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The effect of certain agents on the development of some moulds online

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moulds will he included in articles to follow.

42



in addition to these, note was made of any irregularity in development, a
permanent record of this being made by means of photomicrographs as in
the preceding work. The tabulated results contain the observations cited,
and in addition, the age of the original culture from which the inoculation
was made. A young, vigorous culture, in conjunction with an old control
culture, were used in determining the deviations from the nonnal which
could be ascribed to the effect of the agent.



Results of Germination Tests

Development of first generation in the normal solution. Inoculation from cultures
grown for varying periods of time in solutions of the respective agents.



Cultures from which inoculations were made.



Development in normal solution.



Agents


Number of

days in

contact

with

agent


No


. of days


to


Kind


Per
cent


germi-
nate


form
conidia


mature


Control




14
176
183
210
380
423
590
423
590
7
106
590
7
106
423
590
423
590
423
590

28

26

14
113

14

113

7

106

89
169

91
161

91
161

91
161

91


1
2
2
2
2

3
3


2
3
3
3
4
4
6


3


Control




4


Control . . .




4


Control




5


Control




5


Control




6


Control




8


Salt


5

5
10
10
10
15
15
25
25
50
50

66^
66^

3

5

7

7
10
10
.25
25
30
30

1

1

2

2

5

5
10




Salt








Salt


2
2


3

5


4


Salt


6


Salt




Salt


2
3

2
2
4


4
4
3
3

5


6


Sah


6


Sugar


4


Sugar


5


Susar


7


Sugar




Sugar


2

4
2
2
2
3
2
2
2
2
3


3
8
3
3
3
4
3
4
3
5
6


6


Sugar


9


Potassium nitrate


5


Potassium nitrate


5


Potassium nitrate


4


Potassium nitrate


6


Potassium nitrate


4


Potassium nitrate


6


Potassium nitrate


5


Potassium nitrate


6


Potassium nitrate


7


Potassium nitrate




Allspice


3
2
2
5
3


■5
3
4
7
9


7


Allspice

Allspice


' 5
6


Allspice


9


Allspice seed

Allsnice seed


11


Allspice seed


3


8


9



43



Results of Germination Tests — Continued
Cultures from which inoculations were made. Development in normal solution.



Agents


Number of

days in

contact

with

agent


No. of days to


Kind


Per

cent


germi-
nate


form
conidia


mature



Allspice seed

Cinnamon (infusion)
Cinnamon (infusion)
Cloves (infusion).. . .
Cloves (infusion).. . .
Mustard, ground.. . .
Mustard, ground.. . .
Mustard, ground.. . .
Mustard, ground.. . .
Mustard, ground.. . .
Mustard, ground.. . .
Mustard, ground. . . .
Mustard, ground. . . .
Mustard, ground.. . .
Mustard, ground.. . .
Mustard seed, black.
Mustard seed, black.
Mustard seed, black.
Mustard seed, black.
Mustard seed, black.
Mustard seed, black.
Alustard seed, black.
Mustard seed, white
Mustard seed, white
Mustard seed, white
Mustard seed, white
Mustard seed, white
Mustard seed, white
Mustard seed, white

Acetic acid

Acetic acid

Acetic acid

Acetic acid

Acetic acid

Acetic acid

Acetic acid

Acetic acid

Acetic acid

Acetic acid

Acetic acid

Acetic acid

Cider vinegar

Cider vinegar

Cider vinegar

Cider vinegar

Cider vinegar

Cider vinegar

Cider vinegar

Cider vinegar

Malt vinegar

Malt vinegar



10

lU,

10.

10,

10.

0.



0.

0.

0.

0.

1.

2 ,

2

2

1

2

2

2
25
25
25

1

2

2

2

25

25

25









1
1
1
1
1
1

2
2



1
1
1
1

2






(J




2
2
2
5
5
5









.0

.0
.0
.0
.0
.0
.0
.0
.5
.5
.5
.5
.0
.0
.0
.0
.5
.5
.0
.0
.5
.5
.0
.0
.5
.5
.0
.0
.5
.5



161
423
590
423
590

14

113

183

8

14
113

12

12

118

188

8

8

107

177

7

107

177



107
177
7
107
177
210
380
423
590
210
380
423
590
210
380
210
380
210
380
210
380
210
380
210
380
210
380



3
3



2
2
2
4
5
3
3
7
2
2
2
2
2
2
2



2
2
2
3
2
3



2
2
2
3

2
2

2



2
3
2

2



4
5



3
4
3
9
11
4
4
8
3
3
3
3
5
3
3



3

3
4
4
3
5



3
3
4
5
3
3
4



3
4
2
3



4
3



4
5
5
10
12
7
7
9
5
5
4
4
6
5
4



4
4
6
6
4
7



6
6
6
7
4
5
6



6
6
3

5



6
6



44



Results of Germination Tests — Continued
•Cultures from which inoculations were made. Development in normal solution.



Agents


Number of

days in

contact

with

agent


Nc


. of days


to


Kind


Per

cent


germi-
nate


form
conidia


mature


IVTalt vinpp'ar


1.0

1.0

1.5

1.5

2.0

2.0

0.5

0.5

1.0

1.0

1.5

1.5

2.0

2.0

0.1

0.1

0.1

0.2

0.2

0.2

0.5

4.0

4.0

5.0

5.0

6.0

6.0

4.0

4.0

5.0

5.0

6.0

6.0

4.0

4.0

5.0

5.0

6.0

6.0

1.5

2.0

2.0

4.0

0.1

0.2

0.2

0.2

0.5

0.5

0.5

0.1


210
380
210
380
210
380
210
380
210
380
210
380
210
380
34
132
202
39
138
208
32
25
124
14
113
14
113
25
124
14
113
14
113
25
124
14
113
14
113
31
28
127
100
14
14
423
590
14
423
590
14


3
3


4

5


6


Malt vinpcrar


6






























Tlistilled vinei?ar


2
2
2
3


3
3
4

5


4


Oi'^tille^d vinegar


6


Distilled vinegar


6


Distilled vines?ar


6






uisiiiieu vinegar
























Rntvric acid.


2
3
2
2
3
2


2
5
4

2
5
4


4


Rutvric acid


7


Rntvnr ^cul


6


Rntvric acid


4


Rntvric acid.


8


Rntvric acid


6






Citric acid


2
3

2

2
2
2

2
2
2
2
2
2
2
3
2
2
2
2
2
2
2
2
2
4


3
5
3
5
3
4
3
4
2
4
2
4

v5

7
2
5
3
4
3
2

3
3
3

5 '


4


Citric acid


10


Citric acid


4


Citric acid


6


C^itrir' acid


4


- Ci+ric acid


6


T^actic acid


4


T>actic acid


6


T.actic acid


3


Lactic acid


6


T^actic acid


3


T^actic acid


6


Malic acid


4


Malic acid


10


Malic acid


4


IVlalic acid


7


Malic acid


4


Alalic acid


6


' Tartaric acid


4


Tartaric acid


. 4


Tartaric acid


5


Tartaric acid


6


Benzoic acid


5










































Sodium benzoate


2


3


5







45



Results of Germination Tests— Continued
Cultures from which inoculations were made. Development in normal solution.



Agents


Number of

days in

contact

with

agent


No


. of days


to


Kind


Per
cent


germi-
nate


form
conidia


mature


Sodium benzoate


0,2
0.2
0.2
0.5
0.5
0.5
0.1
0.2
0.2
0.2
0.5
0.5
0.5
0.1
0.2
0.2
0.5
0.5
0.5
0.2
0.2
0.5
0.5
1.0
1.0
0.2
0.2
0.5
0.5
0.1
0.2
0.2
0.2
0.5
0.5
0.5
0.1
0.2
0.2
0.2
0.5
0.5
0.5
0.1
0.2
0.2
0.2
0.5
0.5
0.5
1.0




14
423
590

14
423
590

14

14
423
590

11
423
590

14

14
423

14
423
590
423
590
423
590
9
108
423
590
423
590

14

14
423
590

14
423
590

14

14
423
590

14
423
590

14

14
423
590

14

423

590

8


2


3


5


Sodium benzoate




Sodium benzoate








Sodium benzoate


4


5




Sodium benzoate




Sodium benzoate








Boric acid


1

2


3

4


4


Boric acid


5


Boric acid




Boric a'^id








Boric acid


5






Boric acid






Boric acid








Borax


2

2


3
3


4


Borax


5


Borax




Borax








Bora-X








Borax








Formic acid


3


4


6


Formic acid . .




Formic acid .








Formic acid








Formic acid








Formic acid








Sodium formate


3


7


91


Sodium formate




Sodium formate


3


4


6


Sodium formate .... ...




Salicylic acid


1
1

5
7


3
3

7


4


Salicylic acid .




Salicylic acid


12^


Salicylic acid




Salicylic acid






Salicylic acid








Salicylic acid








Sodium salicylate

Sodium .salicylate

Sodium salicylate

Sodium salicylate

Sodium salicylate


1

1

3

5

1

3

4

2

2

3

3

1

3

53

2


3
3

4
8
3
4
5
3
2
4
7
2
4


4
4
6
9


Sodium salicylate

Sodium salicylate

Saccharin


6
7
4


Saccharin


4


Saccharin


6


Saccharin

Saccharin

Saccharin

Saccharm ...


13
4
6


Sacchaiin


5


7







^ Curled. ^ Surface yellow.



3 Wet curled masses on surface.
46



Results of Germin.\tion Tests— Continvied
Cultures from which inoculations were made. Development in normal solution.



Agents


Number of
days in
contact

with agent


No


. of days to


Kind


Per cent


germinate


form
conidia


mature


Saccharm


1.0

1.0

2.0

0.1

0.2

0.2

0.2

0.5

0.5

0.5

0.1

0.2

0.2

0.2

0.5

0.5

0.5

1.0

1.0

1.0

1.0

1.0

0.02

0.02

0.04

0.04

0.02

0.02

0.04

0.04

0.1

0.1

0.1

0.2

0.2

0.1

0.1

0.1

0.2

0.2

0.2

0.5

0.5

0.5

1.0

1.0

1.0

3.0

3.0

3.0

0.02

0.02

0.04

0.04


107

590

21

14

14
423
590

14
423
590

14

14
423
590

14
423
590

21

28
117

35
134

28
127

25
124

14
118

19

177

8

21

28

21

28

34
132
202

34
132
202

28
127
197

25
124
194

28
127
197
8
107
8
177


3
5
3
3
2
3
7


5


8






Sacchai"in


7
3
2


8


Sodium sulohite


5


Socliiim sulohite


5


^nrli'ii m Qiili^ilii t"P


121














Sodium sulohite


2
3
3

2

2

7^

2

2

3


8
9
4
3
3


12^


Sodium sulohite


133


Conner sulohate


5


Conner sulnhate


4


Conner sulnhate


4






Conner sulnhate


3
3

5


4


Conner sulnhate


6


Conner sulnhate


7






















Alum


2
3

2
3
2
5
2
2
2
1


4

5
2
5
3
6
2
3
2
3


6


Alum


6


Creosote


4


Creosote


6


Creosote .


4


Creosote


8


Formaldehyde (40 per cent). . .
Formaldehyde (40 per cent). . .
Formaldehyde (40 per cent) . . .
Formaldehyde (40 per cent). . .
Formaldehyde (40 per cent). . .
Formaldehyde (40 per cent). . .
Formaldehyde (40 per cent) . . .
Formaldehyde (40 per cent). . .
Formaldehyde (40 per cent). . .
Zinc chlorid


3
6

4-
4


























2
3
2
2
3
3
2
2
2
2
3
2
2
3
3
2
2
2
2


3
5
6
3
6
4
3
5
3
3
6
6
2
5
4
2
3
4
3


4


Zinc chlorid


7


Zinc chlorid


8


Zinc chlorid


4


Zinc chlorid


7


Zinc chlorid


6


Oxalic acid


4


Oxalic acid .


6


Oxalic acid


5


Oxalic acid


4


Oxalic acid


7


Oxalic acid


8


Tannic acid


4


Tannic acid


7


Tannic acid


7


Carbolic acid


3


Carbolic acid


5


Carbolic acid


4


Carbohc acid


4



1 Yellow.



2 Yellow.



^ Yellow.



■* Tiny compact submerged colonies.



47



Results of Germination Tests — Continued
Cialtures from which inoculations were made. Development in normal solution.



Agents



Kind



Per cent



Numljer of
days in
contact

with agent



No. of davs to



germinate



form
conidia



mature



Carbolic acid

Carbolic acid

Carbolic acid

Carbolic acid

Carbolic acid

Carbolic acid

Hydrochloric acid . . . .
H\'drochloric acid. . . .

Nitric acid

Nitric acid

vSulphuric acid

.vSulphuric acid

-Sulphuric acid

-vSulphuric acid

Potassium hydrate. . .
Potassium hydrate. . .
Potassium hydrate. . .
Potassium hydrate. . .
Potassium hydrate. . .
Potassium hydrate. . .
Potassium hydrate. . .

Sodium hydrate

Sodium hydrate

Sodium hydrate

-Sodium hydrate

Sodium hydrate

Sodium hydrate

-Sodium hydrate

Atropin sulphate

Atropin sulphate

Cocain hydroclilorate.
Cocain hydroclilorate.
Morphin sulphate. . . .
Morphin sulphate. . . .
Strychnin nitrate. . . .
Strychnin nitrate ....

Quinin sulphate

Quinin sulphate

Quinin sulphate

Quinin sulphate

Mercuric chlorid

Mercuric chlorid

Mercuric chlorid

Mercuric chlorid



0.1


0.1


0.1


0.1


0.2


0.2


0.5


0.5


0.2


0.5


0.1


0.1


0.2


0.5


0.2


0.2


0.5


0.5


1.0


1.0


1.0


0.2


0.2


0.5


0.5


0.5


1.0


1.0


0.0065


0.0065


0.1625


0.1625


0.1625


0.1625


0.0065


0.0065


0.2


0.2


0.5


0.5


0.01


0.01


0.1


0.1



13

21
127
176

21
127

39
138
138

32

34
132

32

21

14
113

19
118

12
118
188

14
113

19
118
188

12

19

31
130

31
130

31
130

31
130

34
132

34
132

28

127

8

107



1
3
2
4
2
3
4



2
2
2

2
3



2
2
2
6

7



2
3
2
2
2
3
2
3
2
2

2
2
2
3
4



2
8
3

11
2
4

10



2
4
3

5

7



2
4
3



3
5
3
4
3
6
2
5
3
5
5
5
2
5
9



4'
10

5
132

4

8
12



4
5
4
6
9



4

6

6

10



4
6
4
6
4
7
31
6
4
6
6
7
4
7
10



1 Heavier than control. - Surface rough.

As in the previous experiments, the microscopic characters were of
greater value than the macroscopic in determining the deviations from the
normal which appeared in the following generation. In many cases where
growth appeared to be nomial, the minute structure showed more or less
variation, similar to that induced in the organism when in contact with
the agent. That the changes produced by some of the agents were of a
vital character and sufficiently pronounced to cause the protoplast to exceed
the limit of physiological elasticity is evidenced by their transmission to a
greater or less extent to the following generation. It seems possible that

48



if tlic action of the inducing agent were continued, the changes produced
might ultimately become permanent, if they did not finally destroy the
organism, since changes of a less violent character disappear when the
organism is returned to the original conditions, according to Thom.^ His
statement which is based on extensive cultural work is "Many species
dilTer so materially in gross characters when grown upon these different
media that successive cultures, if not known to be pure transfers, might
be supposed to be different species, but w^hen returned to the original media
and conditions these fonns have immediately produced the characters and
reactions first fotind, with a large degree of uniformity." It is probable,
however, that with the development of the organism in a normal environ-
ment, the variations would gradually disappear, and the organism assume
its original form and structure, since according to Pfeffer,^ variations of
this nature being labile only gradually acquire a fixed hereditary character.
In the cases in the original experiinents, where the most pronounced effects
of the agents, swelling and various degrees of disorganization, took place,
similar ^•ariations were present in the following generation, these being
distinct from the changes resulting from the reduced vitality due to the age
of the inoculating spores. In those experiments in which the agents merely
reduced the food supply of the organism, the following generation was
practically normal, no results other than those seen in the control being
evident except in the case of the 15 per cent salt culture, in which the char-
acters were apparently transinitted.

The results of the experiments indicate that the organism when in a
labile condition is susceptible to the influence of external conditions, which
may account to a certain extent for many of the so-called species of this
genus. They also indicate that the organism possesses great adaptability
and resistance to variable and adverse conditions shown in the ability
to develop after having been subjected to the influence of the many diverse
and deleterious substances, some so strong as to prevent development
while the conidia remained in contact with them, others, though per-
mitting a retarded germination, producing extreme changes in structure and
form.

See Plates 58-62.

GENERAL CONSIDERATIONS

The cultures of the organisms in which the various chemicals were used
were made, as nearly as possible, under similar conditions. The stock
medium, an organic solution, was kept at uniform acidity; the containers
were flasks of the same size, so that there were equal surface areas of the
solution; the same amount of solution, 100 c.c, was used in all, so as to
avoid any variations due to the nuinber of spores present in a given volume
of the medium; the cultures were kept shaded, and at an approximately
even temperature. The method of seeding has been described, and was
as nearly uniform as possible.

Preliminary to the work with the agents, the moulds were grown in
moist chambers, with air and without, to determine the effect of a lack
of oxygen on development. The resvilt of the lack of oxygen, but sufficient



1 Cultural Studies of Species of Penicillium, U. S. Dept. of Agr., B. A. I., Bui. 118,
1910.

2 Physiology of Plants, Vol. 3, 1903.

49



food supply, was a reduction of the protoplasm, and an increase in the
number of septa, the cells thus formed becoming rounded^ in many cases.
There was an increase in the formation of fruiting in the Penicillium and a
greater degree of catenulation in the Altemaria. It is stated by Pfeffer^-
that P. glaucum is destroyed or severely injured by the withdrawal of
ox\'gen for a single hour. The effort in this work was to determine the
result of other factors apart from the agents in producing morphological
changes in the organisms.

The macroscopic appearance of the grou^hs of the organisms in the
respective solutions though indicating by retardation and abnormalities of
development the general effect of the agents on the stiaicture, is not to be
depended upon alone for an accurate estimation of the effect. In some
cases where a seemingly nonnal gro\\1;h occurred, the microscopic appear-
ance showed the most pronounced effect in the sweUing, distortion, and dis-
organization of the hyphae, whereas in other cultures with the same agent
in which the gro\^1:h was stunted, the microscopic appearance indicated
a nearly normal development, as if the mould had offered a greater resis-^
tance to the specific action of the agent.

In noting the effects of the various agents on the histological structure,
it was found that an approximate classification might be made. The
effects induced were plasmolysis, stunting, distortion, hardening of the pro-
toplasm, reduction of the protoplasm to a comparatively small number of
refractive granules, disorganization of the protoplasm, disorganization
of the cell-walls, the increased formation of se'jDta thus fonning abnormally
short cells, many short sub-branches, sub-branches developed into more
or less stunted fruiting heads in the Penicillium, and with some agents
a combination of different effects. The disorganization of the proto-
plasm produced a granulation, more or less coarse, with no distinction
between the ectoplast and endoplast. This lack of differentiation gave a
swollen appearance to the hyphae, though in some cases they w^ere less
than the normal size, there were no vacuoles, and apparently little cohesion.
The disorganization of the walls was evidenced by their breaking from the
W'Cight of the cover-glass or in the transference of the hyphae from the cul-
ture into a drop of the culture medium on a slide, and complete disruption
when placed in a drop of distilled water.

The salt, sugar, and potassium nitrate produced plasmolysis resulting
finally in complete arrest of gro'^i:h from starvation. That the organism
was not killed was shown by the germination tests with the Penicillium.
The effect was really a "physiological drought"^ produced by a high con-
centration of a non-toxic salt, which agrees with the statement of Pfeff'er*
that when the osmotic concentration of the culture fluid passes a certain
limit, growth becomes impossible, though no poisonous eff'ect is exercised.

The majority of the spices were innocuous, producing neither retardation
nor abnormal effects, so far as could be ascertained from their gross devel-
opment and microscopic appearance. Those which possessed antiseptic
properties — allspice, cinnamon, and cloves — produced varying degrees of"



1 L. Pasteur: Studies on Fermentation, trans, by F. Faulkner: 105, 1879.
"^Loc. cit.. Vol. I: 536.
^ Osterhout : loc. cit.
* Loc. cit., 421.

50



enlargement, accompanied by disorganization of the protoplasm and walls.
The mustard produced starvation and also disorganization of the proto-
plasm, as indicated by the stunted and distorted hyphae, the swollen and
non-hyahne appearance of the protoplasm, and the tendency in the Peni-
cillium to form fruiting heads almost directly from the hyphae developed
from the germinated spore. The hyphae formed matted tufts, difficult to
separate, and with blunt distorted fruiting heads. A peculiarity of the
mustard was that the active principle causing the abnormal growth pro-
duced the effect when present in minute quantities, as in the 0.1 per cent
solution of the ground mustard and the 1 per cent of seeds. Stevens^
working on P. crustaceum states that "peculiar knotted or twisted hyphae
frequently result from the attempt to grow in a poisonous solution."
Clark used P. glaucum and found that the effect of deleterious agents on the
mycelium was "varied and often characteristic."

The ordinary acids — ^citric, lactic, and malic — which are present in
fruits and vegetables, caused only a slight retardation in development
when as large an amotmt as 6 per cent was used, which is more than occurs
in fruits other than the lemon. More than this amount apparently checked
metabolism, as shown in the reduced and impoverished appearing proto-
plasm. The tartaric acid was much stronger, the 4 per cent solution, while
only slightly retarding development, caused stunting, lesser amounts caus-
ing swelling and disorganization. The other organic acids — ^acetic, benzoic,
boric, butyric, and salicylic — and their sodium salts caused a complete
disorganization of both the protoplasm and walls. There was a de-
cided retardation of development by even small amounts, and in some of
them a yellowing or browning of the protoplasm. In the extreme stages
the hyphae were much swollen, and filled with a coarsely granular, non-
hyaline mass, showing no distinction of ectoplast, and on rupture, spreading
as a disintegrated mass.

The salts of the metals produced much the same results, though not so
extreme in either swelling or disorganization, and with amounts that
were not so close to the inhibitive points. When the larger amounts were
used, and gei-mination much delayed, stunted, curled colonies were devel-
oped with attenuated hyphae, and apparently coagulated protoplasm.
Similar changes resulted with the lesser amounts when germination was
delayed by low temperature.

Swelling by the lesser amounts, and stunting by the greater were also
produced by carbolic and the mineral acids, creosote, mercuric chlorid, and
the alkaloids. Mercuric chlorid and the alkaloids caused disorganization
of the protoplasm, shown at first by the full, granular, non-hyaline appear-
ance with no distinction of limiting membrane and plasma, followed by the
reduction of the plasma to a thin layer, containing few granules.

The alcohol retarded development, producing swelling, accompanied
by distortion and hardening; in some cases a shrinking without distortion
occurred, and in others disorganization. ^

There were also some peculiar effects, such as the formation of fruiting
heads in the Penicillium, formed directly from the primary hyphae and in
the Alternaria a long chain of conidia, the formation of an abnormally large



'■ Botanical Gazette, Dec, 1898.

^ Dujardin — Beaumetz and x^udige: Experimental researches on the toxic power
of the alcohols. 1879.

51



number of septa, the formation of giant cells, and colonies that bore no
resemblance to the ordinar\' development. In some of the badly distorted
colonies which were firm and difficult to separate, and were dry masses on the
surface of the solution, after a time drops of brownish liquid appeared in the
depressions, though the solution was pale amber.

In estimating the effect of the respective chemicals upon the living cell
or proto]:)last, it is necessary to consider the condition, or vital activity,
of the organism, as well as the external conditions which act as indirect
agents in inducing changes. It could not be determined how far the
chemicals used were able to penetrate the cell substance in their original
condition, since plants absorb all^ substances which are in solution whether
useful or not, though some of these may penetrate only through the cell
wall, and not into the living plasma. This is true of some salts which plas-


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