|
:
751
K96
B 398059
nunkel, L. 0.
Physical and chemical,
factors influencing the
toxicity of inorganic salts
{
:
IB'Y
PHYSICAL AND CHEMICAL FACTORS
INFLUENCING THE TOXICITY
OF INORGANIC SALTS
TO MONILIA SITOPHILA (MONT.) SACC.
Chemical Library
ак
751
K96
BY
LOUIS OTTO KUNKEL
SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY, IN THE FACULTY
OF PURE SCIENCE, COLUMBIA UNIVERSITY.
PRESS OF
THE NEW ERA PRINTING COMPANY
LANCASTER, PA.
ว
36620
From the BULLETIN OF THE TORREY BOTANICAL CLUB, 41: 265-293. 13 May 1914.]
Physical and chemical factors influencing the toxicity of inorganic
salts to Monilia sitophila (Mont.) Sacc.
LOUIS OTTO KUNKEL
(WITH TWO TEXT FIGURES)
The many researches of recent years on the relative value of
various media for growing bacteria and fungi, and the almost
equally extended studies of the toxicity of various mineral salts
for these organisms have not so far led to any very careful analysis
of the physiological relations of toxic salts as affected by the media
in which they are tested. This is especially true of the commonly
used organic constituents of culture media, such as the sugars,
proteids, etc. The study of the toxic action of salts when mixed
together in aqueous solutions as compared with their effects when
used separately has led to the very fruitful conception of balanced
solutions, which plays so large a role in modern studies of osmosis,
penetrability, the making of media, etc. The chemical inter-
relations of the salts and organic constituents of ordinary media
are doubtless of fundamental significance in determining their
physiological effects. The chemical relation of a so-called toxic
salt to the medium in which it is offered must be understood if the
real nature of the toxic effect is to be correctly analyzed. The
compounds formed when dilute solutions of salts are mixed with
carbohydrates, proteids, etc., are as yet little understood. The
study of the relative physiological effects of series of such com-
binations may indirectly throw light on the nature of the com-
pounds themselves and indicate the conditions under which they
265
266
KUNKEL: FACTORS INFLUENCING TOXICITY OF
are formed. Of still greater significance, however, is the bearing
of such studies on the actual determination of the relative toxicity
of different substances and the fundamental questions as to the
nature of toxic effects in general.
In a former paper (15) I have already shown that the toxic
action of various nitrates on Monilia sitophila (Mont.) Sacc. is
greatly influenced by the medium in which the fungus is grown at
the time it is being poisoned. For example, ferric nitrate and
aluminum nitrate were found to be much less toxic in peptone
media than in glucose, fructose, galactose or starch media.
Barium nitrate, on the other hand, was more toxic in peptone
media than in glucose, fructose or starch. Through further
experiments which it is the purpose of this paper to report, I have
studied the influence of carbohydrates and peptone on the toxicity
of eleven different chlorides. Observations on the germination
and growth of Monilia in slightly toxic media has led to the
suggestion that an important effect of a toxic substance may be
to diminish the rate of water absorption by the fungus. In the
hope that this suggestion might throw light on the conditions
that obtain in poisoned cultures, I have also studied the influence
of the water supply on the rate and amount of growth which
Monilia makes when growing in various media.
As already noted the discovery of the mutual antagonism which
exists between inorganic salts as regards their toxicity to plants
has led to the conception of balanced solutions. Boehm (3) was
perhaps the first to observe this relation. He found that the
poisonous action of magnesium salts on bean plants could be
counteracted by calcium carbonate. He also noted that calcium
nitrate and calcium sulphate had an ameliorating influence on
the toxicity of sodium and potassium salts. Von Raumer (26)
also observed the great toxicity of magnesium salts in the absence
of calcium. The literature on this subject has recently been
summarized by McCool (21) That such mutual antagonism
exists between a rather large number of salts is one of the best
established facts of plant physiology.
In 1902 Loeb (18) attacked the further problem as to whether
or not the presence of non-electrolytes would effect the toxicity of
inorganic salts. Using the eggs of Fundulus, he measured the
INORGANIC SALTS TO MONILIA SITOPHILA
267
toxicity of sodium chloride and zinc sulphate in the presence of
small amounts of urea, alcohol, glycerine and cane sugar and con-
cluded that these non-electrolytes do not influence the toxicity
of either salt. The toxicity of zinc sulphate, however, was
greatly reduced by cane sugar. This result Loeb attributed to
the formation of zinc saccharate. Judging from what we know
of the conditions under which saccharates are produced, we can
hardly accept this explanation for the case of a dilute solution of
zinc sulphate and cane sugar. Saccharates are obtained in
strongly alkaline solutions. Dilute solutions of zinc sulphate are
quite acid in reaction. I shall discuss this point further in con-
nection with my own observations.
Lidforss (16) had long before observed that the toxicity of
calcium nitrate, sodium nitrate and sodium chloride for germinat-
ing pollen grains was much reduced by the addition of ten per
cent. of cane sugar to the solution. Wassermann and Takaki (33)
found that an infusion made from cells of the central nervous
system of the guinea-pig has an inhibiting action on the toxin
of Bacillus Tetani. Like infusions made from kidney or liver cells
did not counteract the poison. Loew (19) was not able to lessen
the toxicity of magnesium salts for Spirogyra by the addition
of methyl alcohol or glycerine to his solutions. Kahlenberg and
True (13) claim that cane sugar has no effect on the toxicity of
boric acid for Lupinus albus, but that the addition of dextrine to a
mercuric chloride solution greatly reduces its toxicity. They
were unable to precipitate mercuric oxide from such a solution by
means of caustic alkali and assume that the mercury and dextrine
combine to form a complex ion which is less toxic than the mercuric
ion. Winogradsky and Omeliansky (36) found that peptone and
glucose had an inhibiting action on the growth of the nitrifying
bacteria. Duggar (6) obtained better germination of the uredo-
spores of Puccinia Helianthi in distilled water than in one per cent.
peptone solution. Stockard (31) reports that when cane sugar is
added to a solution of lithium chloride or ammonium chloride
the toxic action of these salts on Fundulus eggs seems to be
augmented by the presence of the sugar, although the osmotic
pressure of the sugar-salt mixture is considerably below that of
sea-water.
リ
​268
KUNKEL: FACTORS INFLUENCING TOXICITY OF
Quite recently Ritter (28) has made a study of the effects of
acids on Mucors growing in several different media. He finds
that the toxicity of citric, malic, tartaric, hydrochloric and nitric
acids is much reduced by the presence of peptone in the medium..
When the source of nitrogen is ammonium citrate, ammonium
tartrate, ammonium malate, asparagin or peptone, the toxicity of
the various acids is much less than when the nitrogen is offered
in the form of ammonium chloride, ammonium sulphate or
ammonium nitrate. Grape sugar was found to reduce the toxicity
of tartaric acid to a less extent than peptone.
A review of the literature shows that the influence of the more
complex organic compounds on toxic substances has generally
been neglected by those who have studied the effects of poisons
on fungi. In my further studies on this point I have tested the
influence of carbohydrates and peptone on the toxicity of a number
of different chlorides, and have obtained evidence which suggests
that an important factor in toxicity is the influence of the poison
on the ability of the protoplast to absorb water. I have used as
before the fungus Monilia sitophila in all of my experiments. Went
(34) found that this fungus was able to use as a partial source
of its food supply a rather large variety of organic compounds.
This, together with the fact that it is a rapid grower, makes it
especially well suited to such investigations. In experiments on
the effects of toxic substances on fungi the choice of a standard
by which to judge the degree of the poisonous action is a matter
of some difficulty. One method is to grow the fungus in media
containing the poison in different concentrations and then to
determine the weight of the mycelium produced in the several
media in a given period of time. On account of the difficulty
in getting all of the mycelium out of the culture vessels and freeing
it from constituents of the medium this method was not used. In all
of my experiments, I have taken as the standard for judging
toxicity, the highest concentration of the poison in which any of the
spores germinate after three days of incubation. The spores are
considered to have germinated if they show any visible evidence
of growth when magnified to about one thousand diameters. I
find this method to be both reliable and convenient.
My cultures were grown in pint milk bottles or in two hundred
INORGANIC SALTS TO MONILIA SITOPHILA
269
!
and fifty cubic centimeter Jena flasks. New bottles and flasks
were obtained and a different set was used for each of the salts
tested. Thus the same flask was never used for testing more
than one salt. This prevents errors which might arise through
small traces of one salt being taken up by the glass and carried
into cultures of some other salt. All glassware used in the making
of media or the growing of cultures was first treated for at least
twelve hours with a chromic acid cleaning solution. It was then
thoroughly rinsed with tap water and drained. After draining
for a short time it was again rinsed, first in distilled water and
then in triple distilled water. The water used in the making of
culture media was triple distilled, once from acid potassium
dichromate, once from alkaline potassium permanganate and then
again redistilled. This water gave a resistance of approximately
one hundred and twenty thousand ohms. All of the salts used
were Baker's analyzed chemicals, except in the case of barium
chloride, cobaltous chloride and cadmium chloride, which came
from Kahlbaum. The glucose, saccharose, soluble starch and
peptone were obtained from Kahlbaum; the lactose came from
Merck. Five per cent. solutions of each of these substances
gave the following resistances; glucose, fifty-six thousand ohms;
saccharose, forty-nine thousand ohms; lactose, twenty-seven
thousand ohms; soluble starch, three thousand, three hundred
ohms; and peptone two thousand ohms. A comparison of these
resistances gives a good idea of the relative purity of the organic
substances used.
The concentration of the salts in the various media are ex-
pressed in terms of molar solutions; these concentrations were
made by dissolving some multiple or some fraction of a gram
molecular weight in one liter of solution. Twenty cubic centi-
meters of medium were placed in each flask, and the flask and
medium were sterilized for approximately ten minutes at 100° C.
As soon as the medium had thoroughly cooled it was inoculated
with fresh ripe spores of Monilia. The spores used in inoculating
any given series of media were taken from the same culture.
They were shaken from the culture flask on to a piece of white
paper and were thoroughly mixed by means of a sterile scalpel.
Approximately equal quantities of spores were dusted over the
་།
270
KUNKEL: FACTORS INFLUENCING TOXICITY OF
surface of each medium. The cultures were then incubated in a
room which was kept at a temperature of about 26° C. Observa-
tions were always made after an incubation period of three days.
For the purpose of making careful microscopic observations, each
culture was poured out into a small Petri dish, which could be
placed on the stage of the microscope. All cultures were first
observed under a magnification of about two hundred diameters.
If this observation showed no germination, they were then more
carefully studied under a magnification of approximately one
thousand diameters.
In the tables that follow the plus sign indicates that the spores
had germinated while the minus sign means that there was no
germination in the medium in question. The results thus given
in the tables refer always to the presence or absence of germinated
spores in the different cultures, after an incubation period of
three days. The minus sign does not mean that the spores will
not germinate in the given medium but only that they have not
germinated after three days. If left in the medium for a longer
time the spores might germinate. The highest concentration of a
salt that will allow germination of the spores within three days is
termed the limit concentration for that medium and period of time.
The toxicity of eleven different chlorides has been tested.
Chlorides were selected because they have been much studied by
other investigators and because they are, on the whole, more
soluble than nitrates, sulphates or other common salts. In
order to determine the influence of saccharose, glucose, lactose,
starch and peptone on the toxicity of the several chlorides, I have
used each of these substances separately in testing the toxicity
of each inorganic salt. In distilled water and tap water the spores
of Monilia germinate but produce only a very small amount of
mycelium. Any of the organic substances used, when added to
distilled water, greatly increase the growth, and they also tend to
shorten the time required for germination. Abundant mycelium
is produced in five per cent. solutions of glucose, saccharose, lac-
tose, starch or peptone. When chlorides in sufficient concentra-
tion are added to any of these media the germination of the
spores is completely inhibited. The approximate value of the
limit concentration of the different salts in each medium was
INORGANIC SALTS TO MONILIA SITOPHILA
271
obtained through a number of preliminary experiments. It was
impossible to predict what the limit concentration would be in any
case. My method was to make a guess at this value and then test
the accuracy of the guess through experiments. In some cases
only one preliminary experiment was necessary in order to deter-
mine the approximate value of the limit concentration of a given
salt in one of the media. In other cases, however, several pre-
liminary experiments had to be made before this value was ascer-
tained. The data given in the tables was obtained in the final set
of experiments and shows the great influence of the organic part
of the medium on the toxicity of the different chlorides. The
tables are arranged in the order of the toxicity of the salts tested.
The influence of the medium on the toxicity of potassium chloride
is shown in Table I.
TABLE I
THE TOXICITY OF POTASSIUM CHLORIDE IN SACCHAROSE, GLUCOSE, LACTOSE, STARCH,
AND PEPTONE MEDIA
Saccharose 5%
G.M. Growth
Glucose 5%
Growth
Lactose 5%
Starch 5%
Peptone 5%
Conc.
After
G.M. Conc. After G.M. Conc.
KCI
3 Days
KCI
3 Days
KCI
Growth
After
3 Days
Growth
G.M. Growth
G.M. Conc.
KCI
After
3 Days
Conc.
After
KCI
3 Days
2.3
2.3
2.2
2.I
2.0
I.9
1.8
1.7
1.6
1.5
1.4
+++++1│││
2.2
H H
1.6
1.5
1.5
1.5
1.4
I.4
2.I
I.4
1.3
1.3
2.0
1.3
1.2
I.2
HHHHHH
1.9
1.2
I.I
I.I
1.8
I.I
1.7
1.6
1.5
++++
1.0
.9
.8
.7
+++++
1.0
1.0
.9
.9
.8
.7
.6
76
+++
.8
.7
.6
+++++
I.4
A glance at the table shows that the toxicity of potassium
chloride is decidedly influenced by the medium in which it is
offered. It is more than twice as toxic in starch as in saccharose
or glucose. It is also more toxic in starch than in lactose and
peptone. Its toxicity in lactose is the same as in peptone, but is
about thirty-five per cent. less than in saccharose. It is most
toxic in starch and least toxic in saccharose. The average of the
limit concentrations in the five media is 1.1 molar and shows that
potassium chloride is less toxic than any of the other chlorides
that I have tested; this may be associated with the high nutrient
272
KUNKEL: FACTORS INFLUENCING TOXICITY OF
value of potassium. The toxicity of potassium chloride in starch
media is the same as was found for potassium nitrate in the same
medium.
TABLE II
THE TOXICITY OF AMMONIUM CHLORIDE IN SACCHAROSE, GLUCOSE, LACTOSE, STARCH,
AND PEPTONE MEDIA
Saccharose 5%
G.M. Growth
Conc. After
NH4Cl 3 Days
G.M. Conc.
NH4CI
Glucose 5%
Lactose 5%
Starch 5%
Peptone 5%
Growth
After
3 Days
G.M. Conc.
NHĄCI
Growth
After
3 Days
Growth
G.M.
G.M. Conc.
NH4Cl
After
3 Days
Conc.
Growth
After
་
NH4Cl 3 Days
.80
.75
.70
.65
.60
.55
.50
.45
.40
.35
++11
1.30
1.25
1.0
.9
1.2
.95
I.I
.90
++++
1.20
1.15
Ι.ΙΟ
1.05
1.00
.90
.8
.7
.6
.5
.4
18705
.80
.70
++++
.2
.I
324
| 1++++++
1.0
.85
.9
.80
.8
.75
.7
.6
.5
.4
6543
+++++
.70
.60
.50
.40
.30
+++
Table II shows that the toxicity of ammonium chloride is not
greatly different in the various media, though some differences
are to be noted. It is least toxic in glucose and most toxic in
lactose. The toxicity of potassium chloride in starch is approxi-
mately the same as the toxicity of ammonium chloride in peptone,
glucose and starch. The average limit concentration of am-
monium chloride is .72 molar. When all of the media are taken
into consideration, we see that ammonium chloride is more toxic
than potassium chloride but less toxic than sodium chloride.
Ammonium chloride is a possible source of nitrogen for Monilia
and it may be that for this reason it is less toxic than sodium
chloride.
The toxicity of sodium chloride in the different media as shown
by Table III stands in sharp contrast with that of potassium
chloride. While potassium chloride is least toxic in saccharose,
sodium chloride is most toxic in this medium. It is hard to
understand how two salts that are so much alike chemically should
be so different as regards their toxicity in saccharose media. It
is very interesting to see that potassium chloride, ammonium
chloride and sodium chloride show almost equal toxicity in starch
media, while in saccharose, glucose, lactose and peptone they
INORGANIC SALTS TO MONILIA SITOPHILA
273
TABLE III
THE TOXICITY OF SODIUM CHLORIDE IN SACCHAROSE, GLUCOSE, LACTOSE, STARCH,
AND PEPTONE MEDIA
Saccharose 5%
Glucose 5%
Lactose 5%
Starch 5%
Peptone 5%
G.M. Growth
Conc.
NaCl
After
3 Days
G.M. Conc.
NaCl
Growth
After
3 Days
Growth
Growth
G.M. Growth
G.M. Conc.
NACI
After
3 Days
G.M. Conc.
NaCl
Afier
3 Days
Conc.
After
NaCl
3 Days
.56
1.30
.90
.40
I.20
.80
.44
Ι.ΙΟ
.70
.38
1.00
.60
.32
.90
.26
.20
.14
.08
.02
+++++
.80
.70
.60
.56
.50
1+++++
.50
.40
.30
.20
.10
.05
| | 1++++++
H H
1.5
1.00
I.4
.95
1.3
.90
1.2
.85
I.I
.80
I.O
.75
.9
.8
.7
56
.6
1+++
.70
.65
.60
.55
+++++
differ widely. These differences may be due to the influence of the
salts on the enzymes produced in the different media. Sodium
chloride is least toxic in glucose, starch and peptone, the limit
concentration being approximately the same in these three media.
It is more toxic in saccharose than in lactose and more toxic in
lactose than in glucose, starch or peptone. The average of the
limit concentrations in the different media is .62 molar. When all
of the media are thus taken into consideration we see that sodium
chloride is much more toxic than potassium chloride.
TABLE IV
THE TOXICITY OF CALCIUM CHLORIDE IN SACCHAROSE, GLUCOSE, LACTOSE, STARCH,
AND PEPTONE MEDIA
Saccharose 5%
G.M. Growth
After
Conc.
CaCl2 3 Days
.80
.70
.60
.50
.40
.30
.20
.IO
.05
.01
| | 1+++++++
Glucose 5%
Lactose 5%
Starch 5%
G.M. Conc.
CaCl2
Growth
After
3 Days
G.M. Conc.
CaCl2
Growth
After
3 Days
G.M. Conc.
CaCl2
Peptone 5%
Growth G.M. Growth
After Conc.
After
3 Days
CaCl2
3 Days
I.2
.60
I.I
.80
I.I
.50
1.0
.70
1.0
.40
.9
.60
.9
.30
.8
.50
.8
.20
.7
.40
.7
.6
6543
.4
+++++
.12
.II
.10
.09
.08
+++++
.6
.5
.2
65432
.30
++++
.20
.10
.09
.08
+++++
274
KUNKEL: FACTORS INFLUENCING TOXICITY OF
Calcium is one of the most important plant nutrients. It also
has great value for counteracting the toxicity of other salts. The
above table shows how its toxicity varies in the different media.
Its poisonous action in starch is the same as in saccharose and
only slightly less than in peptone. It is most toxic in lactose and
least toxic in glucose. The average limit concentration is .44
molar.
TABLE V
THE TOXICITY OF BARIUM CHLORIDE IN SACCHAROSE, GLUCOSE, LACTOSE, STARCH,
AND PEPTONE MEDIA
Saccharose 5%
Glucose 5%
Lactose 5%
Starch 5%
G.M. Growth
Conc. After
BaCl2
3 Days
G.M. Conc.
BaCl2
Growth
After
3 Days
G.M. Conc.
BaCl2
Growth
After
3 Days.
Growth
G.M.
Peptone 5%
Growth
G.M. Conc.
BaCl2
After
Conc.
After
3 Days
BaCl2
3 Days
.70
.50
.55
.60
.40
.50
.50
.30
.45
.40
.20
.30
.10
++
.40
.25
.20
.15
.10
.08
+++++
.09
.08
.35
.30
.25
.07
.06
.05
+++
.20
.15
.10
| | │
++++++
.80
.50
.70
.45
.60
.40
.50
.35
.40
.30
.30
.25
.20
.20
.IO
.09
.08
+++
.15
.10
.05
++++
Table V gives the toxic values of barium chloride. Although
barium is much like calcium chemically it is not a plant nutrient.
Barium is on the whole much more toxic than calcium. It is less
toxic in lactose media than in glucose. In this respect it is quite.
different from calcium chloride which is more than five times as
toxic in lactose as in glucose. The average limit concentration is
.26 molar. The toxicity of barium chloride is influenced less by
the different media than that of any of the other chlorides which
have been used. In saccharose, glucose, starch and peptone it
is more toxic than calcium chloride. In lactose media, however,
calcium chloride is more than three times as toxic as barium
chloride. It is worth noting that the toxicity of the five chlorides
as shown by the above tables is, on the average, as great in peptone
as in the other media. Their toxicity is least in glucose and
greatest in lactose. They are more toxic in peptone than in
saccharose or glucose, although peptone is a much more favorable
medium for the growth of the fungus. This shows that in some
INORGANIC SALTS TO MONILIA SITOPHILA
275
cases at least, salts may be more toxic in a medium that is well
suited to the growth of Monilia than in one which is less favorable.
TABLE VI
THE TOXICITY OF FERRIC CHLORIDE IN SACCHAROSE, GLUCOSE, LACTOSE, STARCH,
AND PEPTONE MEDİA
Saccharose 5%
Glucose 5%
Lactose 5%
Starch 5%
Peptone 5%
G.M. Growth
Conc.
FeCl3
After
3 Days
G M. Conc.
FeCl3
Growth
After
3 Days
G.M. Conc.
FeCl3
Growth
After
3 Days
Growth
G.M.
Growth
G.M. Conc.
FeCl3
After
Conc.
After
3 Days
FeCl 3
3 Days
.00050
.00050
.000400
.0013
.050
.00040
.00040
.000300
.0012
.045
.00030
.00030
.000200
.00II
..040
.00020
.00020
.000100
.0010
.035
.00010
.00010
.00009
.00008
.00007
.00006
.00005
-++++
.00009
.00008
.00007
.00006
.00005
++++++
.000080
.0009
.030
.000060
.0008
.000040
.0007
.000020
.000010
.0006
.000008
++
.0005
.0004
+++++
.025
.020
.015
.ΟΙΟ
.005
+++++++
Table VI shows that ferric chloride is far more toxic than the
alkali and the alkali earth chlorides which have been tested.
Although iron has a place among the nutrient elements it is never-
theless very poisonous. Of the alkali and alkali earth salts.
tested, potassium chloride is the least toxic. Iron chloride is
least toxic of the salts of the heavy metals which have been used.
It is very interesting to see that both potassium and iron are among
the nutrient elements. This suggests that there may be some
connection between toxicity and the nutrient relations of the
elements. The effect of peptone on the poisonous action of ferric
chloride is strikingly shown by the table. It is eight times more
toxic in starch and ten times more toxic in lactose than in glucose
and saccharose. In lactose it is more than three thousand times
more toxic than in peptone. The average limit concentration in
the different media is .007 molar.
Copper is known to be very poisonous to many algae and fungi,
and is widely used in the making of fungicides. I find, however,
that the chloride is, with the exception of iron, less toxic than any
of the other chlorides of the heavy metals that have been tested.
As shown by Table VII, cupric chloride, like ferric chloride, is
more toxic in lactose than in saccharose, glucose, starch or peptone.
リ
​276
KUNKEL: FACTORS INFLUENCING TOXICITY OF
TABLE VII
THE TOXICITY OF CUPRIC CHLORIDE IN SACCHAROSE, GLUCOSE, LACTOSE, STARCH,
AND PEPTONE MEDIA
Saccharose 5%
Glucose 5%
Lactose 5%
Starch 5%
Peptone 5%
G.M.
Conc.
Growth
After
CuCl₂ 3 Days
G.M. Conc.
CuCl
Growth
After
3 Days
G.M. Conc.
CuCl2
Growth
After
3 Days
Growth
G.M.
Growth
G.M. Conc.
After
Conc.
After
CuCl2 3 Days
CuCl2
3 Days
.00100
.00095
.00090
.00085
..00080
5
.00075
.00070
.00065
09000*
.00055
! !++++++++
.00100
.000055
.00070
.050
.00095
.000050
.00065
.045
.00090
.000045
09000*
.040
.00085
.000040
.00055
.035
.00080
.00075
.00070
.00065
.00060
.00055
++++++
.000035
.000030
.000025
.000020
.000018
.000015
+++++!
.00050
.030
.00045
.025
.00040
.00035
.00030
.00025
++++
.020
++++
.015
.ΟΙΟ
.005
Its toxicity in glucose and saccharose is approximately the same.
On the whole, cupric chloride is little more toxic than ferric
chloride, its average limit concentration being .004 molar. The
influence of the various media on the toxicity of copper is of special
interest because the salts of this metal are so much used in the
making of sprays. The action of lime on the copper in Bordeaux
mixture is not thoroughly understood although this important
subject has been given considerable attention (see Fairchild, 7).
TABLE VIII
THE TOXICITY OF ZINC CHLORIDE IN SACCHAROSE, GLUCOSE, LACTOSE, STARCH, AND
PEPTONE MEDIA
Saccharose 5%
Glucose 5%
Lactose 5%
Starch 5%
Peptone 5%
G.M. Growth
Conc.
ZnCl2
After
3 Days
G.M. Conc.
ZnCl2
Growth
After
3 Days
G.M. Conc.
ZnCl2
Growth
After
3 Days
Growth
G.M. Growth
G.M. Conc.
ZnCl2
After
Conc.
After
3 Days
ZnCl2
3 Days
.00050
.00045
.00040
+11
.000300
.00080
.000250
.000200
.00075
.00070
.00035
.000150
.00065
.00030
.000100
09000'
.00025
.000050
.00055
.00020
.000010
.00015
.000008
.00010
900000*
.00005
.000004
+++
.00050
.00045
.00040
.00035
++++
+++11
.000300
.050
.000200
.000100
.000080
090000*
.000040
.000020
.000010
.000008
.000006
++++++ |||
.045
.040
.035
.030
.025
.020
.015
.OIO
.005
++
Table VIII shows the toxicity of zinc chloride. It will be seen
that it is most toxic in starch and least toxic in peptone. The
INORGANIC SALTS TO MONILIA SITOPHILA
277
most striking point in this table is the high toxicity of zinc in
starch. In this medium it is more toxic than any of the other
salts tested except mercuric chloride. This observation suggests
that the value of zinc chloride for preserving wood from decay is
based on a similar relation between zinc and the cellulose of the
wood. Starch and cellulose are much alike chemically and it is
rather to be expected that a salt which is extremely toxic in starch
media would also be very toxic in cellulose. The toxicity of zinc
chloride in saccharose and glucose is practically the same and is
greater than in lactose. In lactose, zinc is more than fifty times
less toxic than either copper or iron. The average limit concen-
tration is .0022 molar.
TABLE IX
THE TOXICITY OF COBALTOUS CHLORIDE IN SACCHAROSE, GLUCOSE, LACTOSE, STARCH,
AND PEPTONE MEDIA
Saccharose 5%
Glucose 5%
Lactose 5%
Starch 5%
Peptone 5%
Growth
G.M. Conc.
CoCl2
3 Days
After G.M. Conc.
CoCl2
Growth
After
3 Days
G.M. Conc.
CoCl2
Growth
After
3 Days
Conc.
G.M. Growth
After
G.M. Growth
Conc.
After
CoCl2 3 Days
CoCl2 3 Days
.000501
.00040
.000301
.00020
.00010
.00050
.000080
.00080
.0400
.00040
.000070
.00070
.00009
.00008
.00007
.00006
.00005
+
1+++++
.00030
090000*
.00060
.00020
.000050
.00050
.00010
.000040
.00040
.00009
.00008
.00007
.00006
.00005
+++++
.000030
.00030
.000020
.000015
.000010
.000008
++++
.00020
.00020
.00009
.00008
++++++++1
.0350
.0300
.0250
.0200
.0150
.0100
.0050
ΟΙΟΟ
.0008
++++
The influence of the different media on cobaltous chloride is
quite marked. It is five hundred times more toxic in lactose than
in peptone. In starch it is less toxic than in glucose or saccharose
but more toxic than in peptone. The average limit concentration
is .0020 molar, which shows that, on the whole, it is only a little
more toxic than zinc chloride. The poisoning of the fungus by
cobalt, cadmium and mercury is different from that of calcium,
iron, etc., in that Monilia in nature probably does not come in
contact with any but minimal concentrations of the salts of these
metals. Its resistance to cobaltous chloride, cadmium chloride
1 The growth in this culture was quite limited; only a small per cent. of the spores
had pushed out germ tubes.
278
KUNKEL: FACTORS INFLUENCING TOXICITY OF
and mercuric chloride is, therefore, not due to any acquired relation
of immunity or susceptibility.
ļ
TABLE X
THE TOXICITY OF CADMIUM CHLORIDE IN SACCHAROSE, GLUCOSE, LACTOSE, STARCH,
AND PEPTONE MEDIA
Saccharose 5%
Glucose 5%
Lactose 5%
Starch 5%
Peptone 5%
G.M.
Growth
G.M.
Growth
G.M.
Conc.
After
Conc.
After
Conc.
Growth
G.M.
After Conc.
Conc.
CdCl2
3 Days
CdCl2
3 Days
CdCl2
3 Days
CdCl2
Growth G.M. Growth
After
3 Days
After
CdCl2
3 Days
.00050
.00050
.00050
.00090
.0150
.00030
.00040
.00040
.00080
.0100
.00010
.00030
.00030
.00070
.0050
.00008
.00020
.00020
.00060
.0040
.00006
.00010
.00010
1.00050
.0030
.00004
.00002
+++++
.00008
.00006
.00004
.00002
·+++++
.00008
.00040
.0020
.00006
.00030
.00004
.00002
.00020
++
.00010
++++
.0010
.0008
.0006
.0004
| 1++++++++
.00001
.000008
.000006
10000'
10000*
.00008
The toxicity of cadmium chloride is shown in Table X. It is
most toxic in saccharose and least toxic in peptone. In glucose it
is more toxic than in starch, but less toxic than in lactose. It is
more than three times as toxic in saccharose as in glucose. Its
average limit concentration in the different media is .001 molar.
In saccharose, starch and peptone, cadmium chloride is more
toxic than cobaltous chloride. In glucose and lactose, on the
other hand, cobaltous chloride is more toxic than cadmium
chloride.
TABLE XI
THE TOXICITY OF MERCURIC CHLORIDE IN SACCHAROSE, GLUCOSE, LACTOSE, STARCH,
AND PEPTONE MEDIA
Saccharose 5%
Glucose 5%
Lactose 5%
Starch 5%
Peptone 5%
G.M. Conc.
HgCl₂
Growth G.M. Growth
After Conc. After
3 Days HgCl₂ 3 Days
G.M. Conc.
HgCl2
Growth
After
3 Days
G.M. Conc.
HgCl2
Growth
After
3 Days
G.M.
Conc.
Growth
After
HgCl2 | 3 Days
.000400
.00050
.000200
.000050
.0055
.000200
.00040
.000100
.000040
.0050
.000100
.00030
.000080
.000030
.0045
.000080
.00020
090000*
.000020
.0040
.000060
.00010
.000040
.000010
.0035
.000040
.00008
.000020
.000010
.000005
.000001
+++
.00006
.00004
.00002
.00001
+++++
.000020
.000010
.000008
.000006
.000004
+++++
.000008
.0030
.000006
.000004
.000002
.000001
.0025
+++
.0020
.0015
.0010
++
INORGANIC SALTS TO MONILIA SITOPHILA
279
►
Overton (22) has pointed out that mercuric chloride, which
differs from most salts in being more soluble in ether, lanolin, etc.,
exerts its poisonous action more quickly than the other salts of
the heavy metals and thinks that this fact favors the view that
the plasma membrane is a lipoid layer about the cell. The data
in Table XI supports the evidence for the great toxicity of this
salt in all of the different media used. It is, however, more toxic
in starch and less toxic in peptone than in any of the other media.
It is two hundred times more toxic in starch than in peptone.
It is also more toxic in lactose than in glucose or saccharose.
It is,
as noted, the most toxic of all the chlorides used, its average limit
concentration being .0004 molar. A comparison of the limit con-
centrations of the chlorides of zinc, cadmium and mercury shows
that the toxicity of these salts is roughly proportional to the
atomic weights of zinc, cadmium and mercury. Mercury is
somewhat more toxic than would be expected from its atomic
weight but the relation shown here suggests that a better knowl-
edge of the toxicity of non-nutrient salts in different media may
further support the view that toxicity is related to the periodic
functions of the atomic weights of the elements.
TABLE XII
THE TOXICITY OF VARIOUS CHLORIDES IN FIVE DIFFERENT MEDIA
Saccharose.
4
NH- Na- Ca- Ba-
KCI CI Cl Cl₂
.7 .26 .50
1.0 .80 .70
.6 .50 .12
.8 .7 .80 .50
1.8
·
Glucose.
Lactose.
Starch.
I.7
I.I
•
•
Peptone..
I.I
.7
Cl2
FeCl3 CuCl2 ZnCl2 CoCl CdCl₂ HgCl2
|
.30 .00010 .00090.00040.00009 .00060.00006
.20 .00010.00080.00010.00009 .00020 .00010
.35.00001.00003.00070.00002 .00010.00002
.20.00080.00045.00006.00060.00050.00001
.75 .40 .25 .03500 1.02000 .01000 .01000 1.00500 .00200
Table XII gives the limit concentration of each salt in each
medium used. A comparison of these values brings out some
interesting relations. Taking the average concentrations of the
different chlorides in the various media, we see that the alkali and
alkali earth salts which were tested are most toxic in saccharose
and least toxic in glucose. The heavy metals are most toxic in
lactose and least toxic in peptone. While potassium chloride is
most toxic in starch and least toxic in saccharose, sodium chloride
is least toxic in starch and glucose and most toxic in saccharose.
280
KUNKEL: FACTORS INFLUENCING TOXICITY OF
Ammonium chloride and calcium chloride are most toxic in lactose
and least toxic in glucose. Barium chloride, however, is least
toxic in lactose and most toxic in glucose and starch. Cupric
chloride, ferric chloride and cobaltous chloride are most toxic in
lactose. Zinc chloride and mercuric chloride are most toxic in
starch. Cadmium chloride is most toxic in saccharose. None of
the chlorides of the alkali or alkali earth metals, but all of the
chlorides of the heavy metals are least toxic in peptone media.
In lactose media, ferric chloride is more toxic than any of the other
chlorides; in glucose media, cobaltous chloride is most toxic; in
starch, peptone and saccharose media, mercuric chloride and
cadmium chloride are most toxic. Potassium chloride in all of
the media used is the least toxic of all of the salts tested, except
sodium chloride in a starch medium. Potassium chloride in
saccharose is less toxic, and mercuric chloride in starch is more toxic
than any of the other salts in any of the other media. Reading
from right to left in Table XII, one sees the increasing toxicity
of the different salts. The arrangement of the salts in the order
of their toxicity is different for each of the five different kinds of
media.
In order to obtain evidence relative to the number of free ions
in the various media containing limit concentrations of the same
salt, a number of tests were made of the electrical resistances of
the different media. The results of some of these tests are given
in Table XIII.
TABLE XIII
THE ELECTRICAL RESISTANCE OF SOME OF THE MEDIA
Medium
G.M.
Conc. of
FeCl3
Resist-
ance in
Ohms
G.M.
Conc. of
CuCl2
Resist-
ance in
Ohms
G.M.
Conc. of
CdCl2
Resist-
ance in
Ohms
G.M.
Conc. of
HgCl2
Resist-
ance in
Ohms
Saccharose.
Glucose..
Lactose..
Starch..
Peptone.
ΙΟ
.00010 3,700 .00090
.00010 4,900 .00080
.00001 5,600 .00003
.00080 4,200 .00045 1,600 .00050
.03500
97 .00500
8,900.00006
6,500.00006
14,400
10,100 .00020
.00010
.02000
5,000 .00010
7,100 .00002 12,500
1,200
160 .00200
6,900
200
The tests of electrical resistance were made by means of the
Wheatstone bridge method, a Freas electrolytic cell being used.
The resistances shown by five per cent. solutions of the organic
substances used have already been given. Table XIII shows in a
INORGANIC SALTS TO MONILIA SITOPHILA
281
striking way the low resistance of peptone media and indicates
that Monilia is actually able to endure higher concentrations of
these poisonous substances when it is growing in peptone media
than when it is in any of the other media which have been used.
The table also suggests the probability that the susceptibility of
the [fungus to the same kind of ions varies according to the
medium in which it is growing.
In an attempt to further elucidate the nature of the toxic
action of the various salts on Monilia, I have also attacked the
problem from the standpoint of the hypothesis that failure of the
spores to germinate in a medium containing less than the lethal
dose of a toxic substance is due to the inability of the protoplast
to absorb sufficient water. Careful observation of the appearance
and behavior of spores in toxic media has led to the belief that the
retarding action of a toxic salt on germination and growth is the
result of its influence on the ability of the cells to take up water.
As has already been stated, the highest concentration of a salt
in which Monilia spores show germination after three days, has
been designated the limit concentration for that medium. It is
recognized that this is an arbitrary standard. The limit concen-
trations given in the above tables hold only in the case of an incu-
bation period of three days. Germination will take place in
concentrations which are greater than this limit concentration,
provided the spores are left in the medium for a longer period of
time.
Spores placed in a medium in which the toxic substance is
slightly more dilute than the limit concentration, germinate and
produce mycelia. The rate of growth in such a medium is much
slower than in a non-toxic medium. The most obvious char-
acteristic of a poisoned culture is the long incubation period and the
slow rate of growth. The effect of potassium chloride on the time
of germination of spores of Monilia on a potato medium (thirty-
five per cent. potato cubes) is shown by Table XIV.
While growth becomes visible in twenty-one hours in a medium
containing potassium chloride at a concentration of .62 molar, it
takes thirty-four hours when the concentration is 1.26 molar
and fifty-five hours when the concentration is 1.9 molar. This
experiment illustrates the behavior of the fungus in media which
are toxic but which do not entirely inhibit growth.
"
282
KUNKEL: FACTORS INFLUENCING TOXICITY OF
Incubation Period
21 hours.
TABLE XIV
THE EFFECT OF POTASSIUM CHLORIDE ON RATE OF GROWTH
Concentrations of KCl
0.62 molar.
Concentrations of KCl
Incubation Period
66
66
0.70
22
1.42 molar.
1.50
36 hours.
66
37
0.78
66
23
1.58
46
38
0.86
""
25
1.66
46
40
46
66
0.94
27
1.74
42
"
66
1.02
29
1.82
44
•
""
Ι.ΙΟ
31
1.90
55
1.18
46
"
32.5
1.98
"
59
1.26
1.34
34
35
2.06
63
46
2.14
i
No germination after
20 days.
If now we study the condition of the spores in media that
entirely inhibit germination, we obtain some interesting data.
The spores will remain viable for two weeks or longer in the pres-
ence of so toxic a substance as mercuric chloride, provided the
concentration is slightly below that which causes plasmolysis.
Some spores which had been kept for two weeks on a starch
medium containing mercuric chloride at a concentration of .00005
molar germinated when transferred to potato agar. This shows
that although the spores do not germinate in such a toxic medium,
they are not much injured by it. The toxic substances which I
have used cause serious injury to the spores, only when they are
concentrated enough to bring about plasmolysis. At concentra-
tions less than this, the spores do not germinate but they remain
alive for a long time. At still lower concentrations they not
only remain alive but germinate and make a slow growth.
This slow growth in media containing approximately limit
concentrations of toxic substances seems to me strong evidence
that although the poison is not present in sufficient quantities to
cause plasmolysis, it nevertheless hinders the absorption of water
by the spores and in this way inhibits their growth. This assump-
tion seems fully in accord with the facts above noted. As the
concentration of the toxic substance is increased, the ability of
the protoplasm to absorb water becomes less and less and the time
required for germination longer and longer. A concentration is
finally reached at which the spores are no longer able to absorb
any water from the surrounding medium. Although this con-
INORGANIC SALTS TO MONILIA SITOPHILA
283
centration inhibits germination, it does not kill the spores, even,
after considerable periods of time. When the concentration is
still further increased the water holding power of the spores
becomes less and plasmolysis results.
If as is here assumed the rate of growth of the fungus in a toxic
medium depends on its rate of water absorption, then any means
by which the water content of the mycelium could be lessened
should also decrease the rate of growth. If, for example, Monilia
be placed in a dry atmosphere where the loss of water by the
aerial part of the mycelium would be great, the rate of growth
should be correspondingly decreased. The following experiments
show the effect of a dry atmosphere on the rate of growth of the
fungus mycelium.
The media used in this work consisted of cubes of potato to
which was added enough water to fill the bottom of the culture
vessel to depth of about one half of a centimeter. On such a
substratum Monilia makes very abundant growth. The cultures
were incubated at a temperature of 29° C. This is one degree
below the temperature most favorable for its growth (see Went,
34). Since the effect of drying the atmosphere over cultures of
Monilia varies somewhat with the age of the culture at the time
the drying agent is used, a few remarks regarding the appearance
of the fungus at different stages in its development seem desirable
at this point.
Under the conditions outlined above, growth first becomes
visible to the naked eye after an incubation period of from nine to
ten hours. During the next fifteen hours the mycelium grows so
rapidly that it almost hides the surface of the potato cubes. In the
next seven hours the fungus makes still more rapid growth, rising
from five to ten centimeters above the culture medium. This is
followed by a period of about six hours, during which there is
little visible change. When the cultures are approximately forty
hours old they begin to take on a beautiful pink color and during
the next hour spore formation begins.
If the vapor pressure above cultures that are more than thirty
hours old is unduly lowered, the mycelium withers and fails to
produce spores. An entirely different result is obtained in the
case of young cultures. If five cubic centimeters of a four molar
284
KUNKEL: FACTORS INFLUENCING TOXICITY OF
potassium chloride solution be suspended in an oiled paper bag
above a young culture, it checks the growth of the mycelium to a
remarkable extent. The less concentrated the solution in the
paper bag, the greater will be the mycelial growth in the culture
above which it is suspended. Drying the air by placing small
amounts of calcium chloride over the cultures gives still more
striking results. The mycelium can be kept from rising more
than a few millimeters above the surface of the medium. Table
XV shows the effect of suspending potassium chloride solutions
of different concentrations over cultures kept under conditions
that were otherwise identical. The bags containing the solutions
were placed in the bottles at the time the inoculations were made.
TABLE XV
INFLUENCE OF HUMIDITY ON THE GROWTH OF Monilia
Solution Suspended Above Culture
5 c.c. of 4.0 molar KCl solution.
66
5 c.c. 2.4
""
5 c.c. 3.2
"
66
KCI
66
66
KCI
66
66
KCI
66
.8
KCI
86
66
distilled water
5 c.c. 1.6
5 c.c.
5 c.c.
16
Height of Mycelium After 32 Hours
1.0 cm.
66
1.5
44
3.0
64
3.5
"
3.2
66
4.0
FIG. I shows the six cultures referred to in Table XV. The
photograph was taken several hours after obtaining the measure-
ments used in the table. The culture that made the least growth
is the one above which was suspended five cubic centimeters of
four molar potassium chloride solution. Distilled water was
suspended over the culture that made the greatest amount of
growth. As the rate of loss of water by the mycelium is increased
the rate of growth is correspondingly decreased. By suspending
one gram of anhydrous calcium chloride over alternate cultures
(FIG. 2) the influence of vapor pressure on rate of growth is still
more strikingly shown. The amount of water taken up by the
calcium chloride, however, is very small.
In order to determine the amount of water taken up by calcium
chloride suspended over cultures of Monilia when it causes such
variations in growth as are to be noted in FIG. 2, bags were
weighed before and after being suspended over cultures. The
difference in weight gives the amount of water taken up by the

INORGANIC SALTS TO MONILIA SITOPHILA
285
drying agent. This amount was found to be in ten instances
respectively: 1.32g; 1.45g; 1.40g; 1.32g; 1.38g; 1.43g; 1.39g; 1.36g;
1.30g; 1.37g or an average of 1.33 grams. A similar calcium
FIG. I.
Cultures of Monilia sitophila. This photograph shows the influence of
humidity of the atmosphere on the rate of growth of Monilia. An oiled paper bag,
containing a 4 molar potassium chloride solution, was suspended over the culture
that shows the least growth, while distilled water was suspended over the culture
that made the greatest growth. The series of six cultures shows the effect of drying
the air over the mycelium to different degrees.
FIG. 2.
Cultures of Monilia sitophila. This photograph shows the influence
of drying the air over young cultures of Monilia. One gram of anhydrous calcium
chloride was suspended above the cultures in alternate bottles; the small amount of
growth made by the fungus in these bottles is shown.
286
KUNKEL: FACTORS INFLUENCING TOXICITY OF
?
chloride bag was suspended for the same length of time over a
sterile medium exactly like that on which the Monilia was being
grown. This bag took up only .39 gram of water, showing that
most of the water taken up by the drying agent suspended over
cultures comes from the mycelium of the fungus. These experi-
ments show that the removal of less than one and one half grams
of water from the atmosphere above cultures greatly decreases the
rate of growth of the mycelium. Similar experiments were per-
formed with Mucor Mucedo, Sporodinia grandis and Phycomyces
nitens. In each case, drying the air above young cultures greatly
reduces the rate of growth. Sporodinia grandis and Mucor
Mucedo produce shorter sporangiophores when grown in a culture
over which a drying agent is suspended than when grown in a
moist atmosphere. In the case of Phycomyces nitens the rate of
growth is greatly checked by drying the atmosphere but the final
length of the sporangiophores is not decreased.
These experiments show in a striking manner the well known
importance of water to growth. They also suggest that the slow
germination and the slow rate of growth in toxic solutions of
potassium chloride may be largely due to the lowering of the vapor
pressure above the medium. The same thing is probably true
of all other salts that must be used in considerable concentrations
in order to inhibit growth. But it is quite different with more
toxic substances such as the salts of the heavy metals which
inhibit growth when present in very small quantities. Their
effect on the vapor tension of the medium is, of course, insignifi-
cant. This difference between toxic and relatively non-toxic
substances has not been sufficiently emphasized. It seems to me
that the above experiments offer strong evidence in support of
the assumption that an important factor in many cases of toxicity
is the effect of the poison on the ability of the protoplast to absorb
water. They, at least, show that this factor is sufficient to
account for a large part of the decrease in growth which is to be
observed in toxic media containing relatively large amounts of
dissolved salts.
A salt like potassium chloride when added to a medium in
sufficient quantities to check growth, increases the affinity of the
medium for moisture and in this way makes it difficult for the
INORGANIC SALTS TO MONILIA SITOPHILA
287
;
mycelium to take up sufficient water. By lowering the vapor
tension of the medium it also increases the rate at which water is
lost by the fungus. If instead of adding the salt to the medium it
is dissolved in a small amount of water and suspended above a
young culture the strong solution takes up water from the air.
In this case the salt does not retard the rate at which water is
taken up by the mycelium but does increase the rate at which
water is given off. It is very interesting to see that when the salt
is suspended above the growing culture it causes a decrease in the
rate of growth which is similar to that to be observed when it is
added directly to the medium. There can be little doubt that in
media containing toxic concentrations of potassium chloride, the
retardation in growth is largely the result of the inability of the
fungus to absorb and to retain sufficient water. Though toxic
concentrations of the salts of the heavy metals do not appreciably
lower the vapor pressure above the medium, it is possible that these
salts also act by decreasing the power of the cells to absorb water.
A consideration of the factors which may be involved in bring-
ing about the variations in toxicity in the different media leads to
the suggestion of several possibilities. In the first place it is
known that certain salts react with sugars, starch and proteids.
The resulting compounds if formed in a given medium would be
expected to show properties different from the salt and different
from other compounds that might be formed in still other media.
Relatively little is known of these substances, but some of them
have been studied and the conditions that favor their formation
have been determined.
In a solution of sodium hydroxide, cane sugar is converted into
sodium saccharate (see Thomsen, 32). Saccharates of calcium,
potassium and barium are also known (see Peligot, 24). All of these
saccharates are formed by the action of the hydroxides of the differ-
ent metals on cane sugar. The addition of ammonia to a solution
of cane sugar increases the rotatory power of the solution and has
been taken as evidence that a compound is formed between the
sugar and ammonia (see Wilcox, 35). Copper and iron saccharates
have also been reported (see Graham, 10). They are made by
adding the chlorides to alkaline solutions of cane sugar.
We can
not, however, in the cases before us, attribute the variations in
4
288
KUNKEL: FACTORS INFLUENCING TOXICITY OF
toxicity of different salts in cane sugar media to the production of
saccharates, since the addition of chlorides to a neutral solution
of cane sugar does not furnish the conditions necessary for their
formation. In order to obtain saccharates it would be necessary to
add alkalies to these solutions. There seems to be good evidence,
however, that some of the chlorides do form loose combinations
with cane sugar (see Peligot, 24). A compound represented by
the formula 2C12H22O11.BaCl, has been prepared in crystal-
line form (Gauthier, 8). It may be that such addition com-
pounds are factors in determining the relative toxicity of the
chlorides in saccharose media. Barium chloride is less toxic in
saccharose than in starch, glucose or peptone. It may be that this
rather low toxicity in saccharose is due to the formation of the
compound referred to above.
Compounds of glucose and lactose, analogous to the saccharates,
are also known (Hönig & Rosenfeld, 11, 12). They are formed
by the action of alkalies on these sugars. With glucose, sodium
chloride and potassium chloride form compounds which are
represented by the formulae C6H12O6.NaCl and C6H22O6.KCI
respectively (Gladstone, 9). It may be that the rather low
toxicity of sodium chloride and potassium chloride in glucose
media is due to the formation of these compounds. Starch
has been shown to have the properties of a very weak acid and
to be able to react with small quantities of neutral salts (see
Demoussy, 5). Sodium chloride is less toxic in starch than
in any of the other media except glucose. It may be that some
of the salt has combined with starch to give a compound that is
less toxic than sodium chloride. Potassium chloride, on the
other hand, is more toxic in starch than in any of the other media.
It is possible that this is due to the formation of a complex com-
pound which is more toxic than the chloride.
The addition of any of the chlorides to a five per cent. peptone
solution always causes a certain amount of precipitation. That
the salts are to some extent carried down by this precipitate seems
highly probable. Pauli (23) has shown that neutral proteids
adsorb electrolytes. The antitoxic action of peptone on the salts
of the heavy metals may be in part due to this adsorption. Table
XII shows that the toxicity of all of the chlorides of the heavy
metals is less in peptone than in any of the other media.
INORGANIC SALTS TO MONILIA SITOPHILA
289
Although some of the variations in the toxicity of the chlorides
in the different media may be the result of reactions between the
salts and organic substances, there is good evidence that this can
not account for all of the variations brought out in Table XII.
A study of some of the media from the standpoint of their re-
sistance to the passage of the electric current has shown that there
is considerable variation in the ionic concentration of different
media that contain limit concentrations of the same salt, indicating
that some media do influence the concentration of the ions but
that these differences are not correlated directly with the observed
differences in toxicity. Limit concentrations of ferric chloride,
cupric chloride, cadmium chloride and mercuric chloride in peptone
show in each case a much lower resistance than limit concentra-
tions of these same salts in saccharose, glucose, lactose or starch.
This seems to be strong evidence in support of the view that the
fungus is actually able to resist higher ionic concentrations of these
salts when it is growing in peptone than when it is in any of the
other media tested, thus showing an effect of the peptone other
than that due to its precipitation of a portion of the toxic salt.
Another factor of probable importance in this connection is
the influence of the different media on the production of enzymes.
Went (34) has shown that Monilia sitophila produces a number of
different enzymes and that the organic part of the medium deter-
mines which enzyme will be produced in any given case. The
effect of the various chlorides on trypsin, which the fungus pro-
duces in peptone media, may be quite different from their influence
on diastase produced in starch media. The rather high toxicity
of potassium chloride and barium chloride in starch media may
be due to the infleunce of these salts on diastase. Similar varia-
tions may be brought about by the actions of the poisons on
lactase, lipase and invertase.
My observations show clearly that the organic part of the
medium must be taken into account in studies on toxicity. This
important factor, however, has generally been disregarded by those
who have tested the resistance of fungi and bacteria to poisons.
Stevens (30) tested the toxic action of mercuric chloride and other
poisons on Penicillium crustaceum growing in bread media. In a
study of the toxicity of a whole series of substances for Aspergillus
290
KUNKEL: FACTORS INFLUENCING TOXICITY OF
flavus, Sterigmatocystis nigra, Oedocephalum albidum, Penicillium
glaucum and Botrytis vulgaris, Clark (4) used a sugar beet infusion.
Klebs (14) determined the poisonous influence of various sub-
stances on Saprolegnia mixta growing in a pea infusion. Bessey (2)
tested the resistance of fungi to copper sulphate, mercuric chloride
and other poisons without taking into account the possible influ-
ence of organic materials in the synthetic media which he used.
Pulst (25) measured the toxicity of copper sulphate, zinc
sulphate and nickel sulphate. He tested the action of these salts
on Mucor Mucedo, Aspergillus niger, Botrytis cinerea and Peni-
cillium glaucum in a medium containing sugar and peptone, with-
out regard to the effect of sugar or peptone on the toxicity of the
salts.
Loew (20) has studied the poisonous action of sodium fluoride
on Bacillus mycoides, B. pyocyaneus, B. subtilis and B. prodigiosus.
Using a bouillon medium, he found that these bacteria could endure
approximately one per cent. of sodium fluoride. He, therefore,
disagrees with Arthus and Huber (1) who held that a one per cent.
solution of this salt is deadly to all cells. He also compares the
toxic action of sodium fluoride on Spirogyra communis in an
aqueous solution with its effect on bacteria in a bouillon medium
and notes that the bacteria have a much higher resistance than
the alga.
Ssadikow (29) studied the resistance of Bacillus subtilis to
strychnin salts in bouillon, nutrient agar and nutrient gelatin.
My observations suggest that the high concentrations which this
organism was able to endure might have been fatal to it in a
medium containing no peptone. Renard (27) has tested the anti-
toxic action of different concentrations of each of twelve nutrient
salts on the poisonous effects of eleven different toxic substances,
mostly chlorides and nitrates of the heavy metals. He finds that
in general, the antitoxic action increases with the concentration of
the inhibiting salt. Except in a few cases where the antagonistic
substance is a salt of an organic acid, he has made his tests in
glucose media. The antitoxic coefficients by which he attempts
to express the relations between the several poisons and their
antidotes would undoubtedly have been found to be different for
each medium tested if he had tried other common organic sub-
INORGANIC SALTS TO MONILIA SITOPHILA
291
<
stances. He has not taken into account the influence of the
glucose on the toxicity of the poisons used although in an experi-
ment in which he studies the action of copper salts in the presence
of potassium acetate both with and without glucose, he obtains
evidence that the glucose lessens the toxicity of the copper.
Although Lipman (17) makes mention of the fact that sodium
carbonate is much less toxic to ammonifying organisms in certain
soils than in pure peptone solutions, he has, nevertheless, drawn
conclusions as to the toxicity of various salts after testing them
in a medium consisting of soil and dried blood, without taking
into account the effect of the organic substances on the salts which
the soil must have contained.
My results show that toxicity measurements which are made
without regard to the organic substances in the medium are of little
value as indicating the relative resistance of different organisms.
The bearing of the facts here brought out on a number of practical
problems is evident. They must be reckoned with in considering
the influence of the decomposition products of humus in the soil,
on soil toxins, in the preparation of antidotes, in the chemical
sterilization of water and in the preservation of milk and other
food materials by chemical means.
This work has been done under the direction of Prof. R. A.
Harper, to whom I am very grateful for many valuable sugges-
tions. I also wish to thank Prof. W. G. Marquette for helpful
advice.
COLUMBIA UNIVERSITY
NEW YORK CITY
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Combinaison du saccharose avec quelques sels
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19. Loew, O. Ueber die physiologischen Functionen der Calcium-
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der Zelle ihre vermutlichen Ursachen und ihre Bedeutung für
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88. 1899.
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23. Pauli, W.
Kolloidchemische Studien am Eiweiss. Dresden. 1908.
Untersuchungen ueber die chemische Natur der
Zuckerarten. Ann. Chem. u. Pharm. 30: 69.
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VITA
LOUIS OTTO KUNKEL. Born in Audrain County, Missouri,
May 7, 1884. Early education in the rural schools of Audrain
County, Missouri. Graduated from the Mexico (Missouri)
High School, June 1906; attended State University of Missouri
(Columbia, Missouri), September 1906 to June 1911, receiving
the B.S. degree, June 1909, the A.B. degree, June 1910 and the
A.M. degree, June 1911. Graduate student in Washington
University (St. Louis, Missouri), 1911-1912; graduate student
in Columbia University, 1912-1914.
Assistant in Botany, Missouri University, 1909-1911;
Research Fellow in the Henry Shaw School of Botany, 1911-
1912. Assistant in Botany, Columbia University, 1912-1913;
Research Assistant in Botany, Columbia University, 1913-
1914. Teacher in Missouri University, Summer School, 1912;
teacher in Columbia University, Summer School, 1913.
Elected to Alpha Zeta, Phi Delta Kappa and Sigma Xi, 1909;
Phi Beta Kappa, 1910.
Author of the following articles: A study of the problem of
water absorption; The production of a promycelium by the
aecidiospores of Caeoma nitens Burrill; The influence of starch
peptone and sugars on the toxicity, of various nitrates to
Monilia sitophila (Mont.) Sacc.; Nuclear behavior in the
promycelia of Caeoma, nitens Burrill and Puccinia Peckiana
Howe.
}
UNIVERSITY OF MICHIGAN
3 9015 07502 9945
BOOK CARD
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AUTHOR Kunkel
TITLE
•K96
Physical and chemisal
factors of inorganic salts.
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Medical Library,
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