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The Effect of Certain Agents



on the



Development of Some Moulds



by



K. G. BITTING, M. S.

Bacteriologist, Glass Container Association of America









The Effect of Certain Agents



on tne



th(



De\ elopment of Some Moulds



by



K. G. BITTING, M. S.

Bacteriologist^ Glass Container Association of America






'. c C C

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« X c u r t c






NOVEMBER T920

*JATlONAL CAPITAL PRESS, INC., WASHINGTON, D. C.



During the years in which the writer has been engaged in food research
work, various manufacturers have persistently raised the question of
how spices and other condimental agents may be used as preserva-
tives, and it has been taught frequently that these may be used for
such purposes. During this same period, many samples of food stuffs
were examined which were not sterilized, but which showed peculiar and
abnonnal growths of the organisms present. This led to a study of the
effects of various agents found naturally in, or frequently added, to foods,
and for further comparative work, other well-known substances were also
tested. The results presented represent a summary of studies covering
a period of about fifteen years.

The writer wishes to tender thanks and appreciation to Dr. J. C.
Arthur, mycologist, Purdue Experiment Station, and Dr. R. L. Emerson,
noted toxicologist, formerly assistant chief, Bm-eau of Chemistry, U. S.
Department of Agriculture.



R



433153



THE PHYSIOLOGICAL EFFECT OF VARIOUS AGENTS ON THE DEVELOP-
MENT OF PENICILLIUM EXPANSUM, ALTERNARIA SOLANI, AND

OIDIUM LACTIS

INTRODUCTION

It is well known that many chemicals, drugs, and ordinary household
substances possess preser\'ative ]3roperties to a greater or less degree,
depending to some extent upon how they are used. In order to determine
the physiological effect of some of these agents upon living matter, a series
of experiments was made, using three moulds, which are well known,
Penicilliuni expansum Link, Altcrnaria soLini (E. & M.) Jones and Grout,
and Oidiuni lactis Fresenius. In much of the work of this nature, the tests
have been made on either the low organisms, the bacteria, or the higher
forms of plants and animals, the results being necessarily confined to the
bare facts of retardation or inhibition of development for the former, as
they are too minute to note stiaictural changes other than those of form, and
for the latter arc confined to diagnostic features, as examination of the
cft'ect on the tissues is precluded during life. In the present work the
nature of the organisms selected permits of minute changes in structure
being seen during life as well as the more obvious changes in kind and degree-
of development. In the preliminary work, a yeast was used in conjunction
with the moulds, but the yeast development could not be determined in
turbid solutions nor on some solid media, nor could slight changes in the
microscopic structure Ije recorded readily by means of photomicrographs,
which were used throughout the work for permanent records and for com-
parison. As the elimination of this feature of the work precluded close
interpretation of the results, the work on the yeast was discontinued.

The records for the experiments are given as to the effect of the different
agents used in varying degrees of concentration, usually to the highest
point at which growth occurred. Following is a brief description of the
moulds grown under conditions which jjennit normal development.

Pcnicillmm expansum Link. — The Penicillium in developing forms a
white, felt-like mass, covering the medium on which it is growing. As
development proceeds, the mould changes to bluish green, due to conidial
development, then to olive green, finally to a darker, duller color. Thom^
states that, "The shades of color peculiar to each s]jecies under oft repeated
conditions are easily recognized and are quite reliable."

Alternaria solani (E. and M.) Jones and Grout. — Altcrnaria forms a
white abundant growth at first, which darkens later, due to the darkening
of the hyphal walls and the formation of the brown conidia. During the
early stages of development, the mycelium becomes of a greyish color, due
to the darkening of the reverse side of the mycelium, but later the whole
growth becomes very dark and under some conditions wholly black.



I Cultural Studies of vSpecies of Penieilliuni. Bull. 188, B. A. L, Dept. of Agr ,
16, 1910.

5



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Oidiiim lactis Fresenius. — The Oidium differs from the PenicilHum and
Alternaria in that it remains white throughout its development. During
the early stages of growth, it remains submerged in liquid media, then thin
white patches of film are formed, which gradually spread and thicken to
form a thick, wrinkled scum, that becomes powdery in appearance, as the
hyphae divide in forming the conidia.

The Pcnicillium was isolated from tomato pulp and also from a mild
tomato ketchup; the Alternaria from l^lighted tomatoes; and the Oidium
from mature tomatoes allowed to stand until they softened and rotted.
The Oidium also develoj^s frequently on ]ju1]j, whicli is allowed to stand
after ex]josure to the air.

Medium. — The medium selected was tomato bouillon as it was noted
that the three organisms grew luxuriantly u]jon tliis medium, and also upon
tomato gelatin, as w^ell as upon such tomato products, as pulp and puree.
Before deciding upon the use of this medium, other media tested were the
ordinary nutrient bouillon, gelatin, and agar, and diluted fruit juices, pea
bouillon, and wort. The tomato bouillon proved most satisfactory as it
was slightly acid, gave a seemingly nonnal develo]jment, and could be
obtained readily throughovit the year. The bouillon was made by adding
to a definite volume of cooked, ripe tomatoes an equal volume of water,
boiling for half an hour, then filtering. The filtrate is a clear, amber-
colored liquid, having an acidity of approximately 0.15 per cent, estimated
as acetic acid. In the preparation of the medium in the flasks, calendered
paper was, tied over the cotton plugs during the sterilization, and after inoc-
lation the paper caps were kept over the plugs to keep them free from dust,
and to prevent, to an extent, evaporation.

In the preliminary ex]Deriments both moist chambers and flask cultures
were used, but the results in the moist chambers were not unifonn, neither
was the action of the preservative so pronounced as in the flask cultures, due
undoubtedly to the small amount of solution which could be used, and the
exhaustion of the oxygen in the chamber, and since the latter factor alone
produces either an inhibition or an abnormal development, this method of
culture was abandoned.

Developmental Features. — The features noted in the devclopinent of the
organisms were those showing the influence of the agent tested on the
germination of the conidia, on vegetative development, and on reproduc-
tion, these being manifested in the period required for gcmiination, the
extent of growth in a definite period, the form of growth, the formation of
conidia, the time required for maturation, as evidenced by the chromatic
changes, and any irregularity in form, color, or extent. In addition to
these features, microscopic examination was made of the surface and sub-
merged growths to determine the effect on the protoplasm and cell-walls
and any irregularities of development. The microscopic examination
proved to be much the better method of noting the effects of the agents, for
in many cases irregularities were present which were not a]3parent to the
naked eye, and which would pass unnoticed if only the macroscopic features
were examined.

In drawing conclusions as to the effect of any substance tested, the
visible changes taking place in the protoplasm have been considered in
conjunction with the gross developmental features.

6



Inoculations. — The material used for the inoculations was taken from
young cultures grown on the stock medium. The inoculations were made
with a platinum needle, having the tip hammered flat and smooth. This
was touched to the dry spores in the case of the Penicillium, then tapped on
the inside of the flask to dislodge any excess of spores, then touched to the
surface of the culture liquid, after which the flask was shaken vigorously to
distribute the spores. This method gave a fairly even seeding. With
the Alternaria the mycelium had to be used for seeding, the smallest por-
tions possible being cut with the edge of the flattened needle. While the
inoculating j^ortion varied in size, the resulting germination gave fairly
unifonn results. The Oidium, like the Penicillium, permitted even seeding,
the needle being touched lightly to the powdery -surface.

Temperature. — As the number of cultures was large and, in many cases
where germination was retarded, had to be kept under observation for
months, they could not be cared for in an ordinary incubating oven, so were
placed on shelves in a large case that was protected from light and from
changes in temperature, the temperature being approximately 23'^C.

Agents. — Solutions of the agents under investigation are given in per-
centage terms throughout for uniformity, as gram-molecule solutions could
not be made of the spices and some of the other substances which were
tested. The chemicals used were chemically pure, the spices were tested
microscopically for purity, the salt and sugar were the household forms
without additional purification, and the drugs were obtained from a reliable
source.

Alany tests were made in order to determine the inhibiting point of the
agent, and when this was approximated, tests were made with solutions
varying by slight amounts from one another and in the case of the Alter-
naria inoculations made from cultures of different ages as this organism
showed some irregularities in germination. Many repetitions were made,
but only those made in consecutive order are recorded in this paper. The
same is true of the photographs, many being held as records which are not
shown.

Record of Results. — In recording the results, two sets of tables are used.
In the first table are given the number of days required for germination and
the macroscopic developmental features of the growths in the flasks, the
latter at intervals of twenty-four hours for the first five days. Where the
time required for germination exceeded five days, this part of the
record is necessarily blank. The second set of tables records the average
size of the conidia of the Penicillium after germination (in cases showing
extremes in size, two figures are given, the average of the smaller and that
of the larger) and the microscopical appearance. The descriptions here
are supplemented by the photographs, which should be examined with a
hand lens in order to compare advantageously the normal structure with
that induced by the various agents. The conidia of the Alternaria and
Oidium were not measured. Those of the Alternaria vary very consider-
ably in size (145-370^t X 16-18^1)^ during their development under normal
conditions and those of the Oidium vary both in size and shape, so that it
would be impossible to dififerentiate the extent of the variation due to the
agent.



' F. L. vStevens: The fungi which cause plant disease: 623, 1913.

7



Classification of Agents. — The substances used in the experiments were
classified into the following groups. In the grouping the fundamental
principle of chemical composition was eliminated in order to group sub-
stances which arc used for analogous purposes. The last two groups were
used for comparison.

1. The oldest and most generally u.sed preservatives: salt, sugar,
potassium nitrate.

2. The common spices and aromatics : allspice, celery, cinnamon, cloves,
curry,! garlic, ginger, mace, mustard, paprika, pepper — black and white-
cayenne pepper, sage.

3. vSome vegetable acids found in, or which are added to, foods : \-inegars —
cider, malt, distilled — acetic, butyric, citric, lactic, malic, tannic, and
tartaric acids.

4. Some preservatives which have been, and are, used in foods:
alcohol, benzoic acid, sodium benzoate, boric acid, borax, creosote, formal-
dehyde, formic acid, soditun formate, saccharin, salic\-lic acid, sodium
salicylate.

5. Some substances which have been classed as antiseptics, disinfec-
tants, or as having an injurious eftect upon living matter: alum, copper
.sulphate, ferrous sulphate, lead acetate, sodium sulphate, sodium sulphite,
•stannous oxide, stannous phosphate, stannous protochloride, stannous
;sulphate, zinc chloride.

6. Three mineral acids — hydrochloric, nitric, and sulphuric; and two
common alkalis — potassium and sodium hydroxide.

7. vSottte very active drugs and some poisons: atropin sulj^hate, cocain
hydrochlorate, morphin sulphate, strychnin nitrate, quinin sulphate,
mercuric chloride, carbolic and oxalic acids.

Preservatives. — In the conservation of food the object has been to retain
it in as nearly the original condition as possible, and in attaining this object
various substances have been used, some of them, like salt, since remote
antiquitv. Some of them are used also, as the salt is, not only to aid in the
preservation, but to enhance flavor. Though the use of preservatives
furnishes an easy method of conser\'ation, it is attended with objections,
since the effect on the consumer is not properly understood, nor how long
the preservative will he effccti\'e unless used in excessive amotmts.

FIRST GROUP

The members of this group are the oldest and best known food pre-
servatives, salt and sugar particularh' having been prepared and used
in very ancient times. ^ These have been, and are, used in the flavoring of
foods as well as in the drying, pickling, and preserving, both alone and
combined. The saltpeter has been used to partially restore the color of
meats which has been destroyed in the ])ickHng.



1 Though curry is a mixture of spices, it is vised in practically the same way as the
spices, and for this reason was included in the tests.

2 A. Rolet: Les Conserves de Fruits, 1912; H. AI. Robinson and C. H. Cribb. The
Law and Chemistry of Food and Drugs, 1895; C. H. Gill. British Manufacturing
Industries, 1876.



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Surface covered, olive.

Colonies curled, blue.

Tiny submerged colo-
nics.

Ring thick, surface
nearly covered, green
Thin ring, green.


Slightly less than 7

per cent.
Layer thickened, blue

spots.

Thicker, blue spot.

Surface covered, re-
verse and suljmerged
darkened.

Surface covered, thin,
reverse yellowish.

vSlight increase in size.


Ring % inch, green;
surface colonies.

Increased.


Small, surface
colonies.

Ring barely percep-
tible.


Covered, green ; many
submerged colonics.

Nearly covered, green.

Thin white layer.

Surface covered, colo-
nies united.

Surface greenish, re-
verse dark.

Surface greenish,
v/hite at edge.




Ring }/2 inch, older
part blue.

Few surface colonies.


Ring J4 inch wide,
edge blue.




Nearly covered, blue.

Enlarged, green

Surface nearly cov-
ered with tiny trans-
parent colonies.

Surface nearly cov-
ered with tiny trans-
parent colonies.

Number of sub-
merged colonies in-
creased.

Few tiny surface col.,
sub. increased.

Many tiny submerged
colonies.


Ring, blue spots,
many submerged
colonies.

Many tiny suljmerged
colonies.




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Ring 14 ini^'h. l^'ue,
small surface
colonies.

Thin ring, many sur-
face colonies, blue.

Few tiny submerged
colonies.

Many tiny surface
colonies.

Many surface and
submerged colonies. .

Many tiny submerged
colonies.





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One small submerged
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Surface colonies green-
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Surface slightly
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Surface covered, dry,

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Histological Features



Agents




Size of
germi-
nated








Characteristics of development




Per


conidia




Kind


cent


in M




Penicillium








Control




8.5


Hyphae somewhat irregular in outline near germi-






nated conidia, tapering tips; protoplasm homogene-








ous, many large round vacuoles.


Salt


5


7.6


Hyphae short, distorted, blunt tips; protoplasm




homogeneous, no vacuoles. As growt i becomes








older, development becomes more nearly normal.


vSalt


10


7.6


vSome hyphae have many septa, irregular branching,
many close together, blunt tips or some enlarged,
protoplasm apparently normal, many vacuoles.


Salt


15


7.6


Hyphae have many side branches with narrow angles
to main hyphae, so that all run in same general
direction; protoplasm homogeneous, many vacuoles;
fruiting heads normal in shape, but small.


Sugar


50


7.6


Hvphae shrunken, distorted; protoplasm homogene-








ous, vacuoles appear as pink spots, giving a beaded








appearance.


Potassium nitrate. .


5


7.6


Normal.


Potassium nitrate. .


10


6.7


Hvphae slightly thinner, otherwise normal.


Potassium nitrate.


30


7.6


Hyphae have short stumpy side branches, wall
irregular in outline; protoplasm homogeneous,
many vacuoles.


A Iternaria








Control






Hyphae regular appearance; protoplasm finely








granular.


Salt


5




Hyphae enlarged, thickened appearance, septation
increased; protoplasm normal.


Salt


10




Hvphae shrunken, rounded, distorted, septation de-




creased; protoplasm has coagulated appearance.


Salt


15




Hyphae ragged in appearance, septation decreased;








protoplasm granular.


Sugar


75




Normal size, attenuated appearance, septation in-








creased; protoplasm thin, many vacuoles.


Sugar


50




Hyphae shrunken, septation increased; protoplasm








attenuated, highly vacuolated.


Potassium nitrate..


10




Hyphae much matted, clean appearance; proto-
plasm homogeneous, few vacuoles.


Oidium








Control






Few hyphae, conidia mostly rectangular, protoplasm








finelv granular, large vacuoles.


Salt


5




No long hyphae, septation hastened, protoplasm
finely granular, few small vacuoles.








Salt


10




Hyphae and conidia somewhat shrunken, proto-
plasm homogeneous, large vacuoles.




Sugar


'^S




Septation delayed for 48 hours, protoplasm thin,
indistinct, large vacuoles.








Sugar


50




Hyphae swollen, irregular conidia distorted, proto-








plasm disorganized.


Potassium nitrate..


:>




Hyphae septated irregularly, conidia sciuare ends:
protoplasm granular, elongated vacuoles.


Potassium nitrate. .


10




Hyphae unseptated; protoplasm homogeneous, no
vacuoles.



11



The effect of the salt was to retard growth and to produce a stunted
development, the Oidium being more susceptible to its action than the other
two organisms, no development of the Oidium taking place in the 15 per
cent solution. The colonies of the other two organisms developed very
slowly and with irregular, curled edges. The sugar also retarded and pro-
duced stunted development, causing the Oidium to form a hard brownish
lump. The Penicillium and Alternaria developed thin surface mycelia,
and in the Penicillium particularly there was fonued a proportionatelv
larger amount of spores. In making the 66>^ per cent solution, it was
necessary to heat it over the direct flame in order to bring the sugar into
solution. When this was effected, it was sterilized for the same length of
time as were the others. Growth of the Penicillivtm in this solution was
delayed for so long a time, that the suga^: had formed a mass of crystals,
on which the growth appeared as tiny grey patches. No development
of the other organisms occurred in this strength solution. The salt caused
the hyphae to have a smooth, shining appearance, whereas, the sugar
caused an excessive vacuolation, the vacuoles being regular and promi-
nent, and with a pinkish tint, so that they gave the appearance of a
necklace to the ]iy]jliae.

The potassium nitrate was tested in low per cent solutions first, as it
was supposed from some of the literature^ and also its use in meat pickling,
that it had strong preservative ]3ropertics, but beyond a slight retardation,
the develojoment was apparently normal for the Penicillium, even in the
25 i)cr cent solution where crystals were separating. In the 30 per cent
solution a mass of crystals formed in the bottom of the flask, so that
a mount could not be made without them. In this solution, though the
development was retarded to a greater extent than in the 25 per cent
solution, the surface was covered with a fairly thick mycelium in 4 davs, so
that beyond retarding growth slightly, the nitrate was not one-half so
effective as a preservative as was sodium chloride.

In an article^ on the use of saltpeter in pickling meats, it is stated that
its effectiveness in practice is probably due to its conversion to nitrites
or nitric oxid, though usually its effectiveness is attributed to the osmotic
activity which it induces.

The effect produced on the organisms by the salt, sugar, and potassium
nitrate was merely plasmolysis. So far as could be judged by the appear-
ance under the microscope, and the after effects as evidenced by the
use of the cultures for inoculations into ordinary media, no permanent
injury restdtcd. It will be noted that in all the work the agent tested is not
alone, but in a balanced nutrient solution, which gives, when used alone,
a normal development of the organi.sms.

See plates 1-7. ■

SECOND GROUP

The members of this grou]3, comprise some of the spices and aromatic
substances of ordinary household use. These were first tested in the form
of infusions, made according to the method of the U. S. Pharmacopoeia,^



1 A. Herzen. Deut. Chem. Ges., Ber. 8:822.
^H. Schroeter, Pure products, 1908, 4:199.
^ U. S. Dispensatory, 19th Ed.:6.Sl.



12



and with Penicillium only. With the exception of allspice, cinnamon,
cloves, and mustard, the other aromatics had such slight antiscj^tic value
that they were tested by using them direct. The whole substances were
groiind, 5 grams of each placed in flasks and 10 c.c. of boiling tomato
bouillon added. On cooling, they were inoculated with the Penicillium.
Included with these were whole black and white mustard seeds, which in
household practice are often added to cider, and other home-made bever-
ages in order to preserve them.

The results from the use of the infusions and the whole ground aro-
matics are given in the table, the Alternaria and Oidium being used in the
infusions having the more pronounced antiseptic properties.







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Online LibraryKatherine Golden BittingThe effect of certain agents on the development of some moulds → online text (page 1 of 7)