STUDIES OF ENVIRONMENTAL FACTORS INFLUENCING
IN VITRO GROWTH OF IASID‘IOMYCE'EE5 AND THEIR

ELABORATION OF BIOLQGICALLY
ACTIVE SUBSTANCES

Thesls Ior I’I'ie Degree of DII. D.
MICHIGAN STATE UNIVERSITY

Joseph Aifreci Stevens
1957

 

THESIS

 
 

 

L I Z? R R Y
NIIL‘Ilig3n State
University

 
   

 

fr!

STUDIES OF ENVIRONMENTAL FACTORS INFLUENCING IN VITRO GROWTH OF
BASIDIOMYCETES AND THEIR ELABOBATION OF
BIOLOGICALLY ACTIVE SUBSTANCES

By

Joseph Alfred Stevens

A THESIS

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1957

"Mi/$7
39.50 $5

ACKNOWLEDGMENTS

The writer wishes to express his sincere appreciation
to Dr. E. H. Lucas for his help, interest, and direction
throughout this work, and especially for his encouragement
and patience which greatly facilitated its completion.

The author would also like to express his thanks to
Drs. C. L. San Clemente and E. S. Beneke for their valuable
advice, suggestions, and constructive criticism during the
course of this study. The author is also grateful to others
who assisted him during several phases of this work.

Appreciation also is due to the Division of Experi-
mental Chemotherapy, Sloan-Kettering Institute for Cancer
Research for their cooperation in the sarcoma 180 tests,
and their financial support.

The author is indebted to Mrs. Shirley M. Goodwin
for her assistance in.preparing this.manuscript.

Finally, the author desires to thank the United States
Public Health Service, National Institutes of Health, for

supplying the funds which supported the work done with
Boletus edulis and Collybia radicata var. furfuracea.

MtH—Ii—iWi—H

ii

VITA

Joseph Alfred Stevens
candidate for the degree of
Doctor of Philosophy

Final Exam: November 11, 1957.

Dissertation: Studies of environmental factors influencing in;vitro
growth of Basidiomycetes and their elaboration of
biologically active substances.

Outline of Studies:

Major subject: Microbiology
Minor subjects: Mycology, Parasitology

Biographical Items:
Born, January 3, 1927, Cleveland, Ohio
High School: Benedictine, Cleveland, Ohio, who-19%.

Undergraduate Studies: University of Dayton,
Dayton, Ohio, l9hh-l9h9, B. S.

Graduate Studies: Michigan State University,
East Lansing, Michigan,
19h9-l957. M. S. 1953.

Special Graduate Research
Assistantship, 1952-1955.

Affiliations: Society of the Sigma Xi, Society of
.American Bacteriologists, Mycological
Society of America, and the.American
Association for the Advancement of
Science.

iii

STUDIES OF ENVIRONMENTAL FACTORS INFLUENCING IN VITRO GROWTH OF
BASIDIOMYCETES AND THEIR ELABORATION OF
BIOLOGICALLY ACTIVE SUBSTANCES

By

Joseph Alfred Stevens

AN ABSTRACT

submitted to the School for Advanced Graduate Studies of
Michigan State University of.Agriculture and
Applied Science in.partial fulfillment of
the requirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

Year 1957

Approved __7

ABSTRACT

In l9h8, a program of screening Basidiomycetes for oncostatic
principles was started as a cooperative project between Michigan State
College and the Division of Experimental Chemotherapy, Sloan-Kettering
Institute for Cancer Research, New York City. The ultimate objective
of the screening program was to obtain cultures of organisms showing
oncostatic properties and to determine whether cultures of them would
produce the active material under laboratory conditions.

Dried Boletus gdgli§.z§£3 pinicola sporophores were the first to
demonstrate consistently a tumor-retarding activity. Attempts to secure
a culture of this organism proved fruitful following numerous platings
of Bavarian Soil on nutrient agar plates. Preparations made from this
organism,while grown under varying conditions,at times showed the
presence of'a tumor-retarding substance, but duplication of such re-
sults proved difficult, chiefly because of the slow growth-rate

Collybia radicata gag, furfuracea was the next organism studied.
various nutrilites, and carbon and nitrogen sources were studied as they
affected the growth of mycelium and the elaboration of the tumor-
retarding substances. Once again, several preparations made from this
organismand/or its filtrates demonstrated tumor-retarding principles,
but these results could not be duplicated consistently. The require-
ments of this organism with respect to the production of the tumor-
retarding principle could not be established with certainty despite the
fact that the requirements for ample mycelial growth were satisfied.

V

Calvatia maxima was the final organism investigated. A study of
various temperatures and growth periods disclosed that the tumor
inhibitor which was found in the sporophore of this organism could be
produced consistently and at will by means of laboratory cultures.

The results of the screening program are also reported.

vi

TABLE OF CONTENTS

CHAPTER Page
I INTRODUCTION.............................................. 1
General Review of Literature and History......;........ 2

II METHOD OF BIOLOGICAL ASSAY................................ 7
Experimental Details...................................

Interpremtions Of ASWSOOOOOOOOOO...OOOOIOOOOOOOOOOOO
Discussion of.Assay....................................

\OCDN

III INTRODUCTORY EXPERIMENTS: EXPERIMENTAL WORK WITH
MSIDIOMYCETE 288j....0...|00.00000000000000000000IOOOO 12

IV EXPERIMENTS WITH COLLYBIA RADICATA VAR. FURFURACEA........ 2h

Preliminary Experiments................................ 2h

V THE EFFECTS OF NUTRILITES UPON COLLYBIA RADICATA VAR.
WCMQOOOOOCOOCOOOOOOODOCOCOOOOOOOOOOCOOOO0.00.... 32

RWieWOfLiteratureIIOOOOOOO0.000000COOOOCO0.0.0.0.... 32
mermenmliOOOOOOOOOOOOIOOOOOOO0....COOIOOOOIOOOOIOOO 36

VI THE EFFECTS OF CARBON SOURCES UPON COLLYBIA RADICATA VAR.
REFURACMOOOCDOOOCODOOOOOIOIOOOOOOOOOOIOOOOOOOOOIO0.00 1‘2

Review of Literature................................... h2
mermentalOOOOOOODOOOOOOI..00...OOOO’IOOOOOOOOOOOOOOIO 50

VII SUM DETmMmA-TIONOOOOIOIOOO0.00....OOIOIOIOOOIOOOOOI..00 7h

ReViGWOf LiteratureIOOOOOOOUOOOOO0.0.0.000...0.0.0.... 7h.
mermanmJ-IOOOIOOOO0.0.0....OOOOOIOOOOOOO0.0.00...... 76

VIII TIE WT 0F NITROGEN SOURCEOOCCOOODOO0.000.000.00...... 81

RGVieWOf Literature.0.0.0.0000...0.000.000.0000...O... 81
mermenta100030000000000.9.0....ODIODOOOOOOOOOOOOOIOO 88

vii

 

 

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TABLE OF CONTENTS - Continued
CHAPTER Page

IX MISCELLANEOUS EXPERIMENTS WITH COLLYBIA RADICATA VAR.
mom #hobeOCOOOOOOOOOO0.0000...OOOCOOOOOIOOOCIII 101

X SCREENING OF BASIDIOMYCETE SPOROPHORES FOR TUMOR-RETARDING
PROPmTIBOCOOOOCCOOOCO0.0.0.000...00......DIOOOOOOOIIO 10h

XI EDCPERD‘IENTSWITH CALVATIAMAXIMAfi‘éIIZ...” ...... ..........109
XII DISCUSSION OFRESULITSOOCCOCCCOOOOOOIOOOOCOOD IIIIII 00...... 111‘

Experiments with Basidiomycete #288j................... 11L
Preliminary Experiments with Collybia radicata 1.93.. .
furfuracea #uoer. 11h
The Effects of Nutrilites upon Collybia radicata var.
mrfiiracea............................”Hun-.77... 115
The Effects of Carbon Sources upon Collybia radicata
gag. furfuracea..................................... 119
Sugar Determination.................................... 125
The Effects of Nitrogen Sources Upon Collyjoia radicata
3.7.9.1: furfuracea#hObf............................... 127
Miscellaneous Experiments with Collybia radicata 2g.
mrfuracealOOOQOOOOOOOOOIOOOOOOOOII0.0.0.0...0.0.0.0 128
Screeningfiof Basidionwcete Sporophores for Tumor-
Retarding Properties................................ 128
Experiments with Calvatia maxima#6h2.................. 129
myOOOOOO0.0000000000000000.0....0000000000000.0... 132

BBLIOMHYOOOOOOOOOOOOOIOOOOOOOOOCOCOIOODIOOOO...OOOOOOIOOIOOOO 13h

APPmDm.IOOOOIOOCOOOOOOOOOOOOOOOOOOOOOOO0.0......OOOOOOOOOOOIOO. m

viii

 

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77

I!

TABLE

1.

2.

7.

10.

LIST OF TABLES

Page

Boletus edulis, strain 288j, grown on cellulose sponge in
a poo: oz mEaIImA.0.000.000...OOOIOOOOOOOOOOOOO0.0.9.0...O

Elaboration of a tumor-retarding principle by Colgbia
radicata var ___. _fu_rfi1racea #h06f grown in shake ure at
room te‘weratureo.OOOOOOOOOOOOOOOOOOOO0.0.000 00.000.00.000.

 

The effects of nutrilites added to medium B on the growth
of Gown radicata var. flirfuracea #hOéf, shaken for 3).;

days at room temperature...................................

The influence of varying inocula and the addition of
rmtrilites added to medium E on the growth of Colgybia
radicata var. furfuracea #h06f, shaken for 11; days at room
temperature................................................

 

The effects of rmtrilites added to Lindeberg's nutrient
solution on the growth of Coleia radicata var. furfuracea
#h06f, shaken for 15 days a room temperatTfre..............

Carbon utilization by Collie-is. radicata var. furmracea
#h06f in relation to the tumor-m preperties o?
in vitro cultures, shaken for 11; days at room temperature. .

Effects of addition of carbon sources to medium B upon
Collfliia radicata 321;. mrfuracea #Ii06f in relation to the
tame ibiting properties of _i_n_. vitro cultures, shaken
for 17 days at room temperature............................

Mannose utilization by Colybia radicata var . furflu‘acea
#h06f in relation to the minor-1mg... prOperties of
in vitro cultures, shaken for 19 days at room temperature”

Mannose utilization by Collflia radicata var. furmracea
#I406f in relation to the hmor~m preperties of
in vitro cultures, shaken for 27 days at room temperature.

Significant depression of mouse sarcoma 180 after treat-
ment with filtrates of Colly’gia radicata var. furfuracea

 

23

25

38

I40

I41

52

60

69

72

73

LIST OF TABLES - Continued

TABLE Page

11. Hydrolysis of Trehalose....................................

12. Comparison of sugar content determined for A. campestris
with that of in vitro mycelium of C. radicata var.
mrmracea#thOOOOOOOOOOO0.0.0.0.:00000OD.0.00.0.0...I...

13. Moisture determination offiéc_c§mpestrig,...................

1h. Nitrogen utilization by Collybia radicata var. furfuracea
#h06f in relation to the tumor-inhibiting properties of
in;vitro cultures, shaken for 23 days at room temperature..

15. Effects of addition of nitrogen sources to medium B upon
Collybia radicata var. furfuracea.#h06f in relation to the
or- iEiting properties of in vitro cultures, shaken
for 20 days at room temperature............................

16. Significant depression.of mouse sarcoma 180 after treatment
with filtrates of Collzbia radicata var. furfuraceaj#h06f..

17. Results of mouse sarcoma 180 tests with extracts of
kaidiomceteSOO...OOOOOOCOOOOOOCOOOOOO0.00...I.0.0.0.0....

18. Results of mouse sarcoma 180 tests gith_ in‘vitro cultures
of Calvatia maxima #6h2 grown at 19C ......................

78

80
80

9O

95

100

105

112

LIST OF FIGURES

 

FIGURE Page
I Boletus edulis, strain 288j, grown for 7 days on medium.A
agarOOOCCOCCOOOOOOO....0...’.O.C......OOOIOOOCOOOOOCOCOO... 11‘
II Boletus edulis mutant strain 2883b grown for 1 day on
III—TM ,
medum-A amOCOIIOOCOOOOOO0.00.00.00.00...IOOOOOOOIOOOIOOI 2]-
III Diameter of seven-day-old mwcelial colonies of Collzbia
radicata var. furfuracea #h06f grown‘on medium A agar
p testOOOCOOOOOTOOO‘OO00.00.000.000.0......0.09.00.00.00... 28
IV Diameter of myoelial colonies of Calvatia maxima1¢6h2 grown
- on medium.A agar plates.................................... 110
V

Elaboration of a tumordretarding principle 18 shake

cultures of Calvatia maxima_#6h2 grown at 19 C. Determined
at the Division of Ekperimental Chemotherapy of the Sloan-
Kettering Institute for Cancer Research with mouse sarcoma

180....0......OOOOOOOOOOOOOOI.OOOODOOOIOIOOIOIOIOOOOOOJOOCU 113

xi

CHAPTER I
INTRODUCTION

Knowledge of tumor-retarding properties of plants and their
products is limited. It was not until recently that studies of plant
derivatives in relation to tumor inhibition have been made.

Tne primary purpose of this investigation was to elucidate a
portion of the nutritional requirements of Collybia radicata gag.
furfuracea and to study additional aspects of its physiology as they
affected the elaboration of an oncostatic principle in 23.129. . The
nutritional study included the characterization of essential nutrients
for growth in a semi-synthetic medium. This involved various nitrogen
sources, carbon sources, and nutrilites. Further physiological aspects
pertained to the study of conditions required for growth, pH optima,
and temperature optima, as they accompanied the production of the tumor-
retarding principle.

An ancillary activity in the course of this investigation was the
screening of extracts of Basidiomycete fruiting bodies for tumor-
retarding action; the results will be reported here in part.

The ultimate objective of this work is to provide a basis for
further investigation of the Basidiomycetes with the goal of establish-

ing means of producing, in vitro, further agents active against tumors.

General Review of Literature and History

Long before the age of antibiotics, certain investigators reported
the inhibition of living organisms by others. Brunel (1951) reviewed
antibiosis from the time of Pasteur to Fleming.

Following the advent of penicillin many investigators turned to
screening additional fungi for antibacterial substances. A few sought
out members of the higher fungi for their investigations. .Among these,
Wilkins and Harris (19111:) in England and later Wilkins (l9hS-l9h8)
screened the sporophore extracts and culture broths of 900 species for
activity against Escherichia ggli_and Micrococcus pyogenes 222: aggggs,
They reported that 35 Per cent of the Ascomycetes and 25 per cent of
the Basidiomycetes of the total investigated had some degree of anti—
‘bacterial activity. In addition, they noted that the compdsition of
the substrate, whether natural or artificial, was the most important
factor in producing an antibiotic. Mathieson (l9h6) reported similar
findings employing Basidiomycetes gathered in Victoria. Bose (19h5,
19h?) discussed the presence of antibacterial substances in Polypores.
Hollande (19h5), reported in France that clitocybine, an antibiotic
substance isolated from ClitocXQe‘gigantg§.z§£, candida, was active
against several bacteria, including MyCObacterium tuberculosis,
§g1mon§11a.typhosa and Brucella abortus. Furthermore, he reported that
positive therapeutic results were obtained employing guinea pigs
infected with H. tuberculosis. Contrary to these findings, Robbins,

Hervey and Kavanaugh (19h6) Observed activity by many Basidiomycetes

against .15.. pyogenes 233° w in x__r_i__t_1_'_q but none 3-2. Egg. In a subse-
quent paper, Hervey (19h?) mentioned that sometimes a negative corre-
lation existed between growth of the Basidiomycete and production of
the antibiotic principle.

Robbins (1953) reviewed the work accomplished by his c0dworkers
and himself during the seven previous years on the presence of anti—
bacterial substances in Basidiomycetes. This included papers by
Robbins gt g1. (19h7a, l9h7b), Anchel et a1. (191:8, 1950, and 1952)
and Kavanagh 2£.éls (19b9, 1950a, and 1950b). Melin gt_al, (19h?)
reported an antibiotic agent to be found in the substrate of cultures
of the genus Marasmius, while Dorey §t_§13 (1951) reported similar
findings concerning Coprinus guadrifidus.

Following these screening tests, studies of individual species
were made, and isolation of the various basidiomycetous antibiotics was
attempted. Nancy Atkinson (l9h6a, and 19h6b), following her screening
of 200 species, worked on two different substances she obtained from
Cortinarius rotundispprus and Psalliota xanthoderma. She found that
sporophore extracts of these two organisms were active against a wide
range of bacteria, that the activity of both extracts was unaffected by
the presence of 10 per cent serum, and the toxicity to animals was low.
Later (195h) she reported the attempted purification of the active
principles from aqueous sporophore extracts by paper chromatography.
The erratic results Obtained were proved to be due to exposure of the
extracts to daylight. She also reported that ultraviolet light

inactivated one of the anitbiotics, psalliotin.

.Anchel, Polatnick and Kavanaugh (l950),while working with Pgria_
tgggig, E, corticola and an unidentified Basidiomycete,isolated two
closely related, highly unsaturated and highly unstable antibiotic
substances from each of these organisms.

.Anchel (1952) characterized the antibiotic drosophilin A (produced
bw'DrosOthla substrata) as p-methoxy-tetrachlorophenol. In further
studies, Anchel gt .a__1_. (1955) compared its action with that of phygon,
spergon, pentachlorophenol, hydroquinone, and p-methoxyphenol in tests
against bacteria'and fungi. It was more effective against fungi than
bacteria, and the authors concluded that it was a fairly good anti-
fungal agent.

As was inevitable, the scope of the investigators broadened to
cover antifungal, antiviral, and anti-tumor agents. Utech and Johnson
(1950) screened growth products, expressed juices, or water extracts
of bacteria and fungi for their ability to inactivate tobacco mosaic
Virus. .All the Basidiomycetes tested were active. These workers
reported also that varying the cultural conditions caused a variation
in the production of the inactivators. They concluded that, generally
Speaking, the temperature at which the organism grew best gave maximum
PrOduction of the inactivators. Their attempts at chemical isolation
were rather unsuccessful.

Reilly and Stock (1951) reported that extracts from the culture
filtrates and myoelial pads of.A§pergillus fumigatus inhibited the
ETOWth of Crocker mouse sarcoma 180 in mice. .Additional work by .

Petenman, Hamilton and Reilly (1952) revealed the active portion to be

toxic and either a highly basic protein, or group of proteins, with
isoelectric points near pH 10.0.

Stock gt 2.1. (1919) reported that a material from cultures of
aspergilli had shown appreciable ability to inhibit Crocker mouse
sarcoma 180.

Stock gt. 2.1. (19514) reported that a crude filtrate from a culture
of a Strgptomces _p_. possessed potent anti-tumor activity. The active
substance was characterized as 0-diazoacety1-L-serine. The crystalline
antibiotic was found to be effective against sarcoma 180 in amounts as
low as 1-2 mg ./kg-../day. Ehrlich at al. (1951;) reported a correlation
between the inhibition of sarcome 180 and the inhibition of'a yeast,
ICLoeckera brevis, by azaserine. it .

Lb'fgren, Lfining, and Hedstrb’m (1951;) reported the isolation and
determination of the structure of nebularine from Clitocybe nebularis .
This substance had a very selective bacterial spectrum, inhibiting only

members of the genus Mobacterium. However, the investigators added

 

that in tissue culture tests, the substance retarded cell growth of
sarcoma 180. No effect on sarcoma 180 cells in mice was reported. The
cOutbound was characterized as 9-(D-ribofuranosyl)purine.

In 19h8, a program of screening members of the Basidiomycetes for
oncostatic principles was started as a cooperative project between
HiChigan State College and the Division of Experimental Chemotherapy,
Sloan Kettering Institute for Cancer Research, New York. The samples
were tested for activity against Crocker mouse sarcoma 180 in the manner

described by Stock and Rhoads (1919), Stock (1950), and Clarke (1959'

Among the fruiting body extracts screened (at that time), those of
dried Boletus eduli§_xa£, pinicola consistently demonstrated a tumor-
retarding activity. The ultimate objective of the screening program
was to obtain cultures of organisms showing oncostatic properties and
to determine whether cultures of them would produce the active principle
under laboratory conditions.

Spores of _1_3'.e_d_u_l_i‘s_ ya. pinicola were obtained from habitats in
Central Europe and attempts were made to germinate them on the medium
and in the manner suggested by Khudiakov and Vozniakovskaia (1951).
This was unsuccessful. Soil samples were then obtained from an area
‘where this organism fructified profusely. Following numerous platings,
an organism was isolated whose characteristics were not unlike those
attributed to g, EQEEEE by Melin (1921 and 1923). This culture was

designated 288j.

 

 

 

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CHAPTER II
METHOD OF BIOLOGICAL ASSAY
Experimental Details

The tumor-retarding activity of the prepared samples was determined
at the Division of Experimental Chemotherapy of the Sloan-Kettering
Institute for Cancer Research using the ;1_n_ 1133 assay method reported
by Stock and Rhoads (19149), Stock (1950), and Clarke (1955).

Preliminary to the inhibition test, the toxicity of the sample to
mice was-determined by administering a single dose of the sample at
various levels. The maximum tolerated level guided in selecting the
dOSage for the inhibition test.

Five female Swiss albino mice weighing 18-22 grams were used for
each bio-assay. Uniformly cut sections of Crocker mouse sarcoma 180
(ca. 5 mg. wet weight) were implanted subcutaneously by trocar in the
1‘light axillary region. Therapy was initiated 2).; hours after the im-
Pla-ntation and continued for seven successive days. The sample being
te‘S‘ted was usually injected into the peritoneal cavity. The injections
were administered twice daily. On the day following the last injection
each mouse was weighed and the change in weight from the day of implan-
ta’5101'1 was recorded. The weight change for each member of the group
was averaged and the resulting figure was considered representative for

that group. Tumor diameters were measured through the skin by means

of calipers. The largest diameter was selected for one measurement,
and an axis perpendicular to it was taken for a second measurement.
The two diameters were averaged for each mouse to obtain an average
tumor diameter. This parameter was averaged for the group and will be
referred to as the tumor diameter of the treated mice in future dis-
cussions.

A second group of five mice was employed as controls. To these was
administered an equivalent amount of physiological saline twice daily
for the seven day period.

The criterion for the determination of inhibition was based upon a
comparison between the development of the tumors in the treated animals
and those in the untreated (saline injected) mice. The weight change of the

treated animals was also compared with the weight change in the control group.
Interpretations of Assays

The results of the single-dose toxicity tests were reported as a number
Within the range of 0.5 and 5.0 (at 0.5 increments). The smaller number
represented extreme toxicity accompanying a single dose of 0.5 ml. and
5.0 represented non-toxicity accompanying a single dose of 5.0 ml.

Nmnbers between these two arbitrary limits represented gradationsof
tOXicity.

The weight change was reported as the ratio of the average weight

Change in the treated animals to the average weight change in the
°°ntr°l group .

The following arbitrary scale was employed in the inhibition test

for grading the effects of the samples for tumor-retarding activity:

Marked inhibition (+) ....

Good inhibition (i+) .....

Slight inhibition (i‘) ...

NO effeCt (-) 00.0.0009...

Questionable effect (?) ..

Tumor diameter of treated group
less than 25 per cent of the
value for the control group.

Tumor diameter of treated group
between 25 and 50 per cent of
the value for the control group.

Tumor diameter of treated group
between 50 and 75 per cent of the
value for the control group.

Tumor diameter of treated group
greater than 75 Per cent of the
value for the control group.

This designation was used when
three or more mice died during
the course of the inhibition
test. Most of the samples giving
a questionable effect were re-
tested at a greater dilution.

Discussion of Assay

According to Stock (l95h),sarcoma 180 was chosen as the assay tumor

because of its nearly 100 per cent transplantability, low regression rate,

rfipid growth, lack of specific host requirement and its apparent inter-

mediate sensitivity to several adverse agents.

Up to the time of his

report” approximately 9500 compounds and many natural products had been

Screened against this tumor, with 12 of these giving a (+) result. He

further reported that confidence in the assay arises from the fact that

all 12 of these compounds have shown benefits in the case of certain forms

°f human cancer .

10

On the other hand this assay had a few objectionable operational
feamres when it was employed as a standard laboratory tool for this
project. Although the actual assay ' ' only extends for a period
of seven days, additional time was required for toxicity studies, for
shipping the samples to New York City and for mailing the results back
to this laboratory. Under the most favorable circumstances, this
ammmts to a minimum time interval of three weeks. Such a time
interval between preparation, testing and receiving the results elimi-
nates the possibility of more samples being prepared in a given period.
It also introduces the possibility of the active principle being
destroyed or diminished owing to such factors as time and temperature .
variation between the preparation and the testing of a sample.

Toxicity manifestations create another objection. Certain
Preparations demonstrated a single-dose toxicity not normally associated
With the active principle. This necessitated the giving of a small
dose with the possibility of an ensuing negative result because of
dilution. The test, therefore, becomes merely a qualitative assay
showing only the presence or absence of an active principle. This
Situation is compensated by the knowledge of the tumor diameters.

Another objection arose when it became difficult to distinguish
between significant inhibition and normal biological variation. As a
result of this condition a "significant trend" will be reported in a
few cases, although sufficient activity did not exist for the sample to

be graded "positive." It would be very desirable to have an assay

which did not include the objections mentioned above. A chemical,

bacteriological or seed aesay may possibly contain the answer.

However, at present, no such generally approved method has been

devised .

ll

CHAPTER III

INTRODUCTORY EXPERIMENTS:
EXPERIMENTAL WORK WITH BASIDIOMYCETE 288j

The organism designated by the code number 288j was isolated late
in 1952 by sprinkling one of many soil samples received from Bavaria on
the surface of a nutrient agar plate. These soil samples were obtained
from habitats that normally gave rise exclusively to many Boletus 29.11.23
385;. pinicola fruiting bodies anmlally. The soil samples received were
collected at an elevation of approximately 2500 feet, latitude 119°,
longitude 130 east of Greenwich. The habitats are spruce-fir forests
with occasional pines and deciduous hardwoods.

The organism was isolated and I grew at 27°C. on a modification of
Czapek's (1902) medium, which henceforth will be called "medium A" .

The composition of medium A is. as follows:

MgSO, 0.5 gm-
KHzPO4 1.0 gm.

KCl 0.5 gm.

Feed, 0.01 gnu.
Difco Bacto-Peptone 5.0 gms.
Dextrose 15.0 gms.
Sucrose 15.0 gms.
Difco Yeast Ebctract 5.0 gms.
Difco Agar 15.0 gms.
Distilled water to 1000 ml.

pH 5.6

A "medium B" was also used which has the same composition as
medium A except that it lacks yeast extract. In addition, broth forms

01‘ media A and B were made by excluding agar from the formula-

12

l3

Microscopically, the hyphae of 288j showed clamp connections, one
of the identifying characteristics of Basidiomcetes. The culture
isolated grew slowly: a small (3.0 mm.) pea-sized inoculum grew to an
approximate size of 1.25 cm. in four weeks. Its appearance at first
was undistinguished. However, after two months, the organism's
appearance was that of a small mound, covered with short tapering tufts
(see Figure 1). These corresponded to the characteristic tufts seen
in the photographs of cultures of _B_. @113 grown by Melin (1921).
Because of the similarities of growth-rate and appearance, combined
with the knowledge concerning the source of the soil samples, the isolate
was considered to be a culture of 39921-33133: pinicola.

The late Dr. E. A. Bessey, for many years head of the botany.
department of this institution, was consulted several times and agreed
With this conclusion.

The next step was to attempt to grow this organism in greater
quantity and in less time. The solution became evident when 3 mm.’
Pieces of 288j were grown on the surface of nutrient agar in Petri
dishes; in moving the‘pieces of 288j into the desired position on the
plate, microscopically small fragnents gave rise to entire new colonies.
It followed logically that the organism should be broken up in a sterile
Waring Blendor, using sterile distilled water, in much the same manner
as described by Savage and Vander Brook (191:6) and also advocated by
Dorrell and Page (191;?) in order to obtain a shorter lag period and a
Closer Check of replicates. Burkholder and Sinnott (19145) stated that

this type of submerged shake culture produced mycelial pellets which

 

 

Figure I. Boletus edulis, strain 288j, grown
for 7 days on medium A agar (enlarged 7x).

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15

were either globous or irregular, hirsute or smooth, and either hollow
or solid depending on the size and type of inoculum.

The procedure .for preparing this 'inoculum‘ was standardized in
the following manner: Pea-sized pieces of 288j were grown on 12 slants
of medium A for three weeks. The slant demonstrating a colony size
closest to 15 mm. in diameter was chosen as most desirable for prepar-
ing the inoculum. The colony was removed aseptically from the agar
surface and blended under sterile conditions in a Waring Blendor (Monel
metal) with 50 ml. distilled water for two minutes. Ten ml. of this
inoculum was the amount generally employed to inoculate 125 m1. of
medium A broth in a 500-ml. Florence flask, or the surface .of a Petri
dish of solid medium A. At the end of eight days, the broth cultures
(which were aerated in the manner suggested by Kluyver and Perquin
(1933) by continuous agitation on a reciprocating shaking machine having
a stroke of 0.5 inches and giving 100 one-inch excursions per minute)
would appear to be 50 per cent filled with small spheres of the organ-
ism. In the case of Petri dishes inoculated with one ml. of the blended
SAltimension, the surface of the agar was generally covered entirely with
2883' chlium within 11; days.

After the organism 288j had grown in 125 m1. of medium A for six
days, the nycelia from two 500-ml. Florence flasks were separated from
the broth portions. The broth portions were mixed, and two 85 ml.
33-th8 were removed. One of the aliquants was sterilized employing
a ST (Hercules Filter Corporation) Seitz filter, and the other preserved by

adding 0.85 m1. of a one-per cent merthiolate solution. The treated

 

 

16

solutions were aseptically poured into sterile serum bottles, labelled,
and frozen before shipping. The mycelia, regardless of the amount,
were blended with 85 ml. of distilled water, Seitz filtered, and
bottled.

Forty-seven samples were prepared in this manner. However, a few
variations were attempted. The growth intervals extended from seven
to 2).; days, and one sample was blended with an ascorbic acid solution
(one per cent) rather than distilled water.

None of the samples produced the desired activity. From the first
group of samples, it was ascertained that there ms nothing to be
gained by sterilizing the samples by Seitz filtration rather. than by
adding merthiolate. Consequently, most of the samples were merthiolated.

Thirty-six of these 1;? samples comprised anexperiment to determine
the effect of incubation time upon the elaboration of the active
principle. A flask was removed from the shaking apparatus daily for
18 days, commencing with a seven-day growth period and terminating
With a 2h-day-growth-period culture. Each day a broth sample was
Prepared, and also a sample was made by blending the mcelium with 85
"L1. distilled water. As mentioned previously, when tested all the
results were negative. However, while the samples prepared from the
seven-, eight-, and twelve to twenty-four-day old cultures produced no
effect on the tumor, (i.e. the test and control tumors were approximately
the same size), the samples prepared from the nine-, ten-, and eleven-
dEty-old cultures had a tendency to depress the growth of the test tumor

(though not sufficiently to give a reading of i-). This tendency

....

1?

in.both the broth and mycelium-water samples later prompted the blend-
ing of the mycelium with the culture broth in an effort possibly to
concentrate the active principle rather than dilute it by the addition
of water.

Attempts were made to grow 288j in the liquid form of the synthetic
(chemically-defined) medium (code letter J and described in Appendix)
proposed by Khudiakov and Vozniakovskaia (1951) for growing four
Boletus species. The growth in this medium was exceptionally slow.

A growth period of 53 days was required to produce the same amount of
288j mycelium that was observed using medium A within eight days. This
sample, mycelium blended with broth, also produced a negative result.

At this time, 2883 was also grown in an altered version of medium A.
The nitrogen and carbon ratios (normal being 1:10) were adjusted to be
1:5, 1:20, and l:h0. Two sets of these media were inoculated. One set
was grown for 19 days and the second set for 83 days. The extracts
were prepared by blending the mycelium with the culture broth. However,
both sets were negative. The mycorrhizal relationships of many boleti
have been recognized by, among others, Pennington (1908), Melin (1921,
1923), Melin and Nilsson (1952); in addition Rennerfelt (1950) reported
about the growth of various fungi on sawdust. It was, therefore,
decided to investigate the effect of a onedper cent birch sawdust
extract, and a one-per cent fir and spruce sawdust extract. It was
theorized that possibly these wood extracts might contain a substance

‘dfiflh would support the elaboration of the oncostatic principle by the

18

organism, since species of this genus are known to grow in association
with these trees.

0n the first attempt, using 3.5 ml. of a one-per cent fir and
Spruce sawdust extract added to 125 ml. of liquid medium A, a 1‘- result was
obtained. Culture 288j was grown for 21 days in this medium, and the sample
we prepared by blending the rycelium with the culture medium. Dupli-
cations and variations (21 in all), however, failed to reproduce the
initial result. The symbiotic effect of organisms had been known for
some time. It was decided to grow 288j in combination with other
organisms isolated from the same soil because of the work reported by
Zeller and Schmitz (1919) and Porter (1932) on the stimulatory and
antagonistic effects which different microorganisms have upon fungi.
For this purpose, three Strgptomces species were chosen: #252(0)2,
#288k, and #311. These were inoculated singly, along with 288j, into
liquid medium A and grown for 21 days in the usual manner. The results
were all negative. It was then decided to investigate the growth of
28813 on solid media. Six Pyrex preparation dishes, containing 100 ml.
of medium A agar were inoculated in the manner previously described.
These were grown for 2b, 141;, 147, 51, 5b, and 62 days at 27°C. The samples of
the cultures of the first five growth periods were prepared in the following
manner: 1) the mycelial mat was removed and blended with 85 ml.
distilled water, filtered, sterilized and bottled, 2) the agar on which
the organism had grown was extracted using 170 ml. distilled water,
sterilized and bottled. The culture representing the longest growth

Period was made into one sample by blending the mycelial mat together

19

with the agar with 170 ml. distilled water; the preparation was
sterilized and bottled. All of these 11 samples produced negative
results.

In an experiment using positively ionized air (produced by a
"Wesix" ion generator suspended in a flask) above 288j growing on the
surface of agar one positive result was obtained. Two samples were
prepared as described above, one of the mycelium, and the second an
extract of the solid medium. The latter sample produced a i- effect.
These experiments were repeated later, but with negative results.

Sterile quartz sand in a Petri dish thoroughly moistened with
medium A broth was inoculated with a pea-sized piece of 288j mycelium,
and incubated at 27°C for 60 days. At the end of this time a sample
was prepared by extracting the mycelium with water. Although the Sloan-

Kettering method of evaluating tumor inhibitors showed a negative result,
a Significant retardation of the tumor was noted.

At this time, 288j was also grown on one-inch cubes of du Pont
cellulose sponge bathed in medium A broth in Pyrex "preparation dishes" .
These dishes were taped with one-inch masking tape to prevent contami-
nation and the escape of moisture, and incubated at 2700. The samples
were prepared by blending the mcelium with the culture broth. Three
samples, 8919, 8920, and 8921 (see Table l) were prepared from cultures
3mm in this manner for 60 .days. The appearance of the broth of
8amole 8919 was normal, i.e. golden-yellow. Sample 8920 was slightly
darkened, and sample “8921 was dark brown in color. All three of these
”“9193 produced a i-' effect. Two samples (8956 and 8957) which were

lnoculfitted at the same time as 8919 were prepared 30 days later, but

 

 

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produced negative results. One of these (8957) also demonstrated slight
pigment formation. Eight additional samples were prepared in an attempt
to reproduce the initial positive results with no avail. These covered
a growth period which ranged from 26 to 75 days.

Three test tubes, about two months after 2883' was first isolated,
showed rather unusual growth. The colonies in these tubes developed
long tapering tufts, quite unlike those ordinarily seen. Their color
was light peach, and their length approximately one centimeter. The
colonies grew rapidly upon isolation, and gave off an odor not unlike
that associated with Saccharonwces cerevisiae and other common yeasts.
All attempts to isolate or observe a contaminant from these cultures
ended negatively, and .to this day the cultures designated 288jb, 2881c,
and 288jd (see Figure 11.) grow as pure cultures. These cultures were
shown to the late Dr. E. A. Bessey, and after extensive observation he
concluded that, even though he observed structures similar to yeast
cells, the cultures were to be regarded as spontaneous mutants of 2883'.
Accordingly, seven samples were prepared employing 288jb grown in shake
cMllture and also on cellulose sponge cubes, but the desired effect was
lacking. Five samples incorporating growths of 288jc and 288,jd gave
Silnilar negative results.

The association of the yeast-like odor with the cultures of 2883b,
2883c, and 288jd recalled that Fries (191.1, and 19143) has shown that
Some Yeasts exerted a stimula'tive action on spore germination of
BaSidiomycetes. Eleven yeasts which were isolated from the Bavarian

soil that originally yielded culture 2883' were investigated for any

21

 

 

Figure II. Boletus edulis, mutant strain 2883b,
grown for 1 day on medium A agar tenlarged 3x).

22

stimulatory effect they might have upon this organism. The yeasts were
inoculated in the immediate vicinity of seven-day-old cultures of 2883‘,
but no stimlatory effect was observed. In addition, the eleven yeasts
were grown separately in medium A broth for four days at room temperature

on the shaker. None of the samples produced from these cultures gave

rise to a tumor-retarding principle.

23

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CHAPTER IV
EXPERDIENTS WITH COLLYBIA RADICATA VAR. FURFURACEA
Preliminary Experiments

Owing to the slow rate of growth of the organism 2883' and its
apparent inconsistency in producing the tumor-retarding principle, it
was decided to investigate _i__n_ 13.1332 cultures of Basidiomycetes which
grew more rapidly.

After we had obtained tumor retardation with water extracts of
fresh Collybia radicata gar. furfuracea sporophores, a flask containing
culture broth A was inoculated with a culture of this organism (code
designation #14061‘) and grown on a shaker at room temperature until the
entire flask was filled with mcelium (30 days). At the,end of this
Period, a sample was prepared (#9130) by blending the wcelium with the
Culture broth. The result (Table 2) indicated that the tumor-retarding
princiPle was produced _i}_1_ E1332 . A second sample was prepared in the
same Banner, this time employing a 21-day-old culture. This (#9158)
313° Produced a :5. result. A third sample was prepared, again in the
Same manner, but this time employing an. 8-day-old culture. This sample
(#91714) did not show the anti-tumor activity. A fourth sample was
Prepared, again in the same manner, this time using a 16-day-old culture.
This Samlple (#9179) produced the tumor-retarding principle. Assuming
that the nutritional requirements were adequately fulfilled, it was

2h

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26

decided to find out whether there was a time factor involved in the
production of the active principle.

Seven flasks of culture broth A were inoculated with 1406f and
grown on the shaker at room temperature. A flask was taken off the
shaker, and a sample prepared, at the end of 3, 6, 9, 16, 20, 23, and
27 days, respectively. None of these samples demonstrated the presence
of the active material.

A carrot broth was also investigated for its ability to
1) support the growth of h06f and 2) produce the active principle.

A sample produced from the mcelium and broth of said culture failed

to indicate the presence of any active material. What was especially
interesting about this sample was the fact that it was started from

the Same inoculum which produced the active principle in sample #9158
(Table 2). It can be seen that, even though the carrot medium supported
the growth of the organism h06f, it failed in some way to stimulate

the PrOduction of a tumor-retarding principle.

At this point it might be appropriate to explain the identification
0f Organism h06f. The sporophore was found in Sanford Woodland, part
Of the Michigan State University campus. It was 25 cm. high, the cap
light brown, velvety to semidviscid, and 10 cm. across. The spores
were White and measured 9-10 microns x 13.5-16.1 microns. It was
tentatilrely identified as Collybia longipes, even though Dr. E. A.
Bessey Pointed out that Q. longipes specimens are extremely rare in
Michigan. This first identification was based upon three observations:

1) the cap was brown and velvety, rather than the beige-light tan and

2?

viscid cap generally ascribed to g. radicata, 2) its height, and 3) the
size of the spores was intermediate between the two species, and
definitely closer to those of Q. longipes. However, Smith (191:9)

makes note of the variety furfuracea in listing the characteristics

of Q. radicata and these fit the specimen perfectly. A discussion
with Drs. Bessey and Smith concerning identical specimens convinced
the author that the proper designation should be 9. radicata 1'93:
figi‘uragea. Apparently, however, some Ehlr0pean mycologists have

circumvented this difficulty by placing the species radicata in the

genus Mucidula, while retaining the species longipes in the genus

00min .

Collybia radicata var. furiuracea #h06f grew at temperatures rang-
ing from 120 to 28°C., with the optimum temperature, range between 200
and 26°C. This was determined by placing 7.0mm. plugs on triplicate
medium A agar plates at temperatures of 12, 16, 20, 2h, 26, 26.5, 27,
and 28 degrees C. and recording the diameter of each colony. The
aVeraged resultant values are shown diagrammatically (Figure III).

A second specimen of g. radicata gag. furfuracea obtained from
the same area which yielded h06f and designated #h67. Three samples
were prepared using this new isolation but none were active.

Because of the stimulatory effects noted by Hawker (1936), Fries
(19143), Melin (191.6), and Oddoux (1953) when natural extracts are
incorporated into basal media, a medium was formulated which contained
53 such water extracts. To the dry ingredients of medium A, 10 ml. of

““311 extract was added, and the final volume brought up to one liter

28

 

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29

by the addition of distilled water. This medium was designated Super A.
The 53 extracts were prepared and chosen on the basis of previous
reports of their stimulatory action as well as some which were chosen
because of their natural associations or encounters with basidiomycetous
mcelia in the forest soils. They included 10 per cent solutions of
the following: wheat, barley, rye, lentil, white navy‘bean, and corn
seeds; grass, hay, nettle, fresh alfalfa and alfalfa hay; maple, elm,
and oak leaves, Douglas fir, spruce, and pine needles; plum, apple,
cherry, strawberry, red raspberry, peach, blueberry, and coconut fruits 3
coconut milk, spinach, asparagus, lettuce, corn, carrot, peas, and
toIIIELto vegetables; beef duodenum, liver, stomach lining, pancreas,
and hog kidney*; Biopar A, B, c, D, E, F”; dried skim milk, dried egg
yolk, corn steep water, dried yeast—strain G, Anheuser-Busch-, culture
media of bacterium #157, Alternavrvia 532. #175A, Penfiicillium a. #233K,
Afigerglus g2. #1458, and Fusarium £9: #BlhE.

Both h06f and 1167 were grown in Super A medium,but the samples
Prepared from these cultures gave no indication of any tumor-retarding
activity.

Since ionized air was found to have some apparent effect on the
Production of a tumor-retarding principle in a 2. 29.11.1133 culture
(Cf. p. 19), a similar attempt was made with culture h06f grown on the
Shaker in medium A, using positively as well as negatively ionized

air. No active principle was found in either instance.

‘_

* - .—
“Viobin Corporation, Monticello, Illinois.
Liver fractions, Armour and Company, Chicago, Illinois.

30

In trying to explain the inconsistent elaboration of the tumor-
retarding principle by 1406f, it was theorized that perhaps an accumu-
lation of toxic metabolic products occurred in the medium in which the

culture grew. The assumption was that these toxic products in the

Sample might interfere with the activity. In an attempt to test this

possibility, three 500-ml. Florence flasks were modified by attaching
an arm at a 15° angle at the base of the neck of each flask. At the
Point where the base cf the armature met the Florence flask, a circular
Stainless-steel screen was sealed into permanent position. The purpose
Of this arrangement was to allow decanting the broth and replacing it
With fresh sterile culture medium.

For the purposes of the experiment, the culture medium was changed
every three days. The decanted broths from the three flasks were com-
bined, andm85 ml. aliquant was tested. Thirteen successive culture
madia were prepared. At‘the end of the thirteenth growth period the
I'Weelium from the three flasks was removed and blended with 85 m1. of
the last culture broth. With the exception of the sample prepared at
the end, of the twelfth growth period, ‘all the broth samples were negative.
The broth sample of the twelfth growth period as well as the mcelial
extract made at the end of the thirteenth growth period produced a i”
I‘esult.

Owing to the inconsistency of the elaboration of the tumor-retard-
ing meterial, it was decided to investigate some of the other isolates

obtained from the same pileus. Originally seven separate isolates had

been obtained, and these were designated 1406a, 1406b, 11060, 1406‘5: 14069:

‘3

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31

1106f, and h06g. Work was started with h06f because it was the fastest
gowing culture. Its relatively speedier growth-rate is unexplained
since all seven isolations were from the same pileus. In checking the
six remaining isolates, 12 samples were prepared from these cultures
grown in four different media .* Only one of these, h06b (#9232 in
Table 2) produced a positive result. When this isolate was again gown
in the same manner, there was no indication of the presence of any
tumor-retarding material.

When the production of the tumor-retarding principle by h06f and
the other 1:06 isolates proved inconsistent, it was decided to investi-
gate other sources of the same and similar species. The Centraalbureau
voor Schimmelcultures Baarn (Holland) furnished cultures of Collybia
loggipes and Mucidula radicata (designated by our code numbers 151 and
1150 respectively). The nomenclature of organism #1450 (H. radicata) is
EUJ‘Opean, and corresponds to the American Collybia radicata. Both of
these cultures were grown on the shaker in medium A for 27 to hO days,
but no tumor-retarding principle was detected in any of the four
samples prepared from these cultures. In addition, organisms 1:50 and
1451— were gown in a malt extract broth for 68 days, and the latter in
m9dium J for the same period of time, but no tumor-retarding principle

Was detected in any of the samples prepared from these cultures.

‘

4%.

The four media employed (formulae in Appendix) were:
Medium A (Basal medium plus 0.5 per cent yeast extract)
Medium J . (Khudiakov's synthetic medium)

Medium G (Sodium-caseinate medium)

Medium B (Basal medium)

CHAPTER V

THE EFFECTS OF NUTRILITES UPON COLLYBIA RADICATA VAR. FURF‘URACEA

 

 

Review of Literature

The term "nutrilite" was coined by Williams (1928). In a later
paper, (19111), he discusses the development and defines the meaning of
the term. It refers to a number of substances present in yeast
extracts which were formerly known under various names. By definition
it is now meant to "include those organic substances which in minute
amounts function in a similar manner for other types of orgailisms as
the vitamins function in 9% nutrition."

Within the limits of this definition, we are able to include,
besides some of the vitamins, such compounds as the nucleic acids and
their derivatives.

It was in the same sense that Derrick (19149) employed this term.
“illiams (191.1) went on to say, "The term 'nutrilite' like the term
'Vitamin' is a convenient one for designating organic nutrients which
are effective in minute amounts, but the distinction between an ordinary
m"tl‘l’tent and a nutrilite is not, so far as we can see, a very funda-
mental one."

Some vitamins are known to play an important part in the growth
of bacteria, yeasts, fungi, higher plants, and animals. deitamins

nay be either essential for gow'th, stimulatory, or inhibitory.

32

33

Countless papers have appeared on this subject, but only the literature

dealing with members of the Basidiomycetes will be reviewed.

Fries (1938) reported that most members of the Polyppraceae require
thiamine for growth, and that biotin and inositol had no effect upon
this group. He also found that various bacterial culture extracts
apparently contained a growth-stimulating agent which exerted, when
used in very small quantities, a marked effect upon the growth of some
polypores. The substance was identified as being, at least partially,
aneurin (thiamine).

Rennerfelt (19%) while working with only a single member of this
group, m annosus, reported that it required thiamine for growth.

He stated that the addition of 0.1 gamna of thiamine per 50 ml. of
madium produced optimal growth. Yeast extract, he said, produced the
same yield.

Fries (l9h8) mentions that thiamine is indiSpensable for several
Basidionnroetes and that biotin is indispensable for some Basidionvcetes.

Melin and Lindeberg (1939) reported that Boletus elega__ns barely
gr 9" in a mineral-dextrose solution containing ammonium tartrate. The
afidition of thiamine to this basal medium increased growth seven-fold,
while the addition of yeast extract increased the growth ten-fold.

Melin and Nyman (1910), using the same basal medium, reported that
m 111th and g. variegatus would not grow in this medium unless
”118.1an was present. They also stated that g. piperatus grew slightly
in the medium and that a five-fold increase in growth was observed when

thiamine was added. Continuing with their work on boleti, they reported

3b

that 2' Earmlatus grew in the basal medium but addition of thiamine
brought about a noticeable increase in growth. g. viscidus would not
grow in the basal medium, and the addition of thiamine, although it
improved growth,was not adequate. In a, later study \l9hl),they found

that certain strains of Q. Earmlatus had an absolute demand for thiamine,
and other strains of the same organism, while able to grow without
thiamine, are strongly stimulated by it.

Melin and Norkrans (l9h2) generalized that the mycorrhiza-forming
fungi (e.g. boleti) require thiamine for growth. However, one of the
Wcorrhizal fungi they studied was inhibited by thiamine and its
moieties, especially pyrimidine. This organism, they suggested, manu-
factures enough thiamine for its own use, and any excess is inhibitory.

Treschow ( 19%) reported that both the white and brown strains of
the cultivated mushroom Psalliota bispora* required either thiamine or
biotin for growth in synthetic media. .

Lindeberg (l9hl) stated that Marasmius androsgeus would not grow
in a synthetic medium containing thiamine unless biotin was added. He
confirmed this in a later paper (1914b) and also reported that thirteen
Species of Marasmius tested were heterotrophic for thiamine. In the
same paper he reported that some strains of 1:11.. perforans had an
absolute demand for thiamine while other strains of £1. aforans were

able to grow without thiamine, but were strongly stimulated by its
addition.

—*

t-
Treschow's terminology for Aflicus cmestris.

35

Leonian and Lilly (1938) found that Collypia tuberosa would not
grow on a mineral-dextrose medium containing ammonium nitrate and two
per cent agar. Good growth was obtained upon the addition of thiamine
or yeast extract to the agar medium. Growth would also occur when
amino acids plus thiamine were added to the basal medium.

Marczynski (l9h3) reported that when thiamine was added to his
vitamin-free basal medium, the growth of Collzbia velutipes would in-
crease four hundred per cent (figured by dry weight). Biotin, in large
doses, was also highly effective, while riboflavine and pyridoxine
were ineffective. He also reported that yeast and malt extracts were
more active in small amounts (5 mg: ../26 m1. of medium) than a combi-
nation of thiamine, biotin, pyridoxine and riboflavine. He suggested
that these results indicated the presence (in the extracts) of a factor
or factors other than the vitamins investigated, as yeast extract
caused a 7500 per cent increase in dry weight. Marczynski theorizes
that the results obtained through the use of relatively large doses of
crude products such as yeast extract, malt extract, and Bacto-Peptone
Suggest the action of assimilable nitrogen. He also points out that
Shall amounts of agar result in an increase in the production of dry
matter, possibly through its physical rather than chemical properties.

Lindeberg (l9h6a) working with three strains of Collybia dgophila
3330M that they would only grow in synthetic media in the presence of
b°th thiamine and biotin, and that they were absolutely unable to
Synthesize biotin. In another paper (19h6b) he reported that thiamine

“mated the growth of three species of Collybia: 1) ambusta,

36

2) bumacea, and 3) velutipes. Fifty micrograms of thiamine per liter
was all that was necessary to increase the dry weight from 1-2 grams
(control flasks) to 30-100 grams.

Hawker (191th), in a review article on the effect which vitamins have
upon fungi, reported that, while some Basidiomycetes require thiamine or
biotin, none was found to require inositol, pyridoxine, or pantothenic
acid. In conclusion, Lilly and Barnett (1951) reported that a fimgus
may either. require vitamins for growth, synthesize them, or else not
require them at all.

After reviewing the cited literature, judging from the scOpe of
organisms covered (saprophytes, parasites, and mycorrhizal-forming
fungi), it would appear that most Basidiomycetes require one or more
deitamins.

In all the experiments cited the objective was the fulfillment
of nutritional requirements using the weight of mcelium produced as
the indicator. In the experiments described here the primary objective
was the production of the anti-tumor principle which had been found in
SPm‘ophores. However, since there seemed to be a logical relationship
beWeen the amount of nycelium produced and the elaboration of the
”the. Substance, the rmtritional experiments, to some extent, paralleled
the experiments of others.

Experimental

Forty ml. of basal medium B was placed in each of 63 Erlenmeyer

f
laaks (125 m1. volume). To this was added the amount of nutrilite

37

listed in Table 3. These flasks were then sterilized in the autoclave
at 15 pounds pressure for 15 minutes. While the flasks were cooling,
the inoculum of #h06f suspension was prepared by blending a quantity of
nycelium with sterile distilled water in a sterile (monel metal) Waring
Blendor. The density of the inoculum was determined with a Conco-
Photelometer with blue filter. Each flask was inoculated with one ml.
of this inoculum and grown for 31; days on the shaker. At the end of
this time, the contents of each flask were emptied on tared filter
papers, dried for 21; hours at 95°C., and then weighed. The amounts of
mitrilites added were chosen on the basis of information obtained in
two preliminary experiments. The results appear in Table 3.

A second, more limited experiment was also set up. The procedure
was the same, with a few exceptions. The growth-period in this experi-
ment was limited to 11; days. A single flask of each nutrilite studied
was inoculated with one ml. of inoculum. However, three inocula of
different densities were used. The results appear in Table 1;.

For the third experiment in this series, )40 ml. of Lindeberg's

solution B (see appendix for formula) was employed as the basal medium.
All nutrilites were added to this medium in the amount of 1.0 ml.
The growth-period was limited to 15 days. Each flask was inoculated
with 1.0 m1. of a blended h06f suspension (22 per cent transmission at
550 “91)- The results of this experiment appear in Table 5.

There were no samples prepared for testing for the presence of an

on
ecstatic principle from the flasks reported in Tables 3, )4, and S.

 

38

Table 3

The effects of nutrilites added to medium B on the growth of Collflia
radicata var. furfuracea #h06f, shaken for 3h days at room temperature.

 

Weight of

Amount add ed to

 

Nutrilite* each 1.0 ml. of dried mycelium

..l , medium B in mg. W

Biotin 10 m1. . 2110

Biotin 1.0 ml. 310

Biotin 0.1 m1. 300
Calcium pantothenate 10 ml. 220
Calcium pantothenate 1.0 ml. 280
Calcium pantothenate 0.1 m1. 270
Inositol 10 ml. 200
Inositol 1.0 m1. 330
Inositol 0.1 ml. 3140
Para-aminobenzoic acid 10 ml. 220
Para-eminobenzoic acid 1.0 m1. 260
Para-«aminobenzoic acid 0.1 m1. 320
Aconitic acid 1.0 ml. 290
Aconitic acid 0.1 m1. 270
Aconitic acid 0.01 ml. 270
Adenine 1.0 ml. 260
Adenine 0.1 m1. 21:0
Adenine 0.01 ml. hlo
B12 1.0 ml. 210
B12 0.1 ml. 260

B12 0.01 ml. 21.0
B12 0.001 mi. 2140
Choline chloride 1.0 m1. 290
Cholme chloride 0.1 m1. 2140
choline chloride 0.01 ml. 1.00

Folic acid 1.0 ml. 160

F0140 acid 0.1 ml. 160

ggfic acid 0.01 ml. 170

C 8.01
d 0'001 ml. 30°Continued

\ ___

 

*
concentration of all nutrilites was 1 mg./ml. except in the case of
: fOlic, acid, and B1,, where it was 1 microgram/ml.

biotin

Table 3 - Continued

*

39

 

u ' '

 

1
Amount added to

Weight of

 

Nutrilite* each to ml. of dried mycelium
_¥ medium B in mg:
Nicotinic acid 1.0 ml. 320
Nicotinic acid 0.1 ml. 250
Nicotinic acid 0.01 ml. 230
Nucleic acid 1.0 ml. 330
Nucleic acid 0.1 m1. 290
Nucleic acid 0.01 ml. 310
Nucleinic acid 1.0 ml. 260
Nucleinic acid 0.1 m1. 320
Nucleinic acid 0.01 ml. 270
Pyridoxine HCl 1.0 ml. h60
PYI'idoxine H01 0.1 ml. 360
Pyridoxine H01 0.01 ml. 360
Ribose 1.0 ml. 310
Ribose 0.1 ml. 370
Ribose 0.01 ml. 360
Thiamine HCl 1.0 ml. 1480
mimine H01 0.1 ml. 1.30
Thiamine H01 0.01 ml. h20
Adenine triphosphate 0.1 m1. 0 280
Adenine triphosphate 0.01 ml. 280
Adenine triphOSphate 0.001 ml . 300
Desoxyribormcleic acid 0.1 m1. 250
Desoxyribomicleic acid 0.01 ml. 1190,
Desomibomicleic acid 0.001 ml. 330
Riboflavine 0.1 ml. 350
Riboflavine 0.01 ml. 350
Riboflavine 0.001 ml. 280
Controls:
Basal medium (Medium B) 220
medium 270
Basal medium 250
38-1 madium . 280
32:: In"3dium and 0.5% yeast extract (Medium A) 1010
medium and 0.5% yeast extract 1020

 

110

Table )4

The influence of varying inocula and the addition of nutrilites added
to medium B on the growth of Collybia radicata var. furfuracea #h06f,
shaken for 114 days at room temperature. W

#

 

v—v

 

 

‘9 * Per cent fifiWeight of
Nutrilite Amount transmission dried mycelium
_ _ __ added of inoculum in mg. m
Aconitic acid 1.0 ml. 16 160
Aconitic acid 1.0 ml. L1 200
Aconitic acid 1.0 ml. 55 190
Desoxyribonuleic acid 0.1 ml. 16 2).;0
Desoxyribonuleic acid 0.1 ml. 141 220
Desoxyribonuleic acid 0.1 ml. 55 210
Nucleic acid 1.0 ml. 16 260
Nucleic acid 1.0 ml. bl 230
Nucleic acid 1.0 ml. 55 250
Pyridoxine 1.0 ml. 16 380
Pyridoxine 1.0 ml. 1.1 210
Pyridoxime 1.0 ml. 55 330
Riboflavine 0.1 ml. 16 1400
Riboflavine 0.1 ml. bl 250
Riboflavine 0.1 ml. 55 230
Ribose 1.0 ml. 16 180
Ribose 1.0 ml. hl 210
Ribose 1.0 ml. 55 230
Thimnine 1.0 ml. 16 31.0
Me 1.0 ml. 1.1 250
Huamine 1.0 ml. 55 390
controls:
Basal medium (Medium B 16 21.0
Basal medium 111 210
Basal medium 55 170
3:12:11 medium and 0.5% yeast extract (Medium a) 16 290
39.3 1 medium and 0.5% yeast extract hl 3140
a medium and 0.5% yeast extract 55 210

h

x-
cone"311tt1f‘v!3.1::ion of all mitrilites was 1 mg./m1-

Table 5

bl

Tine effects of nutrilites added to Lindeberg's nutrient solution on the
growflm.of Collybia radicata 222: gurfuraceaa#h06f, shaken for 15 days
at room temperature . W

 

 

 

:===================::____..;==:v zz~ BfifimfififEfil‘ 1_::£::
Nutrilite dried mycelium
v_ in mg. ‘fifi

B12 100
Biotin 100
Folic acid 090
Ribose llO
Nicotinic acid 080
Inositol O90
Nucleic acid 110
Calcium.pantothenate 100
Choline chloride 100
Thiamine 100
Riboflavine 100
Pyridoxine 100
Adenine 110
Adenine triphosphate 100
Aconitic acid 090
Para-aminobenzoic acid 080
Desoxyribonucleic acid 090

090

n

Nucleinic acid

Controls:

Basal medium.(Lindeberg's solution B) 080

Basal medium
Medium B
Medium.A

110
270
290

*
concentration of all nutrilites was 1 mg./ml. except in the 0886 0f

12: biotin, and folic acid, where it was 1 microgram/ml.

CHAPTER VI

THE EFFECTS OF CARBON SOURCES UPON
COLLYBIA RADICATA VAR. FURFURACEA

Review of Literature

Carbon, along with nitrogen and inorganic salts, is known to be an
essential requirement for life. The role of the carbon source is
generally that of supplying energy to the organism. It is also known
that Species of fungi vary in their ability to utilize different carbon
sources for growth and biosynthesis.

'- Investigations on the availability and utilization of carbon
Sources by fungi have appeared throughout a period beginning in the
late decades of the 19th century and were intensified within the last
_ 25 years. There are m reasons for this latter fact. This kind of
jmeatigation has become more fruitful with the appearance of pure
chemical compounds, especially the vitamins, whose specific effects
could now be recognized and studied.

A far smaller proportion of papers have been written on the use of
carbon Sources by Basidiomcetes compared to what has been reported for
members of the Myxomrcetes, Phyconwcetes, Ascomycetes, or the Fungi

II"Perfec t1 ,

Dagger (1905) was the first to attempt to grow cultures of Aga__r10us
W in media of known composition on gray filter paper to which

Various carbon compounds were added (along with a mineral mltrient

142

143

solution and nitrogen source). He reported that the common sugars,

starch, mannite, tartrates, and lactates were unsuccessfully used as

carbon sources .

Styer (1928) mentioned that,when Ascampestris cultures were grown
on a paper medium with mineral nutrients and an ammonium salt, they

required the addition of no other carbonaceous material. Both Styer and

Duggar concluded that the additional carbon sources were superfluous,
and that cellulose played the most important part in the carbon nutrition
of this flmgus.

However, in a later paper, Styer (1930) showed that A. cmeiuis

 

would not grow in any medium of nutrient salts and soluble organic sub-
stances when the total concentration was greater than 0.2M. According to

this paper, he grew the mcelium on silica gel plates to which he added

various carbon sources by absorption. He obtained the best gowth

When xylose, glucose, maltose, peach gum, pectin, wheat bran and straw,

01‘ El‘amllated peat moss were employed as carbon sources. Cellulose,

he reported, failed to support vigorous growth. He theorized that
the reason for Duggar‘s conclusion that sugars failed to support the
EI‘OW'th 01‘ A... cmestris was that the concentration of the culture
medium was greater than 0.2M.

Lutz (1925) reported the successful culture of 27 species of
B"“E’idim'WC:etes in a solution of xylose, maltose, ammonium salts and
minerals.

Since the writer knows of few publications that appeared after

1 . .
93S Clea-11118 specifically with the effect of carbon sources on the

his

nutrition of Basidiomcetes, he had to refer to a great extent to
papers dealing with other nutritional aspects of this group of fungi.
The data on carbon sources in these papers are more or less incidental.

LaFuze (1937) reported that ljpjypprus betulinus, £21933 pinicola,
and Polystictus versicolor utilized various pentoses, hexoses, and
polysaccharides .

Findlay (19111), while investigating the rate of destruction of wood
by some wood-rotting fungi, reported the utilization of sucrose and

glucose .

Treschow (19th) reported that, while investigating the ability of

 

Eialliota bispora (the commercial strain of what was called A. campestris)

 

to utilize pentoses, hexoses, polyhexoses, salts, and pectins, he
obtained the best yields with mlose, apple pectin, calcium malate,
arabinose, glucose, calcium oxalate, fructose, galactose, mltose, and
sucrose in that order.

Derrick (19149), experimenting with 142 species of wood-destroying
Basidiomycetes, reported that she attained maximum growth in media
containing 16 per cent glucose.

Khudiakov and Vozniakovskaia (1951) were able to grow four boleti
on a medium employing glucose as the carbon source.

Lilly and Barnett (1953) reported on the rate and amount of growth
0f 57 fungi upon 12 sugars. The following pertinent information on
Basidiowcetes appeared: Col_._lybia velutipes utilized fructose, glucose,
sucrose, mannose, maltose, and cellobiose best in that order; m

8albellus utilized mannose, xylose, glucose, and maltose best in that

1:5

order; Polyporus versicolor utilized glucose, mannose, cellobiose,
fructose, and maltose best in that order; Lenzites saepiaria utilized
maltose, mnnose, glucose, and fructose best in that order; Lenzites
trabea. utilized cellobiose, maltose, glucose, lactose, and mannose best
in that order; and Schizophyllum conmnme utilized glucose, mannose,

and fructose best in that order. This information was repeated in the
second part of the Handbook of Biological Data (Albritton, Editor, 1953).

Oddoux (1953) reported the successful growth of 21;? species of
Basidiormrcetes (out of 508 species attempted) on a medium using glucose
as the sole carbon source.

Regarding ascomycetous fungi, Hawker and Chaudhuri (l9h6) reported
that the mcelial growth of five species increased as the concentration
0f glucose or fructose increased (up to 10 per cent) in the medium.
They also noted that the response of these organisms to more complex
carbohydrates was either the same as to glucose and fructose or else of
a nature similar to "starvation type growth," i.e., very poor growth.
The? suggested, these differences in response could be partly explained
by the rate at which the particular fungi could break down a complex
carbohydrate to hexose.

Some of the information published concerning lower fungi is perti-
nent and may have a bearing on this investigation.

Czapek (1902, 1902a, 1903), while observing the effects of various

Mtrogenms sources upon Agergllus niger, reported that the addition

of three Per cent sucrose increased the yield of mcelium in all cases
except one,

 

 

146

Schade (19h0), and Schade and Thimann (1910) reported the unusual
finding that Leptomitus _I_L_a_c_t_e_u_s was unable to utilize carbohydrates.
This organism used either dl-alanine or 1-leucine as its sole source of
carbon and nitrogen.

Margolin (l9h2), in a thesis which appears to be the forerunner of
the experiments reported by Lilly and Barnett (1953), grew 22 fungi on
common hexoses, mannitol, glycerol, dextrin, and some organic acids.

All of the fungi tested were able to grow well on glucose. Twenty of
the 22 grew well on fructose. Most of the fungi grew as well on mannose
as on glucose.

Van Neil (19th), in a review paper concerning the physiology of
microorganisms, stated "A mnnber of experiments have indicated the exist-
ence of microorganisms which are able to decompose some polysaccharides
but which apparently do not attack the constituent hexose units.

A 391929111 study .of such cases has, without exception, shown that the
interipr'e‘tation of the experimental results was at fault. One of the
most Persistent claims, pertaining to the decomposition of cellulose

by a group of bacteria unable to utilize glucose, was resolved recently
by Stanfiler who emphasized once again the long known but often forgotten
pmdnc‘bion of toxic products during the heat-sterilization of glucose
Selutions. There is at present not a single authenticated instance on
record of microorganism which can attack some polysaccharide but not
its hydrolytic products."

There are some reports in the literature concerning the preference

b
y or“mi-81118 of polysaccharides over monosaccharides and disaccharides,

h?

and vice versa .

LaFuze (1937) reported that ~the~~wood-rotting fungi which he studied
utilized polysaccharides more readily than monosaccharides. Steinberg
(1939), in his review paper, suggested that this apparently greater
availability might be due to the difficulty encountered in purifying
polysaccharides .

Findlay (19111) reported that the wood-rotting fungi he studied
utilized glucose more readily than polysaccharides.

Although some of these papers appear contradictory, they may all
be correct since it must be kept in mind that each of these investi-
gators studied different organisms. However, am' dogmatic statement
Chilling the existence of organisms that are able to utilize other
carbohydrates but not glucose should be studied carefully for misin-
terpretation of results.

The lower rate of utilization of galactose by many types of
°r83nisms has also been reported. Horr (1936) found that Aspergillus
9.1% and Penicillium glaucum 1) produced less nycelium, 2) showed a
I'ed'uction in spore germination, and 3) showed irregularities in the
formation of hyphal threads when galactose was used in the medium
instead of glucose. He thought that these effects were not due to the
toxic na.ture of galactose or to its oxidation products but rather to
the l) unavailability, or 2) slow absorption of galactose, which he
regarded as a poor source of carbon for fungi.

Edgecombe (1938) reported that five out of the six fungi he
studied grew much better in media employing glucose or starch as the

car
bon Source rather than galactose or sucrose.

 

 

 

h8

Margolin (19142) reported that only 12 out of the 22 organisms he

studied utilized galactose .

In discussing the differences in avail-

ability of the four monosaccharides he studied-«glucose, fructose,

mannose, and galactose-«he pointed out that they have the same empirical

formula, C6H1206, but that there are significant differences in their

molecular configurations, which appear below.

H
C
H-C-OH
HO-C
H-C-OH
H-C-OH
H-C-OH
H

ll
0

I
:11

d -gluco se

These differences may appear to

H
H-C-OH

C=O

HO-C-H

H-C-OH
H-C-OH
H-C-OH

HI

d-fructose

H H
C=O C=O
HO-C-H H-C-OH

HO-C-H HO-C-H

H-C-OH HO-C-H

H-C-OH H-C-OH

H-C-OH H-C-OH
H H

d-mannose , d-galactose

be only slight. Margolin pointed

Wt: however, that these structural differences gave fructose its ,ketone

(Want-Y, and glucose, mannose, and galactose their aldehyde properties.

He went on to state that the difference between galactose and glucose

is not great, merely a reversal of a hydroaqu group and a hydrogen

atom on the fourth carbon atom. Margolin asked "Why then should

galactose be so less effective than the other three in supporting the

WWI of fungi?" He answered his question by stating that the lower

four carbon atoms in the molecular configuration of glucose, fructose,

and mannose are of the same configuration, while galactose is different,

 

.

149

and that with the exception of galactose, any of the three sugars could
be regenerated from either form.

Before closing the discussion of literature concerning carbon
sources, a review of papers dealing with the effects of autoclaving
should be added .

Lewis (1930) reported that a medium containing four sugars and
various nitrogen sources failed to support the gowth of Phytomonas
malvaceara after being autoclaved for 15 minutes at 15 pounds pressure.

Tanner (1933) stated that,when polysaccharides were sterilized in
a steam sterilizer, much more hydrolysis occurred than when an autoclave
was employed.

Davis and Rogers (1939) reported that some sugar solutions
(especially fructose, glucose, arabinose, and lactose) changed in optical
rotation and/or reducing power after being autoclaved for 30 minutes.

Englis and Hanahan (1915) found that the autoclaving of glucose
solutions in the presence of a phosphate buffer at a pH of 6.h-6.6
gave a censiderable conversion to ketoses, mostly fructose.

Nielsen and Eistrup (1939) and Hartelius and Nielsen (19111) reported
the f0I'mation of yeast growth substances by heating sugar with organic

acids, ammonium tartrate, or ammonium hydroxide.

Since there is the possibility of the tumor-retarding principle,
biosynthesized at times by Q. radicata y_a_._r_. furfuracea being in part
a °arb°hydrate complex, it should be stated that Derrick (191.9) in her
”19313 r ePOrted that the addition of simple sugars (hexoses) to the

medium increased the yields of certain polysaccharides as metabolic

50

products. Werkman and Wilson (1951) however, state that the addition

of simple sugars to the medium does not increase polysaccharide products.
Experimental

The discussions of Margolin (l9h2) and Lilly and Barnett (1953)
convinced the writer that his method of evaluating carbon sources should
be to supply the same quantity (grams per liter) of carbon in each case.
Since the concentration of carbon (excluding the Bacto-peptone) in
basal medium B had proved in the past to support luxurious growth of
many organisms, this figure was arbitrarily chosen as the standard.

In one liter of medium E, 15 gm. each of glucose and sucrose
contain a total of 12.3 gm. carbon. The carbon sources to be tested

were made up to contain the same quantity of carbon as existed in

medium B .

.

Thirty-three different carbon sources were investigated. Each
carbon source was set up in triplicate as follows: The amount of
the carbon source that would yield 11.92 gm. of carbon was weighed and
dissolved in 100 ml. of distilled water. Ten ml. of each stock solu~
tion was placed in a separate 125-ml. Erlenmeyer flask. Twenty m1. of
double~strength medium B (minus the sucrose and glucose it normally
contained) was added to each flask. Finally, the volume was brought to
1‘0 ml- by adding 10 ml. of distilled water. In the case of succinic
acid this procedure was varied because of its relative insolubility.
The amount calculated to yield 14.92 gm. of carbon would not dissolve

1“ 100 ml- of distilled water, but would in 200 ml. Consequently, 20 ml.

51

of this solution was mixed with 20 ml. of the double-strength medium B
(minus the- sugars) .

After thorough mixing the flasks were plugged and autoclaved for
15 minutes at 15 pounds pressure (12100.). They were inoculated with
one ml. of a suspension (preparation previously described) of hoof (17
per cent transmission at 550 wand placed on the shaker at room
temperature (kept at 25°C. by means of an air conditioner). They were
harvested after 1).; days, and filtered using tared filter papers. The
mcelium was dried for 2h hours at 96°C. and the dry weight of the
norcelium calculated.

The broth portion, following filtration, was bottled, after addi-
tion of 0.01 per cent merthiolate, and shipped to the Division of
EXPGrimental Chemotherapy of the Sloan-Kettering Institute for evaluation.

‘ While each sample was being filtered, the pH, color and appearance
0f the mcelium as well as the medium were recorded.

Three of the carbon sources (honey, ribose, NaHCOa) reported
Within this first experiment were actually investigated at a later date.
HOWVeI', since the conditions were virtually unchanged, the results are
1mlvldmi with this group of data, rather than listed in a separate
table. These data appear in Table 6.

Unlike the first experiment which was set up to study the utili-
zation (and subsequent effects) of various carbon sources by g. radicata

13' W the second experiment was designed to study the effects
°f “rm“ Sources, incorporated in addition to the main supply of carbon,

11an h06f growing in medium B. With this in mind, all of the 33

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previously used carbon sources,as well as seven other compounds, were
investigated. Five ml. of each of the 33 stock solutions (preparation
previously described) except ribose was placed (in triplicate) in
separate 125-ml. Erlenmeyer flasks. Twenty ml. of double-strength
medium B was added to each flask. Finally, the volume was brought to
I40 ml. by adding 15 ml. of distilled water.

In the case of succinic acid, 10 ml. of the stock solution was
added to 20 ml. of double-strength medium E and made up to hO ml. by
adding 10 ml. of distilled water.

The seven new carbon sources investigated (and ribose) were divided
into two groups.

Group I included n-acetyl glucosamine, glucosaminic acid, d+ glucosa-
mine-HCl, trehalose, and ribose. These were present in the amount of
0.333 gm. per flask. One gram of each compound was weighed and
dissolved in 60 ml. of double-strength medium B. Twenty ml. of this
solution was placed into each of three 125-ml. Erlenmeyer flasks, and
20 ml. of distilled water was added.

Group II included inulin, uric acid, and alpha-d-glucose penta-
acetate. Because of their relative insolubility, 0.01. gram of these
compounds was dissolved in 60 ml. of double-strength medium B and 60
ml. of distilled water. This total (120ml.) was then divided into
three 140 ml. aliquots which were put respectively into three 125-ml.
Erlenmeyer flasks.

After thorough mixing, the pH, color, and the appearance of the

medium in each flask were determined. The flasks were then plugged

 

nu...

 

59

and autoclaved for 15 minutes at 15 pounds pressure (12100.). Each
flask was inoculated with one ml. of a suspension (preparation
previously described) of h06f (21 per cent transmission at 550 up) and
placed on the shaker at room temperature (kept at 25°C.) .

The flasks were removed after 17 days, and the contents filtered
using tared filter paper. The mycelium was dried for 2).; hours at 95°C.
and the dry weight of the mycelium was calculated.

Eighty ml. of the broth portion of each flask, following filtration,
was methiolated, bottled, and shipped to the Sloan-Kettering Institute
to be tested for the presence of a tumor-retarding principle.

While each sample was being filtered, the pH, color and appearance
of the nycelium as well as the medium were recorded.

The data pertinent to carbon experiment 2 are to be found in
Table 7.

Table 6 shows that, when mannose and glucose were employed as the
carbon source in medium B, _C_J_. radicata var. furfuracea biosynthesized
a tumor-retarding principle. Confirmation of these findings was
essential.

The experiment was modified quantitatively by increasing the total
volume threefold (to 120 ml.), and also by changing the vessel (to a
500-1711. Florence flask).

Four flasks, two containing mannose and two containing glucose,
were inoculated with a 1406f suspension, and placed on the shaker for
19 days at room temperature. At the end of this period, eight samples

were Prepared from the four flasks in the following manner:

 

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The contents of the two flasks containing mannose were mixed and
the culture broth separated from the mycelium by filtering through
Sargent filter paper #500. Two samples each were prepared from the
mycelium and the broth. An 85-1111. aliquant of the broth was bottled
and merthiolate added. Similarly, an 85-ml. aliquant was bottled after
it had been rendered sterile by filtering through a Seitz-filter pad
(Hercules Filter Corp. type ST). ’

The mcelium obtained from the two flasks was extracted by blend-
ing it in a Waring Blendor for two minutes with distilled water (1:1),
and filtered through Sargent paper #500. An 85-ml. aliquant of this
extract was bottled, and merthiolate was added. Similarly, an 85-011.
aliquant of this extract was bottled after it had been rendered sterile
by filtering it through a Seitz-filter pad.

F0113? samples were similarly produced from the contents of the two '
£133“ containing glucose. The two methods of preservation were
emPlOYed to determine what differences, if any, would occur in the
avaluflting tests conducted by the Division of Ebrperimental Chemotherapy,
Sloan‘Kettering Institute for Cancer Research.

Neither the weights of the mcelia, nor changes in color, appear-
ance, or PH were recorded; the biosynthesis of the tumor-retarding
Principle Was the only criterion of interest.

A 18JT‘ger experiment, with further variations was conducted subse-
quently. In this case, 1.061" was grown in one liter of medium in
Fernbmh flasks (capacity 2.8 liters) for 23 days on the shaker at room

t8 .3
mperature . The media employed were "A" and "B" with l, 3, 5: and 7

68

per cent glucose substituted in place of the usual sugars. Eight
samples were prepared by extracting the mycelium with the culture broth
it grew in, bottling an 85 ml. aliquant, and adding merthiolate.

An 85 ml. aliquant of each of the extracts prepared using medium
"A" was mixed with 85 ml. of absolute ethanol, and placed in the
refrigerator for one hour. The precipitate that formed was centrifuged,

and dissolved in 85 ml. of distilled water, bottled, and merthiolate
was add ed .

The mycelial mat (of the flasks containing medium B) which remained
on the filter paper following the extraction process was blended with
absolute ethanol (1:1), placed in large evaporating dishes and allowed
to evaporate to near dryness. Distilled water was added to bring it to
the original volume, the mixture blended in a Waring Blendor, filtered,
bottled. and merthiolated.

The remaining portion of the eight extracts was frozen and stored
in that state, Upon thawing, it was observed that there were two
distinct fractions in each beaker: l) a mucilaginous portion, and
2) a liquid portion. Samples of each of these fractions were prepared
by bowling 85 ml. aliquants, and adding merthiolate.

All Of the preparations described up to this point, with one
exception, Were negative according to the evaluation of the Sloan-
Kettering Institute. The mcelial extract of 1061' grown in mannose
(

fir . ..
St Serles described) was given a i rating. This result is

rep°rt9d in Table 8

69

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70

Additional experiments were performed with mannose as the substi-
tuted .sugar. The conditions of the first experiment were identical

with those described for the second. glucose experiment with the follow—
ing exceptions: l) the concentrations used were 1, h, and 8 per cent

mannose, and 11/2 per cent each of mannose, glucose, and galactose;

2) the growth period was 27 days.

Eight samples were prepared by extracting the mycelium with the

culture broth it grew in, filtering, bottling an 85 ml. aliquant, and

adding merthiolate. The remaining portion of the eight extracts was

fl‘Ozen and stored in that state. Samples were prepared from the

mucilaginous portions and the liquid portions after thawing the l, h,

and 8 per cent mannose in medium A extracts.
One final experiment concerning the effects carbon sources had
upon Collibia radicata var. furfinracea was performed. A suspension of

h06f was used to inoculate ten BOO-ml. Florence flasks containing 125

ml. 0f medium A. The cultures were grown on the shaker for 21 days at

room temperature. At this time, the remaining culture broths were

decanted, and replaced with sterile 125-ml. quantities of three per
cent, mumse, three per cent glucose, three per cent galactose, one-
half of one per cent glucosamine hydrochloride, and one-tenth of one
per Cent ribose solutions respectively (duplicate flasks of each carbon

S‘011I'ce were made). These flasks were again placed on the shaker. One

set was remved after 12 days, and the second set after 26 days. An
extract was prepared from each flask by blending the wcelium in a
“firing Blendor with the carbonaceous solution it grew in. This extract

was then filtered and bottled, and merthiolate was added.

71

All of the preparations in this second series of experiments, with

two acceptions, were negative according to the evaluation of the Sloan-

Kettering Institute for Cancer Research. The extracts prepared from

h06f mycelium grown in Fernbach flasks in the one and the eight per

cent mannose (substituted in one liter of medium A) concentrations were
given a i‘ rating. These data are reported in Table 9.

Certain preparations of the first two carbon experiments gave
negative results according to the system of tumor evaluation of the

DiVision of Experimental Chemotherapy, Sloan-Kettering Institute for

Cancer Research. However, the consistent depression in tumor growth

observed, when some constituents were incorporated into the culture

medium, prompted a statistical analysis of the results. The F test

from an analysis of variance performed on observations made on the

indiVidual mouse tumors (data supplied .by the Division of Experimental
Chemotherapy, Sloan-Kettering Institute for Cancer Research) disclosed
Significant depression of mouse sarcoma 180 by filtrates of the media
when either citric acid or honey served as the sole carbon source

(Table 6), and by filtrates of the media when mannose and invert sugar

served as additional carbon sources (Table 7). When subsequent experi-

ments with carbon sources (see pages 67-71) showed that previously
actlbre materials gave negative results. according to the Sloan-Kettering
evaluation system, it was found to be of interest to subject the
reeults to statistical analysis. Two of the preparations, the filtrates
containing glucose and marmose as sole carbon sources, were shown to

depress significantly the growth of mouse sarcoma 180 (Table 10).

72

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73 ..

Table 10

Significant depression of mouse sarcoma 180 after treatment with fil-
trates of Colllbia radicata var. furfuracea #hOéf.

v

Turner size expressed as per

 

Carbon Sources in Medium cent of control tumor
Citric acid, 3% 77*

Honey, 3% 80*

Mannose, 3% 76*

Glucose, 3% 82*
Additives to Medium B gsee Amendixl

Manmse, 1.5% 81*

Inth sugar, 1.5% 78%):—

M

h

e:-
0.05 level of P
0.01 level of P

CHAPTER VII
SUGAR DETERMINATION
Review of Literature

Because of the correlation between the biosynthesis of the tumor-
retarding principle and the presence of certain sugars in the culture
medium, it appeared to be of interest to know the sugar content of
Q. radicata 133° mrfuracea mycelium produced in 1.1.3.19:

The possibility was considered that a supply of a simple nutrient
like glucose would enable the fungus to synthesize the active material
at an accelerated rate.

The chemical composition of fungi has been previously reported.

Gussow and Odell (1927) cite the following information as the composition

 

“*1 w campestris:
Water 93-95%
Nitrogen substances 7.8g
Non-nitrogen substances b,%

Small amounts of mitritive salts and sugars.
The Handbook of Chemistry and Physics (Hodgman, 1950) lists the

f0Hindi-'18 data for "mushrooms" under "Composition and value of foods":

Protein 3.5% by weight
Fat 00% by weight
Carbohydrates 6.0% by weight

The Handbook of Biological Data, (2nd Part, Standard Values in

Nut '
rition and Metabolism, Albritton, Editor, 1953) lists the f°11°mg

7h

 

75

data for mushroom (Aflicus comestris):

Energt value 16 calories/100 g.
water 91 g./lOO g.
Protein 2.11 g./lOO g.
Fat , 0.3 g./lOO g.
Total carbohydrate 11.0 g 100 g.
Crude fiber 0.9 g./100 g.
Ash 1.1 g /100 g

Because of discrepancies in the composition figures, and also to
serve for comparison, the total sugar content of the commercial mush-
room, A. cmestris was first determined. The Michigan Mushroom Company,
Niles, Michigan, supplied us with the fresh sporophores used for these
experiments.

‘ The first attempts at determining the total sugar content (normal.
acid hydrolysis, boiled for 10 minutes) seemed to indicate that this
1‘11an contained no sugar.

Bourquelot and Herissey (190).; and 1905) established the presence

of trehalose (generally) and cane sugar (sometimes) in mushrooms. They
WPorted that Boletus edulis, g. aurantiacus, and Cortinarius em
contained one per cent trehaloses‘ _B_. bit-c3223 and Amanita ,muscaria two
per cent trehalose; and Russula delica,,1_i_. Queletii, and Paxillus
W three per cent trehalose.

Immori‘ (1925) showed that the sugar contained in most fungi is
trehalose, and stated that it could be splitonly with great effort.

He 8“seemed that for complete hydrolysis, the material should be boiled
for six hours with five per cent sulphuric acid.

Davis and Rogers (1939) reported trehalose to be one of those
Sugars that belong to the stable group; i.e., one not affected by

aut -
ocmvlhe for 30 minutes at 120°C.

76

Experiments to determine what method of hydrolysis would be most
suitable were made. One method, using the enzyme trehalase, was con-
sidered. Bourquelot and Herissey (1901;) reported finding trehalase
present in the caps of Boletus edulis, .13: aurantiacus, Q. badius, and
Cortinarius elatior. Iwanoff (1925) stated that trehalase, though
present in most fungi, varies in concentration in different parts of
the fruiting body and also sometimes is not present, and then appears
in later stages of growth.

Myrbé'ck and Brtenblad (1936, 1937a and 1937b) while describing the
isolation of this enzyme from yeast reported that one gram of trehalose
dissolved in 10 ml. of the crude enzyme preparation was 57 per cent
hydrOlyzed at the end of 1;? hours. The amount of work this entailed,
and the relatively low yield obtained during the long period of hydroly-
31" Prompted the search for an easier and more accurate method. This

method used acid hydrolysis with and without pressure .66
Experimental

Twenty-five mg. of trehalose hydrate was added to each of twelve
100ml. volumetric flasks containing 50 ml. of distilled water. Two
"11- 0f concentrated hydrochloric acid was added to each flask. Six of
these flasks were then placed in a bath of boiling water. Two flasks

were removed at the end of 90, 180, and 360 minutes. The remaining six

 

f
32131}? e and S‘lPeTViSiZiOYl Of this experiment by Dr. E. Benne and techni-
turalelp extended by Mr. w. s. Brammell, both of the Michigan Agricul-

Experiment Station, are gratefully acknowledged.

77

flasks were placed in an autoclave (15 pounds pressure, 121°C.) and

two flasks each were removed at the end of 30, 60, and 90 minutes.

then all 12 flasks were cool they were made nearly neutral with sodium
hydroxide, and distilled water added until a volume of 100 ml. was
reached. The solution was then filtered through Whatman filter paper
#hZ. A SO-ml. aliquot was transferred into a hOO-ml. beaker containing
25 ml. of a stock copper sulfate solution and 25 m1. of alkaline
tartrate solution. The contents of the beaker were covered with a
watch glass and heated on asbestos gauze over a Bunsen burner. The
flame was adjusted so that the contents began to boil in four minutes,
and the boiling allowed to proceed for exactly two minutes. The hot
solution was immediately filtered through an asbestos mat in a
Porcelain Selas crucible using suction. The precipitated cuprous oxide
“is washed thoroughly with hot water, and then dried for two hours at
1050C . The Selas crucibles were removed, placed in a desiccator, and
were weighed after sufficient cooling. A blank determination was also
Performed.

Since trehalose hydrate (M01. wt. 378.310 was used, the material
actually contains only 90.h8 per cent of anhydrous trehalose (M01. wt.
3,4231) . With this correction in mind, therefore, each volumetric
flask is Observed to contain 22.62 mg. of anhydrous trehalose. Table
11 shows the results of this experiment.

Th53% data indicate that the most efficient method of hydrolyzing
trehaloSe is that which applies pressure in an autoclave for 30 minutes.
The time required in this case is l/l2 of that needed to obtain compar-

abl
3 results with acid hydrolysis in a bath of boiling water.

78

Table 11

Hydrolysis of Trehalose
(sugar determined as glucose)

 

 

Sample Per cent
Boiled number Grams Per cent . averaged
90 minutes 1 0.013hl 56.32 57 h
90 minutes 2 0.0139h 58.55 ’
180 minutes 3 0.01870 78.51: 80 2
180 minutes 1; 0.019h7 81.77 '
360 mirmtes 5 0.02281. 95 .93
360 minutes 6 0.02166 90.97 93 '5
AutOClaved
30 minutes 7 0.02280 95.76 98 1
30 minutes 8 0.02390 100.38 ’
60 Minutes 9 0.02288 96.10 97 L
60 mirmtes 10 0.02350 98.70 '
90 minutes 11 0.02332 97.914 96 1
90 munites 12 0.022hh 91:25 '

‘

Three samples of mushrooms were analyzed for total sugar using the
methOd described. The following kinds of mushroom tissue were divided
into analytical portions:

a) slices of context, i.e., the spongy tissue in the cap exclud-
ing the gills, of 9;. campestris.

b) slices of whole nmshroom (9:. cagpestris).

0) in vitro mycelium of g. radicata var. furfuracea (washed three
times with distilled water) .

Fifty an. of each material was put into a Waring Blendor and blended

f
or four minutes with 100 ml. of distilled water. The slurry was

79

transferred to a 250-ml. volumetric flask containing two ml. of
saturated lead acetate solution (to precipitate the protein present),
mixed thoroughly, and allowed to stand for at least 15 minutes.

Approximately two gm. of solid potassium oxalate (used to precipi-
tate the excess lead present) was added to each flask, and sufficient
water added to bring the volume up to 250 ml. The contents were mixed
thoroughly to dissolve the salt and allowed to stand at least 30 minutes.

A portion of the extract was filtered into an Erlenmeyer flask
through S. & S. #588, 12.5 cm. folded filter paper. A 50-ml. aliquant
was Pipetted into a lOO-‘ml. volumetric flask, two ml. of concentrated
h.Ydl‘oczhloric acid was added, and hydrolyzed in an autoclave for 30
minutes at 15 pounds pressure (12100.).

After the flasks were removed from the autoclave and permitted to
cool, they were made nearly neutral with sodium hydroxide solution,
the volume was brought to 100 ml. using distilled water, and the solu-
tion mixed. The contents were filtered through Whatman filter paper
#112 and a 50-ml. aliquot was transferred to a hOO-ml. beaker containing
Ifli‘hlil’lg‘s solution. The evaluation of reducing sugars was then
performed as previously described. A blank determination was also made
and Sllb‘tzracted. Corrected figures appear in Table 12.

The solid and moisture content of g. gayestris was determined by
weigl'lil’lg four samples of the fresh whole mushroom, drying them for 2).;

hours at 9900, and reweighing the samples. The resulting calculations

are Seen in Table 13.

80

Table 12

Cornparison of sugar content determined for A. campestris with that of
_i_n_ vi___1_:__ro mcelium of _C_3_. radicata var. furfuracea #H06f.

h
_
_¥~ m ____v
w

Glucose, determined

 

 

 

Sample Number Grams Per cent Per cent averaged

,1}; estris

con'tfl%—"ex 1 0.0127h 0.255 0 252

context 2 0 . 012110 0 . 2178 °

A- nggestris,

whole mushroom 3 0.03081 0.616 0 608

whole mushroom 1; 0.03000 0.600 '

C radicata, var. furfuracea,

in vitro mat 5 0.0335]. 0.670 O 682
6 0.03h67 0.693 '

Table 13

Moisture determination of g. campestris

k

__*
———— ‘fi—v

 

Sampie Weight of fresh weight of dried Per cent Per cent
Ember mushroom mushroom solids moisture
1 b.6975 0.3637 7.71. 92.26
2 9.7808 0.6128 6.26 93.717
3 15.0mm - 1.5025 9.98 90.02
1* 31.9511 2.3330 7.30 92.70

w—v

Average 7 .82 92 .18

CHAPTER VIII
THE EFFECT OF NITROGEN SOURCES
Review of Literature

Robbins (1937), following a survey he made of the literature up to
that time, suggested that all organisms could be classified into four
groupings on the basis of their nitrogen requirements: Group I to
contain the nitrogen-fixing organisms, which could also assimilate
nitrate, ammonium, and organic nitrogen; Group II to contain the
nitrate-ammonium users-those unable to utilize gaseous nitrogen but
living on nitrate nitrogen and also able to assimilate ammonium and
Organic sources of nitrogen; Group III, the ammonium users, utilizing
0’11)? ammonium and organic nitrogen sources; and Group IV, the organic-
“.itI‘Ogen users, which could grow only when nitrogen was supplied in a
°°mPlex form.

The above reference, as well as many .of those which follow, was
removed from the normal chronological sequence to give greater lucidity
to this review. In general, chronological order is maintained only
1113015 fir as the various individual genera or natural groupings are
°°ncerned.

Czapek (1902, 1902a, and 1903) studied the effects of various _
inorganic and organic nitrogen sources upon the growth of Agpergillus
£523 With and without three per cent sucrose added (and a solution of
mineral salts). He stated that, while 5. Egg; could utilize nitrates

81

82

and ammonium salts, mycelial proteins are most easily synthesized from
amino acids and those substances which most nearly resemble the amino
acids. For example, he explained the high utility of acetamid as a
nitrogen source by the fact that its structure approaches that of an
amino acid. This work is generally recognized as a pioneering contribu-
tion in the nitrogen nutrition of the fungi.

Klotz (1923) reported that amino nitrogen was most readily assimi-
lated by the fungi he studied. He concluded that the factors influencing
the nitrogen content of the fungous mat are the nitrogen and carbon
sources of the medium, the length of incubation, the rate of growth,
and the hydrogen ion concentration.

Duggar (1905) is credited with being the first to culture success-
fully A_. carmestris _i__n_ 33339.. He reported the organism indicated a
Preference for proteins, ammonium salts, and nitrates in this order.

Styer (1928) reported that ammonium salts, urea, glycine, asparagine,
Peptone, and proteins were all good nitrogen sources for A. campestris,
although the more complex forms gave a slightly denser growth than did
the inorganic nitrogen compounds. Later (1930), following extensive
subs"vequent experiments, he substantiated his original findings by
de°1aring ammonium nitrate, leucine, nucleic acid, nucleoprotein,
glutenin, casein, and albumin to be the better nitrogen sources for
this oI'ganism.

Treachow (191.1.) found 9;. campestris unable to assimilate nitrate
nitrogen while able to utilize ammonium nitrogen, eSpecially the more

Complex Salts. He added that amino acids were the best source of

83

nitrogen, especially asparagine and glutamic acid. However, he found
that when both ammonium salts and amino acids are present in the
medium at the same time, the fungus was unable to select freely the
more suitable source of nitrogen.

Humfeld (19148) grew A. caigestris in submerged culture in media
incorporating either asparagus butt juice or press juice from pear
water as the main substrate.

- Humfeld and Sfiigihara (19149) reported that this organism could utilize
ammonium, urea, amino acids, and some of the more complex forms of
Organic nitrogen.

Later (1952), while studying the nutritional requirements of A.
.Cgmestrisfihey used a medium employing urea as the nitrogen source.
They reported they chose urea "because its utilization does not change
the pH of the medium sufficiently to have an unfavorable effect on
growth, Also, when utilized, it does not leave any residual radical
in solution."

Hawker (1936) reported that she grew Collybia velutipes on a
mineral-dextrose medium containing potassium nitrate.

Leonian and Lilly (1938) stated that _C_. tuberosa did not grow on a
mMoral-dextrose medium containing ammonium nitrate unless amino acids
and 1Thiamine were added. This work illustrates well that some of the
earlier reports of organisms being unable to utilize some forms of
nitrogenous compounds (as compared to yeast and malt extracts) may

have been due to the species“ need for vitamins which were unknown at

the time the investigations were performed.

814

Lindeberg (19h6b) reported that Q. ambusta, Q. butEacea, and
g. Elutipes grew on a medium employing ammonium tartrate as the
nitrogen source. Robbins (1950) reported that only one of two Collybia
ER. grew well on a medium containing asparagine as its nitrogen source.
Norkrans (1950), while studying 17 strains of nine Tricholoma 5 .,found
that ammonium and organic nitrogen compounds could be assimilated by
all species, but only T. w could utilize nitrate nitrogen. Later
(1953) she confirmed these findings by‘using an ammonium salt as the
nitrogen source in a basal medium which supported the growth of seven
Ma. Oddoux (1953) reported the growth of 21;? species of
Basidiomycetes (out of 508 species attempted) on a medium whose chief
nitrogen source was ammonium chloride.

Melin and Lindeberg (1939) reported that they grew Boletus elegans
in a medium containing ammonium tartrate as its nitrogen source. How
(Who) confirmed the ability of E. elegans to utilize ammonium nitrogen
by grOWing it upon a medium containing ammonium chloride as the nitrogen
source. Melin and Nyman (1910) grew _l_3_. ganulatus, E. luteus, Q. WES.‘
3315., E. piperatus, and .}_3_. viscidus in a medium employing ammonium
tartrate as its nitrogen source. The last mentioned species, however,
did not grow as well as the other four. Khudiakov and Vozniakovskaia
(1951) reported the growth of g. luteus, g. variegatus, _1_3_. luridus, and
E' m on a medium containing ammonium nitrate, tryptophane, and
casein 1'1.‘Ydrolysate among other nutrients. They concluded
that these four species were aminoheterotrophic. Melin and Nilsson

(

1952) Proved that g. variegatus in mycorrhizal connection with trees

 

85

could assimilate ammonium nitrogen by using a labelled (N15) salts and
later isolating it from the root tissues, stem, and needles of a pine
seedling. Lutz (1925) grew 27 species of Basidiomycetes in a solution
containing two ammonium salts as the nitrogen source.

La Fuze (1937) while studying the nutritional characteristics of
301yporus betulinus, £93923 pinicola, and Polygtictus versicolor; found
that amino and ammonia nitrogen was better utilized than amide, nitrate,
and nitrite nitrogen. He also observed that 1) the best growth occurred
on proteins containing relatively large amounts of glutamic acid and
tryptOPhane, and 2) the amino acids which contained phenyl and disulphide
radicals retarded growth.

Leonian and Lilly (1938) reported that generally, of 25 organisms
tested, growth improved when amino acids were substituted for ammonium
nitrate in the medium. Among the amino acids used, l—aspartic acid and
d‘glutamic acid wereutilized best by Cgprinus laggms and Pleurotusa
corticatus while Collybia tuberosa utilized d-arginine and l-aspartic

m,

acid best.

Lindeberg (19hl) grew Marasmius androsaceus on a medium employing
alumoniumn tratrate as the nitrogen source. Later (191m) he stated that
only .11- ;‘glvobulbillosus out of 13 species of W that he studied
could utilize nitrate nitrogen (KNO3). All 13 species utilized ammonium
Chloride and asparagine, the latter better than the former. These two
nitrogen sources were superior to alanine, leucine and urea which were
g°°d Sources for all species; glycine was utilized by only one species

and a‘("eJ""=“-"13'-<ie by two. He theorized that asparagine was better than

86

ammonium salts because the NH3 escapes from the medium leaving the Cl-
ion, which unites with H+ ions to produce an unfavorable environment
for the fungi.

Derrick (19h9) while studying the nutrition and physiologr of L2
wood-destroying Basidiomycetes found that, while none of the organisms
required organic nitrogen, they generally grew better in organic
nitrogen sources than in ammonium salts. She concluded that, as
Previously reported by La Fuze (above) and others, as the nitrogen
COMpounds became simpler, the resulting growth was less. She also
rF—‘POrted that the organisms she studied did not utilize either nitrates
01‘ nitrites, or ammonium chloride in the absence of succinic acid.

In general, she stated, maximum growth and maximum rate of growth was
attained in malt extract medium rather than in the synthetic medium she
used , even though the nitrogen content of the latter was three times as
large.

In a later report of this work, Jennison, Newcomb, and Henderson
(1955 ) further elaborated on the growth-supporting ability of amino acids
compared with casein hydrolysate to be as follows for 15 species of
"OOd-rot fungi: Casein hydrolysate, DL-valine, glutamine>DL-aspartic
acid, L-glutamic acid, L-arginine, L-asparagine, glycine, L-dproline,
DL"alpha alanine, DL-ornithinei-hydroxyproline, L-tryptophane,
DL-serjne, DL-threonine, L-leucine, DL-phenylalanine, DL-methionine,
DL‘iSOIeucinei-histidine, DL-norleucine, L-tyrosine, L-cystine>beta
Alanine, L-lysine, and L-cysteine. Additional experiments seemed to

l“dicfitte that the differences in utilization of amino acids was related

to molecular structure other than isomerism.

87

Lilly and Barnett (1953) in studying the utilization of sugars by
fungi employed a medium containing asparagine as the nitrogen source.
They grew 57 fungi on this medium, approximately 10 per cent of which
were Basidiomycetes.

Hacskaylo, Lilly, and Barnett (1951;) in an extensive report on
the growth of fungi in nitrogen sources, determined the rate and amount
01' growth of 25 species of fungi (1)4 of which were Basidiomycetes)
when they were supplied with nitrate, ammonium, and organic (asparagine)
nitro gen. They reported that nitrate nitrogen was used slowly by some
0f the Basidiomcete‘s tested—with one exception, Polypgrus distortus--
and poorly, if at all, by the others. .From the standpoint of rapidity
and the amount of mycelium produced, asparagine and ammonium sulfate
Supplemented with fumaric acid were about equal in value and superior
to either ammonium sulfate or potassium nitrate. They theorized that
the steps in the reduction of nitrate to ammonia were the limiting
factors, or that adaptive enzyme formation was required for utilization
0f nitrate nitrogen by these species. They added that the latent ability
or an organism to utilize nitrate nitrogen may be easily overlooked
when short times of incubation are employed.

In an earlier paper Leonian and Lilly (19140) stated that various
Organic acids (especially four-carbon dicarbqurlic acids or their salts)
render the less favorable sources of nitrogen such as arginine and
Wonium nitrate very readily available so that often as much as 1000
per cent increase or more in growth is effected.

Ev 3118 and Butts (19149) while studying the inactivation of amino

“ids by autoclaving determined that none of them was significantly

88

destroyed when autoclaved alone. However, whenever the single amino
acids were autoclaved in a solution containing sucrose, over 15 per

cent of the amino acid was destroyed (except for cystine).
Experimental

In line with the procedure used in the carbon-source studies, a
similar approach was thought to be the best method of obtaining infor-
mation concerning nitrogen utilization by 9. W gag. furfuracea.

The concentration of nitrogen in basal medium B (source: Bacto-
PeP‘tone) had been proved previously to produce a relatively luxurious
growth of many organisms. This figure was, therefore, arbitrarily
chosen as the standard. Calculation of the quantities needed was
accomplished in the following manner:

In one liter of medium B there are five gm. of Bacto-Peptone. The
per Cent of nitrogen by weight was calculated to be 16.16. The resultant
calculation indicates that there are 808 mg. of nitrogen in one liter.
of medium 13, or, 32.32 mg. of nitrogen in 110 ml. of medium B.

A tom of 25 different nitrogen sources was investigated. Each
nitrogen source was set up (in triplicate) as follows: The amount of
the nitrOgen source that would yield 323.2 mg. of nitrogen was weighed
and dissolved in 100 m1. of distilled water. Ten ml. of each stock
SOlution was placed in a separate 125-m1. Erlenmeyer flask. Twenty ml.
of double~strength medium B (mimis the Bacto-Peptone it normally con-
tained) was added to each flask. Finally, the volume was brought to

ho
’"1- by adding 10 ml. of distilled water. In the case of casein

89

hydrolysate, malt extract, dried skim milk, and yeast extract, this
procedure was varied because the nitrogen concentrations of these
sources were unknown. The figure of 16.16 was used as the arbitrary
percentage of nitrogen in these substances. Consequently, in the case
of these substances, 0.20 gm. of each of the substances was placed in
a separate 125-m1. Erlenmeyer flask (in triplicate). To this was added
20 ml. of double-strength medium B (minus its usual nitrogen source)
and 20 ml. of distilled water.

Procedure of preparation and testing was identical with that used
in investigating the utilization of carbon sources. The exceptions to
this procedure were: 1) the suspension of 1406f produced 29 per cent
transmission at 550 mm, 2) the growth period on the shaker was 23 days,
and 3) the drying temperature was 97°C.

Seven of the nitrogen compounds employed in the first nitrogen
exPalf‘iment (nitrogen-utilization) barely supported the growth of 1406f.
COI’ls'BCIllently, their broths were not shipped to the Division of Experi-
mental Chemotherapy, Sloan-Kettering Institute for testing. The data
Pertinent to this experiment appear in Table 11;.

The second nitrogen experiment was designed to study the effects
01" nitrogen sources, incorporated in addition to the main supply of
nitrogeh, upon h06f growing in medium B. The same sources of nitrogen
were tested as in the preceding experiment. The procedure of prepara-
ti” and testing was identical with that used in investigating the
addition of carbon sources. The exceptions to this procedure were:

1
) the Shepension of u06f produced 17 per cent transmission at 550 1'31:

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2) the growth period on the shaker was 20 days, and 3) the drying
temperature was 96°C. The data pertinent to this experiment appear in
Table 15.

The slight retardation of the test tumor by the broths containing
dried skim milk as a nitrogen source (Tables lb and 15) led to further
experiments. A suspension of Collybia radicata var. flirwfliracea was used
to inoculate two SOC-ml. Florence flasks containing 125 ml. of medium A.
The cultures were grown on the shaker for 21 days at room temperature.
At this time, the remaining culture broths were decanted, and replaced
with sterile 125-ml. quantities of three-per-cent dried skim milk
solution. These flasks were again placed on the shaker. One flask was
removed after 12 days, and the remaining flask after 26 days. An extract
was Prepared from each flask by blending the mycelium in a Waring Blendor
with the dried skim milk solution in which it grew. This extract was
then filtered, bottled, and merthiolate added. No weights of the
m5"“91111m, nor changes in color, appearance, or pH were recorded; the
biosZYII‘ILhesis of the tumor-retarding principle was the only criterion of
interest. These preparations were negative according to the evaluation
0f the Sloan-Kettering Institute for Cancer Research.

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used to inoculate four SOD-ml. Florence flasks containing 125 ml. of
medium E minus the usual sugars and peptone. In lieu of the peptone,
this medium contained one-half per cent dried skim milk. In addition,

one
flask contained one-per-cent each of glucose, mannose, and

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galactose; one flask contained three per cent mannose; one flask con-
tained three per cent glucose; and the final flask contained three per
cent galactose. A similar set of four flasks was set up; these con-
tained one-half per cent yeast extract in addition to the aforementioned
compounds.

All eight flasks were placed on the shaker for 31 days. At this
time, extracts were made in the manner previously described. No weights
of mycelia produced were recorded, since the production of the tumor-
retarding principle was the main objective. According to the Sloan-
Kettering Institute's evaluation reports, it was not present in any of
these eight samples. However, statistical analysis of these results
disclosed significant depression of the test tumor in the sample pre-
pared from the flask containing one-half per cent yeast extract plus
onedper-cent each of glucose, mannose, and galactose.

The F test from an analysis of variance performed on Observations
made on the individual mouse tumors also disclosed significant depression
of mouse sarcoma 180 by filtrates of the media when dried skim milk
served either as the sole nitrogen source (Table lb) or when added to
the basal medium (Table 15). The preparations showing significant

tumor depression are listed in Table 16.

100

Table 16

Significant depression of mouse sarcoma 180 after treatment with
filtrates of Colgzbia radicata var. furfuracea #uoer.

A

 

 

 

 

 

Nitrogen sources in medium Tumor Size expressed
as per cent of
control tumor'

Dried skim milk, 0.5% 78*

Dried skim milk, 0.5%, plus 1% each of mannose, w

glucose, and galactose, and 0.5% yeast extract 81“

Addedfito Medium B_§see.Appendix)

Dried skim milk, 0.25% 81*

*0.05 level of P

CHAPTER IX

MISCELLANEOUS EXPERIMENTS WITH COLLYBIA
RADICATA VAR. FURFURACEA # 0 f

 

Several additional lines of interest were investigated. It seemed
advisable to test a variety of heterogeneous materials for no particular
cogent reason other than to complete the rather exhaustive investigation
of various nutrients. Guirard gt El: (l9h6a, l9h6b) reported that the
presence of sodium acetate stimulated the growth of some microorganisms.
Consequently, sodium acetate was added to medium A in four levels (0.5,
1.0, 2.0, and h.0 per cent) before and after autoclaving. Since Roland
and Heiner (1955) reported that Basidiomycetes can readily perform
oxygenation and other biomodifications of steroids, cholesterol,
lecithin, and oxgall were likewise added to medium A at various levels.
Dried egg peptone was substituted at several levels for Bacto-peptone.
Two of these preparations showed tumor-retarding activity but further
investigations along these lines yielded negative results.

Also investigated at this time was the possible stimulatory effect
which vitamins, other than those previously tested, might have upon
the elaboration of the active material. Vitamins A, E, and D in various
forms were added to medium A. Although a few preparations showed
activity when evaluated by the Sloan-Kettering Institute, they were

all adjudged ineffective when they could not consistently be reproduced.

101

102

Bottomley (191b,) reported that an extract of Sphagnum peat which
had undergone partial decomposition by Azotdbacter species stimulated
the growth of microorganisms in.a manner unmatched by ordinary peat
extracts. 'With this in mind, "bacterized peat" extract, as well as an
extract of an.A§gtObacter 323 were added to medium.A. Neither affected
the growth or the production of the tumor-retarding material.

Although the initial and final pH levels had been observed and
recorded in the carbon and nitrogen experiments, it was of interest to
determine how a change of pH during the active growth.phase might
affect the organism. .Accordingly, after 19 days of growth in medium A,
the'beer from the flasks was decanted and replaced with solutions whose
pH ranged from 3.0 to 11.0. .At the end of an additional lb and 28 days
on the shaker, the pH of all these flasks was in the vicinity of pH
5.5-8.0. .All the flasks showed a slight increase in the amount of
mycelium present and none showed evidence of a tumor-retarding principle.

Large volume experiments were also conducted. Organism #h06f was
grown in one liter of medium A in Fernbach flasks on the shaker and
also stationary. It was also grown in three liters of medium A in five-
gallon Pyrex carboys on the shaker and in stationary culture. None of
the samples prepared from these cultures were active.

Another set of experiments where h06f was grown in Fernbach flasks
and Pyrex carboys employed 1) cheesecloth strips and milk filter discs
hanging from the necks of these vessels and 2) stainless steel screening
rolled into a sphere inside the containers. The organism grew well in

all cases. Some of the samples prepared demonstrated tumor-retarding

103

properties but when replicate experiments were run, the results were
not duplicated .

A final experiment, employing kinetin riboside, Ns-benzyl-NS-
nethyladenine, and the culture beer of Gibberella fuiikurgi, determined
that none of these growth stimulators affected either the growth of

hoof or its ability to produce the tumor-retarding material.

CHAPTER X

SCREENING 0F BASIDIOMYCETE SPOROPHORES
F0R”TUM0R~RETARDINGVPR0PERTIES

Since Collzbia radicata 225: furfuracea #h06f proved to be no more
consistent than organism.#288j in producing the tumor-retarding principle,
the screening of additional Basidiomycete sporophores was continued.
Extracts were prepared by either steeping slices of the sporophore in
distilled water, or by blending the sporophore with distilled water in
a.Waring Blendor for two minutes. Table 17 shows the result of this
investigation. The ratio of sporophore to water is given in each case,
as is also the method employed in making the extracts. Previous to
bottling, the extracts were filtered through Sargent No. 500 filter
paper, and merthiolate was added to give a final concentration of one
part per 10,000.

The sporophores were identified; in instances where identification
0f the Species was impossible the generic identification is reported.
When working with fresh specimens that were in good condition, tissue
cultures were attempted, using the context portion of the pileus.

Though many of these were contaminated, many pure cultures were obtained.

It should be noted that the screening program covered several
seasons. The culture of Q, radicata.Z§£, furfuracea was chosen for
investigation following the experiments on organism #288j because of
all the sporophores whose extracts contained tumor-retarding principles,

it grew fastest. Boletus Frostii #393 apparently contained a stronger

10h

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108

tumor-retarding material, but the culture was extremely slow-growing,
and eventually ceased to grow. Cultures of Boletinus cavipes, Boletus
gd_u_1_i_._s_ pinicola, mum septentrionale, 5121.111". §E_. #11116, and Polyporus
niggicans could not be obtained owing to the presence of parasitic
microorganisms, or else the dearth of viable cells.

Thirteen of the 31 extracts prepared from the fresh sporOphores
and three of the four extracts prepared from dried sporophores showed
the presence of tumor-retarding principles in the mouse sarcoma 180‘

tests made by the Sloan-Kettering Institute for Cancer Research.

"'1'

CHAPTER XI
EXPERIMENTS WITH CALVATIA MAXIMA-#6h2

‘Water extracts of fresh sporophores of Calvatia maxima strains
#6h2-6h7 had shown the-presence of an oncostatic agent in mouse sarcoma
180 tests at the Sloan-Kettering Institute. Cultures of all six of
these isolations were viable, and strain.#6h2 was chosen for ig_zit§2.
work. The primary interest was the same as in the Collybia experiments,
namely to determine whether the tumor inhibitor encountered in sporo-
phores wpuld also be produced consistently i§;Xli£23‘ This was found
to be the case when preparations made from cultures of #6h2 were grown
in medium A. The main problem now appeared to be not one of nutrition
but one of determining the optimum environmental conditions under which
the tumor-retarding material would be produced consistently.

A preliminary study established that Q. m #6h2‘ grew at
temperatures ranging from 80 to 28.500., with the optimum temperature
range between 200 and 26°C. This was determined by placing 3.5 mm.
plugs on triplicate medium.A agar plates at temperatures of 8, 12, 16,
20, 22, 2h, 26, 28, 30, and 37 degrees C. and recording the diameter
of each colony. The averaged resultant values are shown diagrammatically
(Figure IV).

The elaboration of the tumor-retarding material was investigated

by using shake cultures of #6h2 at five temperature levels, 16, 19, 22,

109

110

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111

25, and 28 degrees C. The 190C. experiment is described and serves as
an example of the temperature study.

Thirty-five 500-ml. Florence flasks containing 125 m1. of medium A
broth were inoculated with 10 ml. of a suspension of g, maifim§.#6h2
abcording to the procedure previously described, and placed on the
shaker in a controlled temperature room at 19°C. Starting with the
fourth day, and subsequently at four-day intervals (until the 36th day),
three flasks were removed from the shaker. Samples were prepared in
the manner previously described by blending the mycelium with the
culture beer and were shipped to the SloanéKettering Institute for :
evaluation. The results of the individual series were averaged
(Table 18)and plotted (Figure V). The eight excess flasks were inocu-

lated as alternates in case any flasks should become contaminated.

112
Table 18

Results of mouse sarcoma 180 tests with in vitro cultures of Calvatia
maxima #6142 grown at 190 C.

W

 

Sample Days * Tumor diam. in cm/ Per cent Per cent
Number 01d Effect diam.of control tumor Change Average
1 h 1.00/o.89 +12.3
2 h - o.89/o.87 +2.2 +7.1 ,
3 1. - 0.93/0.87 +6.8
h 8 - 1.0h/l.28 418.8
s 8 -_ 1.2o/l.28 -6.3 -18.o
6 8 i 0.91/1.28 -29.0
7 12 i" 0.73/l.15 -36.6
8 l2 - 1.0h/l.15 -9.6 -l9.2
9 12 - 1.02/1.15 -11.3
10 16 i: 0.68/0.91 - 5.3
11 16 i_ o.68/b.91 -25.3 -25.7
12 16 i 0.67/o.91 -26.h
13 20 i: o. 71/1.16 -38.8
1h 20 i o. 75/1.10 -31.9 -29.3
15 2o - 0. 91/1. 10 -l7.3
16 2h i: o.75/1.3S ~uu. S
17 2h i_ 0.90/l.35 ~32. h ~39.S
18 2h i 0.79/l.35 -h1. S
19 28 i_ o.76/l.os -27.7
20 28 i 0.55/1.05 -h7.7 -36.6
21 28. i o.69/1.oS 03h.3
22 32 -_ l.06/1.38 —23.2
23 32 i_ 1.01/l.38 -26.9 -27.6
2). 32 i 0.93/1.38 am
25 36 - 0.9h/0.9h o
26 36 - 0.76/o.9h -l9.2 -9.6
27 36 0.85/O.9h -9.6

 

*The information in these columns was obtained from the Division of
EXperimental Chemotherapy, Sloan-Kettering Institute for Cancer

Research .

113

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CHAPTER XII
DISCUSSION OF RESULTS
Experiments with Basidiomycete #2883

Evidence had been obtained through the experiments conducted upon
Basidiomycete #2883 that the tumor-retarding principle can be elaborated
by the mycelium in_zit£2, The control of environment, however, which
is indispensable for this purpose, has not been accomplished as yet and
will require further investigation.

Preliminary Experiments with Coll bia
radicata var. glrfuracea # 0 f

While trying to discover an explanation for the inconsistent
elaboration of the tumor-retarding principle, it was noted that the
flasks had been inoculated with a culture of h06f which was the fourth
consecutive transfer on artificial media the organism had undergone.
Derrick (19h9) mentioned that often Basidiomycete cultures slow down
their growth rate or die after the second through the ninth consecutive
transfer on laboratory media. This may be part of the answer to the
organism's apparent loss of the ability to produce the tumor-retarding
principle 135m. However, it should be noted that in the case of
this organism there was no apparent decrease in the rate of growth,
merely a decrease in the measurable quantity of the production of the

tumor-retarding material, i.e., a physiological change had occurred.

11h

115

The Effects of Nutrilites upon Collybia
radicata gar. filrijlracea

0f the three experiments reported using nutrilites, the last
(Table 5) is perhaps the most important. Lindeberg‘s solution B, being
a purely synthetic medium, was formulated with the idea in mind that
amr response by the test organism in the presence of a nutrilite could
be easily demonstrated.

The results of this experiment indicate that h06f will grow with-
out rmtrilites present. None of the 18 nutrilites tested elicited a
response better than that seen in the control flasks containing Lindeberg's
solution B. Certainly there was nothing like the 15- to 25-fold
increase Lindeberg reported for Collybia ambusta, Q. butyracea, or
Q. velutipes when thiamine was added. The conclusion that could be
drawn, therefore, is that h06f neither requires nor is stimulated by
the nutrilites tested. This, of course, brings up the question of why
h06f grows better when Bacto-Peptone or Bacto-Yeast Extract is present
in the medium. Although there is no positive proof, we can suggest a
possible answer. Marczynski (1983) thought that this might be due to
the presence of more assimilable nitrogen. Fries (19118) said, "The
(yeast) extract in question may serve as a buffer to the solution, it
may contain metals, which are present in the solution in insufficient
amounts, and it may contain carbon-, nitrogen-, or sulphur-compounds
which are more easily assimilated. There is, however, no doubt that

the effect in many cases is brought about by growth factors of a sort

as yet unknown ."

116

These are but a few explanations regarding the stimulatory action
of Bacto—Peptone and Yeast extract. Fries' idea concerning the unknown
growth factors is entirely plausible when we consider the definition of
a growth factor, namely "a substance which is essential for optimum
growth and is effective in minute quantities."

Other investigators have also reported on the effects of unnamed
growth factors. Robbins ( 1939) reported the presence of unknown growth
factors in agar, brown sugar, potatoes, corn meal, and oatmeal. These
unknown substances stimulated 1) the growth and 2) spore germination
of Phycomces Blakesleeanus. In a later paper (19141) he reported the
presence of the same factors (now labelled Z1 and Z2) in Neo-Peptone
and Difco Agar. 21 was found not to be identical with biotin, panto-
thenic acid, glutamine, or para-aminobenzoic acid. Z2 was found not
to be identical with glutamine or para-aminobenzoic acid.

Melin (19146) reported that forest litter extracts increased the
in 233:; growth of L‘actariugs, deliciosus up to 60 times. He suggested
that the active principle in litter extract might be identical with
Robbins' factor 2, and theorized that it might be hypoxanthine. He
tested species of the genera Boletus, Lactarius, Bacillus, Rhizopogon,
Morchella, Psalliota, ClavariaJ Clitocybe, Collybia, Excena, and
Strgpharia (all mycorrhiza-forming fungi), and found that most species
required thiamine, and a few needed biotin in addition. Litter extracts
increased growth“ 150-300 per cent in some cases. He also reported that
some leaf extracts (maple, birch, beech, oak, aspen, and pine) inhibited
growth. Some were intensified in their inhibitory action after being

autoclaved .

Hawker (1936) reported thato while Collybia velutipes grew and
fruited in light. on a mineral-dextrose medium containing potassium

nitrate and 1.5 per cent agar, the addition of a lentil extract in-
creased fruiting.

None of these investigators associated their unidentified growth
factors with either thiamine or riboflavine.

0n the other hand, Robbins and Schmidt (1938) have proved that
Neo-Peptone, a product of the Digestive Ferments Company contains a
small amount of thiamine or its intermediates (about 0.00002 per cent,
enough to cause growth of excised tomato roots). Thus, if five grams
of Neo-Peptone were added to one liter of medium, 100 micrograms of
thiamine would be present in that quantity of medium.

An analysis furnished us by the Difco Company (Detroit, Michigan)
on their product Bacto-Peptone (see Appendix) shows it to contain 2.50
micrograms of pyridoxine, 0.32 micrograms of biotin, 0.50 micrograms of
thiamine, 35.00 micrograms of nicotinic acid, and 1.4.00 micrograms of
riboflavine per gram.

Since five grams of this product comprise part of the formula of
basal medium B, it can be seen that this medium contains 2.50 micrograms.
of thiamine per liter. This is far below the amount of thiamine re-
ported by other investigators as being necessary for the growth of
certain basidiomycetous mycelia.

An analysis furnished by the Difco Company on their product Bacto-
Yeast Extract (see appendix) shows it to contain 20.0 micrograms of

pyridoxine, 1.1; micrograms of biotin, 3.2 micrograms of thiamine,

I ‘1."

118

279.00 micrograms of nicotinic acid, 19.00 micrograms of riboflavine,
and 0.3 micrograms of folic acid.

Since five grams of this product comprise part of the formula of
our medium A, this medium therefore contains 16 micrograms of thiamine
per liter. This is less than one-third of the amount of thiamine that
Lindeberg reported was necessary to stimulate the growth of Collybia
ambusta, _C_. butyracea, and g. velutipes 15- to 50~fold.

The [conclusion one nmst draw then is that none of the nutrilites
when used singly has any stimulatory action upon culture #1406f.
However, it would be wise to note that in Bacto-Peptone and Bacto-Yeast
Extract there are not single. nutrilites present, but rather several in
combination with several amino acids, several salts, proteoses, and
peptones. When they are present in proper balance they may (and only
then) exert an effect which is not seen when they are investigated
singly.

Lewis (1953) believes that a balance exists between the substances
that promote and those that inhibit the growth of an organism. He also
pointed out that it is important to remember that, while one nutrient
may be essential or stimulatory to one species of a specific genus, it
may actually inhibit a second species of the same genus.

Some of these ideas may help explain the results of the first
nutrilite experiment reported (Table 3). Thiamine, riboflavine,
pyridoxine, ribose, and nucleic acid all show at least a 20 per cent
increase in dry mycelial weight over the control flasks (Basal medium E) .

The same nutrilites, when added to Lindeberg's solution B, (Table 5)

119

produced no stimulatory effects. Perhaps it is the new balance which
the Specific nutrilite affects in the presence of the assorted nutrients
(see Appendix for ingredients of basal medium B and analysis of Bacto-
Peptone) that produces the stimulation. These same nutrilites (except
ribose) repeat this stimulatory effect under the similar conditions
reported in the secondexperiment (Table 14).

Skoog (19514) reported that evidence exists to implicate nucleic
acids directly or indirectly as key substances in the chemical regulation

of growth. This may explain the action of the nucleic acid preparation

used (Table 3).

The Effects of CarbOn Sources upon Collypia
radicata var. furfuracea

Considering that all the carbon sources tested were'present in an

amount calculated to yield an equal supply of carbon, some interesting
results were observed when the results were tabulated. Judging from
the first experiment (carbon utilization) the trisaccharides apparently

were most conducive, as a group of carbon sources, to the production of

mycelium by 2. radicata var. furfuracea. The disaccharides were next

best, followed closely by the monosaccharides (the hexoses being better

than the pentoses). The single polysaccharide tested, dextrin, was

next best. The lowest yields were obtained with polyhydric alcohols,

NaHCOa, the single inorganic salt used, and the organic acids.
There are many possible explanations; an examination of the

components of the poly-, tri-, and disaccharides may be informative.

120

 

 

Poly:(tri-L£r di-lLsaccharide . Simple Sugars Present
Dextrin ‘ Glucose

Melezitose Glucose (2), Fructose
Raffinose Glucose, Fructose, Galactose
Honey Glucose, Fructose

Invert sugar Glucose, Fructose

Lactose Glucose, Galactose

Maltose Glucose (2)

Sucrose Glucose, Fructose

Trehalose Glucose (2)

.It1is difficult to comprehend why, for example, melezitose produces
a greater mycelial yield than either of its hydrolytic products (glucose
and fructose) especially when the organism must first produce an enzyme
to hydrolyze this trisaccharide. The same holds true for maltose,
sucrose, and invert sugar. Perhaps the explanation may lie in the
consideration that the biochemical mechanism which enables the organism
to break down the molecule furnishes a stimulus which continues to act
after the initial hydrolysis, and thus makes the organism capable of
utilizing the resulting monosaccharides at a faster rate than if those
carbon sources were available originally in that form.

As previously stated, among the monosaccharides the hexoses were
better utilized than the pentoses. The best of these was galactose,
followed by glucose, mannose, fructose, and sorbose, in this order.

The appearance of galactose as the hexose best utilized by g. radicata

121

z.a_r_. furfuracea was certainly unexpected and rather difficult to explain

 

in view of the findings reported by other investigators concerning vari-
ous fungi. It can only serve to illustrate the variability of preferences
among fungi.

Among the pentoses, xylose appeared to be the best utilized,
followed by l-arabinose, rhamnose, ribose, and d-arabinose, in this
order. The result obtained with xylose appears to confirm the findings
of Lutz (1925), Styer (1930), and Treschow (l9hh).

Disregarding groupings, the (ten) carbon sources best utilized by
g. radicata _v_a_1_‘_. furfuracea were melezitose, galactose, sucrose, xylose,
invert sugar, maltose, glucose, mnnose, l-arabinose, and raffinose, in
this order.

The small amount of mycelium produced by culture #h06f when poly-
hydric alcohols and organic acids were used as carbon sources is
attributed to the respective high and low pH values observed in the
culture media.

As shown in Table 6, the quantity of mycelium produced is not
necessarily accompanied by the production of the tumor-retarding
principle. Only two carbon sources, both hexoses (glucose and mannose),
enabled the organism to biosynthesize measurable concentrations of the
active principle (as determined by the Sloan-Kettering Institute for
Cancer Research).

In order to obtain more information concerning the effect of the
media a statistical analysis of the results was made. It became evident

that some of the media supported the organism in elaborating the

122

tumor-retarding principle although according to the Sloan-Kettering
rating this did not constitute tumor retardation. Significant depression
of the test tumor was noted at the 0.05 level of probability when the
filtrates of the culture media containing citric acid or honey (as the
carbon sources) were injected into the mice. '

The reason why the additional system of evaluation was used to
supplement the Sloan-Kettering method was that in our tests the same
compounds and concentrations of certain nutrients retarded the test
tumors in repeated consecutive experiments. Since it appeared that
some of these replications consistently depressed tumor growth (without
bringing about a depression to the level where the 1'- rating of the
Sloan-Kettering Institute could be given), it was felt that a statisti-
cal analysis would be helpful.

The results of the second experiment (carbon sources added to the
basal medium) indicate that the disaccharides, as a group, were best
utilized by culture #h06f judging from the amount of mcelium (produced.
The monosaccharides were next best, closely followed by the poly-
saccharides tested. The trisaccharides and polyhydric alcohols followed,
and the organic acids and sodium bicarbonate were least useful.

Invert sugar, sucrose, and lactose were the best of the disaccha-
rides, in this order.

Among the monosaccharides, the hexoses produced more norcelium than
the pentoses and the others. Fructose was the best of the hexoses,
followed by galactose and glucose. The yields produced by the pentoses

were very nearly similar. However, xylose and d-arabinose were the

best in this group.

123

The only significant growth produced by a member of the other
monosaccharides was provided by alpha-d-glucose-tpentaacetate.
Melezitose, dextrin, and glycerol were the most important of the tri-
saccharides, polysaccharides, and polyhydric alcohols, respectively.

Among the organic acids used, uric acid produced twice as much
mycelium as the next best carbon source.

Disregarding the individual groupings, the added carbon sources
best utilized by hoof were invert sugar, sucrose, fructose, lactose,
galactose, glucose, dextrin, mlose, d-arabinose, rhamnose, sorbose,
honey, glycerol, ribose, maltose, uric acid, l-arabinose, mannose,
alpha-d-glucose-Jpentaacetate, melezitose, and trehalose, in this order.

Only three of the 10 carbon additives tested made culture #h06f
biosynthesize a tumor-retarding principle. These were galactose,
malonic acid, and sodium bicarbonate.

When statistical analysis was applied to the remaining data, it
became apparent that the filtrates of the additives, mannose and invert
sugar, caused a significant depression of the test tumor at a 0.05 and
0.01 level of probability respectively.

When the findings of the first carbon experiment revealed that
mannose and glucose enabled culture #hOéf to biosynthesize a tumor-
retarding substance, confirmation was essential, as reported previously.
The activity of these broth replications did not reach the level on
which a positive rating, according to the Sloan-Kettering method of
evaluation, could be based. However, statistical analysis of the data

showed both of these preparations to be significant at the 0.05 level

“I."

12h

of probabilit . In addition, a preparation made by extracting the

nycelium of culture #hOéf (grown in mannose) with water, was given a 35
rating (Table 8).

Although none of the additional experiments with glucose yielded
a tumor-retarding principle (as recorded by the Sloan-Kettering method
or statistical analysis of the data supplied by that institute), i"
ratings were obtained for two preparations made from additional experi-
ments with mannose (Table 9). None of the other preparations proved to
have present a mmor-retarding material. Even though at times the test
tumor appeared depressed, statistical analysis of these'data did not
show significance.

Finally, it should be pointed out that three hexoses seem to play
an important role in the biosynthesis of the tumor-retarding principle:
galactose, glucose, and nannose. In contrast to these, fructose--
utilized by h06f not quite as well as the others-never gave any indi-
cation of being involved in the biosynthesis of a tumor-retarding
principle. This may be ascribed to the fact pointed out by Margolin
(19142) that the first three are aldehydes, while fructose is a ketone.
However, it should be kept in mind that the molecular configurations
of glucose, fructose, and mannose are so very similar that they can be
regenerated from either form.

The one very apparent fact that resulted from this investigation
of carbon sources is that mannose, more consistently than glucose or
galactose, though not always, caused _C_. radicata var. furfuracea to

biosynthesize a tumor-retarding principle. Perhaps future experiments,

125

where the extracts could be prepared in larger volume and then concen-
trated by low-pressure evaporation, will prove the contention that this
organism can produce such a material in 23.33. Certainly the results
that were obtained, and especially the statistical data, seem to indicate
the. presence of such a principle. A concentration of the extracts

(as suggested above) may intensify the weak action of such a material

to a degree where the Sloan-Kettering method of evaluation will always

record a i- rating.

Sugar Determination

. Since there was a correlation between the biosynthesis of the tumor-
retarding principle and the presence of certain sugars in the culture
medium, it appeared to be of interest to know the sugar content of
Q. radicatam. furfuracea mycelium produced _i_n m. The possibility
was considered that a supply of a simple nutrient like glucose would
enable the ftmgus to synthesize the active material at an accelerated
rate. - '

Hydrolysis ‘of trehalose was observed to occur more quickly (within
30 minutes) and thoroughly (yielding 98 per cent) under pressure in an
autoclave then when boiled for six hours (yielding 93.5 per cent) as
suggested by Iwanoff (1925).

The carbohydrate content of A. campestris as determined by the
method employed was found to be approximately only one-tenth the figure
reported in the Handbook of Chemistry and Physics (Hodgman, 1950); the

figure obtained for the _ip_-4vitro produced mycelium of g. radicata var.

126

furfuracea was similar to that determined for A. cangestris. The figure

 

obtained for these two materials apparently was more in line with the
evaluation presented by Gussow and Odell (1927) who stated that "small
amounts of nitritive salts and sugars" were contained in A. camgestris.

The carbohydrate content of the context was determined to be less
than half of that found for the whole mushroom. No explanation, other
than to compare this variation with similar variations occurring in
distinctive tissues of higher plants, is attempted.

The tissue of g. radicata Lag. mriuracea grown An 3.7.1.353. appeared
to have a sugar content comparable to that of the commercially culti-
vated A. campestris. Not only have the results of previous tests shown
that the elaboration of the active principle is not affected by the
presence of excess sugars, but it was noted that no excess sugar was
accumulated in the tissue.

Tne moisture content of A. cafimpvestris was difficult to obtain
accurately because evaporation occurs while the weighing takes place.
It is easy to understand the variation occurring in the literature
concerning moisture content when one observes this fast drying. The
results obtained by different investigators may have differed because
of the evaporation that occurred between the time the specimens were
collected and the time they were evaluated. Another possibility for

the varying figures is the moisture content of the specimens at the

time of collection .

127

The Effefiiéiiitiiyflf; 563132;: 2.”? B36~gL-§° b“

Using the weight of the mycelium produced as the criterion, the
individual nitrogen sources best utilized (as established in Table lb)
by g. radicata 1a; . furfuracea were yeast extract, malt extract, dried
skim milk, DL-serine, and L-asparagine, in this order. As a group,
the miscellaneous nitrogen sources were far better utilized than the
amino acids or inorganic forms investigated. In this respect the
findings reported here correspond with those of LaFuze (1937) and
Derrick (191:9).

When the nitrogen sources studied were employed as additives
similar results were obtained, judging by the dry weights of the mcelia
produced. The best single nitrogen sources were yeast extract, dried
skim milk, malt extract, urea, L-qnethionine, DL-serine, L-leucine, and
ammonium tartrate, in this order. Again the miscellaneous group of
nitrogen sources produced more mycelial growth than did the amino acids
or the inorganic sources. However, in this case, the difference was
not as great as it had been previously.

None of the "beers" shipped to the Sloan-Kettering Institute for
evaluation contained enough of a tumor-retarding principle to receive a
rating of i". The slight retardation of the test tumor evoked by the
"beers" when dried skim milk was used as either the sole nitrogen
source or a nitrogen additive was not duplicable. Similarly, the
results obtained using extracts of the combined carbon- and nitrogen-

sorurces media were negative. Although two of the eight samples showed

128

a. slight retardation of the tumor, the results were judged to be
inconclusive as they were not readily duplicated.

In any case it appears safe to state that the nitrogen sources
studied were not used by _c_. radicata 1735' furfuracea to produce the
tumor-retarding principle either directly or indirectly.

Miscellanigljlscaibatpz‘éme—rgi 51:11:21 egollybia

None of the final experiments with _C_. radicata 19.3.. furfuracea;
demonstrated activity of a nature that was reproducible. However, one
item of major interest was observed in the experiments where cheese-
cloth strips and milk filter discs were suspended from the necks of
the flasks. The strips and discs were supported in such a manner that
the bottom edge was submerged in medium A. A suspension of culture
#hObf was used to inoculate the entire length of the strips and discs.
What proved of interest ms the growth of the nycelia along the upper
portions of the strips and discs. This nycelium was not unlike that
of the context of g. radicata m. furfuracea sporOphores. It was firm
and "snapped" apart when bent, quite unlike the cottony nycelia seen
growing on the surface of agar plates or broth cultures, and also very
different from the flaccid, wet spheres observed in shake cultures.
The samples prepared from these nycelia, however, were also negative.

Screening of Basidiomcete Sporophores for
Tumor-Retarding Properties
These preliminary tests cannot determine in any manner whether the

active compound is the same in all cases. Certainly the intensities of

129

the tumor-retarding principles vary, as evidenced by a comparison of
the results obtained with Bole’ms Frostii or Calvatia maxima and those

obtained with Collybia radicata var. fluflracea or Hydrmm septentriomle‘

 

(Table 17) .

Experiments with Calvatia maxima #6h2

While determining the temperature range and optimum temperature
for the growth of 9. w #6142, a very marked effect was noted in the
28-3o°c. range. This organism remained viable at 28.5%., producing
a colomr approximately one-third of that observed when it was grown in
the 22-2600. range. The thermal death point of the organism apparently
was between 29 and 30°C. The growth that occurred in the lower
temperature range-«although not spectacular-dues fairly uniform.

As shown in Figure V and Table 18, the tumor-retarding principle
becomes evident in the samples prepared from the eight-day culture and
gradually intensifies until a i“ rating is observed in the samples
prepared from the 16-day cultures. Figure V demonstrates (in the 8-
to 16-day area) the validity of the author's contention in employing
statistical analysis on some of the carbon and nitrogen experimental
data presented earlier. It was stated then that certain preparations
gave an indication of possessing tumor-retarding preperties, even
though the evaluation by the Sloan-Kettering Institute resulted in a
negative rating.

At 19°C., 2. w #6142 demonstrated maxinnlm production of the

morretarding principle at 21; days. The active material gradually

130

lessened-«although still evident in the samples prepared from the 32-day
cultures-«mtil it was no longer evident in the 36—day cultures,
possibly suggesting autolysis.

The 19°C. temperature ms found to be the optimum condition for
the production of nycelium coupled with the elaboration of the tumor-
retarding principle. For this reason only the 19°C. study is presented.
In the other four temperature experiments, the tumor-retarding principle
first appeared in the samples prepared from the 2h- or 28-day cultures.

The original purpose of the work undertaken was the A}; m
production of tumor-inhibitory principles by Basidiomcetes. The
principle first encountered in Boletus edulis sporOphores was thought
to be suitable for experimentation. As this study progressed it
became obvious that the difficulties, although they were not considered
insurmountable, were of such nature that a solution of the problem
would require more time than could be allowed for the performance of
a thesis research.

The substitution of a different organism which had also shown
tumor-«retarding properties. in sporophore preparations was therefore
considered. .Collybia radicata m. furfuragga was selected. It should
be emphasized that this selection was made on' the basis of information
at hand at that time and that it represented the best possible choice
available. As in every experimental study a risk had to be taken
which, as it turned out later, became quite critical. The extensive
cultural studies made with this organism failed to produce conclusive

information as to its exact requirements, and it remains to be seen

131

whether this problem can be solved at all. The sporadic production of
active extracts shows that the tumor-retarding principle can be pro-
duced in cultures of the organism. That it has been impossible to
produce it consistently may be due to experimental shortcomings of
which the writer has not become aware. It is conceded that on the
basis of the experience acquired during this work further attempts
might be successful. Such hope, however, was insufficient assurance
that the thesis research could be crowned by positive findings.

Out of this consideration—and it may be said that the decision was
not an easy one-nanother substitution of the test organism was made.
The choice of Calvatia maxima turned out to be a fortunate one. It could
have been another failure, of course, and in that case undoubtedly a
further attempt wouldhave been made to find still another organism
which would satisfy the requirements. If this research, therefore, may
be found lacking a deliberately outlined experimental procedure which
could be assessed beforehand to produce results, its unusual nature
should be taken into consideration. On the other hand, it perhaps
proves that persistence is as useful in research as experimental skill.

With the Calvatia. maxim experiments it has been shown—and it is
believed for the first time-that a tumor inhibitor originally observed
in sporophore extracts could be produced at will and consistently in
laboratory culture.

It can be presumed that the results of this study will serve the
purpose of stimulating in. 31.359. production of tumor inhibitors found

in the sporophores of other Basidiomcetes.

132

Summary

Tests conducted at the Division of Experimental Chemotherapy,
Sloan-Kettering Institute for Cancer Research disclosed that some
aqueous extracts of Basidiomcete sporOphores contained tumor-retarding
substances. Experiments designed to develop methods of producing these
biologically active substances under controlled conditions by laboratory
cultures were described.

Boletus edulis 331;. pinicola, #288j, was the first organism investi-
gted. Stationary and shake cultures were grown employing various
media and environmental conditions. Preparations made from these
cultures at times showed the presence of a tumor-retarding substance,
but when duplication was attempted the results were inconsistent.
Attempts to reach the goal with this organism were unsuccessful largely
because of the difficulties of growing it with sufficient ease.

Collybia radicata gag. furfuracea, #h06f, was the next organism
investigated. It was chosen chiefly because of its relatively fast
growth rate. Various mltrilites, and carbon and nitrogen sources were
studied as they affected the growth of nycelium and the elaboration of
the honor-retarding substances. Preparations made from cultures grown
in media incorporating mannose or glucose at times indicated the
presence of a tumor-retarding substance. However, these results could
not be duplicated consistently. Attempts to reach the goal with this
armism were also unsuccessful because the requirements of this
organism with respect to the production of the tumor-retarding principle

133

could not be established with certainty in spite of the fact that the

requirements for good mycelial growth were satisfied.
Calvatia maxima, #6h2, was the final organism studied. _ An investi-
gation of various temperatures and growth periods disclosed that the

minor inhibitor which occurs in the sporOphore was produced consistently

by laboratory cultures.

13h

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Van Niel, C. B. Recent advances in our knowledge of the physiology of
microorganisms. Bact. Rev. 83225-2314 (191414).

Werkman, C. H. and Wilson, P. W. Bacterial physiology. Academic Press,
Inc., New York, N. Y. 707 pp. (1951).

Wilkins, W. H. Investigations into the production of bacteriostatic
substances by fungi; cultural work on Basidiomycetes. Trans. Brit.
Wool. Soc. 283110-1114 (19145).

. Investigations into the production of bacteriostatic substances
by fungi; preliminary examimtion of more of the larger Basidiomycetes
and some of the larger Ascomycetes. Ann. Applied Biol. 33:188-190

(19h6a).

. Investigations into the production of bacteriostatic sub-

stances by fungi; preliminary examination of the fifth 100 species,
all Basidiomycetes, mostly of the wood-destroying type. Brit.
Jour. Exp. Path. ELMO-1142 (l9146b).

1143

Wilkins, W. H. Investigations into the production of bacteriostatic
substances by fungi; preliminary examination of the sixth 100
species, more Basidiomcetes of the wood-destroying type.

Brit. Jour. Exp. Path. 28: 53-56 (19h7a).

. Investigations into the production of bacteriostatic sub-
stances by fungi; preliminary examination of the seventh 100
species, all Basidiomycetes. Brit. Jour. Exp. Path. 28:2h7-252
(l9h7b).

. Investigations into the production of antibacterial substances;
preliminary examination of the eighth 100 species, all Basidionwcetes.
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. Investigations into the production of bacteriostatic sub-
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and Harris, G. C. M. Investigations into the production of
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Basidiomycetes. Ann. Applied Biol. 3152619270 (191.414) .
Williams, R. J. Nutrilites. Science 61:60? (1928).

. Growth-Promoting nutrilites for yeasts. Biol. Rev.,
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VIII. Mixed cultures. Ann. Missouri Bot. Gard. 9183-192 (1919).

APPENDIX

.E"

Medium.A

Agar

MgSO4.7H20

KCl

KH'2PO4

FeSO4-7H20

Glucose

Sucrose
Bacto-Peptone
Bacto-Yeast Extract

Distilled water:

pH

11414

15.0 grams
0.5 gram
0.5 gram
1.0 gram
0.01 gram

15.0 grams

15.0 grams
5.0 grams
5.0 grams
bring up to 1000 ml.
5.6

Basal medium (Medium B)

Agar
MgSO4.7H20
KCl

KHz'PO4
FeSO4.7H20
Glucose
Sucrose

Bacto-Peptone

Distilled Whter:

pH

15.0 grams

0.5 gram

0.5 gram

1.0 gram

0.01 gram
15.0 grams‘
15.0-grams

5.0 grams

'bring up to 1000 ml.
5.6

1&5

Sodium caseinate medium (Medium G)

Sodium caseinate
Glucose

KgHPO4

MgSO4.7H20

FeSO, (0.1% solution)
Peptone

Glycerine

Agar

Distilled water to

pH not adjusted, usually 7.3 before autoclaving.

2.0
1.0
0.2
0.2
1.0
1.0
5.0
15.0

1000

gram
gram
gram
gram

n1.

gram
gram
gram
m1.

1&6

Khudiakov's medium (Medium J)

Glucose
NH4N03
KH2'P04
MgSO4
CaCl2

NaCl

Casein hydrolysate containing a 0.5%

tryptophan solution
Agar
Fe012
CuSO4
Na 23407
NagMOO4
MnCl2
ZnSO4
Nicotinic acid
Para-aminobenzoic ac id
Riboflavine
Calcium pantothenate
Biotin
Thiamine

Distilled water:

1h?

15.0 grams
1.0 gram
1.0 gram
0.5 gram
0.1 gram

0.1 gram

5.0 grams
15.0 grams
0.6 mg.
0.23 mg.
0.0h5 mg.
0.0h3 mg.
0.0h6 mg.
b.300 mg.
1.000 mg.
1.000 mg.
1.000 mg.
1.000 mg.
1.000 mg.
1.000 mg.

bring up to 1000.ml.

Note: The agar content was modified to read 20.0 grams, to
give a firmer more solid medium when this was desired.

The casein hydrolysate was modified to read 5.00 ml.
casein hydrolysate and 0.025 gram tryptophan.

1&8

Carrot Medium

Carrot slices 200 grams
Distilled water 500 ml.
Steam for 30 minutes, then filter through cheesecloth.

Add distilled water to bring volume up to 1000 m1.

Agar 20 grams

lh9

Fries Mineral Medium

Ammonium tartrate 5.0 grams
Ammonium nitrate 1.0 gram
KHzPo4 1.0 gram
NaCl _ 0.1 gram
CaCl2 (anhydrous) 0.1 gram
MgSO4.7H20 0.5 gram
Sucrose 15 .0 grams
Distilled water: bring up to 990 ml.

Autoclaved at 15 lbs. pressure for 12 minutes.

10.0 ml. of the following vitamin solution added after cooling:

Inositol 140 mg.
Para-aminobenzoic acid 0.3 mg.
Thiamine HCl 0.3 mg.
Pyridoxine HCl 0.14 mg.
Biotin 0.005 gamma

Distilled water: bring up to 1000 ml.

150

Lindeberg's Nutrient Solution B

Glucose 20.0 grams
Ammonium tartrate 5.0 grams
KH2P04 1.0 gram
MgSO4.7H20 0.5 gram
FeCl3 solution (Fe conc. 1/500) 0.5 ml.
ZnSO4 Solution (Zn conc. 1/500) 0.5 ml.
MnCl2 (0.1 M solution) 0.5 m1.
CaCl2 (0.1 M solution) 5.0 ml.
Distilled water 995.0 m1.

pH after autoclaving is 5.5

151

Typical analysis of Bacto-Yeast Extract*

Per Cent

Ash

Total N
Chloride
Total Suphur

PPM

Lead
Arsenic
Manganese
Zinc
Copper

Per Cent

Phosphorus
Iron

SiO2
Potassium
Sodium
Magnesium
Calcium
.Arginine
Aspartic acid
Glutamic acid
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phylalanine
Threonine
Tryptophane
Tyrosine
Valine

.144”me 28: saw

Pyridoxine
Biotin
Thiamine
Nicotinic acid
Riboflavine
Folic acid

1—‘O\OO

16.00
0.11
7.8

88.00

19.00

0.89

0.028
0.052
0.0142

0
W
N

00
(DI-“W

OO

O\

c?Chcncrha;acnbxwaggcr0u+4~u

O O O
\O

ch)c)uJhJC):‘anJC)h0ChUfiCDC)CDC)
c>cn

fAnalysis furnished by Difco Laboratories, Incorporated, Detroit 1,

Michigan

Typical analysis of Bacto-Peptone*

Total Nitrogen
Primary Proteose N
Secondary Proteose N
Peptone N
.Ammonia N

' Free.Amino N (Van Slyke)
Amide N
Mono-amino N
Di-amino N
Tryptophane
Tyrosine

Cystine (Sullivan)
Organic Sulphur
Inorganic Sulphur
Ph03phorus
Chlorine

Sodium

Potassium
Calcium
Magnesium
Manganese

Iron

Ash

Lead

Arsenic

Zinc

Copper

SiO2

.Arginine
Aspartic acid
Glutamic acid
Glycine
Histidine
Isoleucine
Leucine

Lysine

Methionine
Phenylalanine
Threonine

valine
Pyridoxine
Biotin

Thiamine
Nicotinic acid
Riboflavine

fAnalysis furnished by Difco Laboratories, Incorporated, Detroit 1,

Michigan.

16.16%

0.06%

0.68%

15.38%

0.0h%

3.20%

0.h9%

9.h2%

1.07%

0.29%

0.98%

0.22%

0.33%

0.29%

0.22%

0.27%

1.08%

0.22%

0.058%

0.056%

nil

0.0033%

3.53%
15.00 ppm

0.09 ppm
18.00 ppm
17.00 ppm
0.082%

8.00%

5.90%

11.00%

23.00%

0.96%

2.00%

3.50%

h.30%

0.83%

2.30%

1.60%

3.20% ,
2.50 gamma/gm.
0.32 gamma/gm.
0.50 gamma/gm.
35.00 gamma/gm-
h.00 gamma/gm.

152

a ‘27:.

Date Due

 

Demco-293

Thesis

Stevens, Joseph A.
Studies of enviro

factors influencing ...
growth of basidiomycetes.

nmental

1’Thesie

1 Stevens J

1 Ph.D. . oseph A.

7 Studies of environment-

factors influenting ... K
growth of basidiomycetes.

 

 

 

 

 

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