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S'FUEZ‘iES ON THE MORPHOLCQECAL
AND E’Z‘E‘t’flOLOGiCAL
CHAEQACTERS OF A NEW SPECEES
OF THE GENUS PAECSLOM‘KCES

Tim-sis {0: fits Degree cf M; S.
MiCHEGAN STATE CGLLEGE
Hsie, Ian-Yak

E947

I'HIEIS

Llrtl .‘ II

Thisistocertifgthatthe

thesis entitled

Studies on the Norphological and Physiological
Characters of a; New Quotes of the Genus Paecil-

omyee 3.
presented by

Hsie J en-yah

has been accepted towards fulfillment
of the requirements for

Me 80 degree in heteriology

 

STUDIES ON THE MORPHOLOGICAL AND
PHYSIOLOGICAL CHARACTERS OF A
NEW SPECIES OF THE GENUS
PAECILOMYCES

By
HSIE, JEN-YAH

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirement
for the degree of

MASTER OF SCIENCE

Department of Bacteriology and Public Health

1947

TH S1

(

CONTENTS

Page

I. Introduction ----------- ; .............. ---- 1
II. Review of Literature ------- — ............. - 5
III. Materials and Preparations -------------- -- 12
IV. Morphological Characteristics - ------------ 17
V. Physiological Characteristics ------------- 52
(A) Biochemical Reactions ---------------- 52

(B) Thermal Death Point in Moist Heat ---- 54

(C) Thermal Death Point in Dry Heat ------ 35
(D) Resistance to Ultraviolet Light ----- - 57
(E) Resistance to Desiccation -d---------- 58
(F) Antibiotic Tests --------------------- 59

(G) Determination of Optimum Temperature - 40

VI. Discussion - -------------------- ----------- 42
VII. Summary ------- - ------------- - ........ ----- 47
VIII. Acknowledgement ----------------------- ----- 49
IX. References ----------------------------- --- 50

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- 1 -

INTRODUCTION

This organism was obtained from the stock rats by
Dr. Margaret A. Ohlson and Mrs. Annanell C. Jubb of
the Department of Home Economics of Michigan State
College in East Lansing, Michigan. They had made the

following observations:

"This organism was observed in blood smears from
the stock rats. A later check of all stock and exper-
imental animals revealed that 50-75 % of these animals
were infected. Ingredients in the diet were examined mi-
croscopically with negative results. Old a1d non-fertile
stock animals were anesthetized with chloroform and or-
gans and blood cultured on maltose agar medium. The
organs were sectioned (1-6 microns) and stained with
Wright's stain. Sections made of tissues from other an-
imals were negative. A variety of organisms showed on
the culture plates, and those from the kidneys and spleen
were the only ones to take the stain as did the organisms
in the blood. The growth on the plates was transferred
several times with.no results. Maltose agar medium was
used throughout. The tubes were left for nearly two
months, and were examined before they were discarded.
Subcultures of organisms from kidneys were of the size,

shape md color of those from the blood. These were

transferred to sugar broth tubes and from these to corn-
meal agar medium. Broth tubes produced growth of or-
ganisms with the color and shape of those found in the
blood, but they were slightly larger. Also, a mycelial
mass, which was tough and difficult to break, was found

on the surface of broth tube.

"Four rats from another laboratory (Sprague-Dawley)
were inoculated intraperitoneally with blood containing
the organism from one of our rats. These rats were kept
separately, and the food, dishes, etc. were sterilized.
When the four injected rats failed to show the organism
in their blood they were reinjected with the pure cul-
ture. After four weeks the organism was found in the
blood. Five months later, it was not found in two of the
injected rats. The control rat in the stock colony room
had the organism when examined at the end of four weeks.
None of the rats manifested pathological manifestations."

The stock culture grown on cornmeal agar medium re-
ceived from Dr. Ohlson and Mrs. Jubb was transferred to
various media for morph010gica1 and physiological studies.
According to its colony characteristics, microscopical
features and physiOIOgical manifestations this organ-
ism.is classified as a new species of the genus

Paecilomyces.

 

-5-

REVIEW OF LITERATURE

A review of the literature reveals that a large

number of organisms with the characteristics of

Paecilomyces have been found, among which 14 species have

 

been identified. These vary in shade of color of colony,
color in the substratum; in floccosity; in the arrange-
ment of the sterigmata and the size of the conidia. No
one at present claims to know this genus well enough to
establish scientific lines of relationship among them.

In regard to the descriptions and figures Wh13h appear
in the literature, this structural type seems to be cos-
mopolitan and is described under the different generic

names Corolium, Spicaria, Penicillium, Paecilomyces,

 

 

 

 

and Eidamia e

In 1907 Bainier obtained an organism, Paecilomyces

 

varioti, and described the genus, translated and amend-

ed by Thom, as follows: "Genus related to Penicillium

 

and Aspergillus, distinguished by sterigmata, short-

 

tubular or more or less enlarged, tapering into long
conidium bearing tubes, mostly curved or bent slightly
away from the axes of the main sterigmatic cells; ster-
igmata variously arranged, partly in verticils and branch-
ing systems suggesting Penicillium, partly irregularly

 

arranged upon short branchlets, partly arising singly

- 4 -
along the fertile hyphae; conidia in chains, ellipti-

cal, never green."

In 1910 Thom recorded the cultural data of the
Penicillium divaricatum which should be classified as

 

a species of Paecilomyces as it was pointed out later by

 

the same author and summarized as follows: "Cultivated
in gelatin or bean agar, yellowish brown (avellaneous)
never green, broadly spreading in the substratum; super-
ficial growth consisting mostly of trailing fertile
hyphae, becoming powdery in appearance when mature; re-
verse of colony not discolored; fertile hyphae septate,
usually short, mostly creeping; conidial fructification
either terminal or on short branches of creeping or
partially erected hyphae, consisting of separate ster-
igmatic cells, of verticils, or of series of verticils
of branchlets and sterigmata irregularly distributed
along the fertile hyphae; sterigmata 15 to 20 by 5 mi-
crons, with long acuminate tubes usually bent from the
axis of the cell and broadly divergent at the apices,
bearing long chains of conidia; conidia elliptical or
fusiform, 5 to 7 by 2.5 to 5 microns, yellowish to brown-
ish, swelling in germination to 10 microns and producing=
2 or more tubes; does not liquefy gelatin; litmus re-

action alkaline."

-5-

In 1912 Sepp reported an organism, Corollium

 

dermatophagum, found first upon rotten leather and later
common upon miscellaneous substrate in the laboratory;
liquefies gelatin; colonies at first veil-like, sug-
gestive of Penicillium aromaticum, show yellowish brown
or greenish yellow colors, later becoming dense and
wrinkled, with reverse dirty green; Optimum tempera-
ture for growth 58 to 40 degrees 0., maximum above 45
degrees C. Sopp also reported extensive experimentation
upon different substrata in which it produced malic

and citric acids, catalase, chymosin, pepsin, and
diastase, as well as small amounts of alcohol, ether

and other aromatic substances.

In 1913 Bainier and Sartory reported an organism
which colonises on licorice sticks, fruiting about 6
days, at first pale yellow then in deeper shades to
dark orgnge yellow; the penicillus is rarely terminal,
usually borne upon irregularly produced short branches
of creeping or ascending hyphae enlarging slightly from
base to apex and bearing single verticil of sterigmata
with their conidial chains, or more or less biverticil-
late by the develOpment of part or all of the sterig-
mata into secondary branches with their verticils of ster-
igmata; sterigmata 5 to 6 or more in the verticil, 15
to 20 by 7 to 9 microns; conidia variable in size mostly

6 to 8 by 3.5 to 4 microns, smooth, in chains, yellowish,

swelling to double size inggermination and sending out

several tubes. They named it Penicillium repandgm, but

 

according to Thom's opinion that the description men-

tioned above would place it among the Pgecilomngs

 

strains rather than the strictly pgpiggllate species.

In 1921 Saito reported an organism, Penicillium

 

mandshuricum, appearing on wine yeast generally as a

 

mass of olive green or olive yellow spores, occasion-
ally as aerial hyphae having a threadlike appearance,
in artificial media developing a thick membrane which
bears a large number of spherical and smooth chlamydo-
spores; stalks very tiny, 170 to 180 microns, irregular;
spore bearing branches one to three, irregular, each
beating a chain of spores; conidia smooth, ellipti-

cal to ovate, generally 6.5 by 4.5 microns, some as
great as 10 microns; Optimum temperature 56 degrees 0.;
gradually liquefies gelatin and starch media; secretes
as enzymes maltase, protease, peroxidase and catalase.
According to Saito this fungus bears a striking resem-

blance to Penicillium olivaceum, but may be separated

 

by the difference in their optimum temperature, the
latter preferring 23 to 25 degrees C. In the opinion
of Thom, his strain No. 4279 received as Penicillium

 

mandshuricum in 1918 from Saito is a strain near Paecil-

omyces varioti Bainier.

In the same year, Moesz reported an organism,

Spiggria fimet§£$§ found in horse dung in the field

 

at Teteny near Budapest. The Latin diagnosis translat-
ed by Thom is as follows: "Effused, rosy, powdery;
sterile hyphae creeping, branched, septate, about 5
microns in diameter; fertile hyphae suberect, septate,
irregularly branched; branches frequently bearing di-
chotomous branchlets; ultimate branchlets (sterigmata)
with apex acute producing conidia ellipsoid, hyaline,
smooth, 6.5 to 10 by 5 to 6 microns and in long chains."
Marchals (1921) reported an organism, Penicillium

 

flavum, found upon fruit, apples, pears, cherries, etc.,

in Belgium: colonies flavous rarely in age verging

toward ochraceous; conidiophores erect, simple or funi-
culose, septate, 145 to 190 by 4 to 5 microns, branched
above in 5 to 4 series; sterigmata frequently in 5', scarce-
ly attenuate at the apex, 15 to 19 by 2.5 to 3 microns;
conidia ovoid hyaline, smooth, frequently one to several,
guttulate, 4.2 to 6 by 5 to 4 microns.

Pollacci (1921) reported an organism, Paecilomyces

 

burci, isolated by G. Burti from an experimentally pro-
voked nodule. In Sabouraud's broth and the same broth
with peptone and glucose at 18 degrees C., colonies
cottony in 2 to 3 days with delicate, short sterigmata
bearing yellowish white, little conidia in chains,
avellaneous; mycelium, septate, creeping or ascending,

6 to 7 microns in diameters; conidiophores erect, sep-

- 8 -

tate, hyaline, 50 to 110 microns in length, bearing
small branches;'conidia globose, rarely elliptical,

pale fuliginous, 4 to 5 microns in diameter.

In 1925 Biourge described an organism, Paecilo-

myces aureo-cinnamomeum, colonies on wort gelatin yel-

 

lowish to fulvous, cinnamon, reverse at first yellow

then cinnamon brown; odor none; conidiophores 5 t0 4
microns in diameter; penicillus up to 60 microns long,
with.walls smooth; branches irregularly produced; met-
ulae 16 to 20 by 2 to 4 microns, irregular in number;
sterigmata single or paired, 13 to 30 by 1.5 to 2.5
microns, producing long slender tubes bent from the median
line; conidia elliptical varying greatly in size 2.4 to
4.5 by 2.4, to 6.4 by 5.6, and also 5.8 microns.

Shaposhnikow and Manteifel (1925) reported a lab-
oratory contaminant growing at 40 degrees C. under re-

duced oxygen tension to which the name Penicillium

 

arenarium was given on account of its sandy color. In

 

artificial media as undiluted wort, glucose with 5 to

5 % of peptone and media a>ntaining a total nitrogen up
to 0.2 % in contrast with the usual 0.05 to 0.08 %,
mycelium white, 15 microns in diameter, twining, sur-
face of colony olive sandy or sometimes greenish, even
rose, light yellow, dark olive green to blackish, dark
blue green in depressions between buckled areas; re-

verse characterless; conidiophores 150 by 5 to 15 microns,

-9-

on short sde branches of mycelium, having an indefinite
tree-like branching; metulae when present varying in
number, and bearing 2 to 4 sterigmata, separated by
septa from metulae, or entire structure non-septate(?);
sterigmata few sometimes borne directly on the stalk,

25 by 5 to 6 microns, or even 75 to 100 microns in length;
conidia in extremely long chains, lemon-shaped with dis-
tinctly pointed ends, large, 10 to 11 microns in diamet-
er. No perithecia were found. The Optimum tempera-
ture was 55 to 40 degrees C. In cultural experiments
this organism tolerated 0.5 % acetic acid, 12 % citric
acid and 20 % of tannin.

Horne and Williamson (1925) reported an organism,

gidamia catenulata, isolated from a seasoned specimen of

 

the heartwood of Quercus robus. The mycelium consists
of colorless septate branched hyphae, varying from 5 to
11 microns in width. These hyphae are usually thin-
walled, but thick-walled hyphae do occur when the fungus
is grown on certain media, such as potato glucose agar
or pure agar. Two kinds of spores are produced, hya-
line macrospores and conidia. The macrospores are borne
directly on the hyphae or at the end of a short branch,
or may be intercalery. They vary in size from 7.5 by
8.5 to 14 by 10 microns, or may be 18 microns in diamet-
er. These hyaline spores occur on all the culture media

employed when kept at 20 or 25 degrees 0.; they are less

- 10 -

numerous or absent at 50 degrees C. The optimum temp-
erature for growth proved to be 50 degrees 0., moderate
growth occurred at 25 degrees C., and least at 20 de-
grees C. This organism inoculated on Bramley's seedling
apples at 20 degrees C. for eight days showed a surface
growth of mycelium.and conidiOphores at the place of
inoculation, but no parasitism at the living cell occurr-
ed. Growth on seasoned pine, chestnut, beech and oak

was prolific, and penetration soon took place. In view
of their descriptions and the figures this Species is

better classified under the genus Paecilomyces rather

 

than Eidamia.

In 1927 Gilman and Abbot transferred some Species

of Paecilomyces such as P. divaricata t0 the genus

 

 

Spicaria and gave the following generic descriptions:
"ConidiOphores erect, septate, usually freely branched,
branching often in whorls but also irregular; each
branchlet bears a terminal fructification composed of

a verticil of divergent metulae on which are borne a
verticil of divergent phialides (sterigmata); heads
divergent and seldom penicillate; conidial chains usu-
ally long; conidia hyaline, round ovoid, elliptical, or

elongate."

In 1950 Kennelly and Grimes reported a new species,

Paecilomyces hibernicum, found during the examination of

 

moulds from butter. The characteristics are as follows:

- 11 -

The conidial fructifications are either terminal or on
short side branches of the main hyphae, and consist of
sterigmata variously arranged, either separate, in verti-
cils, or branching systems resembling a Penicillium head.
Sterigmata vary very much in the same culture; they may

be of the shape characteristic of the genus Paecilomyces,

 

or may be scarcely distinguishable from conidiophores;
again they may show a series of swellings and constric-
tions. Conidia are elliptical, 4 by 2.6 microns, at
first hyaline, later pink and densely powdering over the
mycelium. It partially liquefies gelatin, with evolu-

tion of gas and an acid reaction.

In 1941 Szilvinyi reported 157 species of Mucoraceae

 

and Fungi_Imperfecti isolated from soil 1950-1952 near

 

Lunz, Austria, and cultivated for 8 years. Among these

species two new Species of Paecilomyces, namely Paecilo-

 

myces austriacus and Paecilomyces sulflavus were listed

 

 

with descriptive notes and illustrated figures. The
conidiophores of the former are 15-20 microns in length;
sterigmata, 12-15 by 2-5 microns; conidia, 5 by 2 mi-
crons. The size of the sterigmata of the latter are
11.5-12.5 by 1-1.5 microns; the conidia, 1.1 by 2.5-

5.5 microns.

- 12 -

MATERIALS AND PREPARATIONS

Modified Sabouraud's Medium.

Sixty-five grams of Difco Bacto-Sabouraud Dextrose
agar was suspended in 1000 ml. of cold distilled water.
The medium was boiled for a minute in order to dissolve
it. The solution was then distributed into tubes (for
making slants) or flasks (for making plates), and steri-
lized in the autoclave for 20 minutes at 15 pounds pres-

sure (121 degrees C.). The final reaction of the medium

was about pH 5.6.

Sabouraud's Broth

Difco Bacto-Dextrose - ----------------- 40gms.
Difco Bacto-Peptone ------------------- 10 "
Distilled water - ------------------ ---- lOOOml.

Ten ml. of this solution was dispensed to each
tube. It was then autoclaved for 20 minutes at 15
pounds pressure (121 degrees C.). The final reaction

of this broth was about pH 5.6.

Czapek's Medium (modified by Dox & Thom)

 

Sucrose ------------------------------- 50.00 gms.
Sodium nitrate - -------------------- --- 2.00 "
DibaSic potassium phosphate - --------- - 1.00 "
Magnesium sulfate --------------------- 0.50 "
Potassium chloride - ------------------ - 0.50 "

Ferrous sulfate ...................... _ 0.01 H

- 15 -

Agar --------------------------------- - 15 gms.

Distilled water ---------------------- ~1000 ml.

The ingredients were melted in the autoclave and
then filtered through cotton. The solution was distri-
buted into tubes (for making slants) and flasks (for
making plates), and sterilized in the autoclave for 20

minutes at 15 pounds (121 degrees 0.).

Plain Nurient Broth.

 

Difco Bacto-peptone ------------------ -- 5.0 gms.
Difco Beef Extract -------------------- - 5.0 "
Sodium chloride - ---------------------- - 5.0 "
Distilled water -------------------- ---- 1000 ml.

. The ingredients were dissolved by heating, filtered,
adjusted to pH 7.4, tubed and then autoclaved for 20

minutes at 15 pounds (121 degrees C.).

Beef Extract Agar Medium

 

Difco Bacto-Beef Extract -------------- - 5.0 gms.
Difco Bacto-Peptone -------------------- 5.0 "
Sodium chloride --------------- -----—---..6.0 "
Agar -------- -------------------------- - 15.0 "
Distilled water - ---------------------- - 1000 ml.

The ingredients were dissolved in the autoclave, fil-
tered through absorbent cotton and brought back to vol-
ume, 1000 ml. It was then adjusted to pH 7.2 and steri-
lized in the autoclave for 20 minutes at 15 pounds (121

degrees C.).

- 14 -

Carbohydrate Fermentation Medium

 

Difco Bacto-Peptone --------------- -- 10.0 gms.
Sodium chloride - -------------------- 5.0 "
Distilled water - ------------ - ------- 1000 ml.

The solution was boiled for five minutes, adjusted
to pH 7.6 to 7.8, cooled, and filtered through paper.
Andrade's indicator-was added. Five tenths of the vari-
ous carbohydrates which are not easily decomposed by
autoclaving was added. In the case of heat labile car-
bohydrates such as maltose, the carbohydrate solution
was separately sterilized by filtration with Seitz fil-
ter, and then added to the sterile solution in Durham's
fermentation tubes to which Andrade's indicator had been
previously added. Ten ml. of the solution was dispensed
to each of Durham's fermentation tubes, and sterilized

by autoclaving.

Starch Agar Medium

Soluble starch ------------ ------- ----- 5.0 gms.
Difco Bacto-Beef Extract ---------- ---- 5.0 "
Peptone - -------------- ----- -------- ~-- 2.0 "
Dibasic potassium phosphate --- ----- ~-- 1.0 "
Sodium Chloride - -------------- ~------- 5.0 "

Distilled water ----------------------- 1000 ml.
The ingredients were dissolved in autoclave, filtered

through absorbent cotton, adjusted to pH 7.2 and then 15

--

- 15 -

m1. dispensed into each of the large test tubes.
They were sterilized in autoclave for 20 minutes at 15

pounds (121 degrees 0.).

Nutrient Gelatin Medium.

 

Difco Bacto-Nutnient Gelatin --—-- ----- -~ 128.0 gms.

Distilled water ------ ----------------- -- 1000. ml.

It was dissolved by warming to about 50 degrees C.
and then sterilized by autoclaving. The final pH of

the medium was about 6.8.

Carrot Slant Medium.

 

A cork borer of the same diameter as that of the
test tube was used to pierce the fresh carrot for making
carrot cylinders. Each of the carrot cylinders was
obliquely cut to make two carrot slants. A piece of
cotton was put at the bottom of the test tube and wetted
with distilled water. Then the carrot slant was put in-

to the test tube, plugged and sterilized in the autoclave.

Slide Cultures.

 

Two pieces of filter paper were put into the Petri
dish. A clean slide was put on the filter paper; a
cover glass, on the slide. Then it was sterilized in a
dry oven for 15 minutes at 180 degrees C. Two pieces of
paraffin were melted on the slide to make two parallel

lines. The distance of these two lines was of about the

- 15 -

length of the cover glass. When the melted paraffin was
about to harden the cover glass was mounted on it to
make a chamber of about 1/10 mm. depth. A tube of
sterile Modified Sabouraud's medium or Czapek's medium
was melted and cooled to about 48 degrees C. It was
then inoculated heavily with the conidia of the organism.
A capillary pipette was used to transfer a minute drop
of the suspension into one of the open sides between

the cover glass and the slide. Due to the capillary at-
traction the suspension spread between the slide and
cover glass. A few drops of sterile distilled water
were added to the filter paper in the Petri dish to keep

the moisture adequate.

- 17 -

MORPHOLOGICAL CHARACTERISTICS

Growth was studied in Sabouraud's broth, Modified
Sabouraud's agar medium, Czapek's medium, plain nutrient
broth, beef extract agar medium, carrot slant and slide

culture.

In Sabouraud's broth this organism formed much sub-
merged growth of loose texture and a superficial pale
yellowish film.¢f 2-5 mm. thickness after 48 hours' in-
cubation at 57 degrees C. The color of the superficial
films gradually turned to yellowish green, dirty green
and ultimately chocolate brown after keeping at room
temperature for two months. While in the plain nut-
rient broth, the superficial film was thinner and mani-
fested avellaneous color instead of green, dirty green
and chocolate brown. In both cases the films consist-
ed mostly of densely interwoven vegetative mycelium,
germinating spores and some aerial hyphae with very few
fruiting structures and conidia. The germinating spores
were round or pear-shaped with.well-marked granulated
contents. They varied in size from 6 by 7 to 15 by 15
microns. Occasionally the size may reach 17 microns in
diameter. The superficial film in Sabouraud's broth was
so densely interwoven with mycelium that it was very hard
to break. The width of these vegetative hyphae grown

in Sabouraud's broth were usually larger than those in

- 18 -

plain nutrient broth, the former varying from 5.5 to
6.5, the latter varying from 5.2 to 5.8 microns (figs.
9 & 10).

When it was transferred on Sabouraud's agar plate
and incubated at 57 degrees C. for 24 hours the giant
colonies were formed. The rapidly growing colonies
measured in average about 15 mm. in diameter after 24
hours’incubation at 57 degrees 0., thinly covered the
medium, and gradually became grayish green and powdery
due to the abundant production of conidia after keeping
at room temperature for three weeks. When grown at room
temperature for 48 hours the thin and minute avellaneous
colonies were the result, but slowly enlarged and turned
to yellowish green, greyish green, and powdery after

four weeks.

When grown on Czapek's medium at 57 degrees C. for
24 hours a scanty growth of tiny avellaneous colonies
were observed. After 72 hourS’incubation they turned to
green color also. If the cultures were kept at room
temperature from the beginning the pale yellowish colonies
gradually changed to an avellaneous, dirty green, and
ultimately chocolate brown color after keeping for about

a month.

When grown on beef extract nutrient agar medium the

colonies were thinner and smaller. The color of the

-19-

colonies was at first pale yellowish, then turned to
avellaneous, and ultimately pale brown after about a
month. The color has never been green, greyish green

or chocolate brown even after four months.

When grown on carrot slant for 24 hours at 57
degrees C. a green thick felt was usually formed. The
color gradually changed to greyish green, brownish green,
and ultimately chocolate brown after standing at room
temperature for two weeks. Ascospores have never been

found.

The vegetative mycelium was a complex network of
septate branching, thin-walled hyphae. The entire veg-
etative mass was submerged in the substratum. The sur-
face growth consisted of aerial hyphae arisen like the
stems of a field of wheat. The submerged mycelium was
usually massed within the first few milimeters below
the surface. The width of the vegetative hyphae varied

from 2.5 to 6 microns (fig. 5).

The superficial growth consisted of aerial mycelium
and conidial fructifications (figs. 1, 2, 4, 5, 6, 7, and
8). The former consisted of colorless septate branched
thin-walled hyphae varying from 5.5 to 8 microns in
width. The aerial hyphae erected or partially erected
or crept on the surface or looped partly below partly

above the surface of the medium. Conidial fructifica-

-20..

tions were either terminal or on Short branches on erect,
partially erect or creeping hyphae, consisting of sterig-
matic cells arisen from conidiOphores, their branches,
branchlets, or metulae as occasionally seen, or directly
from fertile hyphae. Many conidiophores arose perpendi-
cularly from the fertile hyphae. The sterigmata were
irregularly distributed singly, in couple (conidiophores
bearing dichotomous sterigmata) or in verticil. Their
size varied from 5.5 to 6.8 by 14 to 24 microns, some
with long tapering tubes bent from the axis of the ster-
igmatic cell and broadly divergent at the apices bearing
long chains of conidia. The conidia were elliptical,
varying in size from 5 to 4.5 by 4.5 to 7.4 microns.
Under certain conditions, for instance, in an old cul-
ture the conidia were not rarely disposed in groups:

the conidium at the end of a chain tilted over until

it rested sideways on the one below, and this process
continued until all the conidia were arranged in a

group near or at the tip of the sterigmata (figs. 6

and 8, a).

The color exhibited in cultures of this Species
was due to the innumerable colored conidia produced on
the fertile hyphae. The pigment of the conidia was
soluble in ether, leaving the liquid yellowish green,
avellaneous or pale brown in accordance with the orig-

inal color of the conidia; it was insoluble in chloro-

form, alcohol, benzene, and distilled water. When sus-
pended in alcohol, benzene, and distilled water the con-
idia sank slowly; while in chloroform, they rose slowly

to the surface, leaving the liquid uncolored.

 

 

 

Fig. 1
Slide Culture
Incubated at 57 degree C. after 20 hours.
Magnification: 100 times.
a. Aerial hyphae. b. Czapek's medium.
0. Fig. 2

 

- 25 -

 

 

 

 

Fig. 2
Slide Culture
Incubated at 57 degree 0. after 20 hours.
From Fig. l c.
Magnification: 450 times.
a. Conidiophore b. Sterigmata c.
tube of the sterigma d. Conidium

Tapering

 

- 24 -

 

 

Fig. 5
Slide Culture
Incubated at 22 degree C. after 40 hours

Magnification: 100 times
a. Vegetative mycelium b. Aerial hypha

c. Czapek's medium

 

 

-25..

 

Fig. 4
Slide Culture
Incubated at 22 degree 0. after 4 days.
a. Aerial hypha b. Fig. 5

 

 

 

Fig. 5
Slide Culture
From Fig. 4
Magnification: 450 times
a. Conidiophore
b. Sterigma
c. Conidium

 

 

 

Fig. 6
Slide Culture
Incubated at 22 degree 0. after 6 days.
Magnification: 100 times
a. A group of conidia b. Conidiophore
c. Dichotomous sterigmata

 

 

 

 

 

 

 

Fig. 7
Slide Culture
Incubated at 22 degree 0. after 7 days.
Magnification: 100 times
a. ConidiOphore b. Dichotomous sterigmata

c., d. Conidial chains.

 

-29..

 

 

 

F180 8
Slide Culture
Incubated at 22 degree 0. after 8 days.
Magnification: 100 times
a. A group of conidia gathering at the tip of
the sterigma
b. Sterigmata arranged in verticil

\" 0 '
Lemma e
‘3“. M“

 

 

 

 

F180 9
Broth Culture
Grown in Sabouraud's broth at 57 degree 0. after
48 hours. Ioox
a. Germinating Conidium b. Vegetative hyphae

-__
o

 

 

 

 

Fig. 10
Broth Culture
Grown in plain nutrient broth at 37 degree 0. after
48 hows o '09 X
Showing the interwoven vegetative mycelium.

PHYSIOLOGICAL CHARACTERISTICS

Biochemical Reactions

 

For testing the biochemical reactions of this or-
ganism a standard 100pful of 4 mm. diameter of the con-
idia was inoculated into each of the following fermen-
tation tubes. The nutrient gelatin medium and starch
medium were stabbed with the same organism. All of
the tubes were incubated at 37 degrees C. for five days
and the results recorded daily with the exception of
the starch medium culture which was tested after 5

days' incubation as shown in the following table:
Table 1.

Biochemical Reactions 22.2 New Species 23 Paecilomyces

 

 

 

 

m B333}; Incubation Period in Pm
l 2 3 4 5
Dextrose A A A A A
Maltose O O A A A
Sucrose O O 0 O O
Lactose O O O 0 O
Mannitol 0 O O O 0
Salicin O 0 O O O
Xylose O 0 O O O
Inositol 0 O O O 0

-55-

Sorbitol O O O O O
Arabinose O O O O O
Dulcitol O O 0 O O
Rhamnose O O 0 O 0
i292

Nutrient Gelatin - - - - -
Starch' +
Remark: A ' Acid. 0 I No acid or gas.

No liquefaction of gelatin.

* : Hydrolysis of starch.

As indicated in the above table no acid or gas was
produced on any of the carbohydrate broths except in
dextrose and maltose where a trace of acid was manifested.
In the case of dextrose the Andrade's indicator turned
to pale pink color after incubating for 24 hours 81d
then became deeper pink after two days; while in maltose,
the pink color did not appear until after the third day

of incubation.

No proteolytic action was manifested by this or-
ganism as indicated by the fact that gelatin was not

liquefied after five days' incubation.

After incubating at 57 degrees C. for five days a

-34-

few drops of Lugol's solution was added to the starch
medium. No blue color was observed, while the non-
inoculated control tubes developed distinct blue color.
It is assumed that the starch had been hydrolyzed furth-

er than the stage of red erythrin.

Thermal Death Point in Moist Heat

 

 

Procedure: Sternberg's method of determining the
thermal death point in moist heat was used throughout
this test.

Results: Table 2

Thermal Death Point of E_New §peci§gwg£ Paecilo-

 

 

myces in Moist Heat.

 

 

 

Exposurg_Time - 10 min. Incubation Period in Hours
I“. -—-.-- Set A 'TSeE—ITI'

Temperature 0 C. gig—”g 23 gi-‘g :73
52 ------------ + + + + + +
54 - -------- --- e + + + + +
56 ------------ + + + + + +
58 - ----------- + + + + + +
60 ----- - ----- - + + + + ¢ ¢
62 - ---------- - f + + + + +
64 - ----------- + + + + + +

Remark: + : Growth - - No growth

The results show that the thermal death point of
this organism in moist heat is 68 degrees C. for 10

minutes.

Thermal Death Point in Dry Heat

Procedure: The procedure used in this test was based
upon Dr. W. L. Mallmann's Laboratory Manual of Antiseptics
and Disinfectants. The 24-hour Caapek's medium slant
culture of the organism incubated at 37 degrees C. was
prepared. It was suspended with sterile physiological
saline. Eight sterile cover glasses and 16 strips of
sterile filter paper were dipped into the suspension of
organisms. These were dried rapidly and aseptically in
a sterile Petri dish. Into each of 8 sterile Petri
dishes were placed two strips of the inoculated filter
paper and one inoculated cover glass. The Petri dishes
were heated at the different temperatures as shown in
Table 3. The strips were then removed and dropped se-
parately into the tubes of Sabouraud's broth and plain

nutrient broth. Each one of the cover glasses was broken

- 55 -

into two peices and drOpped separately into the tubes
of Sabouraud's broth and plain nutrient broth. All of
these tubes were incubated at 57 degfiees C. for three
days and results were recorded at the intervals of 24

hours.

Results: Table 3.

The Thermal Death Point of a New Species of Paecilomyces

 

 

 

 

 

 

in Dry Heat
Exposure Time 10 min. Incubation Period in Hours
Sabourand s Broth ’PlaIfi”Nutrient Broth
Temperature 0C. Est A Set B Sat A Set B
24-13—72' 23"18_72' 21—18—72
'70 + + + 1- 1- + + 4- 1- + 1- 1»
80 + + + + + + + r + + + +
100 + + + + + 1- 4- -r + 1- 4- i
120 + + + ¢ + 1 o e + + o +
140 - + s - - + - - + - - +
160 - - - - - - - - - - - -
180 '- - - - - - - - - - - -
Control - - - - - - - - - - - -

Remark: Set A. Inoculated filter paper.
Set B. Inoculated cover glass.

+ : Growth. - = No growth.

The filter paper strips dipped in the organism sus-
pension and heated at 140 degrees C. for 10 minutes showed
a trace of growth after 48 hours' incubation in Sabouraud's

broth or after 72 hours' incubation in plain nutrient broth:

while the cover glasses, after 72 hours' incubation

in both kinds of broth. All of the filter paper strips
and cover glasses heated above 160 degrees C. for 10
minutes showed no trace of growth, therefore the thermal
death point of the organism in dry heat was estimated to
be approximately 160 degrees C. for 10 minutes.

One of the most important factors which affects the
thermal death point of an organism is the coagulation of
proteins. Proteins coagulate at lower temperature in the
presence of moisture: for instance, albumin coagulates at
56 degrees C. in the presence of 50 % of moisture, and at
160-170 degrees C. in the absence of moisture. This ac-
counts for the wide difference in the thermal death points

in dry and in moist heat.

Resistance to Ultraviolet Light

Procedure: Nine sterile Czapek's medium plates were
prepared. With a wax pencil, a 2-inch square on the bottfim
of each plate was marked. A sterile cotton swab was dipped
in the physiological saline suspension of the organism.
With the inoculated swab the 2-inch squares on all nine
plates were uniformly smeared, and then exposed directly
to the light of a quartz mercury lamp of llO-Watt at a
distance of four inches with the cover of the Petri dish
removed. The time of exposure was 103.80, 120, 180, 240,
500 and 600 seconds respectively. All these cultures were

incubated at 57 degrees C. for 48 hours.

- 58 -

Results: Table 4.

Ultraviolet Light Radiation Test.

 

Time of Radiation in Seconds. No. of Colonies after 48

 

Hours' Incubation.

 

10 --------------------------- numerous
50 - -------------------------- numerous
60 ----------------------- ---- numerous
120 --------------------------- 3,500
180 --------------------------- 2,408
240 - ------------ -é-- ---------- 800
500 -------e-- ---------------- - ' 146
600 --------------------------- 32

Control

No radiation ----------------------- numerous

The results show that this organism has marked re-
sistance to ultraviolet light radiation. When it was ex-
posed directly to a quartz mercury lamp of llO-Watt at a
distance of four inches even for 60 seconds no perceptible

decrease in number of colonies could be observed.

Resistance to Desiccation

 

Procedure: A physiological saline suspension of a
48-hour Czapek's medium culture of the organism was pre-
pared. About 200 strips of filter paper and cover glasses
were dipped in the suspension and dried in sterile Petri

dishes. At 24-hour intervals, two pieces of the inoculated

- 39 -

filter paper and the cover glass were planted in Sab-
ouraud's broth and incubated at 37 degrees C. for three

days to determine its viability.

Results: The organism remained alive after desiccation

for 40 days.

Antibiotic Test

 

Procedure: A sterile wire 100p was dipped into the
bacterial suspension of the test organism and rubbed back
and forth on the surface of an agar plate, making suc-
cessive streaks as close togeter as possible. A central
straight streak of the fungus suspension was made, cross-
ing the bacterial streaks. It was then incubated at 57
degrees C. for three days and the results were observed

at intervals of 24 hours.

Results: Some strains of Staphylococcus aureus,

 

Salmonella enteritidis, Salmonella paratyphi, Salmonella

 

pullorum, Salmonella typhimurium, Salmonella abortivo-

 

 

eguina, Salmonella choleraesuis and Salmonella Schott-
muelleri were used for antibiotic tests with this new

species of Paecilomyces. The results were all negative.

 

All of these test organisms were obtained from the
Department of Bacteriology and Public Health, Michigan

State College.

Igtermination of Optimum Temperature

Procedure: Eight plates and eight slants of hodified
Sabouraud's agar medium and beef extract medium were pre—
pared. A standard loopful of 4 mm. diameter of this
organism was inoculated on the surface of each tube and
plate. The duplicate slants and plates were incubated
at the different temperatures as shown in Table 5. The
growth was observed and recorded at intervals of 24 hours

for three days.

Results: Table 5.

Growth Rate at Different Temperatureg

 

 

 

 

 

Incubation Period in Hours. Temperature CC:
24 ' 4 22 so 57
”Hodified Sabouraud's Slant A - + ++ +++

" " " B - + ++ +++
" " Plate A - + ++ +7?
'1 n H B _ + +4 *7*
Beef Extract Agar Slant A - - + ++
ll “ s: n B _ _ + ++
" ” " Plate A - - + ++
I! u H n B _ _ + ++
48
'IIHOdified Sabouraud's Slant A - + r++ +++
" " " B - + +++ +++
" ” Plate A - + +++ +++
" " " B - + ++T +++
Beef Extract Agar Slant A - + +++ +7?
u " u n B _ + ++ +¢+
” " " Plate A - + ++ +++
u n n H B _ + 4* +++
72 '
——Modified Sabouraud's Slant A - f+ +++ ff?

" " " B - +1 s++ +++

" " Plate A - +¢ +++ +++
" " ” B - ++ +++ +++
Beef Extract Agar Slant A - ++ +++ +++
" " " " B - ++ +++ +++
" " " Plate A - + ++ r++
" " " " B - + ++ +++

Remark: .9 - No growth + = Slight growth

++: Fair growth +++ 3 Heavy growth

“

 

- 41 -

Abundant growth was observed on the modified
Sabouraud's medium after incubating at 57 degrees C. for
24 hours; moderate growth at 50 degrees C.; and slight

growth at 22 degrees C.

When transferred on beef extract medium this organism
showed more scanty growth, and no trace of growth could be
observed when incubated at 22 degrees C. until the second
day. At 4 degrees C. no trace of growth was detected in

any of the media after three days' incubation.

The results show that the Optimum temperature for the,
growth of this organism is about 37 degrees C. or higher.
No attempt has been made to determine its precise optimum

temperature.

DISCUSSION

In view of its morphological and physiological
characteristics this organism belongs to the class

Deuteromycetes, order Hyphomycetales (or Moniliales)

 

 

 

family Mucedinaceae (or Moniliaceag), genus Paecilo-

mZCGSQ
According to Vuillemin's classification which is

 

 

in general use by French mycolOgists, this organism

belongs to the class Fungi Imperfecti because of non-

 

sexual reproductive structure, order Conidiosporales

 

because of reproducing conidia, suborder Phialidineae

 

because of reproducing conidia borne upon phialides or
sterigmata. But according to Saccardo's classification
which is followed by most mycologists today, this organ-
ism belongs to the order Hyphomycetales, frequently call-

 

ed Moniliales, because of conidiOphores are produced

 

neither in pycnidia (SphaerOpsidales) nor upon acervuli

 

(Melanconiales) but are formed from superficial hyphae

 

over the entire surface of the colony; family Mucedinaceae,
because of mycelium is colorless in contrast to the

family Dematiaceae whose mycelium is dark or smoky such

 

as Hormondendrum, Cladosporum, Alternaria etc. It

 

 

should be classified under the genus Paecilomyces, be-
cause it is distinguishable from Penicillium by its

longer tubular sterigmata, many tubular processes being

- 43 -

bent away from the axis of the sterigmata, and by great-
er irregularity of the branching which is only in part
verticillate.

Up to the present time only 14 species, possessing

the characteristics of the genus Paecilomyces, have

 

been reported. Generally Speaking, very few studies on

the physiology of Paecilomyces have been carried out.

 

For convenient review the following table of morpholog-
ical and physiological comparisons of the various

species of Paecilomyc§§_is compiled. This organism is

 

distinguishable from the other known 14 species of

Paecilomyces by the size of sterigmata and conidia,

 

optimum temperature, and some biochemical reactions.
Since this organism showed no morpholOgical and

physiological characteristics in common with either one

of the known 14 species, it is therefore considered to

be a new species of Paecilomyces and the name Paecilo-

 

Eyces michiganesis is proposed. Because of lack of

 

evidence as to real natural relationship among these
species the names of these species in the following

table are arranged alphabetically.

 

Table 6
Morphological and Physiological Comparisons of
Various Species of Paecilomyces.

 

 

Strains Size of Forms of Size of Color of Optimum Biochemical
Sterigmata Conidia Conidia Conidia Tempera— Reactions
(Microns) - .7. r (hicrons) _____ ture(3§;) _

Penicillium 25 by 5-6 lemon- 10-11 35-40

aurenarium shaped

Shaposhnikov &
Manteifel (1923) ..

 

Paecilomyces 15-50 by ‘
aureo- 1.5-2.5 elliptical 2.4-4.5
cinnamomeum by 2.4 to g
(Biourge)Thom 6.4 by 3.6 . |
(1923) ___ m __ "w" 1,”- m

Paecilomyces 12—15 by oval 3 by 2
austriacus 2-5

Szilviny(194l)

Paecilomyces 10-12 in globose, 4-5(Thom: almost
burci length ellipsoi- 6-8 by colorless
Pollacci(l92l) dal 2 5

 

Eidamia l
catenulata 8

5 by elliptical 4- by pale cream, 30 hydrolyses
Horne & 2 5 yellow, olive, starch in-
Williamson avellaneous, verts su-
(1925) cinnamon crose decom-
brown, etc. poses protefll

and asparagin

Corollium 50-60 by oval 9-10 by 58-40 liquefies
dermatophagum 10-12 5-4 yellowish gelatin
Sopp (1912) A __brown

Penicillium elliptical
elegans '
Corda (1858) _¥

Spicaria ellipsoid 6
fimetaria 5-
Moesz(192l)<_

Penicillium 13-19 by ovoid 4.2-6 by
flavwm 2.5-3 3-4

El. & Em.

Marshal (1921)

Paecilomyces elliptical 4 by pink partially
hibernicum, 2.6 liquefies
Kennelley & gelatin
Grimes (1930)

Paecilomyces elliptical 6 5
mandshuricum to ovate 4 5 56 liquefies

Saito (1921) gelatin &
starch media

Paecilomyces 5.5-6.8 elliptical 5-4.5 yellow, 57 ferments

michiganensis by by yellowish green, dextrose &

(1947 n.sp.) 14-24 4.5-7.4 avellaneous maltose,
pale brown hydrolyzes

starch

Paecilomyces 15—20 by 5.5-4 by yellowish

repandum 7-9 6-8

Bainier &

Sartory (1913)

Paecilomyces 11.5-12.5 oval l
subflavus by 1-1.5 2
Szilvinyi

(1941)

Paecilomyces 15-20 by elliptical 5-7 by yellowish gelatin'

varioti 5 or 2.5-5 to brownish not lique-

Bainier (1907 fusiform fied
(Synonym:Pen -

Gillium divarication Thom) fl-~;

 

-45..

The so-called "macrospores" observed by Horne and
Williamson have never been found in the writerls culture
even those incubated at 22 degrees C. The above-men-
tioned authors indicated that when kept at 20 or 25 de-
grees 0. these hyaline macrospores occurred on all the
culture media employed. However, the term "macrospores"
can not be used as a definite determinative criterion,
because they are of transformed units, homologous to
sterigmatic cells and conidia under certain conditions

as demonstrated by Kits and Wai and other investigators.

That this organism can utilize inorganic instead of
organic nitrogen as the only source from which to build
protoplasm, as most of the molds do, is clearly indicat-
ed by the abundant growth on Czapek's medium.which is an
organic nitrogen free medium, 1.6., sodium nitrate is
the only source of nitrogen. Carbohydrate compounds
greatly enrich the growth of this organism, and pro-
bably contribute directly or indirectly to the produc-
tion of green pigment of the conidia as was the case
when grown on modified Sabouraud's and Czapek's media
in contrast to the avellaneous color develOped when
grown on beef extract medium.

Ordinarily this organism may be saprophytic, but
under certain conditions, 1.6., when it enters the ani-
mal body in sufficient numbers either through eXperi-

' mental injection or natural infection, it may exist for

- 46 -

certain periods of time, without causing pathological

manifestations.

SUWIA BY

(1) Up to the present time 14 species possessing

the characteristics of the genus Paecilomyces have been

 

reported in literature.

(2) The salient morphological features and some
physiological characteristics of a new species of Paecilo-

myces are described, and the name Paecilomyces michiganes-

 

$2 is proposed.

(5) The organism was found to produce acid but no
gas in dextrose and maltose broth and to hydrolyze
starch. It doe. not ferment sucrose, lactose, mannitol,
salicin, xylose, inositol, arabinose, dulcitol and

rhamnose nor does it liquefy gelatin.

(4) The thermal death point of this organism was
determined to be 68 degrees C. for 10 minutes in moist

heat and 160 degrees C. for 10 minutes in dry heat.

(5) Ultraviolet light radiation of llO-Watt quartz
mercury lamp at a distance of 4 inches for 60 seconds
failed to reduce the number of organisms; a small num-

ber of organisms survived an exposure of 600 seconds.

(6) This organism was viable after desiccation for

40 days.

(7) It manifested no antibiotic properties toward

Staphylococcus aureus and several salmonellae.

 

(8) The optimum temperature of this organism
seems to be about 57 degrees C. No attempt has been

made to determine its precise Optimum temperature.

(9) The pigment of the conidia is soluble in
ether, insoluble in chloroform, benzene, alcohol and

distilled water.

- 49 -

ACKNOWLEDGEHEN"

The writer is indebted to Dr. Henrik J. Stafseth
and Dr. E. Bessey for valuable advice and suggestions
during the period of this work and to Mrs. Betty H.
Vincent and Miss Lisa Neu for their constant kind
assistance. The writer also wishes to thank Dr. Mar-
garet A. Ohlson and Mrs. Annanell C. Jubb for having
allowed me to use a part of the record of their previous
work with this problem. Thanks are also given to Dr.
Phillip A. Hawkins for his help in taking one of the
microphotOgraphs. The writer is also grateful to Drs.
W. L. Mallmann and E. D. Devereux, Mrs. Vera Bleil and
Mr. L. Chilson for their essential help in one way or

another.

REFERENCES

Bainier, G. .
1907. Paecilomyces varioti Bainier. Bul. Soc. Mycol.
France 23:26-27.

Bainier, G., and Sartory,A.
1913. Penicillium repandum. Bul. Soc. Mycol. France
29:367.

Biourge, Ph.
1923. Les moississures du groupe Penicillium Link.
La Cellule 33: fasc. 1. pp.213-214.

Buchanan, E. & Buchanan, R.

1927. Bacteriology. Macmillan Co. N. Y.
Castellani, A. '
1928. Fungi and Fungus Disease. Amer. Med. Assoc.
Chicago 0

Corda, A. C. I.
1838. Icones Fungorum, Abbildungen der Pilze und

Schwamme Pregue Tomus 11, fig. 74, p.18.

Gilman, J. 0., and Abbot, E. V.
1927. A summary of the soil fungi. Iowa State Coll.

Jouro 5010 1(3) 3225-3450

Henrici, Q. T.
1930. Molds, Yeasts, and Actinomycetes. John Wiley &

Sons, N. Y.

Horne, A. S., and Williamson, H. S.
1923. The Morphology and physiology of the genus
Eidamia. Ann. Bot. 37(147):393-432.

Kennelly, V. C. E., and Grimes, M.
1930. Paecilomyces hibernicum-new species. Sci.
Proc. Roy. Dublin Soc. 19(40.47):513-516.

Marchal, E1 and Em.
1921. Penicillium flavum. Bul. Soc. Roy. Bot. Bel-
gique, T. 54(Ser. 2 T. IV) p. 129.

McCulloch, E. C.
1945. Disinfection and Sterilization. Lea & Febiger,
Philadelphia.

- 51 -

Moesz,
1921. Spicaria fimetaria. Botanikai Koslemenyek
19:58.

Pollacci, Dino.
1921. Haplogeaphium De Bella Marengo. Atti. lst.
Bot. Univ. Pavia, 2 ser., 18:125-129.

Saito, Kendo.
1921. Investigations of sorghum brandy, kaoliangchim
(Trans. title). South Manchuria Railway Co.,
Cintral Laboratory, Report No. 6:11-12.
(In Japanese)

Sartory, A.
1920-1923.Champignons Parasites de 1'Homme et des
Animaux

Shaposhnikov, V. and Manteifel, A. -

1923. A Morphological, physiological and biological
study of Penicillium arenarium n. sp., in con-
nection with citrus fermentation (trans. title)
Trans. Sci. Chem.-Pharmacéut. inst.(moscow)
5:1-64 (In Russian)

Sopp, Olav JohanaOlsen.

1912 Monographie der pilzgruppe Penicillium mit
besonderer Berucksichtigung der in Norwegen
gefundenen Arten. Videnskapselskapets Skrif-
ter. 1. Mat.-Naturv. Klasse 1912 No.11. pp.
208.

Sternberg, G. M.
1896. A textbook of Bacteriology. pp.150-151. W.
Wood & Co. N. Y.

Stevens, F. L.
1927. Plant Disease Fungi. Macmillan 00. N. Y.

Szilvinyi, A. V.
1941. Mikrobiologische Bodenuntersuchungen im Lunzer
Gebeit 111. Die Schimmelpilzflora. Zentralbl.

Thom, C.
1910. Penicillium divaricatum. U. S. Dept. Agri.
Bur. Anim, Ind. Bul. 118:72. .
1930 The Penicilia. Williams & Wilkins Co.,

Baltimore

Vuillemin, P.
1910. Materiaux pour une classification Rationelle
des Fungi Imperfecti. Compt. Rend. Acad. des
Sci. 150:882.

1912. Les Champignons. Essi de classification 0.
Doin et Fils, Paris. p.236.
1925. Classification Mormale, Classement Anxiliaire,

et Groupement Pratique des Champignons.
Compt. Rend. Acad. des Sci., 180:102.

Waksman, E. A.
1932. Principles of Soil Microbiology. Wilkins Co.
Baltimore.

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