I ' i I- 77-2426 JONES, Jeanette, 1950- GROWTH AND MORPHOLOGY OF HORTAE (BRUMPT) FONSECA AND A R & LEAO: THE CAUSAL AGENT OF BLACK . The Ohio State University, Ph.D., 1976 Botany

Xerox University Microfilms,Ann Arbor, Michigan 48106

© 1976

JEANETTE JONES

ALL RIGHTS RESERVED Growth and Morphology of Piedraia hortaa (Brumpt) Fonseca and Area Leao

The Causal Agent of Black Piedra

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree

Doctor of Philosophy in the Graduate School of

The Ohio State University

hy

Jeanette Jones, B. Sc., M. Sc,

The Ohio State University

1976 Reading Committee Approved by

J. A, Schmitt, Chairman M* 0. Garraway Adviser V, Raghavan Department of Botany R. L. Seymour C. E. Taft ••..the fungi are not degenerate organisms which are on their way out in a scheme of evolution, and so of little economic importance and scientific interest. The fungi, on the contrary, are progressive, ever changing and evolving rapidly in their own way so that they are capable of becoming readily adapted to every condition of life. We may rest assured that as Green Plants and Animals disappear one by one from the face of the globe, some of the fungi will always be present to dispose of the last remains.

B. 0. Dodge (1939)

ii ACKNOWLEDGEMENTS

I am particularly indebted to Dr. J. A Schmitt for his guidance and support when they were needed most. A special thanks is extended to the members of the reading committee, Dr. J. A. Schmitt, Dr. M. 0.

Garraway, Dr. V. Raghavan, Dr. C. E. Taft and especially Dr. R. L.

Seymour for their critical reading of this manuscript and their help­ ful suggestions during the final wording of the manuscript.

Gratitude is also extended to Dr. R. Vanbreuseghem, Institute of Tropical Medicine, Antwerpen, for providing the isolates of , and the specimens used in this study.

Appreciation is extended to the: various individuals in the Depart­ ment of Botany, both faculty and graduate students, for their suggestions and/or technical assistance.

Appreciation is also extended to my parents, Willie and Alice Jones, my brothers, and sisters, relatives and friends, who have been under­ standing, thoughtful and encouraging during my graduate studies.

Thanks are extended to my undergraduate adviser, Dr. W. D. Moorehead and the many people not mentioned by name who have contributed to making this manuscript possible.

iii Vita

Sept. 19, 1950 ...... Born, Fort Valley, Georgia

1972 ...... ».B. Sc., The Fort Valley State College, Fort Valley, Georgia

1972 Instructor of Biology Fort Valley State College

1972 ...... Research Assistant Southeastern Forestry Exp. Laboratory, Macon, Georgia

1972-73 ...... University Fellow, Dept, of Botany, The Ohio State University, Columbus, Ohio

1973-75 ...... Graduate Teaching Associate The Ohio State University Columbus, Ohio

1975-76 ...... Dissertation Year Fellow The Ohio State University Columbus, Ohio

1976 ...... Consultant, Medical and Other Health Professions Career Day Fort Valley State College Fort Valley, Georgia

Publication

Jones, Jeanette and J. A. Schmitt, The Effects of Chlorination on the Survival of cells of . Mycologi’a (in press)

Field of Study

Major field: medical mycology

Studies in medical mycology, Professor J. A. Schmitt

Studies in general and aquatic mycology, Professor R. L. Seymour

Studies in fungal physiology, Professor M. 0. Garraway

iv TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS 00a0*0*000a00#*a000»0a00a00a000«00a«a0»a0#000 ill

VITA 00*0*OMO«OOIOMOOO«OMDOOOOOOMO*OI*ll»OOIOO««»«MOOOtOO IV

LIST OF TABLES #00oo oooo»oo*ooooo*o*o**o*o*o*«n**oo«**o**o*o*o VX

LIST OF FIGURES ...... viii

LIST OF PLATES ...... c.a...... ix

INTRODUCTION «a*aaoo*o*o*»«*eoo»ooo**«**o**a»*oao»»o**»»»os*oo i

MATERIALS AND METHODS...... 4

RESULTS AND DISCUSSION......

Chapter

™ VITRO INVASION OF H A I R ...... 12 A, Introduction Ba Hyphal Structure C. Hyphal Development on Hair

H o NUTRITION AND TEMPERATURE 28 Ao Preliminary Studies 1* Carbon Sources 2* Hydrogen ion concentration B* Temperature ..... Co D® Amino Acids E0 Fatty Acids F* Vitamins

CONCLUSIONS 0 0 0 0 aocooaoooooooooaoooaoooaaooaooooooooooaooaoo»

LITERATURE CITED Oooooooooaooaooaooooooaoaaooooaoooaaoaoooooo

V LIST OF TABLES

Table Page

1. Growth of P. hortae on from Afro-American and Caucasian females, artificially infested, with observation at various incubation times at 25 C ...... •.••»<> 23

2. Dry weight of JP. hortae in basal medium with various carbohydrates added at 5.0 g/1 concentration after 8, 12 and 18 days incubation at 25 C •••••... o ...... *• 29

3. Dry weight of P. hortae in basal medium with various carbohydrates added at 10.0 g/1 concentration after 8, 12 and 18 days incubation at 25 C 30

4* Effect of initial pH on the growth of P, hortae in arginxne-maltose medium after 8 and 12 days incubation at 25 C 33

5. Effect of various temperatures on the growth of P. hortae in malt extract medium after 8, 12 and 18 days incubation at various t e m p e r a t u r e s ••••»•••• 37

6. Effect of various keratin concentrations (percentages) on . the growth of JP. hortae. measured as dry weight (mg), and the final pH at a 25 C incubation temperature...... *c 43

7* Growth of P. hortae on basal medium with maltose as the carbon source and five organic nitrogen sources supplied at three concentration levels with 8 days incubation at 25 C .••....«•» 45

8. Growth of P. hortae on basal medium with maltose as the carbon source and five organic nitrogen sources supplied at three concentration levels with 12 days incubation at 25 C ...... 46

9. Growth of P. hortae on basal medium with maltose as the carbon source and five organic nitrogen sources supplied at three concentration levels with 18 days incubation at 25 C ...... *

10. Effect of various nitrogen sources on the final pH value of the culture medium with maltose as the carbon source and five organic nitrogen sources supplied at three concentration levels with 8 days incubation at 25 C...... 11* Effect of various nitrogen sources on the final pH value of the culture medium with maltose as the carbon source and five organic nitrogen sources supplied at three concentration levels with 12 days incubation at 25 C **•• 49

120 Effect of various nitrogen sources on the final pH value of the culture medium with maltose as the carbon source and five organic nitrogen sources supplied at three concentration levels with 18 days incubation at 25 C *.** 50

13* Growth of P*. hortae after 12 and 18 days incubation at 25 C on basal medium with, arginine supplied at three concentrations with/without the addition of maltose.***** 52

14, Effect of the addition of fatty acids and a vegetable oil to SDA at various concentrations (percentages) on the growth of P, hortae at 25 C «0.* 0 62

15. Growth of P. hortae on basal medium containing three con­ centrations of arginine supplemented with biotin and/or thiamine with 12 days incubation at 25 C ••••••••o******* 66

16. Growth of I\> hortae on basal medium containing three con­ centrations of arginine supplemented with biotin and/or thiamine with 18 days incubation at 25 C *..,.,..,....0** 67

17, Growth of P* hortae on basal medium containing three con­ centrations of arginine supplemented with biotin and/or thiamine with 24 days incubation at 25 C •••.•••,.•••*••* 68

vii LIST OF FIGURES

Figures Page

1. Effect of hydrogen ion concentration on the growth of P. hortae in arginine-maltose medium after 8 and 12. days incubation at 25 C ...... 35

2. Effect of various keratin concentrations (percentages) on the growth of P. hortae, measured as linear extension (cm) at various incubation times (days) at 25 C ...... 40

3. Effect of various keratin concentrations (percentages) on the growth of P. hortae, measured as dry weight (mg), with incubation at 25 C ...... 41

viii LIST OF PLATES

: Plate Page

.1. Stereoscan electron micrographs of Pe hortae cells cultured in malt extract medium at 25 C and observed at various magnifications.•..••••••• 15

II. Stereoscan electron micrographs of P. hortae cells embedded in an amorphous substance and observed at various magnifications...... 17

III. Photomicrographs of P. hortae cells on human hair segments observed at various magnifications. 19

IV. Stereoscan electron micrographs of artificially infested hairs observed at various magnifications. 22

V. Stereoscan electron micrographs and X-ray point analysis of P. hortae cells on hair obtained from a Bolivian source...... ,..,...... ©.. 26

VI. Photomicrographs of P. hortae cells cultured in basal medium with arginine and lysine as the nitrogen sources incubated at 25 C 54

VII. Photomicrographs of P. hortae cells cultured in basal medium with cystine and tryptophan as the nitrogen sources incubated at 25 C ..••••.••••••• 56

VIII. Photomicrographs of JP. hortae cells cultured in basal medium with histidine and keratin as the nitrogen sources incubated at 25 C ••••••••••.••• 58

IX. Photomicrographs of JP. hortae cells and crystals produced in basal medium with arginine and cystine as the nitrogen sources incubated at 25 C ...... 60

ix INTRODUCTION

Piedraia hortae^- (Brumpt) Fonseca and Ar§a Leao (1928) is the eti­ ological agent of black piedra, a fungal infestation of the hair shaft, characterized by dark, hard, irregularly placed and shaped nodules

(ascostromata), hence, the name ''piedra11, which is Spanish for stone.

Microscopic examination of the mycelium reveals dark, thick-walled, closely septate hyphae containing numerous chilamydospores or swollen, irregularly shaped cells.

According to Castellani and Chalmers (1919) the has been known in Colombia since remote times, but was not described until 1878 by Dessene. Subsequently, Behrend (1890) reported cases of black piedra in Colombia on hairs of the head being caused by Trichosporum giganteum

(Behrend). However, several investigators in Brazil examined cases of black piedra, and in 1911 Horta described spherical cysts in nodules of

Brazilian piedra, each of which contained ciliated bodies. The presence of these cysts led Brumpt in 1913 to separate the causal agent of Brazil­ ian "piedra” under the name Trichosporum hortae from the Colombian form known as Trichosporum giganteum. Fonseca and Area Leao (1928) suggested that the Colombian form was too poorly described to determine if the cysts reported by Horta (1911) were indeed present, and that the cysts were really asci with fusiform . These authors

later obtained cultures of piedra and from these observed black, dry colonies covered with aerial hyphae which corresponded to Brumpt9s

^Orthography in accordance with proposal made by: Borelli, D. 1959. El nombre correcto es Piedraia hortae. Derm. Venezolana 1; 357.

1 T, hortae. In old cultures of T, hortae, they observed fusiform elongated spores similar to those present in the nodules of Brazilian

"piedra". Based upon these observations, they established the new genus Piedraia based on P. hortae (Brumpt) Fonseca and Area Leao,

Since 1928, subsequent investigators have reported cases of this mycotic infestation and its occurrence in various parts of the world

Langeron, 1929; Langeron and Vanbreuseghem, 1965; Brumpt and Langeron,

1934; Boedyn and Verbunt, 1939; Lewis and Hooper, 1948; Kaplan, 1959;

Van Uden et al,, 1963; Moyer and Keeler, 1964; Fischman, 1965, 1973;

Emmons et al,, 1970; Ajello, 1964, 1971; Takashio and De Vroey, 1975),

These accounts, however, were restricted to reporting the case and describing the condition. Several workers have suggested or proposed new species of Piedraia for cases of black piedra endemic to their geographical regions. However, these taxa have been found to possess morphological features not that different from those of the type species and have been reduced to synonomy with P. hortae (Brumpt and Langeron,

1934; Dodge, 1935; Simons, 1954; Emmons et al,, 1970; Ajello, 1971;

Conant et al,, 1971). Recently, observations of the ascosporic state and its morphological features utilizing transmission electron micro­ scopy have been reported (Chong et al,, 1975). There is, however, little information presently available concerning the life cycle or nutritional requirements of JP. hortae (Dodge, 1935; Ajello, 1971).

Delamare and Gatti (1928) used cultures of P, hortae obtained from

Paraguay for some nutritional studies. The results of their study indicated that the could utilize glucose, maltose, lactose, levulose, and glycerine agar media. They also reported that growth

at room temperature was slow and almost nil at 37 C. The only other

investigation dealing with the nutrition of this fungus was that by

Takashio and Vangreuseghem (1971). They reported that the in vitro

production of ascospores by P. hortae is considerably enhanced by-

transferring the fungus from one concentration of an agar medium to a medium of lower concentration.

Some general conclusions established by previous investigators

are that cells of JP. hortae form nodules on hair of the scalp, that it

is usually restricted to tropical and semi tropical regions, and that

it grows very slowly in the laboratory on agar media at room temperature.

There are, however, several questions which remain unanswered because

of the lack of information concerning the nutrition of this organism.

Why does this fungus grow only on the hair? Does it utilize hair as

its sole source of nutrients? Why is it found predominately in warm

climates? These are the questions that have been addressed in this

study. This study provides insight into those factors influencing

the geographical distribution of the fungus and its occurrence on hair. MATERIALS AND METHODS

I, Maintenance of fungal cultures

All experiments were carried out using an isolate of JP. hortae

and hair specimens, obtained from the laboratory of Dr. R. Vanbreuseghem

(The Institute of Tropical Medicine Prince Leopold, Antwerpen). The

cultures were maintained on Sabouraud dextrose agar (SDA) at 25 C and

subcultured monthly.

II. Inoculum

Ascospores of P. hortae are rarely obtained in the laboratory (Ajello,

1971), thus, the methodr of inoculating experimental media with 0.5 ml

aliquots of- and hyphal fragments of the test fungus was used.

III. Measurement of growth

To determine the dry weight of the fungus, three flasks were re­

moved from the experiment at the various time intervals, separated

from the medium by vacuum filtration on oven dried, tared Whatman #1

filter paper and washed with 10 to 20 ml portions of distilled water

to remove any residual medium. The mycelium was oven dried at 70 C for

at least 24 hours, then weighed. Dry weights were determined with a

single pan Mettler balance and the values rounded to the nearest milligram*

Each value reported is the mean of three replicates.

IV. In vitro invasion of human hair

Hair invasion by P. hortae was examined by the methods of Ajello

and George (1957). Five centimeter segments of adult human hair from a

Caucasian female and an Afro-American female were autoclaved (121 C,

4 for 15 min. ) and placed in petri dishes containing sterile distilled water to which two drops of sterile 10 % or malt extract were added. The medium was inoculated with the test fungus, and these cultures were incubated at 25 C. After various intervals, infested hair segments were removed and processed for ordinary light, and scanning electron microscopy.

V. Specimen preparation for microscopy

For better interpretation of the scanning images and specific identification of different structures or ornamentation, segments of mycelium and in vitro infested hair specimens were freshly mounted in lactophenol and examined using the ordinary light microscope.

Observations with the scanning electron microscope were made using mycelium and infested hairs from the malt extract enriched medium.

The mycelium was fixed in 3.0 per cent gluteraldehyde for one hour.

The tissue was rinsed with 0.1 M phosphate buffer (pH 7.2) and post fixed in 1.0 per cent osmium tetroxide. Fixed material was later rinsed with distilled water and dehydrated through a graded series of ethanols. The mycelium was then transferred through a graded series of amyl acetate and dried by the critical point drying method, using

liquid carbon dioxide as a transition fluid. Specimens were vapor coated first with carbon, then with gold on a rotary stage of a vacuum evaporator, rotating at an angle of 45 degrees to the vapor source.

The critical point dried and air dried specimens were mounted with con­ ductive silver paint and observed with a Cambridge S4-10 Stereoscanning

electron microscope, at 10 - 20 Kv. VI. Temperature

Mycelium of the test fungus, maintained on malt extract medium

was washed several times in triple distilled water, and homogenized

using a Waring blender. Aliquots containing 0.5 ml of chlamydospore

and hyphal fragment suspensions of P. hortae. were inoculated into

250 ml Erlenmeyer flasks containing 50 ml aliquots of malt extract

medium, A minimum of 10 replicates were maintained for each temperature

level. The temperature levels tested were 18, 22, 25, 30, and 37 C.

At different incubation times, three replicates were used to obtain

the tissue dry weight. Dry weights were determined as previously des­

cribed in the section on the measurement of growth.

VII. Hydrogen ion concentration

The pH of the liquid medium was determined at the time of harvest.

The mycelium was separated from the test medium by vacuum filtration and

the resulting medium tested.

To determine the pH of the solid medium, the colony plus the agar

medium for each treatment was macerated and placed in 10 ml of distilled

water. This was left for at least two hours with occasional stirring,

and then measured with a Corning glass electrode research pH meter.

For those experiments designed to test the growth of the fungus

on medium at different pH values, arginine-maltose medium was used.

Forty-five ml of medium were distributed equally into 250 ml Erlenmeyer

flasks, and the pH adjusted before autoclaving. The adjusted pH values

were 2, 4, 6, 8, and 10, and are considered the initial pH values.

Each flask was inoculated with 0.5 ml aliquots of cells of ]?. hortae, incubated at 25 C and observed at various intervals. The pH value of the medium at the time of harvest is considered the final pH. The values reported are the mean value of three replicates.

VIII. Fatty acid and vegetable oil experiment

The following oil and fatty acids were used: Castor oil, a vegetable oil; two unsaturated fatty acids, oleic and linoleic acid; and one saturated fatty acid, stearic. The oil and fatty acids were incorporated without prior sterilization in SDA medium containing

0.05 mg/1 of chloramphenicol. The percentage concentrations employed were 0.5, 1.0, 2.0, 3.0, 5.0 and 10.0. In order to ensure a uniform suspension of these substances in the medium, thorough swirling was done before the agar medium solidified. Control plates did not contain oil or fatty acids. However, all plates were inoculated with the test fungus and incubated at 25 C for at least two months. The results were recorded as growth observed or no growth observed.

IX. Keratin

Hair keratin (ICN Pharmaceuticals, Inc.) was used in these experi­ ments. The keratin was added, at five different concentration levels, to 3.0 per cent purified Bacto agar, autoclaved, and distributed into sterile disposable petri dishes. Controls containing only 3.0 per cent purified Bacto agar were maintained throughout the experiment.

All of the media were inoculated with the test fungus, and incubated at 25 C. At least ten replicates were maintained for each concentration level. Observations on growth were made at frequent intervals and colony diameters were measured. Using three replicates, the fungal colonies were separated from the agar medium, and the dry weights determined as previously described in the section on growth measurement.

X. Nitrogen experiments

The inoculum was obtained from cultures of P. hortae maintained in 10 % malt extract medium. The mycelia were removed from the liquid, placed in sterile tubes and centrifuged at 2,400 X g. The supernatant was decanted and the remaining mycelium washed several times with sterile distilled water to remove all traces of the malt extract medium.

The contents were later placed in a sterile Waring Blendor and homogen­ ized into a hyphal fragment and chlamydospore suspension. This served as the best inoculum source, because it is difficult to obtain an a- bundant or average number of spores from cultures of P. hortae, in addition to the fact that the organism is a very slow grower in the laboratory. The test medium was inoculated with 0.5 ml aliquots of the fragmented, hyphal suspension and incubated on shakers oscillating at 100 cycles/minute at 25 C.

The basal medium contained a carbon source, a nitrogen source, macronutrients, and micronutrients. The exact amount of each consti- uent of the medium is as follows:

Maltose 10.00 g

MgS04 .75 g

KH2p04 1.50 g

CuCI2 0.1 mg

MnCl2 0.1 mg

ZnCl2 0.1 mg

Fe(S0)4 0.1 mg NaWoO^ ...... 0.1 mg

All constituents were dissolved in one liter’of triple distilled water.

The nitrogen sources varied with the particular parameter tested and were added to the medium at several concentrations. The nitrogen sources were;: arginine, lysine, histidine, cy'stine, and trypto­ phan. The carbon source and the micronutrients were autoclaved se­ parately, while the nitrogen and macronutrients solution were adjusted to a pH of 6.5 and autoclaved together. After the addition of the other constituents, the resulting pH of the medium remained the same.

The nitrogen sources were added to the medium at several con­ centration levels and the necessary replicates were maintained for each experiment. Cultures were observed at frequent intervals and the tissue incubated in liquid cultures was harvested by passage through Whatman#l filter paper. A final pH of the culture medium was recorded.

XI. Carbon experiments

The type of inoculum for these experiments was treated thevsame as that used for the nitrogen experiments. The nitrogen source for this test medium was arginine at a 2.0 g/1 concentration. The macronutrients and micronutrients were added at the concentration levels stipulated in the nitrogen experiments. The carbon sources varied and were added to the medium at 5,0 and 10.0 g/1 concentration levels. The carbon sources were provided in the form of glucose, fructose, sucrose, and maltose. The necessary replicates were maintained for each experiment..

Observations were made at several incubation times and the tissue har­ vested as reported earlier. A final pH of the culture medium was re- 10

corded.

XXI. Vitamin experiments

The inoculum source was obtained and treated the same as in the

nitrogen experiments. The basal medium constituents are the same as

listed under ':h- nitrogen experiments. The magnesium phosphate-nitro-

gen solution was adjusted to a pH of 6.5 with either 5 N HCl or 5 N

NaOH before autoclaving. The micronutrients, carbon source, and the

vitamins were autoclaved separately. The concentration of vitamins

used was 0.1 mg/liter.

The culture vessels used were 250 ml pyrex Erlenmeyer flasks to

which 45 ml of medium was added. Each flask was inoculated with 0.5

ml aliquots of the mycelial-spore suspension and incubated at 25 C.

The time of incubation varied and is reported in connection with the

experiment. Several replicates were prepared for each treatment. The

amount of growth was determined by harvesting replicate flasks and

weighing the mycelium as described in the section on growth determination.

Each datum is the average of three single dry weight determinations.

The final pH of the culture medium was determined at the time of har­

vest.

XIII. Analysis of Data

For the most part, each experiment had at least three replicates.

The results listed in the tables and figures represent the mean value

of the replicates plus or minus the standard deviation.from the mean*

The latter was computed with the formulas: ( Downie and Heath) ( X. - X ) Standard i=l Deviation N - 1

\ c x. r - n ( x >

N - 1

Standard Deviation Mean Deviation 'i Number of samples RESULTS AND DISCUSSION

I. IN VITRO INVASION OF HAIR

A, Introduction

Since the site of infestation by J?* hortae is animal, hair, the ability of the fungus to grow in vitro on this substrate is of parti­ cular interest. The in vitro growth of P. hortae on hair would con­ clusively demonstrate growth of this fungus on hair.

Various species of ( a genus of fungi that infests hair) were found by Roberts (1894) to partially degrade animal hair, including h&man; some species destroyed the cuticle and cortex simul­ taneously, whereas others attacked the cortex first, and had little or no effect on the cuticle. According to English (1952), keratino- philic fungi have three morphologically different growth phases: one parasitic and two saprobic. The parasitic phase has long been known and is quite distinct from either of the saprobic phases. In the sapro­ bic phase, and sometimes ascospores are produced, the difference between these two phases being the specialized mycelium developed on the keratinous substrate. One type, referred to as

"eroding myceliuniy is composed of closely packed masses of cells of short, wide celled mycelium, whether on the surface of the substrate or embedded between layers of keratinized cells. These masses of cells lift the scales of the cuticle and usually grow longitudinally along the hair shaft. The other type, referred to as perforation pegs or organs, is composed of hyphae that penetrate into the hair. This

12 13 penetration is perpendicular to the length of the hair* The con­ tinues to grow inside the hair and in so doing perforates it* The formation of perforation pegs by hyphae of Microsporum and Trichophyton species on hair has been reported!! (Vanbreuseghem, 1952),

B, Hyphal Structure

Ultrastruetural characteristics of the hyphae of P, hortae are depicted in Plates I and II, Numerous chlamydospores, both terminal and intercalary, and variations in the shapes and sizes of the hyphae are indicated on the photomicrographs, Masses of cells of £, hortae, sometimes referred to as pseudoparenchyma (Plate II), are often embedded in a brownish, sticky, amorphous substance,

Co Hyphal Development on Hair

Hyphal development on artificially infested hairs was com­ pared to naturally infested hairs. The naturally infested hairs were obtained from a Bolivian source^ whereas the artificially infested hairs were obtained by inoculating the hairs with the test fungus in petri dishes containing distilled water, hair segments and malt or yeast extract. Usually within a week, mycelium was observed floating in the medium,shortly after which it developed on the hair segments.

Generally, by two weeks, the mycelium became matted around the hair and was readily visible to the naked eye (Plate III), Microscopic observation of the fungal cells did not reveal any perforation organs reported for some groups of keratinophilic fungi(Davidson and Gregory,

1934; Vanbreuseghem, 1952), Plate I

Stereoscan electron micrographs of J?. hortae cells cultured in malt extract medium at 25 C and observed at various magnifications* a, Stereoscan electron micrograph of I>. hortae of the filamentous growth and numerous swellings throughout the hyphae. Critical point dried X500 b. Stereoscan electron micrograph of P. hortae. Higher magnification of A. Note the differences in the size of the hyphae and the chlamydospores. X2,000 c* Stereoscan electron micrograph of the hyphae of P. hortae0 Note the intercalary chlamydospores and the architectural features of the swollen cells. Critical point dried XI,000 d. Stereoscan electron micrograph of cells of P0 hortae that no longer have the filamentous or hyphal orientation. Note the cells which appear smooth on the surface and some that have a rough appearance. Critical point dried XI,000

14

Plate II Stereoscan electron micrographs- of TP, hortae cells embedded in an amorphous substance and observed at various magnifications a. Stereoscan electron micrograph of Py hortae cells embedded in an amorphous substance which acts as a cement, holding the cells together,. Note the cells appear embedded in this matrix (see arrow), X 500 b, Stereoscan electron micrograph of clumped cells of P* hortae. Note the substance which has been referred to as amorphous,. Higher magnification of a, X 2,000

16

Plate III

Photomicrographs of P. hortae cells on human hair segments observed at various magnifications. a. Photomicrograph of P. hortae cells on human hair segments in water, (ordinary light microscopy). b. Photomicrograph of P. hortae cells on human hair segments in water. Note the aggregation of cells and the associated pigmentation. X220

18 'I 20

Artificially infested hairs viewed with the light microscope gave some indication of the shape and clustering of the cells (Plate III)*

However, the same hairs viewed with the scanning electron microscope

(Plate IV) gave a more detailed picture of the intercalary chlamydospores

and the assumed direction of growth of the fungus around the hair shaft.

The length of time required for P* hortae to attach to the hairs

from the Afro-American female varied (Table 1)* Generally, it took

a much longer incubation time before the fungus was observed on these

hairs than hairs from the Caucasian female,. In addition, observations

of the fungus on the hair were made mainly of the infested hairs from

the Caucasian female because they were colonized more rapidly, less

pigmented, and easier to distinguish the fungus on brown hair*

Growth of the fungus on the artificially infested hairs differed

from that observed on the naturally infested hair specimen of JP* hortae,

even when the hair itself is the culture medium (Plates III, IV).

Similar observations were made by Page (1950) and Vanbreuseghem (1952)

for Microsporum gypseum on infested hair* The masses of organized

hyphae forming the characteristic pseudoparenchyma of P. hortae was

observed (Plate III) and the proliferation of growth was conspicuously

present in vitro and absent in vivo* Artificially infested hairs re­

vealed numerous chlamydospores and abundant mycelium, whereas, the

mycelium on naturally infested hairs lacked chlamydospores and was

composed of masses of smaller cells(Plates III, IV)* Plate IV

Stereoscan electron micrograph of artificially and naturally in­ fested hairs observed at various magnifications* a* Stereoscan electron micrograph of JP, hortae cells on the surface (cuticle) of adult human hair, artificially infested*- Note the characteristic clustering or clumping of the cells at the lower end of the hair shaft, and the numerous chlamydospores 0- Air dried X 500 b* Stereoscan electron micrograph of naturally occurring nodule formed by I?* hortae cells* The hair specimen was obtained from a Bolivian source. Air dried X 100

Co Stereoscan electron micrograph of IV hortae cells on hair artifici­ ally infe-sted* Note portions of mycelium growing underneath the scales of the hair,(see arrow ). Air dried XI,000 d^ Stereoscan electron micrograph of naturally occurring nodule formed by cells of IV hortae0 Higher magnification of B* Note the cells densely packed, and the shape of the particles around the cells (see arrow ).- XI,000

21 22

®rr-gs3ss«c,* 23

Table !• Growth of P0 hortae on hairs from an Afro-American female and a Caucasian female, artificially infested, With observation at various incubation times at 25 C.

• Incubation time (Days)

Afro - American Caucasian

Treatment 7 14 21 28 7 14 21 28

cl Control ------■■ mm

Malt extract - - + + + + +

Yeast extract - - - + - + + +

Controls did not contain malt or yeast extract

+ indicates growth on hair

- indicates n o growth on hair . 2/»

Chlamydospores and hyphae of the fungus on the hair closely

follow the edges of the scales of the cuticle (Plate IV). Portions

of the mycelium also appeared to have grown between the scales of

the cuticle of one hair (Plate IV - c). Thus, the action of the fungus

on the hair is not apparently the result of the penetration of specia­

lized mycelium (perforation peg) of the fungus into the hair shaft.

It appears that the cuticle is the only part attacked by simple contact with masses of compact hyphae. Support for this observation has been

reported in transmission electron microscopic work done with naturally

occurring nodules of cells of P. hortae (Chong, et al., 1975).

Crystals were observed around the cells and in the naturally

occurring nodules of JP. hortae (Plate V). Crystals have also been

reported in other groups of fungi pathogenic to man, Keratinomyces

ajelloi, , , and Aspergil­

lus species, which are also keratinophilic (Benedek, 1961). A great

variety of crystal forms were produced in cultures of these dermato­

phytes. The chemical composition of the crystals was identical, al­

though their crystallographic forms were different. One substance

observed was calcite (CaCO^) which had over 300 crystallographic

forms.

The chemical composition of the crystals observed in cultures of

P. hortae were determined by point analysis with the x-ray analyzer of

the scanning electron microscope. Comparison of the results of the point Plate V

Stereoscan electron micrographs and X-ray point analysis of P. hortae cells on hair obtained from a Bolivian source.

a. Stereoscan electron micrograph of naturally occurring nodules formed by cells of J?. hortae. X 1,000

b. Stereoscan electron micrograph - X-ray point analysis of the hair only, as shown in a. Note the presence of only the iron peak.

c. Stereoscan electron micrograph - X-ray point analysis of P. hortae cells shown in a. Note the presence of the calcium and iron peaks.

25 91 analysis of P. hortae cells and the unknown particles (Plate V-c) with results from the analysis of hair only (Plate V-b), revealed

the presence of calcium ions where the crystals are present. The point analysis also strongly suggests that in some way, the fungus has produced a detectably high concentration of calcium ions.

This study of the in vitro infested hairs with light and scan­ ning electron microscopy has provided some information about the

the fungus and also indicates the direction for investigating the remaining questions raised in this study. These observations have

also established the method by which the fungus infests the hair,

simple contact with cuticle uplifting. In addition, observations of

the naturally occurring nodules with the scanning electron microscope

revealed some unexpected findings. There is preliminary evidence

that the presence of large amounts of calcium in and around the cells

£• hortae suggests that the fungus is producing calcium crystals, which could, account for the hard, crusty state of the nodules. II. NUTRITION AND TEMPERATURE

A* Preliminary Studies

Because there are no reports in the literature concerning

the growth of I*. horta. in a defined medium, it was necessary to determine exact cultural conditions for the experiments designed to resolve the questions raised in this study. Those factors under consideration were carbon sources and optimum pH range0

1) Carbon sources

Delamare and Gatti (1928) reported the growth of P. hortae on several carbon sources supplied in agar medium. However, the establish­ ment of a single carbohydrate as an optimum carbon source in defined medium has not been determined. The results of various carbon sources

on the growth of P. hortae at various incubation times are listed in Ta­ bles 2,3, It is notable that the maxium amount of mycelium was obtained with 10,0 g/1 concentration of maltose, after eighteen days incubation.

It is even more interesting to note that with glucose as the carbon

source, the fungus grew very slowly since most of the previous investi­

gators (Delamare and Gatti, 1928; Fischman, 1965; Dodge, 1935) cultured

the fungus on Sabouraud dextrose agar, which contains glucose as the

carbon source. Very likely, reports of the slow growth of the organism

could be due to the type of carbon source used.

The growth of _P, hortae on maltose and sucrose is not

common for most fungi (Lilly and Barnett, 1951), The hydrolytic

I ■ . i ■ : • , , . -. • ■ 29 29

Table 2* Dry weight of P. hortae in basal medium with various carbohydrates added at 5.0 g/1 concentra­ tion after 8, 12, and 18 days incubation at 25 C.

. Incubation Time (Days) Carbon source 8 12 18

Control^ 6.9 8.5 10.3

Glucose 4.6 8.2 18.4

Fructose 6.2 10.4 28.4

Sucrose 8.0 12.3 20.0

Maltose 11.5 15.3 23.6 i

3 ■ controls contained no carbohydrates 30

Table 3*. Dry weight of P. hortae in basal medium with various carbohydrates added at 10.0 g/1 concentration after 8, 12, and 18 days incubation at 25 C.

Incubation Time (Days) Carbon X Sources 8 12 18

Control a 6.0 7.9 10.9

Glucose 2*9 6.3 17.7

Fructose 4.8 9.7 25.2

Sucrose 6.0 10.1 19.2

Maltose 12.6 18.7 31.2

0 control contained no carbohydrates 31 enzymes necessary for the hydrolysis of these oligosaccharides are - evidently synthesized by the fungus.

The fungi, both pathogenic and non-pathogenic, vary in their ability to utilize the various carbon sources, Bodin (1899) found that did not utilize sucrose but assimilated glucose and maltose. Trichophyton mentagrophytes was found by «Bodin (1902) to utilize glucose more readily than lactose or maltose, Tate (1929) reported that none of the he studied could utilize sodium acetate, formate or lactose as carbon sources, Steinberg (1939) found that glucose, fructose, mannose and sucrose were all effective in the nutrition of niger*, And finally, some investigators have found that strains of Saccharomyces cerevisae, when first isolated could not utilize glucose. However, the strains became adapted to glucose after long exposure to the sugar (Skoog and Lindegern, 1947),

The availability of certain carbohydrates depends upon the pro­ duction of the necessary extracellular hydrolytic enzymes, A carbo­ hydrate and its hydrolytic products are not necessarily equivalent in all respects*. Hawker (1950) reported the amount of mycelium produced by Melanospora destruens varied when this fungus is grown in equivalent amounts of glucose, fructose, mixtures of glucose and fructose, and sucrose. More mycelium was produced from glucose than from an equiva­ lent amount of sucrose, and this was true whether the concentration of these sugars were high or low. While a fungus may utilize an oligo­ saccharide and its hydrolytic products, it does not necessarily follow 32 that both are used with the same efficiency for all purposes (Lilly and Barnett, 1951).

The maximum amount of mycelium was obtained on the maltose medium, while on glucose, a hydrolytic product of maltose, growth was relatively slow. Likewise, growth was much better, in terms of dry weight, on fructose than on sucrose, the former being a hydrolytic product of sucrose.

Thus, on the basis of this experiment and on the basis of the reports of previous workers, maltose at a 10.0 g/1 concentration seems preferable as a carbon source for the optimal growth of ]?. hortae in test medium.

2) Hydrogen ion concentration

A second factor for priminary investigation was the optimal pH for the growth of P. hortae in defined medium. In this

experiment, the pH was varied from 2.0 to 10.0 (Table 4).

Piedraia hortae was cultured in arginine-maltose medium having initial pH values of 2.0, 4.0, 6.0, 8.0, and 10.0. After various incubation periods, the dry weights and final pH values were

recorded. These data indicate a significant drop in the pH of the medium initially adjusted to a pH of 10.0. If a fungus is able to

grow in an alkaline medium, such as this, the carbon dioxide could

conceivably lower the pH. Organic acids may also be produced,

while carbon dioxide from the air will be absorbed by the alka­

line medium (Burnett, 1968). The pH drop from 10.0 to 6.7 could be 33

Table 4.. The effect of initial pH on the growth of JP. hortae in arginine-maltose medium after 8 and 12 days in­ cubation at 25 C.

Initial 8 Days Incubation 12 Days Incubation pH Dry Weight3 Final pH Dry Weight Final pH

2.0 4.7 2.2 10.4 2.25

4.0 10.9 6.0 14.4 6.4

6.0 9.1 6.2 21.0 7.0

8.0 9.2 7.65 18.5 7.61

10.0 4.2 7.25 16.5 6.7

dry weights are the mean value based on three replicates (mg) 34 due to one or more of these factors*

The pH, of course, affects enzyme activity, which in turn affects growth. In general, there is a striking correlation between the optimum pH range for most enzymes and the optimum pH range for growth* Haldane (1930) found all but 9 of 105 enzymes to have optima pH values between 4 and 8, Most fungi, including JP. hortae, have pH optima between these limits* Correlation of the pH values of the medium with the rate and amount of growth of JP. hortae may be obtained by comparing the growth curve (Fig. 1) obtained at the same time for the organism. Maximum growth was obtained at pH 6.0, although good growth was obtained at both pH 4*0 and 8.0.

From the data in Table 4,. it is evident that the pH may change considerably during incubation periods. These changes could be due to the changes in the amount of acids and bases in the media.

Fungi produce acids from non-acidic nutrients, such as carbohydrates

(Burnett, 1968). Among the acids produced is pyruvic acid which accumu­

lates in nutrient solutions in which most fungi grow and often accounts

for the early depression of the pH. The eventual utilization of the pyruvic acid causes the pH of the nutrient solution to rise* This pat­

tern is evident in Table 4. . The maximum production of mycelium was

obtained from medium with an initial pH of 6.0, which had been raised

to 7.65 during twelve days incubation.

As nearly as can be ascertained from these data, the chemical Dry Weight (mg) 40 30 20 10 0

Fig. 1* The effect of hydrogen ion conentration on the 1*on Fig. ionconentration of hydrogen effect The 2

rwho . ote in arginine-maltose medium P. hortae of growth after 8 and 1225incubationdaysandat C.8 after Hydrogen ionconcentration Hydrogen 4

6 _____ incubation days twelve

ih as incubation days eight 8

10 35

36

changes in media due to alteration of the pH, whether imposed from'

the outside or caused by the fungus, affect metabolic processes.

The pH of a culture medium changes during the growth of the fungus,

and these changes affect the composition of the medium and thus,

the fungus.

Therefore, on the basis of this experiment and other reports

concerned with hydrogen ion concentration, medium to test the growth

of JP. hortae was adjusted to pH 6.5 prior to autoclaving.

B. Temperature

Temperature has been recognized as an important natural

factor governing the geographical distribution of the fungi (Bisby,

1943; Pirozynski, 1968). The temperature must be favorable not only

for growth, but also for the production and germination of the spores

(Cochrane, 1958). Among those external factors which influence the growth of fungi, temperature greatly influences the rate of growth, probably owing to its effect on the chemical and physical processes

involved in growth.

The characteristic effect of the different temperatures on

the rate of growth of JP. hortae is listed in Table 5. The dry weight

of cultures incubated for various periods of time in malt extract medium at different temperatures are reported. Three recognizable

temperature points are evident* At the lowest temperature tested 37

Table . 5. Effect of various temperatures on the growth of P. hortae in malt extract medium after 8, 12 and 18 days incubation at various temperatures (mean dry weight plus or minus mean deviation)#

Incubation Time ( Days ) Incubation Temperatures 8 12 18

18 C 19.3+ 3.9 23.0 + 0.3 29.6 + O'. 5

22 C 31.0 + 3.4 32.1 + 1.0 33.0 + 0.5

25 C 49.9 + 1 . 1 53.2 + 0.6 58.4 + 0.8

30. C 31.1 + 4.2 33.3 + 0.7 35.0 + 2.3

37 C 11.4 + 0.8 12.3 + 2.1 21.2 + 0.5

a mean dry weight based on three replicates 38

(18 C), growth occurred at a slower rate, perhaps due to the suboptimal temperature which is not enough for the induction and/or depression of enzymes in the medium. At 25 C, the optimum temperature, growth occurred at a relatively constant rate, perhaps due to an adequate temperature level for the induction and/or depression of enzymes in the medium. At the highest temperature tested ( 37 C ), growth is decelerated, but not ceased, due to the increase in the temperature which most probably inactivated to some extent the enzymes involved in growth.

Pathogenic fungi have been divided conveniently into groups on the basis of the temperature range of growth in vivo (Nickerson, 1947).

Those organisms causing superficial skin infections inhabit an environ­ ment which reflects, to some extent, atmospheric temperatures, and that even in vivo, the organism is subjected to the temperature ranges dictated by its geographical distribution,

1 • With P. hortae, the probable reason for the optimum growth being obtained at 25 C could be associated with the geographical distri­ butional of the organism, considering the fact that the fungus is limited to the tropical and semi- tropical regions of the world, where the atmospheric temperature is usually 25 - 26 C ( Fischman, 1965).

Thus, on the basis of this experiment, a 25 C temperature is optimal for the growth of P. hortae on malt extract medium. 39

This could also indicate that temperature is a factor contributing-to

the distribution of this fungus in the tropical and serai tropical re­ gions of the world.

C. Keratin Utilization

The natural habitat of JP. hortae is hair, which is composed

primarily of keratin ( Savill, 1952 ). Block and Vickery (1931)

emphasized the chemical sta bility of keratin in their definition that

"keratin is a protein which is resistant to digestion by pepsin and

trypsin, which is insoluble in dilute acids and alkalines, in water

and inorganic solvents, and which, on acid hydrolysis, yields basic

amino acids." In addition, keratin has been characterized as contain­

ing high concentrations of amino acids - arginine, lysine, histidine,

tryptophan, and cystine (Block and Vickery, 1931). According to Savill

(1952) the fully keratinized hair cortex contains only a small amount

of free amino acids. This may suggest a reason why this fungus does

not penetrate the hair shaft, but instead, surrounds the cuticle of

the hair as confirmed in electron microscopic work done by Chong, et

al. (1975).

Experiments were performed to determine the effect of varying

concentrations of hair keratin on the growth of P. hortae. When sup­

plied in the various concentrations, there is evidence from dry weight

measurements and linear extension data, that keratin stimulates and

supports the growth of JP. hortae (Figs. 2,3). The stimulatory effect Fig*2 Linear extension (cm) Linear extension (cm) 4 0 3 0 2 5 4 2 1 5 1 3 The effect of various concentrations of keratin ofconcentrationskeratin of effect various The tvros nuain ie (days);days, times B 30 - incubation A various at 0dy, 0-dy, n 100days. and -days,D -- C90 days, 60 - B the growth of thegrowth _P. linearextension,as hortae, measured Keratin concentration^) Keratin Keratin concentration Keratin , 0 .3 0 .31 .07 .03 .010, (7.) •H dl •H 4J 6 o ♦rl o d (U X d M M

40 on

(7.) dry weight (mg) 20 10 15 0 5 Control i* 3.. . Fig*Effect ofconcentration(percentages) keratin on Concentration of Keratin (percentages) with incubation25at C. with theofgrowthP. dry ashortae, measured weight .0187 .0375 .075 .31 41 (mg),

42 is much more observable in terms of dry weight, when the hair keratin

is supplied at 0.075 per cent. Generally, the dry weight of the fungus

increased with the increase in the amount of keratin added to the medium. In addition, the final pH of the medium became more alkaline

(Table 6).

The tendency for the pH to become more alkaline is a feature of

the dermatophytes as a group ( Nickerson, 1947). Mallinckrodt-Haupt

(1927, 1933 ), on the basis of studies on the pH changes produced by dermatophytes and certain saprobic hyphomycetes in various

culture media put forward the generalization that true dermatophytes

tend to change the media alkaline and saprobic hyphomycetes tend

to turn the media acid. This feature might have a real bearing on

the affinity of dermatophytes for keratin. Support for this idea is

also gained from studies of keratinolytic bacteria by Crewther,

McQuade and.Pressley ( 1955), who noticed that a number of strains of Bacillus subtilis degrade wool in the same order as they increase

the pH of the growth medium.

The fact that P. hortae'changes the medium alkaline when keratin is present, is a factor of considerable importance in contributing to its keratinolytic properties. - i--

Thus, on the basis of-these experiments, it has been demonstrated that Ey hortae can utilize keratin as its sole source of nutrients.

This implies that P* hortae not only grows on the hair, but that it is 43

Table 6, Effect of various keratin concentrations (per­ centages) on the growth of P. hortae, measured as dry weight (mg), and the final pH , at a 25 C incubation temperature (mean dry weight plus or minus mean deviation).

Keratin Dry Weight Final pH Concentra tion (mg)

Control^ 2.2 + 1.1 6.6

•0187 8.6 + 2.3 7.0

.0375 11.9 + 1.3 6.95

.075 12.4 + 1.6 6.8

.31 11.0 +_2.3 6.4

controls contained purified bacto agar without the addition of keratin capable of utilizing the major constituent of hair, keratin.

D. Amino Acids

A review of the literature of previous work on the utilization

of various nitrogen sources by P. hortae revealed little or no agree­ ment as to which promoted optimal growth ( Delamare and Gatti, 1928).

Thus, a series of experiments were done to test those organic compounds

of human hair keratin as nitrogen sources for P. hortae in a defined medium* As previously stated arginine, lysine, histidine, cystine,

and tryptophan are released in abundance upon acid hydrolysis of hair

keratin. Because the site of attack by P. hortae is the hair, these particular amino acids were tested for their utilization as nitrogen

sources by the organism.

The results of these experiments are listed in Tables 7 - 9.' •

Piedraia hortae grows well with the various nitrogen sources tested.

However, some specific trends were evident. In the arginine medium, the

dry weight increased with increasing concentration of arginine. Cystine

has a similiar effect on the organism, though, the length of time to

demonstrate the effect was more for the cystine than for the arginine

source* This trend was evident for all the nitrogen sources except

histidine and lysine. The dry weight of the tissue increased with a

decrease'in the concentration of these nitrogen sources. However,

in general, the dry weight of the tissue growing in the various nitro­

gen sources increased with increased incubation times ( Table 9)*

Tables 10- - 12 list the final pH values of the medium after 45

Table 7.. Growth of ]?. hortae on basal medium with maltose as the carbon source and five organic nitrogen sources supplied at three concentration levels with 8 days incubation at 25 C (, mean dry weight plus or minus mean deviation)

Nitrogen Nitrogen concentration g/1 Sources 0.1 0.5 1.0

Arginine 5.4 + 0.2 6.3 + 0.2 7.5 + 0.3

Lysine 4.5 + 0.4 4.5 + 0.3 2.8 + 0.5

Histidine 4.3 + 0.4 5.6 + 0.3 2.9 + 0.6

Cystine 4.2 + 0.2 4.6 + 0.4 6.8 + 0.2

Tryptophan 4.3 + 0.4 2.4 + 0.3 3.4 + 0.8 46

Table 8». Growth of P. hortae on basal medium with maltose as the carbon source and five organic nitrogen sources supplied at three concentration levels with 12 days incubation at 25 C (mean dry weight plus or minus mean deviation)

Nitrogen Nitrogen concentration g/1 Sources 0*1 0.5 1.0

Arginine 7.0 + 1.2 7.1 + 1.2 11.8 + 0.3

Lysine 5.3 + 0.2 6.0 + 0.2 3.1 + 0.1

Histidine 5.1 + 0.1 3.1 + 0.2 5.4 + 0.3

Cystine 6.7 + 0.2 8.3 + 0.3 10.7 + 0.3

Tryptophan 5.6 + 0.1 4.6 + 0.2 4.3 + 0.2 47

Table 9. Growth of JP. hortae on basal medium with maltose as the carbon .source and five organic nitrogen sources supplied at three concentration levels with 18 days incubation at 25 C.( mean dry weight plus or minus mean deviation)

Nitrogen Nitrogen concentration g/1 Sources 0.1 0.5 1.0

Arginine 8.6 + 0.2 9.1 + 0.1 16.8 + 1.9

Lysine 16.0 + 0.1 6.1 + 0*1 5.7 + 0.2

Histidine 6.9 + 0.1 16.6 + 0.3 7.7 + 0.3

Cystine 9.8 + 0.6 10.3 + 0.5 18.3 + 2.1

Tryptophan 7.3 + 0.5 11.8 + 0.2 6.7 + 0.2 48

Table 10, Effect of various nitrogen sources on the final g pH value of the culture medium with maltose as the carbon source and five organic nitrogen sources supplied at three concentration levels with 8 days incubation at 25 C,

Nitrogen Nitrogen concentration g/1 Sources ' 1*0 0.5 0,1

Arginine 4.9 5.3 5.9

Lysine 5.8 5.7 5.1.

Histidine 5.6 5.7 5.9

Cystine 5.5 6.1 6.3

Tryptophan 5.6 6.0 • 6.2

- - . - ......

a initial pH adjusted to 6,5 49

Table 11* Effect of various nitrogen sources on the final pH . value of culture medium with maltose as the carbon source and five organic nitrogen sources supplied at three concentration levels with 12 days incubation at 25 C.

Nitrogen concentration Nitrogen g/1 - Sources 1.0 0.5 0.1

Arginine 4.1 5.0 5.8

Lysine 5.5 5.8 5.5

Histidine 6.1 5.7 5.9

Cystine 5.6 6.7 6.1

Tryptophan 6.1 6.5 5.8

a initial pH adjusted to 6,5 50

Table 12® ( Effect of various nitrogen sources on the final pH value of the culture medium with maltose as the carbon source and five organic nitrogen sources supplied at three concentration levels with 18 days incubation at 25 C.

1 Nitrogen concentration Nitrogen g/1 Sources i.o 0.5 0,1

Arginine 3,7 3.9 4.9

Lysine 5.6 5.0 5.6

Histidine 5,5 5.8 502

Cystine 4.9 6.4 5.5

Tryptophan 6.0 6.1 5.6

a initial pH adjusted to 6,5 the various incubation times on’ the five nitrogen sources at different concentrations* In general, the final pH of the media dropped considera­ bly from the adjusted pH of 6,5 to 4,9 for arginine, and 5,5 for cystine.

The pH of the remaining media also dropped, but the changes were most evident in the arginine and cystine media, With these amino acids as the sole nitrogen sources, the pH is decreased in all instances. This relationship between the pH and the nitrogen sources suggests that some metabolites are being produced in the media, depending on the amino acid present. The pH of the nutrient solution also reflects the utili­ zation of certain ions from the medium.

The amount of mycelium produced when arginine was’ the nitrogen source, was greater at the minimum incubationtime. Thus, arginine was used as both the nitrogen and carbon source* Table 13 list the results of this experiment. In all cases, regardless of the concentration employed, when arginine was used as a dual source for both carbon and nitrogen, there was a reduction in the amount of . mycelium, obtained.

Thus, based on these experiments, there is every indication that the fungus utilizes the amino acids of hair keratin. In addition microscopic observation of the mycelium from the medium with the various amino acids revealed morphological features that were similar to those observed when the fungus was grown on hairc There were numerous chlamy-

dospores and abundant masses of hyphae.(Plates VI - VIII), Crystal

like particles were only observed from the arginine and cystine media,

(Plate IX). Table 13* Growth of JP. hortae after 12 and 18 days incubation at 25 C on basal medium' with arginine supplied at three concentrations with/without the addition of maltose (mean dry weight plus or minus mean deviation)

WITH MALTOSE WITHOUT MALTOSE Incubation Arginine concentrat.ion g/1 Arginine, concentration g/1 Time (Days) 1.0 2.0 4.0 1.0 2.0 4.0

12 7.2+ 0.2 8.5+ 0.3 9.9+ 0.1 4.8 + 0.2 6.9 +0.6. 8.8+0.5

‘

18 10.9+0.8 15.3+ 0o7 21.0+ 1.2 8.0 + 1.6 7.2 +0.4 9.1+0.8

Ln to Plate VI

Photomicrographs of P, hortae cells cultured in basal medium with arginine and lysine as the nitrogen sources incubated at 25 C. a* Photomicrograph of P* hortae cells cultured in arginine- maltose medium incubated at 25 C. b* Photomicrograph of JP, hortae cells cultured in lysine- maltose medium incubated at 25 C.

53

Plate VII

Photomicrographs of P. hortae cells cultured in basal medium with cystine and tryptophan as the nitrogen sources incubated at 25 C. a. Photomicrograph of P* hortae cells cultured in cystine- maltose medium incubated at 25 C. b0 Photomicrograph of P. hortae cells cultured in tryptophan- maltose medium incubated at 25 C.

55 56

6 Plate VIII

Photomicrographs of JP* hortae cells cultured in basal medium with histidine and keratin as the nitrogen sources incubated at 25 C. a. Photomicrograph of P* hortae cells cultured in histidine- maltose medium incubated at 25 C*

b0 Photomicrograph of JP* hortae cells cultured in medium with keratin as the sole nutrient source*

57 58 Plate IX

Photomicrographs of P* hortae cells and crystals produced in arginine and cystine medium with maltose as the carbon source incubated at 25 C. a* Photomicrograph of P, hortae cells cultured in arginine- maltose medium at 25 C. Note the crystals b. Photomicrograph of 1?. hortae cells cultured in cystine- maltose medium at 25 C* Note the crystals

59

E, The Effect of Fatty Acids and Vegetable oils

The natural habitat of hortae is the hair of the scalp

(Fischman, 1973; Horta, 1911)* However, there has been no report of the occurrence of this fungus on the scalp. Thus, the question was raised - why doesn°t I?. hortae grow on the scalp? There are

some groups of fungi ( Microsporum and Trichophyton species) that grow on both the hair and scalp. Why,then, doesn°t P. hortae 'infest

the scalp?

Experiments were designed to test the effect of various

fatty acids that are secreted by the sebaceous gland, that may be

found on the scalp and a vegetable oil found in commercially prepared

oils and dressings commonly used on the hair and scalp. The fatty

acids tested were oleic, linoleic, and stearic, and the vegetable oil

tested was castor oil. These substances were incorporated in SDA at

various concentrations because this medium is known to support the

growth of P. hortae and so that the results obtained could be compared

with the results obtained in a similar investigation with fungi infesting

the hair ( Hajini et al,, 1971),

The results of this study are in Table 14, All concentrations,

ranging from 0,5 to 10,0 per cent, of the test substances completely

inhibited the growth of this fungus in vitro, at 25 C, while growth was

observed on the SDA control plates which did not contain the fatty

acids or vegetable oil. 62

Table 14, Effect of the addition of fatty acids and a vege­ table oil to SDA at various concentrations (per centages) on the growth of ]?. hortae at 25 C,

Concentrations(7») of the Substances Added

Substances .05 1.0 2.0 3.0 5.0 10.0 Added to SDA , a Control + + + + + +

Castor oil - am m m - m m -

Oleic acid m - m m - m m -

Linoleic acid - - - m m - -

Stearic acid - m m - - - -

+ growth observed

- no growth observed a controls consisted of SDA without the addition of oil or fatty acids 63

The presence of these fatty acids on the scalp could account

for the limited reports of epidemics of black piedra in the

endemic areas# In addition, the application of vegetable oils and

the presence of these fatty acids on the scalp and hair could account

for the fact that this particular fungus does not pose a real threat

or problem to many groups of people who have a common practice-of

applying oils to the scalp for cosmetic reasons*

Hajini. et al.( 1971) reported the effect of hair oils and fatty

acids on the growth of dermatophytes of the genera Trichophyton and

Microsporum. They found that mustard oil and unsaturated fatty acids

in concentrations of 1*0 per cent and above completely inhibited

the growth of the organisms tested, Hendrix ( 1970 ) reported the

poor growth of Phytophthora parasitica, var* nicotianae (Hendrix and

Apple) on oleic,ftriolein and tristearinj while the addition

of mineral oil to the medium stimulated growth.

Moyer and Keeler ( 1964 ) reported the presence of P. hortae

on the head hairs of an albino girl at the island of Ailigande, San

Bias, Panama, They also observed an albino moon-child man, who was

a therapeutic chanter, and found that his hair was completely covered with black- piedra nodules. According to Moyer and Keeler, the chanter had

been very careful not to put oil on his hair, as is the custom with

many of the young Cunas, because it was well known in their tribe

that oil would remove the fungus. The albino Indian had cultivated 64

black piedra conscientiously in his hair for thirteen years for the

purpose of making it dark.

These data and reports suggest that the fatty acids and vegetable oils have an inhibitory affect on the growth of P. hortae

at 25 C. From the data listed in Table 14, it appears that the

levels of concentrations of the fatty acids and the vegetable '_oil were inhibitory for the growth of P. hortae.

F. The Effect of Vitamins on growth

Semi - synthetic medium, such as malt extract, contain a variety of vitamins' and other growth substances. Organisms una­ ble to synthesize these components grow well on semi - synthetic medium^ and do not reveal these deficiences unless they are grown on

a chemically defined medium ( Lilly and Barnett, 1951 ). Growth

°f P. hortae on malt extract and Sabouraud dextrose agar suggested

that the addition of vitamins to the medium could affect the growth

of the organism favorably* This observation motivated this particular

study in that the effect of vitamins on the growth of P. hortae has

no bearing on the questions raised in this study. However,

experiments designed to demonstrate the effect of these substances

on the growth of this organism will provide the impetus for a more 65 detailed investigation of this nature.

Vitamins have been shown to affect the growth ( Fries, 1965; Skula and Bais, 1971), reproduction (Garraway, 1973), and other metabolic processes ( Lilly and Barnett, 1951 ). In some cases, vitamins may accelerate a particular process or inhibit the process at a given concentration* The two vitamins tested were biotin and thiamine, both contain sulfur, which is reported present in mycelium and spores of fungi ( Cochrane, 1958 ).

With evidence that indicates growth of this organism might be enhanced with the addition of those vitamins, and considering the fact that cystine, a sulfur containing amino acid, is formed from the hydrolysis of hair, the natural host of P. hortae, experiments were performed to determine the effect of thiamine and biotin on the growth of this organism. The results are presented in Tables .15 -

17, and indicate that the initial growth of the organism, in terms of dry weight, was better when biotin was added to the medium.

Generally, during the early growth of the organism, as the weight of mycelium increased, the pH of the medium decreased. Thiamine was most effective upon prolonged incubation, which could result from the activation of enzymes which are involved in growth.

The concentration of the nitrogen source present also had some 66

Table 15* Growth of JP. hortae on basal medium containing three concentrations of arginine supplemented with biotin and/or thiamine with 12 days incubation at 25 C, (mean dry weight plus or minus mean deviation)

Arginine concentration g/1 Vitamin Added 1.0 2.0 4.0

Control 7.2 + 0.2 8.5 + 0.3 9.7 + 0.1

Biotin 18.7 + 1 . 3 7.6 + 0.2 12.1 + 4.4

Thiamine 14.5 + 0.3 6.0 + 0.6 12.8 + 0.8

Biotin + Thiamine 5.0 + 0.0 6.9 + 0.6 9.2 + 1.2

Controls contain only arginine-maltose medium without the addition of the vitamins 67

Table 16. Growth of P. hortae on basal medium containing- three-, concentrations of arginine supplemented with biotin and/or thiamine with 18 days in­ cubation at 25 C. ( mean dry weight plus or minus mean deviation)

ArginirLe concentration g/'1 Vitamin Added 1.0 2.0 4.0

Control3 10.9 + 0.1 15.3 + 0.5 21.0 + 0.6

Biotin 28.6 + 1.5 16.7 + 1.0 21.6 + 6.6

Thiamine 15.0 + 1.1 13.0 + 0.9 12.4 + 1.6

Bio tin + Thiamine 14.2 + 1.1 12.9 + 0.8 . 9.9 + 0.4

A Controls contain only arginine-maltose medium without the addition of the vitamins 68

Table 17*, Growth of P. hortae on basal medium containing three concentrations of arginine supplemented with biotin and/or thiamine with 24 days incubation at 25 C. (mean dry weight plus or minus mean deviation)

Arginine concentration g/1 Vitamin Added 1.0 2.0 4.0

23.7 + 0.9 28.3 + 1*6 31.9 + 1.0 Control3

Biotin 42.0 + 1.2 40.0 + 1.2 35.4 + 2.5

Thiamine 95.3 + 1.0 54.3 + 0.7 33.0 + 1.0

Biotin + Thiamine 38.2 + 1.4 31.3 + 0.9 31.0 + 0.6

aControls contain arginine-maltose medium without the addition of the vitamins 69 effect on the dry weight of mycelium. As noted in Table 17, the' enhancement of mycelial growth is most demonstrable in the medium with a 1,0 g/1 concentration of arginine with thiamine added.

Generally, during the early growth phases, both vitamins had some effect on the growth of the organism, with thi-.;aine being most effective with prolonged incubation. The effect of these vitamins was somewhat masked when they were both added to the medium* CONCLUSIONS

The investigation addresses some questions that have been raised about the fungus, P*^ hortae. Does this fungus grow on hair?

What does it utilize from' the hair? Is it actually using the hair as its sole source of nutrients? Why is it found mainly in the tropical regions of the world?

The following conclusions were made from this study:

1, Observations of in vitro infected hairs with the light and scanning electron microscopes have established the fact that

P* hortae grows on the hair,

2, The method by which the fungus invades the hair is by simple contact and cuticle up lifting,

3, Large amounts of calcium ions in and around the cells of I?, hortae suggest that they produce crystals which account for the hard, crusty nodules, characteristic of the fungus,

4, The production of these crystals and the amorphous substance inwhich the cells are embedded, probably serve to hold the cells on the hairs, and that the hard nodules formed by the fungus may afford some protection for both the spores and the mycelium,

5, Temperature is an important natural factor governing the geographical distribution of this fungus. The maximum amount of mycelium was obtained with incubation at 25 C, which is also the temperature that is usually in the tropical and semi tropical regions of the world,

70 where cases of black piedra are reported.

7. Growth on human hair keratin suggests that P. hortae utilizes keratin as its sole source of nutrients.

8. Fatty acids and a vegetable oil incorporated in a medium known to support the growth of P. hortae completely inhibited

the growth of the organism.

9<> The superiority of one nitrogen source over another varied. The decreasing order of utilization of nitrogen sources by P. hortae were arginine, cystine, histidine, tryptophan and

lysine.

10. Thiamine considerably enhanced.the growth of P.,hortae., LITERATURE CITED

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