soil or
air, Since then considerable work has been done in Europe
on the subject and in both Europe and America on the allied
subject of the toxicity of smoke. Some of the recent work on
both questions has been done in this country by Crocker and
his students, of Chicago University, who have demonstrated
the very large role played by ethylene in producing the re-
sult 8 observed. They have secured remarkable result 8 with the
use of illuminating gas and ethylene on some very common
plants. They have found, for instance, that the almost in-
finitesimally small amount of one part of ethylene in 2, 000,
000 parts of air causes closing of carnation flowers in twelve
hours (l) and that the even smaller ratio of one part in
10, 000, 000 parts causes nastic curvatures in cast or bean seed-
lings (7). Besides being a matter of very great general and
theoretical interest, the work on toxicity of gases to grow -

ing plants, of which the results just mentioned are probably
as remarkable as any reported to date, has had important
practical applications as well. Thus the greenhouse man has
learned that it is not a good plan to have gas piped into
the greenhouse, especially if connections be not made with the
greatest care. Thus, also, workers in the physiology of the
higher plants have adopted the plan of keeping illuminating
and other gases away from all laboratories in which the work
i 3 conducted. -
With the bacteria and fungi, however, there have not
been reported thus far any cases where such remarkable sen-
sitivene 55 to the chemically more inert organic gases haſe
been exhibited. In fact, very little work has been done with
these gases in this field, and, in most cases reported, gases
were used in the pure condition, very few or no attempts
having been made to determine the lower limit of toxicity.
It became, therefore, a matter of considerable independent
interest and some practical importance as affecting labora-
tory practice to determine as nearly as possible the lower
limit of toxicity of illuminating gas and its separate con-
stituents toward several of these organisms. It was to ef-
fect this sort of a determination and to investigate any
allied questions that might come up that the experiments
forming the basis of this paper were conducted.

H I S T O R T C A L
A number of investigations have been carried out to
determine the reactions of phanerogams to low concentrations
of different gases and a smaller number to determine the more
fundamental matter of the effect on *fare processes. They
have often been concerned in the first instance with smoke
injury; and the results in general have tended to show, as
would be expected, that mineral acid oxides or the oxides of
toxic elements, as, for instance, arsenic, are decidedly
toxic under conditions of much dilution in the air. They
have also shown, as mentioned earlier in this paper, that
certain plants show a remarkable sensitiveness to certain
quite inert gases. This work will not be reviewed further
here, as it has only an indirect bearing on the problem in-
vestigated.
When we come to the lower plants, however, we find that
comparatively little has been done along this line. Quite
early in the history of bacteriology the question of the
Oxygen relation was worked out and convenient methodºper-
fected for the study of this relation with reference to any
particular organism. This latter involved a study of hydro-
gen and carbon dioxide as determining their availability for
displacing the air in anaerobic culture conditions; but the

matter of other gases, especially in less concentrations than
purity, appears not to have seemed to be of importance. There
4.
have been some pieces of work done, however, which have a
suggestive or direct bearing on the problem and are there-
fore worthy of mention here.
Perhaps the earliest published paper of this kind was
by Tassinari (15). In this case, the effect of tobacco smoke
on several bacteria, including both pathogenic and non-
pathogenic species, was investigated. The exposure to the
smoke was made by means of a clever bit of apparatus in which
a drop of the culture was held on a fragment of linen and the
smoke drawn past it for a little while. The strip was then
dropped into sterile media and the time required for develop-
ment noted. The check cultures developed uniformly in 12 to
24 hours. The smoked cultures with only one exception were
delayed from 24 to 100 hours in development or had failed to
develop at all at the end of 8 to 12 days. -
Percy Frankland (4, 5) investigated the effect of hydro-
gen, carbon dioxide, carbon monoxide, nitrous oxide, nitric
oxide, sulphuretted hydrogen, and sulphurous anhydride on
Bacillus pyocyaneus, and the spirilla of Koch and Finkler.
In carbon monoxide the spirilla produced colonies sparsely
while B. pyocyaneus produced none until later exposed to the
air. Nitrous oxide hindered the development of colonies but
did not prevent it, while nitric oxide, sulphuretted hydrogen,
and sulphurous anhydride each prevented the development of
colonies not only while the media": exposed to the gas but
+4 Co
also after ++s return to the air.

Krause (9) observed that Bacillus pyocyaneus would
grow in an atmosphere of illuminating gas or hydrogen sulphide
but would not produce pigment under those conditions. When
later exposed to atmospheric air the cultures produced the
usual pigment.
Edwin F. Smith (14) has made the atatement that the
small amount of carbon monoxide present where the oxygen has
been removed by the potash-pyrogall ol method of conducting
anaerobic cultures is harmless to many bacteria but that he
has reas on to think that it is injurious to others, even if
it does not entirely inhibit growth. The grounds for his
suspicion were not given.
moltech (11) studied the effect of tobacco smoke on
certain phanerogams and microorganisms. He found that the
movements of Chromatium Vinosum (Ehrenb.) Winogradsky,
Begºñas SQ = , and Spirillum sp. were stopped by the smoke.
The experiments were carried on by means of hanging drops
in a small chamber. The growth of Phycomyces nitens was
slowed down. His work showed that nicotin will not cause
reactions in the “phan erogamic plants used, Vicia satival,

Pisum sativum, and Cucurbita Pepo, similar to those caused
by the smoke, but that pyridin and carbon monoxide will each
do it. This result is reinforced by the added ob servation.
that the smoke from burning paper, wood, or straw will give
rise to the same reaction.
Mūnz (12) has recently succeeded in isolating from
garden soil, ditch water, river ooze, and leaf fragments of
various water plants certain bacteria which are capable of
utilizing methane as a source of carbon and of energy. The
writer regrets that he has not had the opportunity of read-
ing Irºnz's work. The note given above was made from an ab-
stract in the Zeitschrift für Botanik. The original paper
is a dissertation at Halle, and dºo the war, was not
available for examination.
The published work with fungi and algae is even less
abundant than with bacteria. A few pieces of work are worth
mention, however. Molisch (11) has shown, as was mentioned
above, that the growth of Phycomyces nit ens is slowed down
by smoke; and Thom (16) has reported that in an atmosphere
of carbon dioxide no one of the species of Penicillium with
which he worked developed within a week but that development
set in after the tubes were rest or ed to the air,
Richards and MacDougal (13), working with Nitella, found
that it could live in carbon monoxide of 80% concentration but
it was somewhat paler than the check in air .
Working with certain other algae Woycicki (19) has shown
that illuminating gas will induce certain remarkable altera-
tions both in the shlºſºe of the cell and in its internal
structure. He worked with species of Spirogyra, Cladophora,
and Mougeotia and found that in many cases curious outgrowths



of the cells were produced which often resembled holdfast s
the filaments usually became broken up into small pieces or
even into the individual cells and the contents of the cells
became more or less disorganized according to the strength
of the gas. The cells were often killed. In the case of
Cladophora fract a var. horrida the sensitiveness was seen to
be much less than for Spirogyrg and with certain strengths of
the gas this species produced a plano spores. It was found also
that the laboratory air of ten contains enough gas to induce
alterations in the algae and that carbon monoxide and acety-
lene are capable of calling forth the changes.
Langdon (10) has recently made the somewhat remarkable
discovery that free carbon monoxide occurs in the floats of
a Pacific marine alga, Nereocystis luetkeana, sometimes to
the extent of 12% of the enclosed gases. The range was found
to extend down to 1% and to average about 4%. This is in-
teresting in view of the generally accept ed belief in the
poisonous nature of this gas to plants, since it shows that
at least some plants capable of conducting photosynthesis
contain tissues which are tolerant of quite large amounts
of this substance.
I N V E S T I G A T I O N
ORGANISMS USED AND GENERAL METHODS
The organisms used in the study here reported consisted
of bacteria and fungi. Of the se, a number, Bacillus subtilis
B. & Fey, easis (Zehrn, a na rearm.) ſtig, ("ruber of Kiel),
Conna B. pyocyaneus Gessard, B. rubidus Eisenberg, and Sarcina

lutea Schröter were obtaired from the Dept. of Bacteriology
of the University of Michigan. A number of bthers, Bacillus
garot ovorus Jones, B. melonis Giddings, B. gampestris Pammel,
B. mycoides Flºgge, B- solanisaprus Harrison, B. radicicola
Beyerinck (of red clover), Bacterium stewarti Erw. Smith, and
Bact. tumefaciens Erw. Smith were securéd from the American
Museum of Natural History through the Botanical Dept. of the
University of Michigan. The following fungi were used: –
Oidium lactis Fregenius, obtained from the Dept. of Bacteri-
ology, rerºsolated from the air in the laboratory,
Penicillium sp. , isolated from moldy bread, E. pinſºphilum
Hedgcock, P. camemberti. Thorn, P. roqueforti Thorn, and P.
expansuſ, Link, all of which were obtained from Miss Margaret
B. Church through the courtesy of Dr. Chas, Thorn, Fusarium
radici cola Wollenw: , Gleosporium cingulata. Atkinson, Endothia
parasitica (Murr.) P. J. & H. W. And..., and E. fluens (Sow.)
S. & S., all of which were received from Dr. Lon A. Hawkins,
The cultures were carried on ordinary 1% glucose, 1%
pept one, 0.3% beef extract agar, with 0.5% sodium chloride
and 1.5% agar, except that some of the experiments with B.
rubidus were carried out on autoclaved potato slants. The
color which it develops on this substratum makes the medium
of some value. The reaction of the agar varied with differ -
ent batches from nearly zero to slightly over +l on Fuller's
scale, but was usually about +0.8.

The exposures of the bacteria and some of the fungi to
the gases were made in test tubes confined in air tight
chambers. This method has the disadvantage that it is prae-
tically impossible to get quantitative data by its means
such as could be obtained by using Petri plates and counting
colonies. On the other hand the development can be followed
better from day to day in tubes than in plates within a
larger vessel. The air tight chambers used consisted dºf four
Novy jars and a number of bell jars with tubulature at the
top which were fitted with two hole rubber stopper's holding
tubes for the introduction of gas. sºlaces in a base
composed of a heavy crystallizing dish with a layer of plaster
of Paris about 2 cm. thick, impregnated with paraffin, in
the bottom. The plaster of Paris was poured in the dish in
the wet condition and was prevented from breaking the dish
by putting paraffined corrugated paper around the edge of the
dish to take up the expansion. The chambers were sealed by
running melted paraffin between the base of the bell jar and
the side of the dish.
The gases were introduced from a Hempel gas burette,
+++++++g the ga's 3+, by means of the pressure of a few centi-
meters of water during the earlier part of the work. However
in most of the experiments the gas was allowed to enter di-
rectly and its amount was measured by reading pressures on a
manometer made by inserting the open lower end of a tube of
mercury in a layer of mercury in a bottle. The tube passed
- ºtá, 7c. Cº. 7 ºł. —£, Zz6, , &_v^^ a C º://".
AL-M.

1 O
through the other hole in the stopper. As thus set up the
apparatus constituted a barometer, but it was only necessary
to attach the free tube to any closed receptable in order to
use it as a manometer. When a certain amount of gas was to
be introduced into a given chamber, the refore, the chamber
and the man ometer were connected at the same time to an aspi -
rat or and exhaustion carried out, usually to about 15 cm, of
mercury. They were then connected with the gas container and
gas all owed to ent er until the pressure had risen the calcu -
lated amount on the scale, the calculation being on the basis
that the amount of a gas in a given volume varies as the
pressure. The apparatus was then allowed to finish filling
with ordinary air, after which it was closed and set aside.
The reason for introducing the gas while the pressure was low
was to furnish other means than diffusion to insure it 5 pass -
ing through the cott on stoppers. It is not certain, of course
that the gas within the tubes was of the same compositi on as
that in the chamber, outside the tubes; but with the evidence
y/e/area/
of diffusion through the stoppers,in such cases as that of
smoke and methyl iodide vapor where the aid of air pressure
could not be employed it seems not unreas onable to think that
the gas concentrati on within the tubes at least approached
pretty closely that in the bulk of the chamber. The concen-
trations mentioned in all cases are to be considered not as
giving exact values but merely fairly close approximations.
The actual values were probably slightly lower. When pure
gas of some kind, or at least oxygen free gas, was desired in
-- ~~

11
contact with the cultures one of two or three different plans
was employed. In the case of illuminating gas it was either
allowed to pass through the vessel continuously during the
experiment or it was passed through long enough to displace
the air and then stopped. In the latter case it was usually
renewed daily during the experiment. When washed with
pyrogallol to remove the oxygen the experiment was conducted
in the usual way in a small Novy jar, the exhaustion being
carried out about six times. Foºerſ gases, which had to
be manufactured for the purpose, a battery of eight test
tubes fitted with perforated rubber stoppers containing glass
tubes arranged in wash bottle fashift was used. The gas after
Washing in alkaline solution of pyrogallol was passed through
the battery of tubes. The connections were carefully wired
and sealed with sealing wax and paraffin.
All results here reported unless otherwise stated were
from at least two trials and many of them were checked
several times.
EXPERIMENTAL
l, Illuminating Gas
Source and Composition of the Gas
tº of the work consisted in test s with illumi-
nating gas. Such tests have the disadvantage, of course,
that the gas is a mixture and not a perfectly constant mix-
ture at that ; but its - d the fact that it is


l2
the substance which usually contaminates laboratory air made
it seem worth while to use it. The gas used in Ann Arbor
during the first part of the experiments (winter of 1915 - 16)
was pure coal gas; later (winter of 1916-17 to Feb. 1), it
was a mixture of coal and water gas; and at the last (after
Feb. 1, 1917) it º:-- once more of coal gas only,
except for the 5-day period, Feb. 12-16, during which time a
small amount of water gas was mixed in. The gas before and
l
after the date mentioned analyzed approximately as follows:
*I am indebted to Prof. W. L. Badger of the Dept. of
Chemical Engineering of the University of Michigan and to
Mr. Chas. R. Henderson, Chemist to the Ann Arbor Gas Co., .
for the analyses and data given here concerning the illu-
minating gas used in the se tests.
Before Feb, l After Feb. 1.
C02. . . . . . . ... i - 2 % . . . . . . . . . . . . . . . . e -- e. 0.9 – 2.0 %
CnH2n . . . . . 4 - 5 % . . . . . . . . . . . . . . . . . . . . 3.5 - 4.5 %
02 . . . . . . . . 1 - 2 % . . . . . . . . . . . . . . . . . . . º 0.8 - 1.5 %
CO . . . . . . . . ll -14 % . . . . . . . . . . . . . . . . . . . º 6.0 - 7.8 %
CH4 . . . . . . 25 -30 % . . . . . . . . . . . . . . . . . . . . 30 - 35 %
H2 . . . . . . 40 -50 % . . . . . . . . . . . . . . . . . . . º 35 - 45 %
Nº. about 10% . . . . . . . . . . . . . . . . . . . . 8 - ll 3%
The figures given here are not the result of specific
analyses made for the puſgºose of this study, but instead are
the result of the examination of a large number of 3tudent

13
analyses. However, as the gas was used at various times over
an interval of a year and a half or more it seems that any
analysis more exact than that given above can hardly be of
greater value for purposes of interpreting the results. Or -
dinarily the gas was not washed. It was the intention to do
so, but the pressure in the pipes was not sufficient to drive
it through wash bottles, and one or two preliminary experi-
ment s showed that with the organisms at hanfºia not ex-
hibit the extraordinary toxic properites shown toward some
phan erogams. It was therefore decided that as long as no very
decided toxic properties were shown it was not necessary to
- other in organ ic
remove traces of H2S, NH3, or, gases which presumably might
be present and exert harmful influences,
Effect, on the different organisms.
In giving the results of the tests with illuminating
gas, and with the other gases as well, a brief summary will
be given for each species used, instead of giving a chronolog -
it?al account of the experiments or of giving single experi-
ment 3 in detail.
Bacillus subtilis. This organism was cultured in the
following approximate concentrations of illuminating gas : -
0.5%, 5%, 10%, 25%, 50%, 75%, 85%, and 100%. In ałt these
concentratiº and including 25% the colony development,
both as to abundance and as to character, was practically
identical with the development in air. This normal growth,
14
as is well known consists of a white, often wrinkled, layer
on the surface of the agar. The development in 50% gas and
above, however, was quite different in character. The chief
difference, and perhaps the only one of importance, was the
f! Wvalad, 2k, wºoa a
much smaller volume, of the colony produced. This was very
small, so that the colony never had the opaque character of
normal ones. The development was confined to the area in -
ošulated and did not spread over the surface of the agar
as was the case when development was normal. Occasionally
only pin point colonies were developed, or perhaps nothing
at all until after return to the atmosphere. Complete steril-
ization practically never took place with arl exposure not
to exceed ten days; but it sometimes took a week of more for
development to become evident after return to the air. In
those cases where some development occured in the gas it did
not proceed further when returned to the air, but usually
after the lapse of a variable period of time an area of normal
development began at some point and grew over the slant.
Inoculations made from these colonies grown in gas produced
in the air a colony development differing very slightly or
not at all from the normal in appearance.
Two series of expejºiments were run to test the ability
of the organism to develop continuously in different per-
centages of illuminating gas. In these tests the inoculations
after the first generation instead of being made from air
cultures were made from the percentage of gas into which they

lº
were to go unless no development had taken place in that
concentration. In that case the inoculation was made from the
highest lower concentration in which growth had occurred. In
the course of this work, the organism was carried through 5
transfers in each of 5%, lož, and 25% gas, 3 transfers in 50%,
5 transfers in 85%, 5 transfers in 75%, and 9 transfers in
pure gas. It is quite evident, therefore, not only that the
organism can grow in the gas, but that it can keep + up, Or
an indefinite period under the conditions of these experiments.
To determine if it was the small amount of oxygen present in
the gas that made growth possible some cultures were run in
a small Novy jar which was evacuated six times to about 10 cm.
- *** w8.2k woe-
Of mercury, and, gas, allowed to enter slowly through two or
three wash bottles containing alkaline solution of pyrogallol.
The development was almost as good as in untreated gas, but
there may have been traces of oxygen present. In one case a
culture of yeast in sugar solution was also put into the jar
to absorb any small amount of oxygen that might still be
present. There was no noticeable difference in the develop-
ment, but there may still have been traces of oxygen present
as the yeast culture fermented very weakly if at all, so that
it can hardly be relied on to have removed any oxygen possibly
present. Cultures made directly from air cultures showed an
ability to grow ºn-ºre illuminating gas sº not




slightly superior to that of cultures from pure gas.
16
The growing of the organism in the pure gas seems to have
caused little or no change in it, except in the colony charac –
ter due to the slowing down of growth to a very low point.
This is evidenced by the fact that a culture inoculated, with
the organism from a line which had been carried exclusively
in the air developed as well as another from a line which had
been carried for some time in the gas. It is also evidenced
by the fact that in the first transfer cultivated in the air
after a period of several generations in gas the colony growth
was normal in appearance in all respects. The examination of
stained microscopic mounts supported the foregoing evidence.
Bacteria from air cultures one day and ll days old and from a
gas culture ll days old were stained. The bacteria of the
gas an". *). case resembled those of the one day air
culture more than the 11 day air culture in size, shape, and
especially in the presence of spores. There were only a few
spores in the one-day culture, none in the ll-day gas culture,
but most of the structures in the ll-day air culture were
Spores,
Bacillus pyocyaneus. The culture of B. pyocyaneus used
in this study developed the green color only rarely. The
coi or has therefore not been used as a character on which to
base comparisons, although it has been so used in the past.
The typical colony growth was rather dirty white, semi-trans-
lucent in character, and it was often difficult to determine
whether or not the growth in one tube was more vigorous than




17
->
in another. The organism was first grown in 5%, 50% and
85% gas. The development was quite normal in 5% gas but pro-
ceeded more slowly in the higher concentrations so that where -
as it took about three days for a culture in air to reach its
maximum, it took two to three days longer for the 85% gas
culture to reach the same stage. Attempts at this time to
grow it in pure %luminating gas met with three clear cut
failures and one apparent success, but the apparent success
was perhaps due to failure to displace all the air in the
container. In one of the cases of failure the organism
developed (after a 4-day exposure) when returned to the air,
but in the other two (after exposures of 6 days and 3 days)
it did not develop within periods of 16 and *...* rectively.
These results were apparently in opposition to those of
Krause (9), who reported that B. pyocyaneus will grow in pure
illuminating gas. In later experiments, however, as will be
shown presently, the attempt to cultivate the organism in
pure gas resulted successfully. There was little or no al-
teration in the colony character in the gases, provided a
colony was produced.
It was found also that the organism can apparently be
carried indefinitely in most of the gas concentrations and
perhaps also in pure gas. It was carried through 5 transfers
in 5%, 10%, and 25% gas, 3 transfers in 50%, 5 transfers in
85%, ll transfers in 75%, and 9 transfers in pure gas. In the





unsuccessful trials with pure gas in these two series, the
18
organism always developed on the slant after being exposed to
the air, although sometimes appearing in separate colonies
instead of a streak, as if most of the inoculating bacteria
had been killed. It seemed at first, therefore, that B. pyo-
cyaneus is able, either by direct modification or by the
selection of a resistant strain, to develop the ability to
grow in pure gas. Later trials showed, however, that cultures
devel oped in the gas just as well from inoculations from the
air as from 75% gas or pure gas. These apparently contradic -
tory result s seemed quite unexplainable except on the assump-
tion of a change in the composition of the gas and it was
thought at the time that as A&ºss had taken place.
Later, knowledge was obtained of the variation in the gas com -
position which has already been noted. Upon comparison it,
was found that the successful cultures of the organism began
about Feb. 1, 1917, at the time the change was made to pure
coal gas instead of the mixture of aoal and water gas. The
significant change in the composition of the gas would seem
to have been the drop from about 12 or 13 to 7 or 8% of carbon
monoxide. It should be remarked, however, as will be shown
later, that neither of these coneontrations of Co is of much
significance if the rest of the mixture be atmospheric air.
In fact the organism showed such great tolerance of CO when
mixed with air that one would not expect a difference of only
5 or 6% of the gas to exert any marked effect. It is also
worthy of note that the first failures to grow in the gas

19
occurred during the first period when the gas consisted Of
pure coal gas and had about the same composition as in the
last period. The results, therefore, are even yet unex-
plainable on the basis of any knowledge now at hand.
Bacillus Kieli ensis. This organism grows vigorously,
reaching a maximum in 3 to 4 days and has a very brilliant
dark red color with a surface greenish metallic or coppery
sheen. As the colony ages the sheen gradually disappears,
the colony becomes brickish red and the pigment often diffuses
more or less into the medium. This color responds readily to
cultural conditions and so furnishes a sensitive index for
detecting disturbances with the life processes. It s alt era -
tions are so numerous and complicated, however, that no at -
tempt will be made either to describe them fully or to rºlerº -
ti on the many variations observed. In gas concentrations of
10% and less the development was normal. In 25% it was some-
times normal but more of ten slightly retarded and the color
rendered less brilliant. In 50% gas the metallic sheen was
usually nearly lacking, the color considerably lighter, and
the rate of growth considerably less, so that it took ab-eat
two or three days longer for it to reach its maximum than it
took in air. In still higher concentrations these changes
were progressively more noticeable until pure gas was reached,
in which development was very slight and the colony was color-
less or whitish, with only occasionally a trace of pinkish
pigment. In the gas-air mixtures the color was usually


20
variable, ranging from déep red to light pink or whitish and
sometimes with purplish shades, and a number of these shades
usually occurred in the same streak. The weak colonies de-
veloped in gas of a high concentration grew vigorously when
again returned to the air and devel oped pigment, but never
to the extent shown by a colony grown in air from the start.
In testing its ability to grow continuously in gases
the organism was carried through 5 transfers in 5%, 10%, and
25% gas, 3 transfers in 50%, 5 transfers in 85%, ll transfers
in 75%, and 10 transfers in pure gas. Growth occurred in
pyro gallol washed gas when tubes were inoculated from the pure
gas culture or from an air culture. They were about equal in
amount and grew also in a second oxygen free gas culture. Air
cultures i no culated from cultures carried for several trans -
fers in pure gas developed abundant but not normal pigment,
However, a few transfers sufficed to bring about normal de-
velopment. Where the cultures had been carried in gas for
only two generations the color was normal at the first re -
cultivating in air.
w The examination of the stained preparations showed a
lack of any striking morphological differences between the
treated and unt reated bacteria. In air culture the organism
tends to show shorter, smaller, more c occus-like rods as the
culture grows old. In gas the juvenile shape seems to be
maintained for a longer period of time, probably ſº to the
slowing down of the development.
2l
Bacillus rubidus. - Part of the cultures of B. rubidus
in illuminating gas were made on autoclaved potato plugs.
On this medium it produces a clear orange color which makes
development easy to detect. The organism grew well in 0.5%
and 5% illuminating gas, but did not grow in a strength of
approximately 85%, except possibly slightly in one trial,
although it developed promptly when restored to the air. In
pure gas no development eccurred during the exposure. . In one
case no development followed a 3-day exposure within 18 days,
and in another a 6-day exposure was followed by no development
within 22 days; in a third a very slight development began
5 days after the close of an 8-day exposure.
- Sargina lutea. Sarcina lutea proved to be one of the
more susceptible organisms studied. It was, however, grown
in 5%, 10%, 25%, 50%, 75%, and 85% of illuminating gas.
There was ordinarily no checking of the development in the
- *~~tº wra o cLa e-º-º-o-º-4-
5% concentration, but * ºf £ºlº
i-at-en-sity in ºntration. used, al-e-ºe-tº-º. The air
culture and 5% gas culture would reach their full development
first, and then the others would successively reach approxi-
mately the same stage, although this did not come fully to
pass except very rarely with culture in 50% of gas or above
in the time during which the exposure continued. In the con-
tinuous culture in gases the organism was carried through
5 transfers in 5%, 10%, and 25% gas, 3 transfers in 50%, and
lo transfers in 75%. It was found impossible to secure


22
unmistakable development in pure gas although there were a
few cases of possible very slight development.
Oidium lactis. . The work with Q. lactis revealed the
fact that it +s—apparently—just about &ºts ºf
* º ŽK.
- -
º
º
OYıCII. T. j. Of
It was grown in all cong entration 3 of illuminating gas em-
ployed, including the pure gas both untreated and washed in
pyrogall ol solution; but its behavior in the stronger concen -
trations was rather erratic, Usually the growth under such
conditions was very slight; and sometimes it started from only
a few centers, as if the treatment had partially sterilized
the slant; but at other times it would approach the maximum
reached by a culture in air if left long enough. It was
grown continuously for 5 transfers in 5%, lož, and 25% gas,
3 transfers in 50%, ll transfers in 75%, 5 transfers in 85%
and 5 transfers in 100%. At times the character of the col-
ony development diffeyed between those in large percentages
of gas and those in air or low percent ages of gas but here
again the reaction was not uniform. In some tests the
threads were more *pressed and more water soaked in appear-
ance in a high gas atmosphere and in others the threads Were
more upright and tufted or white velvety in appearance.
Torula sp. - This organism did not show any particularly
remarkable characteristics in connection with these studies.
Normally the colony is deep pink in color and is composed of
quite a considerable mass of material. In gases the toxic







23
ffects are evidenced by a slower or in complete development
of the colony and by a paler color than normal. The organism
grew in all the gas-air mixtures used -- 5%, 10%, 25%, 50%,
75%, and 85% gas -- but not in pure gas. The 5% and usually
the 10% concentration did not show any toxic effect but at 25%
of illuminating gas the development was checked without ex =
ception. At 75% and 85% the development was very slow and
the colony quite pale, sometimes nearly colorless. Normally
an exposure in pure gas was followed by development within a
week when returned to the air, but in a few cases an exposure
Of a week or less seemed to sterilize the material, since no
development had taken place in 13-18 days. In continuous
growth in gas it was carried 5 transfers in 5%, lož, and 25%
gas, 3 transfers in 50%, ll transfers in 75%, and 5 transfers
in 85% gå.5 s
Earlsºn Sps - - This species on the medium used
grows rather rapidly and soon becomes green with the large
number of conidia produced. This color soon changes to some
shade of brown, and later the colony is often overgrown with
hyphae from under neath. The checking effect of unfavorable
conditions can of ten be detected in cultures long after
conidia production by means of the younger appearance of the
treated cultures as compared with the check. Development
occurred in 5%, 10%, 25%, 50%, 75%, and 85% gas but not in
pure gas. It was quite normal in character tº o 50% but was
much slowed down at that concentration. The checking of
24
growth was abserved at 10% but not at 5%. At concentrations
of 75% and above development is slow and does not extend very
far laterally. As a result a cushion shaped or rought ly
hemisphaerical mass of apparently upright hyphae is produced
at each point of inoculation. The entire slant, therefore, often
contained these pulvinate colonies, which sometimes reached a
diameter of 2 or 3 m. m. and became more or less confluent.
Conidia were not produced under such circumstances except in
one or two instances where it is doubtful if the percentage
of gas had been maintained. Even after the return of these
cultures to the air there was only exceptionally any coni dia
production although new cultures inoculated from them and
kept in the air devel oped coni dia, normally. Cultures pre-
vented from developing by being exposed to pure gas grew and
produced conidia as usual in ºf cases upon being returned
to atmospheric air. The species was maintained continuously
in culture for 4 transfers in 5%, 10%, and 25% gas, 3 transfers
in 50%, lo transfers in 75%, and 4 transfers in 85% illumina-
ting gas.
Bacterium stewarti. Bact... stewarti, which is the cause
of a disease of sweet corn, is an aerobic organism which proved
to be one of the most susceptible employed. It grew in 5%,
10%, 25%, 50%, and 75% of illuminating gas; and it was possible
to keep it growing continuously in the se; but the development
in the last concentration mentioned was slow. A distinct


25
checking effect was always shown by 10% and often a slight
one seemed to be shown by 5%. In 75% of the gas the organism
failed to develop at first but later did develop. The first
development occurred at about the time the city gas company
ceased producing water gas as was the case of the first de-
velopment of B. pyocyaneus in pure gas. The circumstance is
probably referable to the reduction of the CO content of the
gas. No development was observed in any concentration used
above 75%; but only rarely did development fail to take place
after removal to the air, although it was usually 5-15 days
in becoming visible and was also usually slight or very slight
in amount.
The following organisms were tested once in each of 25%,
50%, 75%, 85%, and 100% illuminating gas. -
Bacillus carotovorus. -- In pure gas there was slight
development of B. carotovorus in one case at the end of a
6- day exposure but none in the other at the expiration of an
equal period. In both cases, however, prompt development fol-
lowed a return to the air. Growth occurred in all the lower
concentrations, but it was checked in all; and the retardation
was still noticeable in the 25% concentration at the end of the
6-day period.
Bacillus melonis. In one case with B. mel on is the tube
in pure gas showed a very slight development at the end of the
period. In the other, however, no growth was visible although
it became visible soon after the return to the air. Growth
26
took place in all the lower comeentrations but it was much
retarded in all. The retardation was greatest in the higher
concentrations. £3
Bacillus campestris. -- witägempestria also, develop-
ment occurred in all the concentrations of gas used except
pure gå.S., although there was distinct retardat i on in even the
25% concentration. In pure gas there was not growth in one
trial but a possible very slight development in the others
In both cases growth occurred after removal from the gas,
although it took periods of 6 and 5 days respectively for it
to become discernible.
Bacterium tumefaciens. -- There was visible development
of Bact. tumefaciens in all percent ages of gas except pure
gas, although a distinct checking effect was observed in the
lowest concentration used, 25%. No development took place in
pure gas but occurred after returning the tube to the air.
The colonies in this case became visible 5 - 8 days after the
removal from the gas.
Bacillus solani saprus. --- There was a flistinct and c on-
siderable checking of the development of B. solani saprus in
all of the concentrations of gas used, and in the greater ones
the development was only slight . In the pure gas there was no
visible growth, although it did occur following the return of
the culture to the air, in which case it became visible in
1–5 days.
Bacillus radicicola. -- B- radi cicola proved to be one

27
of the more susceptible species. Development occurred in
25% gas, but it was only slight. At 50% concentration and
above development was absent although it occurred following
the return to the air. The periods of time in which the col-
onies became visible in the cultures removed from pure gas
to the air were 12 days and 5 days respectively.
Bacillus mycoides. -- There was at least a vejºy slight
development of B. mycoides in all of the concentrations of gas
used but the 25% strength showed a retarding action, since it.
took three days for the colony to cover the slant from a spot
in oculated at the center while in air the slafºt was covered
in two days. The development remained very slight in the
higher percentages of gas until the tubes were removed to
the air 6 days after the beginning of the tests in each case.
This very slight colony developed in the case of the 50% and
85% gas, where the inoculation was by streak, was very similar
in appearance to that of B. subtilis in concentrated gas.
After being removed to the air normal development began in
from 2 to 5 days and soon covered the slants. It did not
originate all along the streaks, however, but at isolated
points, so that separate colonies were formed as if a partial
sterilization of the slant had been produced by killing the
bacteria between the points where the colonies arose.
A number of fungi were grown in Petri plates and the ef-
fect of different gases noted by measuring the diameter of the


28
colonies at 3-day intervals, -es (in some instances where this
could not be done the sum of two different radii was taken
instead). The medium used was potato, glucose (2%) agar (3%).
It was poured into the plates and the fungus inoculated into
the center of the freshly hardened layer of agar. The plates
were then sealed under bell jars and the gas introduced in the
usual way. The apparatus was taken down every third day to
record data. In the jar containing pure gas the gas was al-
lowed to pass through constantly. This dried the agar in the
first test and may have had something to do with the failure
of any growth to occur in any of the plates after return to
how 2 v Q ſº
the air. In the second test, the gas was passed over water in
a wash bottle before entering the jar, and this prevented ex-
cessive evaporation. The accompanying ºre. the organ-
isms, the treatments, and the results obtained, the measure-
ment s being diameters of colonies in millimeters. It can be
seen from this that the lower limit of toxicity, or at least
of retardation of growth, is between 10% and 25% in most of
the species; but in one, Fusarium radicicola, it is remarkably
high, being at or nearly 50%. None of the values indicating
retardation at 10% or below tºnes than the value
for its check except for Endothia fluens at 10%. The lower
limit for inhibition of E. fluens, therefore, would seem to
be somewhere between 5% and 10%. The retarded cultures, after
being returned to the air, grew at approximately the same rate
29
as the regular air culture, thus demonstrating that after -
effects are usually lacking; except that no development oc-
curred in any case in any plate which had been exposed to
pure gå 5 s
Some of the cultures, including the two species of
Endothia and Fus. radicicola, produced in the toxic amounts
of gas a more compact, velvety, deeper colony, with the
hyphae more erect than in air. It is likely that these
hyphae have interesting morphological characteristics induced
by the treatment but opportunity was not found to investigate
this feature.
It seems clear, therefore, that among the species studied,
there is no example of the extreme sensitiveness to illumina-
ting gas which is displayed by some phanerogamis. The tolera-
tion of the organisms for other gases will be treated in the
parts that follow.
2. Ethylene,
Production and purification of the gas, etc.
The ethylene used in the se experiments was produced by
heating 95% alcohol with c. p. sulphuric acid. It was passed
through wash bottles containing water, sodium hydroxide solu-
tion, and c. p. sulphuric acid respectively and stored in a
gasometer over water until required for use. It was not ana-
lyzed but when it was important that the oxygen impurity be
removed the gas was passed through two or three wash bottles
containing pyrogallol. The exposures to the gas were made in

TABLE SHOWING THE EFFECT OF TIFFERENT CONCENTRATIONS OF
ILLUMINATING GAS ON THE GROWTH OF SEVERAL FUNGI
|
Gas No. of Penicillium Penicillium Penicillium Penicillium Endo thia Endºthia. Fuscºrium Glomerella
trial pinophilum camernberti | rºgue forti eXparl 81 m. paraxitica. # fluens - radici cold cingulata.
No of --- -
mea, Surº €e l 2 3 4 | l 2 3 4. l 2 3 4 l 2 3. 4. L 2 3 4 l 2 3 4. l 2 3 4
ment --- d
-- l ižº Lºs T-13 IEE 13 – 25 -35 T 35T-62T-51 - 18 -37 – - 24 =53 -87 - 3 ºf -2.3 - 24 -45 -66 -90 29 - 54 -80 -
Air 2 6.5-24 -41 -56 ll -23 -32 -43 23 -61 -85 - l6 =33 -50 -68 4 - 5-34 -65 -88 1; -º-; C.s" l'? -47 -73 20 -55 -84
A.Y. 10 -26 -42 -57 12 –24 -33.5-43 25. 5-61 - 5-88 - 17 – 35 =50 =68 14 -44 - 5-76 -88 - e 20 - 5-46 -69.5-90 24 - 5-54 w8-82
- - - ll. -42 -71 -87 5 -44 -71 -º 19 -52 -80
5% gas 2 7 -24 -39 -53 11.5-24 -32 -44 18 - 52 -89 - 15.5-33 -5]. -67 7, 5-35 -67 – 9 O * 42 | = { l l
- 7, 5-29 -51 -73 14 –43 -70 -92 17. 5-50 -78
10% gas 2 6.5-21 -36 -51 ll -22 -29 -41 14 -49 -81 - 15 - 31 =46 -63 7. 5-33 -60 -83 -
- º 9 - 12 =34 =60 23 =44 -65 -87 18 -37 -58 -82
l 10 -l9 -29 - 8 -l 5 -21 -30 17 -44 - 52 -93 8 - 17 - 25 -38 18 -32 -49 -73 5.5-22 -39 -67 14 -41 -66 -85 12. 5-39 -68
25% gas 2 4 -ló -27 -42 7.5-16 –24 -30 || 7.5 - -59 - 8.5-19 -27 -4.3 6 *23 -39 -65 7 -17 -36.5-63.5 la .5-42.5-65.5-86 15 -38 -6.3 -82
AV - 7 -17. 5-28 --42 8 -15 - 5-22. 5-30 12 •44 - 55.5-93 8 -18 -26 -40 - 5 12 -27. 5-44 -69 --
- ----- º 7 - 9 – 21 -45 20 -42 -63 -87 15 -29 -44 -67
l 8 -13 - 22 - 6 *ll -ló -24 8 - 29 -40 - 70 5 =9 - 5-sel 3 = 24 ll. -21 -34 - 56 l, 5–10 -l'? -37 12 -36 -60 -82 9 -26 -4.3 =69
50% gas 2 2 -ll - 5-21 -36 4.5-lo -16 -23 3 -12 - 23 -73 3.5 - 7, 5-12 -29 -l'? - -26 -48 4 * 9 - 5 -19 -41. 16 -39 -61 - 5-84 - 5 12 -27. 5-43 - 5-68
AV . 5 -l2 -21 - 5-36 5 -10.5-16 -23.5 5.5-20.5-36.5-71.5 4 - 8.5-12-5-26-5 11 -19 -30 -52 -
- 1 - 5 - 6 -ll = 30 l6 =39 - 54 -76 10 -19 -30 -53
º: l 4 - 5 - 5 - 7.5–21 4 - 5 = 7 = 2 - 3 - 5 -33 2 - 4 - 5 - 16 8 =l 4 - 20 -42 O = 2 - 6 - 27 7 - 23 -38 -59 5 e 16 -29 -55
75% 2 2 - 4 s 5- 7 - 22 2 - 6 - 9 -18 1 - 4 - 8 -37 3 - 5 - 7 - 22 3 - 8 -13 -39 1 – 4 – 8 - 5-28 - 5 ll a 5-31 -46 -67 - 5 7.5-17. 5-29. 5-54
AV . 3 - 5 *- 7 -21 - 5 3. º 5.5- 8 *18 l. 5* 3 - 5 - 6 - 5-35 2, 5* 4 - 5 =6 -19 5.5-ll =16 • 5-40 - 5
- | 0 - O - O - 0 O = 0 = 0 = 0 O = 0 = 0 = 0
- l 0 - O - O - O 0 - O - O - O 0 - 0 - 0 - 0 0 - O - O - O Q - 9 - 9 - 0 || 0 – o – o – o 9 - O - O - O O = 0 = 0 = 0
100% gas 2 0 - O - C - O 0 - O - O - O 00 - 0 - 0 - Q O = 0 - O - O 0 - 0 -- 0 - 0 \ 0 - 0 - 0 - 0 0 - O - O - O O = 0 = 0 = 0
AV . 0 - O - O - O 0 - O - O - O O O = - 0 = 0 9 = 0 = 0 = 0 0 - 0 - 0 - 0 ||
- |
day in tervals of exposure to the gas, the fourth is after an
| additional 3 days out of the gas and in the ordinary air .
| Measurements represent diameters of colonies in millimeters.
The first three measurements in each case were made after 3-&







3i
the manner already described; but it is doubtful * *
oxygen free conditions were attained in any test. The concen-
trations used were approximately 0.4%, 4-5%, 20%, 40%, 50%,
60%, 85%, and 100% or nearly 00; and were the same rºle
organisms used unless otherwise noted.
Effect on the different organisms

Bacillus subtilis. -- With B. subtilis there was little
or no checking of development up to a concentration of 50 or
60% and no very great checking in any cancentration. The
appearance of the colony in all cases was quite normal.
There were no examples of the very thin colony growth produced
under certain other conditions.
Bacillus pyocyan eus. -- Ethylene exhibited very little
inhibiting effect on B. exocyaneus. The appearance of the
cultures was quite the same as that of cultures in air for all
the lower concentrations. In concentrations of 85% or more,
however, there was sometimes a discernible retardation of the
development. It is possible that this occurred always, as
differences in colony development are hard to detect with this
organism under the conditions used, and may have been over-
looked. -
Bacillus Kieli ensis. There was little inhibiting effect
exerted by ethylene on B. Kieli ensis. The organism grew vig -
orously and in all the concentrations except the very highest


produced almost as large a colony growth as in air. There was,

32
in fact, often no detectable difference up to an 85% concen-
tration, although at a higher figure retardatioſ.nd paleness
in the color of the colony were observed. With conditions
approaching absence of oxygen in the ethylene surrounding the
culture the inhibition and paling were quite marked although
not equaling that produced by raw illuminating gas, which it -
self contains about 1% of oxygen.
Bacillus rubidus. -- The concentrations in which B.
rubidus was tested were 0.4%, 4–5%, 85%, and approaching 100%.
In the first three the culture was on autoclaved potato, in
the last on agar. It grew in all, , and when growing on the
potato produced the characteristic orange yellow pigment freely,
sarcina lutea. -- This organism was tested in 4%, 20%,
40%, 60%, 85%, and approaching 100% ethylene. It grew well
1%ll of these, but more or less inhibition could be detected
at 60% and higher, increasing in general with the concentra-
tion although in some individual trials the development at a
concentration of 85% was almost as vigorous as in air. The
color of the culture was never much affected.
Oidium lactis. -- This organism grew well in all the
gas concentrations used, the cultures being scarcely distin-
guishable up to 85% from those in air. At greater concentra-
tions slight inhibitive effects were not, ed.
Torula sp. -- This yeast grew well in all of the tests,
the cultures being scarcely distinguishable from a culture in
air at as high a concentration of the gas as 60%. At 85% and

33
higher the growth was checked and the color paler, so that at
nearly 100% the growth was quite slow at first and the colony
practically colorless or whitish. In a few days, however, the
development became greater and the color darker, although not
as dark as the culture in air until after the return of the
Culture to the air,
Penicillium sp. -- This species of green mold was grown
in 4%, 85%, and a concentration approaching 100% of ethylene.
The development was after the normal fashion and was nearly
or quite as vigorous at 85% as it was in air, while the dele –
terious effects of the more nearly pure gas operated only to
delay the natural course of development for a day or two.
The following species were grown, two trials each, in
only 50% and 85% of ethylene: -
Bacterium stewarti. -- The growth of Bact, stewarti in
both concéntrations of ethylene was good, In the higher gas
content there was slight inhibition; but in both tests the de-
velopment was fully equal to that in air by the third or fourth
day,
- Bacillus carotovorus. -- The development of B. carot ovorus
while exposed to ethylene was good in all cases tested. In
50. of the gas it was quite equal to the air culture and in
85% it was only slightly less vigorous.
Bacillus melonis. -- In one trial the growth of B. melonis
in both ethylene contents was about equal to that in air. In


the other there was slight inhibition in the 85% concentration.

34
Bacillus campestris. -- The tests seemed to show a
slight inhibition of B. campestris at 50% of ethylene although
they did not agree especially well on the point. At 85% the
inhibition was somewhat greater but not at all remarkable.
Bacterium tumefaciens. -- With Bact. tumefaciens the
tests showed a clear inhibitive effect at both concentrations
of the gas but greater at 85% than at 50%, and the effect was
maintained until the cultures were removed to the air. -
Bacillus solanisaprus. -- The growth of B. solanisaprus
in the two ethylene-air mixtures used was practically equal
to that of the same organism in air.
- Bacillus radici cola. -- The development of the check
culture of B. radicicola in air and of the cultures in 50%
and 85% ethylene were practically identical in both tests.
Bacillus mycoides. -- In the first test with B. mycoides,
where the inoculation was made in a streak, no difference could
be made out between the growth in air and in the ethylene. In
the second test, however, where the slant was inoculated at
3, single spot near the center, it to ok about four days for the
colory to spread over the entire slant in 85% of ethylene,
and about three days in 50% ethylene while in air the inva-
Si on was complete in two days.
It seems quite clear to the writer from the results men-
tioned above that the presence of 4-5% of ethylene in the
illumingting gas used is totally inadequate to account for the
effect of the gas on the bacteria and fungi studied.

35
3. Carbon Monoxide.
Production and purification of the gas, etc.
The carbon monoxide used in these studies was made by
heating crystallized potassium ferrocyanide with strong c. p.
sulphuric acid. The gas was bubbled through a strong solution
of sodium hydroxide and stored over water or weak sodium hydrox-
ide solution until needed for use. It was not further purified
except that it was passed through two wash bottles of freshly
prepared pyrogallol solution when it was important to have all
oxygen removed. The tests were made in the manner already
described for the other gases. The concentrations used were
5%, 10%, 25%, 50%, 75%, and a concentratiºn approaching 100%.
Effect on the different organisms.
Bacillus subtilis. -- The effect of carbon monoxiºle on
B. subtilis was to induce in all the higher concentrations the
very thin sort of colony described for the higher concentrations
of illuminating gas. As in the case of illuminating gas, also,
the colony did not undergo change to the normal air type when
returned to the air, but often a normal colony would start up
at some point on the slant and progress over the slant from
that point. The colony was normal in type and vigor to a con-
centration of 10%; at 25% and above the modified colony devel-
oped, the vigor being successively less as the gas approached

36
purity. Using this organism as a measure of toxicity, there-
fore, carbon monoxide would appear to be something like twice
as toxic as illuminating gas.
Bacillus pyocyaneus. -- This organism grew in all the
concentrations of carbon monoxide used although its develop-
ment was slowed down to some extent in the higher percentages.
It was hard to make out any place at which the inhibition set
in, º the inderinite tint of the colonies, although it
seems likely that it should be placed at 25% or 50%. There
was no apparent change in colony type.
Bacillus Kieli en sis. -- B. Kieli ensis was found capable
of growing in all the test conditions with carbon monoxide.
The first clear cut response to the gas was at 50%, where re-
tardat ion of the growth and modification of the color took
place. At 25% development was usually normal but the color
was sometimes rendered slightly paler. As the concentrations
increased the inhibition of growth and the interference with
color production increased until as the condition of purity
of the gas was approached the colony growth produced was very
slight and practically without color. It will be noted that
if we take B. Kieli engis as a test organism carbon monoxide



seems to be just about as toxic as illuminating gas, or per-
haps even slightly less so.
Bacillus rubidus. -- In the case of B. rubidus a single
successful series of cultures in carbon monoxide showed a re-
tardation in the rate of development at 10%; at 25% it was

37
quite marked; at 50% and 75% the development was very slight
at the end of a 5-day period; and at 100% there was no visible
development. After return to the air vigorous growth took
place in 2 to 3 days. -
Sarcina lutea. -- Saroj na lutea proved quite susceptible
to the influence of carbon monoxide. The development was
normal or nearly so to 10%. of the gas but at 25% the inhibitive
effect was clearly noticeable. At 50% and above the develop-
ment was very slight during an 8-day period of exposure. At
a concentration approaching purity of the gas there was very
little if any discernible growth.
Oidium lactis. -- Carbon monoxide exerted a checking ef-
fect or the growth of 0. lact is at a concentration as low as
10% but it was very slight. The development was good even at
nearly 100% and only slightly atypical in character. There
wes rather more tendency in the gas for the hyphae to grow up -
ward and assume something of a tufted character.
- Torula sp. -- This organism grew in all the concentra-
tions of the gas used, although slight inhibition occurred at
25% and perhaps at 10% and the development was very slight
at a Co content approaching purity. The decrease in the
growth was accompanied by a decrease in the depth of color of
the dolony, so that in the case of the greatest inhibition
the colony was practicelly colorless. The colonies in the gas
up to 75% reached a maximum quite as great as that in air but

38
took a few days longer, while for colonies in an atmosphere
containing more co neither this maximum nor the typical in -
tensity of color was reached. Upon return to the air, however,
these conditions were attained.
Penicillium sp. -- The lowest CO content at which growth
was checked was lož, increasing gradually up to the greatest
used. At this point the development was very slow. It was
also very slow at 75%, although more vigorous than at the
greater concentration; but contrary to the condition with
illuminating gas conidia were produced.
From these results it would appear that carbon monoxide
is approximately as inhibitory in its effects on the organisms
tested as is illuminating gas. It would not seem to be suffi-


ciently toxic to account for the effects produced by the illu-
minating gas.
4. Methane
Production and purification of the gas, etc.

Since methane is usually present to approximately 30%
in the illuminating gas used in these studies it seemed reason -
able to expect tº the effects of the illuminating gas might
be due, at least partially, to its presence. This was rendered
the more likely by the fact that it seemed impossible to attri-
bute the results to ethylene or carb on monoxide or to the two
combined. Accordingly some methane was prepared from methyl
39
iodide by means of the copper-zinc couple. Tests with this
gas gave results corroborative of the hypothesis mentioned
above. The gas as produced had a distinct odor, however, and
it, seemed likely that some unchaged methyl iodide vapor had
been carried over with it in spite of the fact that it had
been passed through a column of zinc for the purpose of de-
composing such traces. The question arose, therefore, whether
the result s were due to the methane or to the methyl iodide.
Accordingly a few tests were run with methyl ibdide vapor; and
the results indicated clearly, as will be shown in a subsequent
section, that the former results might, indeed, have been due
+, o methyl iodide in the gas. It was necessary, then, to run
some tests with methane known to be free from methyl iodide.
is effie–ethe-r-method. The most convenient way to make pure methane
is by teeating aluminium carbide with water as acetylene is
produced from calcium carbide. No aluminium carbide could be
procured, however, owing to the European war; and the method
finally adopted was by heating barium oxide and anhydrous
seau. acetate. This method is said to produce nearly pure
methane, as opposed to the soda-lime method in which the product
has small quantities of ethylene and other impurities mixed
with it. As previous experiments had demonstrated that ethylene
in ºnal quantities is practically inocuous it was felt that
all such impurities incident to the use of barium oxide would
probably be of no consequence; and the results were amply


40
corroborative of the -i-Ges. The gas was stored over water until
required for use. The results from the gas produced from methyl
and arº / or one /r/a/ on Zy

iodide are not considered in the folio wing note; The concen -
trations used were 5%, 10%, 25%, 50%, 75%, and approaching 100%.
Effect on the different organisms
Bacillus subtilis. -- In all the concentrations of methane
used the development of B. subtilis was normal in character
and good in amount. There was some checking of development in
the higher concentrations, extending apparently as low as 25%;
but it was not great and was dissipated at the end of only 3
days except in the nearly pure gas. Even in this the reduction
in development at the end of 6 days was only slight and one day
after return to the air was indi stinguishable.
Bacillus pyocyaneus. -- As was perhaps to be expected
B. pyocyaneus grew well in all the concentrations used, but,
with a slight reduction in the highest concentrations. This,
however, had disappeared by the end of three days,





Bac illus Kieli ensis. -- The development of B. Kieliensis
in methane was good throughout but was slightly less vigorous
in the greatest concentrations of the gas. It was practically
impossible, moreover, to pick any one place at which the first
inhibition could be said to occur. The color was also affected
comparatively little though it was possible to pick on the 25%
concentration as that at which the first color change was
visible.

41
Bacterium stewarti and Sarcina lutea. Contrary to ex-
pectations Bact, stewarti and S. lutea were practically un-
checked in their growth throughout the series of exposures
to methane used.
Oidium lactis. -- The development of Q. lact is was prac –
tically unchecked throughout the series of methane exposures,
In the practically pure gas there was some difference in the
mycelial development in that the hyphae seemed less appressed
to the medium, but this was perhaps due to a difference in the
water content or some other feature of the medium.
Torula sp. -- The growth of this yeast was good in all
the tests with methane although somewhat slowed down in the
highest concentrations. The colony was somewhat pale at 75%
of the gas and more so at the still higher concentration. A
few days after the return to the air the cultures were indis —
tinguishable.
*** *. -- It was impossible to establish any in-
hi bi + i on by methane of the growth of this species of Penicillium.
The development at 75% and above did seem to be somewhat checked,
but the slowing down of the development was only slight at best




-
and probably without significance.
5. Methyl Iodi de Vapor.
The use of methyl iodide vapor was not contemplated in the
original plan of the experiments and it was brought in, as ex-
plained above, to check up the results obtained with methane

42
made from methyl iodide. The chemical was introduced into the
culture chamber by dropping the liquid on a bit of absorbent
cotten supported by a glass rod passing through a rubber stop-
per which was then immediately put in place in the tubulature
of the bell jar which served as the châmber. The liquid then
evaporated and the vap or diffused to all parts of the interior
of the jar. That it diffused through the cottºn plugs is at -
tested by the effects it produced on the cultureS. In the
first trial no measure was secured of the amount of methyl
iodide used, but it was considerably more than in the second,
where the amount was limited to 5 drops. The volume of the
jar was approżimately 3.8 liters. The results with whe di f –
ferent species in these trials were so much alike that it is
hardly necessary to consider each separately. The species

tested in the conditions mentioned above were Bacillus subtilis,

---
lactis, Torula sp., and Penisºn sp. Without exception the
culture was killed in the test with the larger amount of methyl
iodide, i.e., there was no development during a 7-day exposure
to the vapor nor within a period of 24 days after return to the
air, at the close of which time the tubes were discarded. With
the smaller amount of the chemical the development was nearly
normal in all cases except that there was a slight slowing down
of development, visible, however, only for periods of one to
4 days. In addition, , the yeast was slightly paled in color and
B. rubidus (on potato) was clear yellow instead of orange yellow

43
in color,
In a second series of two trials 6 drops of the liquid
were used in the first trial and something more than 10 in the
second. The species used in this series were Bacillus stewarti,
---
Bac f//
solanisaprue, ####m radioicola, and B. mycoides. In this
series again the results were quite uniform.
The organisms were characteristi cally greatly inhibited
at first but soon recovered and developed with great vigor so
that in the first experiment they overtook the air cultures in
- about 4 or 5 days on the average. In the second experiment
where the amount of the chemical was more than twice as great
the inhibiting effect was more permanent and the recovery not
so marked. Thus Bacillus melonie, B. tumefaciens, and B-
solanisaprus were the only ones to recover and develop as fully
as in air by the close of a 6-day exposure. B. mycoides would
probably have shown equal ability except that the inoculation
in this case was made at the center of the slant only. The
colony was quite normal but showed reduced ability to invade
the surface of the substratum in the vapor .
The general effect of methyl iodide vapor, therefore, as
shown by the data presented above, is to sterilize when used in
sufficiently large amounts; but when used in smaller amounts it.
is to induce an initial great retardation of development followed
later by a very vigorous growth.

44.
6. Tobacco Smoke.
Preparation and composition of the smoke, etc.
Inasmuch as smoke of various kinds has been found to be
decidedly toxic to a number of phanerogams it was thought to
be worth while to run a few tests along with the others on the
cryptogams under study. The smoke was produced by connecting
the interior of a bell jar inverted over the culture tubes to
8,11 aspirator by means of a tube through a rubber stopper and
also to a cob pipe by means of another tube. The pipe was filled
with tobacco, "Prince Albert" brand, the aspirator started, and
the tobacco light ed. When the culture chamber became filled
with a white, opaque smoke cloud the aspirat or was stopped and
the connecting tubes plugged. The air in the chamber soon be -
came clear, but the upper surfaces of everything within, and
the vertical surfaces to some extent also, became coated with
a brown layer. This did not extend into the test tubes, and
consequently not to the agar, because it was clearly limited
to the surface of the cott on plugs. Any reactions, therefore,
can not be laid to these condensation products except as the
condensation may not have been complete or as reevaporati on may
have taken place so that the diffusion through the cott on plugs
could occur. In some of the test s the smoke was passed through
one or two wash bottles containing water. In the se cases a
brown oily substance formed in small quantities and came to the
surface in the water, and it took longer to produce the opaque

45
cloud in the culture chamber. No trials were made with smoke
from other substances, although results similar to and quite
as interesting as those secured from tobacco smoke would be
expected.
It is quite impossible, of course, to get any accurate
idea of the chemistry of the mixture called smoke without mak-
ing an analysis. Even then the analysis would have to be from
the actual gases in the culture chamber to give any precises
delineat i on of the conditions. Moreover, the composition of
the gases of ºf the combined gases and suspended matter certain -
ly varies according to the time the smoke has been standing.
work has been done, however, to determine qualitatively the
compounds present. No attempt will be made here to review the
findings of investigators further than to enumerate some of the
compounds which have been reported. Thus Wohl and Eulenberg
(18) reported a series of hydrocarbons of the benzene series
or Orne analogous to it, and in addition formic, propi onic, buty-
ric, valerianic, and carbolic acids, creosote, ammonium chlo-
ride, ammonia, pyridine, pic oline, luti dine, collidine, par-
voline, coridine, and rubidine. Kissling (8) reported that the
mono x ide
strongly poisonous materials are carborºhydrogen sulphide, hy-
drocyanic acid, picoline bases, and nic otine. To this list
Thoms (17) added a phenol boiling at 190° - 200°, a small
quantity of furfural, and a substance boiling at 200°-260°,
containing sulphur and nitrogen and no terpenes. Knight and
Crocker (2, p. 346) have called attention to the presence of

46
ethylene and correlated its presence with the effect of smoke
on some phanerogams.
Effect on the different organisms.
The development of the bacteria in smoke was rather vari-
able, probably ºf to a failure to secure uniform conditions
in the different trials. There was a stronger tendency in
smoke than in the other gases for the colony development to
begin at the bottom of the slant and progress upward after a
preliminary period of no growth. No particular It ea. S. On can be
assigned for this at present, although the general tendency
for growth to proceed in this way in the cultures in all gases
was probably due to the greater thickness of the substratum at
the bottom of the slant and to the heavier inoculation secured
on the first parts of the streak. It would seen that the pre -
vention of growth at the out set would preclude any assumption
that the gases did not diffuse to the bottom of the tube. There
was only one trial with washed smoke for each of the following
organisms,
Bacilius subt ili se -- In the first test there was no vis-
ible development in unwashed smoke till the sixth day; in the e-
second test it became visible on the second day. The retarda-
tion continued throughout the duration of the exposure, however,
with the washed smoke showing the less inhibiting effect.
Bacillus pyocyaneus, -- In unwashed smoke, both trials,



the growth of B. pyocyaneus became visible at one day but the

47
colony had not entirely covered the slant at the end of the
7-day exposure. In washed smoke the slant was covered in 6-
days.
Bacillus Kieli ensis. -- In the first trial the colony of
B. Kieli ensis in raw smoke had just become visible in 6 days.
In the second trial it had become visible in 2 days in both
raw and washed smoke. By the end of the 7-day period, however,
it had covered only a little more than half the slant in raw
Smoke but the whole of it in washed smoke.
Bacillus rubidus. -- The inoculation of B. rubidus for
this test was made on potato and no test was run in washed
smoke. In the other, distinct retardation was evident, as
the treated cultures required 2 days and 4 days respectively
to become visible while the check colony in both cases was vis-
ible the day after inoculation.
Sarcina lutea. -- The development of S. lutea was hin-
dered by the smoke, more so by that which was untreated than
by that which was passed through water; but in all cases the
colony occupied all the inoculated area and had produced an
abundace of material by the close of the 7-day exposure.
Oidium lactis. -- The development of Q. lact is began
promptly in smoke at the bottom of the slant and progressed
gradually upward. The colony in washed smoke covered the
surface of the agar in considerably less time than in the raw
smoke.
Torula sp. -- In neither trial did the colony of the
yeast in raw smoke cover the surface of the agar by the end

48
of the 7-day exposure, although the colony in the treated smoke
did reach that degree of development. As with most of the
others the development proceeded from the bottom upwards.
Penicillium sp. -- The development of this green mold was
retarded in both smoke chambers but more so in the uritreated
than in the treated smoke. The development began at the bottom
of the slant and proceeded upward, conidia being produced normal -
ly. By the end of the 7-day exposure the slant was covered in
the washed smoke and only about half covered in the other.
With each of the following bacteria there were two trials
each of untreated and of washed smoke,

Bacterium stewart i. -- The behavi or of Bact, stewart i
in smoke was quite typical. The washed smoke was less toxic
than the unwashed and the development proceeded from below up -
ward, but was still limited to a rather small area at the base
of the slant in raw smoke when the week's exposure closed.
Bacillus carotovorus. -- The development of B. carot ovorus
began at the bottom in raw smoke and had covered half to 3/4 of
the available agar surface at the end of a week. In washed
smoke, however, the surface was covered in 4-6 days.
Bacillus melonis. -- The colony of B. melonis was visible
within a day after inoculation in washed smoke but it required
2-4 days in the other. In the former condition, , also, the
colony had not occupied all of the slant at the end of a week
in either trial,
- Bacillus campestris. -- In raw smoke it took two days


for the colony of B. campestris to become visible and it had

49
extended upward only about 1 cm. at the end of 6 days. In the
treated smoke the colony was visible in one day and the slanted
agar surface practically covered in 6 days. In the second test
with raw smoke there was no development at all, , but this was
possibly due to some other condition than the presence of the
smoke.
Bacterium tumefa.giens. -- The growth of Bact. tumefaciens
in untreated 5moke did not become visible for two days in the
more vigorous culture of the two and in both cases was still
confined pretty closely to the base of the slant at the close
of the 6-day period. The cultures in the washed smoke grew
more vigorously.
Bacillus solanisaprus. -- The presence of smoke, either
washed or unwashed, proved to be a hindrance to the growth of
B. solani saprus, with the washed smoke, as usual, exhibiting
the less toxicity.
Bacillus radi cicola. -- There was little difference be -
tween the behavior of B. radici cola and the other bacteria in
smoke. In the first trial the colony was still confined to the
lower half of the slant in the raw smoke at the end or the ex-
posure while in the second no growth had taken place. In the
treated smoke the grown was more vigorous and the colony ex-
tended the entire length of the slant.
Bacillus mycoides. -- The presence of either washed O1" url =
washed smoke proved to be an inhibiting fact or to the growth of
B. mycoides, with the raw smoke exerting the more severe

50
inhibition. In the washed smoke the colonies had spread nearly
or quite over the surface of the agar by the time the exposure
was ended but in the raw smoke there was no development in one
case and a colony pretty closely confined to the base of the
agar slant in the other.
It thus appears that tobacco smoke is more or less toxic
to the organisms used. In view of the very complicated and
variable mixture of compounds which constitute smoke it is
hardly worth while to venture an opinion as to which substance
or group of substances exerts the influence.
7. General Observations and Discussion

The experiments as carried out and the method adopted for
reporting them were not chosen to bring out differences in the
reactions of the different species used, but a few general ob -
servations may not be a miss.
It was noted, as was, of course, to be expected, that not
all organisms showed the same sort of reaction or exhibited
the same degree of tolerance to the gases. Thus B. subtilis,
B- Exocyaneus, B. mycoides, and B. Kieli ensis, showed a high
degree of tolerance for illuminating gas. In the case of B.
subtiliš, B- mycoides, and B. Kieli ensis the colony in the high
concentrations was quite different in appearance from the normal
one. In the case of the first two this is due perhaps merely to
the very small mass of material produced, but in the last named

it is associated also with a decrease in pigment production.

However, in the case of B. pyocyaneus and Q. lact is the appear-
ance of the colony was comparatively little altered. Probably
the most sensitive of the species studied were Sarcina lutea,
Bacterium stewarti, and Penicii'ium sp. Among the group of

fungi tested together, Fusarium radioicola was the most re-

sistant while Endothia fluens was most inhibited by the lower
contents of gas, followed by E. parasitica and Penicillium
Pinophilum.

The data of these studies seem to show that the extreme
sensitiveness manifested by certain phanerogams to some of the
chemically more inert gases is not to be found throughout the
whole plant kingdom; but that, on the contrary, many organisms
possess in a high degree the ability to tolerate the se gases.
Incidentally it may be remarked that so far as the results
from the species studied in this investigation can be projected
to cover all species, thei=ef-ere, they indicate that there is
only a small chance that the gas which would escape from the
gas fixtures in a room would be enough to invalidate results
obtained from cultures in that room. It should be remembered,
however, that even phanerogams vary considerably in this re-
gard, as has recently been shown by Miss Doubt (3) and others,
also that at least some algae appear to be quite sensitive, as
is reported by Woycicki (19). It would not be at all surpris-
ing, therefore, and is perhaps to be expected, even, that some
more sensitive bacteria and fungi will yet be found. The known

existence of only a few such would render precautions necessary

52
in bacteriological and mycological work which in the light of
the results here reported seem unnecessary.
The data do not seem to warrant any conclusion that any
of the strains acquired an increased degree of tolerance for
illuminating gas by being cultivated continuously in its pres-
ence. Such did seem to be the case for a time with B. pyocyan-
eus and Bact... stewarti; but when cultures inoculated with the
original mother strain, which had not been exposed to gas at
all, were exposed to the gas along with the supposed acclimated
strain the development of the unacclimated strain was quite
the equal of the other . In fact there was some evidence that
continuous growth in toxic concentrations of the gas weakened
the organism slightly. This was more clearly evidenced with
B. ruber, perhaps, than with any other and was shown by the
fact that the color production was not normal for 3 or 4 trans-
7 razos Xers
fers in the air after several g-e-Hera-ti-e-Hä in pure gas.
It is hardly possible at this time to state definitely
just what causes the inhibiting action of illuminating gas.
Some checks run with hydrogen, carbon dioxide, and air washed
in pyrogeliol indi cated rather strongly that a good part of it
is due to the lack of oxygen even with the facultative anaerobic
species. Not all of the results can be so accounted for, how-
ever, as it does not explain the after-effects, nor why one
gas in a given concentration should produce a greater effect
than another, as, for instance, why a mixture of 25% carbon
monoxide and 75% air should produce almost as great a retarding

53
effect on the growth of B. subtilis as a similar mixture of
50% illuminating gas and 50% air. Certainly no one gase eas
component of the illuminating gas has toxic properties suffi -
cient to account for the results, Ethylene and methane are
relatively iºnous and in additi on ethylene is present in
only small quantities in illuminating gas. Carbon monoxide
proved to be more toxic than the illuminating gas in some
cases; but it also is present in only small quantities. Possibly.
the most reasonable hypothesis for the present is that the re-
sults are the sum of a relatively large effect due to the dilu-
tº i or of the oxygen plus a smaller effect due to the weakly


poisonous properties of some of the component gases, the most
- important apparently being carbon monoxide.
C O N C L U S I O N S

l. None of the species of cryptogams studied, including
13 bacteria and 12 fungi, showed any very marked sensitiveness

ow avº (; -
to, illuminating gas or its components.
2. In the higher concentrations (above 25%) of various
component gases, however, most of the bacteria and fungi used
are checked in growth or wholly stopped. In the latter case
growth will usually take place after exposure to the air,
although often from a comparatively few foci, as if many of the

cells had been killed. Sometimes the culture is entirely ster-
ilized.
***
3. Different species exhibit different degrees of tolera-
tion for the gases, and in general a species which is relatively
54
intolerant of one is relatively intolerant of others.
4. There was no real evidence that the continued culture
of an organism in illuminating gas induced the development of
any increased tolerance for the gas by the strain so cultivated.
On the other hand there was some slight indication that the
vigor of a strain so cultivated was slowly lowered.
5. The colony habit of organisms is often modified more
or less strikingly in the more toxic gases. This is exempli-
fied especially in the color variations of B. Kieli ensis, the
decrease in colony mass and gross appearance in B. Kieli ensis,
B. subtilis, and E. mycoides, and the more compact, upright
arrangement of hyphae in several fungi.
6. Ethylene and me thane are relatively less inhibitory
to the organisms used than is illuminating gas but carbon
monoxide is about equal to the illuminating gas in this respect,
7. The effect of the gas cannot be laid to any one con-
stituent but is probably the sum of the small effect of each
plus the greater effect of a deficient oxygen content.
8. Tobacco smoke is toxic to the organisms on which it
was tried.
9. The vapor of methyl iodide is toxic to the organisms
tested, and if used in sufficient quantity kills the cultures.
10. Incidentally the foregoing results indi cate that the
small amount of illuminating gas often present in laboratory

air is not a menace to scientific result 5 in bacteriology and
my cology.
55
The writer takes pleasure in extending thanks to
Prof. F. C. Newcombe for suggestions and help tendered during
the prosecution of this study, and also to all others who have
helped in forwarding it.


LITERATURE CITED
Crocker, Wm., and Knight, Lee I. Effect of illuminating
gas and ethylene up on flowering carnations. Bot. Gaz.
46: 259-276. 1908. - -
------- ----- Toxicity of smoke. Bot. Gaz. 55 : 337-371. 1913.
Doubt, Sarah L. The response of plants to illuminating
gas. Bot. Gaz. 63: 209 – 224. 1917.
Frankland, Percy. Ueber den Einfluss der Kohlens"ure und
anderer Gasse auf die Entwickelungsfähigkeit der Mikro-
organismen. Zeitschr. Hyg. 6: 13–22, 1889.
- - - - - - - - - - - - - - - On the influence of carbonic anhydride
and other gases on the development of micro-organisms.
Proc. Roy. Soc. Lond. 45: 292–301. 1889.
Girardin, --- Einfluss des Leucht gases auf die
Promenaden und. Strassenbäume. Jahresber. Agrikultur
Z: 199-200, 1864. (Not seen.)
Harvey, E. M. The cast or bean plant and laboratory air.
Bot. Gaz. 56 : 439-442. 1913. -
Kissling, Rich. Der Gehalt des Cigarrenrauches an Nikot in
unter gleichzeitiger Berºcksichtigung der giftig wirkenden
Verbrennungsprodukte des Tabakes. Dingler's Polytech.
Jour. 2443. 64-71, 234-246, 1882.
Krause, Paul. Beiträge zur Kenntniss des Bacillus


pyocyaneus. Centralbl. Bakt. 27:769-775. 1900

10.
11.
12.
13.
14.
Langdon, Seth C. Carbon monoxide, occurrence free in kelp.
(Nerocystis luetkeana). Jour. Am. Chem. Soc. 39: 149-156.
1917.
Molisch, H. ther den Einfluss des Tabakrauches aur die
Pflanze. Teil I, sitzuºber. Kais. Akad. Wiss. (Wien)
Math. Nat. Kl. 120: 3-30. 1911.
Mºnz, E. zur Physiologie der Methanbakterien. Diss.
Halle. 1915. (Abstracted by R. Lieske in Zeitschr. ... Bot.
8: 132-133. 1916.)
Richards, H. M., and MacDougal, D. T. The influence of
carbon monoxide and other gases upon plants. Bull. Torr.
Bot. Club, 31:57-66. 1904.
Smith, Erwin F. Bacteria in relati on to plant diseases.
1 : 1905.
Tassinari, Vincenzo. Experimentalunter suchungen ºber die
Wirkung des Žºaches auf die Mikroorganismen im all-
gemeinen und im besonder en auf die krenkheit sergeºcenden.
Worl. Mitt. Zentralbl. Bakt. 4: 449–453. 1888. -
Thom, Charles. Cultural studies of species of Penicillium.
Bur. An. Ind. Bul. 118; pp. 109. 1910.
Thoms, ---- Constituents of tobacco smoke. (Abstract
in Am. Jour. Phar. 72: 227–228, 1900, of a paper read
by Prof. Thoms before the German Scientists Association.
(süddtsch. Ap. Zt. 1899. 650)).
vº

vº
18. Vohl. Herm., and Eulenberg, Herm. Ueber die physiologische
Einwirkung des Tabaks als *arkotisches Genus smittel, mit
besonderer Berºcksichtigung der Best and thei le des Twº
sons. Archiv der Pharmacie || ºf 7: 130-167. 1871.
(Abs. in Pharmaceutical Jour, and Trans. 3rd ser. 2 : 567–
568, 1872.)
19. Woycicki, M. Beobachtungen äber Wachstums -, Regenerations -
und Propagations-lirscheinungen bei eini/gen fadenförmigen
Chlorophyceen in Laboratoriums - Kultur und unter dem
Einfluss des Leucht gases. Bull, internat - acad. Sci.
Cracovie. Classe sci. math, et. nat. 1909% : 583–667.
1909.

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