F R A G M E N T A MYCOLOGICA

II. ON C A S T E L L A N I ' S " W A T E R C U L T U R E S " AND BENEDEK'S

" M Y C O T H E C A " IN CHLORALLACTOPHENOL.

by

TIBOR BENEDEK

(Mycology Section, Department o/ Dermatology, Stritch School o/

Medicine, Loyola Universitiy, Chicago, Illinois)
(22.XII.1961)

INTRODUCTION
The proper maintenance of even a small culture collection of
dermatophytes and other hyphomycetes is a time consuming, te-
dious, and costly procedure. Through the years and decades, m a n y
suggestions were made to maintain fungus cultures in viable condi-
tion in culture collections, but all had some drawbacks and did not
attain general acceptance. Suffice it to mention only the more
currently used methods. BUELL & WESTON (1947) covered agar
slant cultures with sterile mineral oil. BAKERSPIGEL (1953) suggested
the dispersal of spores in soil. CARMICHAEL(1956) as well as KRAMER
& MIX (1957) advanced the method of deep freezing of fungal
cultures. HARRIS (1954) and more recently, HESSELTINE, BRADLE
BENJAMIN (1960) reported satisfactory results by the freezing-
drying method (lyophilization). SHUH WEI HWANG (1960) proposed
the preservation of fungi at ultra-low temperatures (nitrogen refrig-
eration, --195.5 ° C). It seems unnecessary to herewith repeat the
criticism of these methods as t h e y are generally well known. Many
different suggestions to reach the same goal always indicate that
any one of the methods is not well fit for all purposes. Moreover,
except for the very messy mineral oil method, all other methods
suggested require more or less complicated and expensive equip-
ment, which a small fungus collection cannot afford.

I. CASTELLANI'S " W A T E R CULTURES"

CASTELLANI (1960) recently suggested a new method which is as
simple as it is inexpensive, and ideally fits the purpose for main-
tenance of small culture collections.
256 T. BEN~DEK

CASTELLANI used distilled water in conventional test tubes and he

transferred into it the fungus growth of a dextrose agar (Sabouraud)
culture, taking with it only the minimum layer of the agar necessary
for transfer of the fungus growth. Originally, he sealed these tubes
with Bunsen flame, but later on, used only cotton plugs as with
ordinary test tube agar mediums. Following "inoculation" of these
"water cultures" they were left at room temperature. His "water
culture" experiments included a number of fungi: assorted Candida
species, Sporotrichum anglicum CAST., Glenospora lanuginosa CAST.,
Tr. rubrum CAST., several species of Trichophyton and Microsporon,
Coccidioides irnmitis RIXFORD and GILCHRIST,Blastomyces derma-
titidis GlaCHRIST and STOKES, Cryptococcus neo/ormans SAN~ELICE,
etc. Results have been the same with all species. After twelve months,
all fungi were found alive and grew quite well when inoculated in
glucose agar, producing colonies identical with the original ones,
and the biochemical characteristics (Candida spp.) remaining the
same. After a year, transfers were made on solid medimns and all
fungi had unchanged characteristics. From these transfers, then, a
new series of "water cultures" were made.
The advantages of the "water culture" method as CASTELLANI
found, consisted of the highly reduced necessity of subculturing,
and the apparent prevention of pleomorphism. However, a pleo-
morphic strain transferred to the "water culture" remained pleo-
morphic.

Control Experiments
Now that (a year past) I am using CASTELLANI'S"water cultures"
method in my smali fungus collection, I found this method highly
satisfactory in every respect by saving time in avoiding short term,
continuous tranfers, by reducing the expenses for culture mediums,
etc.
In two respects I modified CASTELLANI'Smethod, but this does
not concern its principle. The two points of change are these: (1)
Instead of distilled water, I arn using commercially available phys-
iologic salt solution (Nat1). 1) (2) I am using, instead of cotton-
plugged, test tubes, small, square, screw-capped medicine bottles ~)
of 30cc contents. They are available in any drug store. They are
filled half with physiological salt solution and sterilized with the
loosened cap on. (The cap material well withstands the heat of
sterilization.) To properly identify the bottles I paste the same
number on the face and on the cap of the bottles. The name of the

1) I t is n o t implied t h a t physiologic s a l t s o l u t i o n is or s h o u l d be e x a c t l y " p h y s i -

ologic" for a n y or all fungi. Distilled w a t e r , o n t h e o t h e r h a n d , is definitely h y p o t o n i c
for all fungi. H o w e v e r , I believe t h a t "physiologic" s a l t s o l u t i o n is a b e t t e r p r e s e r -
v a t i v e t h a n distilled w a t e r for v i a b i l i t y a n d for a n y o t h e r c h a r a c t e r i s t i c s .
~) T h e s e a r e s i m i l a r t o s c r e w - c a p p e d c u l t u r e t u b e s u s e d n o w a d a y s m o r e a n d
m o r e in m a n y m y c o l o g i c a l laboratories.
 FRAGMENTA MYCOLOGICA II 257

fungus species and its accession number m a y be added on another

label placed on the front of the bottle. I keep these bottles in the
refrigerator ( + 5 ° C) with the screw cap, of course, tightened. There
is no evaporation, compared with the test tube container. These
bottles are solid compared with test tubes, so that the probability
of breakage is reduced to a minimum. Moreover, they can be packed
together, side b y side, in a small space, which has also a considerable
advantage.
The fungus species used included strains of Trichophyton, Micro-
sporon, Achorion and Epidermophyton species. The age of cultures,
(always on Sabouraud's dextrose agar, Difco Bacto brand) to be
transferred in "water cultures" made no difference. Transfers were
made from dextrose agar cultures after one week (from quick
growing species), several weeks or, even 2 to 3 months, as the case
m a y be. The preservation of fungus strains remained the same,
excellent quality. Within one year's time, particles were withdrawn
from the "water culture" at intermittent intervals to test the via-
bility and identity of characteristics of the species. These remained
viable and unaltered as to their characteristics.
CASTELLANI's "water cultures" also offer some substantial ad-
vantages in other respects.
1) There are fungus growths, particularly from the scalp, which
carry along with them quick growing bacterial contamination,
(mainly Cocci) and which in repeated transfers on solid mediums
either retard or smother the fungal growth. In order to get rid of
bacterial contaminants, the "water culture" is an ideal medium.
To test tubes containing about 10cc of physiologic salt solution, I
add penicillin 1) and a normal loopful of streptomycin. Following a
24 to 48 hour period, one m a y withdraw culture particles from
"water cultures" after completely eradicating bacterial contamina-
tion. This penicillin-streptomycin " b a t h " is most efficient and ad-
vantageous.
2) Dreaded Acarus infestations of cultures can be eradicated and
prevented from spreading while in "water culture" and under
refrigeration, since the Acari are relatively thermophile.
The term, "water culture" is not alone symbolic b u t is directly
factual. CASTELLANI had the impression that several of the fungi
showed some growth in "water culture". This is correct. I can
confirm that some of these fungi showed definite growth, either on
the submerged particle, (with a magnifying glass one conldsee on the
edge of thin agar particles the radiating growth of fungus) or, directly
upon the surface of the water column (e.g.M. canis, K. a/elloi) such
as the " K a h m h a u t " in certain },east cultures.

~) There are two kinds of water-soluble penicillin in t a b l e t form, which can be

used for this purpose: 1) P e n t i d s SohlbleR Squibb, Penicillin G Potassium, 200,000
u n i t s p e r tablet; 2) SolvetsR Lilly, Penicillin-G 100,000 units per tablet.
IVIycopathoI. et MycoI. App1. X V I I , 3. 17
258 T. ~ E ~ E D ~ X

II. BENEDEK'S "MYCOTHECA" IN CHLORALLACTOPHENOL

Phanerogamists for centuries have had the great advantages of
their "herbariums." For taxonomic and many other purposes they
were able to have recourse to both pressed and dried specimens of
flora to study comparative features of a newly found specimen, for
instance. Somewhere, in one of the great herbariums of the world,
desired specimens may be found.
Cryptogamists, in dealing with microscopic fungi, cannot enjoy
the compounded advantages of a "mycological" herbarium, because
micro-fungi cannot be pressed, nor can they be dried. They would
have lost all characteristic features for any type of comparative
studies.
For large, fleshy fungi, JOSSERAND (1934) suggested the same
procedure of pressing and drying as was customary for phanerogam
specimens; however, he recommended that one "uses these herba-
rium specimens as rarely as possible."
I have conceived a simple and efficient method to give the crypto-
gamists dealing with microscopic fungi an always available "her-
barium", a "mycotheca" preserved in chlorallactophenol.
For the purpose I use 15cc or 30cc size of brown, screw capped
pill bottles, commercially available in any drug store. These are
filled half to three quarters full with chlorallactophenoP) in which
culture particles from any agar mediums, together with their agar
support on which they were grown, are deposited. The great advan-
tage of this procedure is quite obvious. Culture particles can be pre-
served in this manner at any age and stage of the progressive
growth of the fungi with unchanged characteristics. For instance,
in taking particles from a 7 day old culture, it will show all charac-
teristic features of growth at the seven day stage, after 3 months,
five or ten years! All morphological characteristics are "frozen"
spontaneously the moment the fungus particles are immersed in
chlorallactophenol. For general studies or for comparative investi-
gations, particles can be withdrawn from the "mycotheca" and they
are just as fresh and well preserved and unchanged as on the day
they were deposited in the "mycotheca".
I wish to mention one conspicuous advantage. Perithecial derma-
tophytes, (these become each day more and more numerous) and
other hyphomycetes, if kept in artificial culture, very often lose
their ability to form perithecia. If such perithecial stages are pre-
served in chlorallactophenoI, particles which are withdrawn from
the "mycotheca" can be studied, compared, after years as the case
presents itself.

1) Phenol cryst.
Lactic acid AA 10.0
Glycerine 20.0
Aq, dist. 10.0
 FRAGMEi'qTA M Y C O L O G I C A II 259

How much material and which series of developmental stages

should be preserved from a certain fungus strain, is entirely up to
the individual purposes of the investigator.
One vial of fungal material is, of course, not an inexhaustible
quantity. According to the rarity of the specimen, several vials and
a larger amount of material can be preserved in one's "mycotheca.'"

Summary
CASTELLANI'S "water culture" method for microscopic fungi was
re-examined and confirmed in its every detail. It is an ideal method
for, at least, the smaller culture collection, in order to avoid contin-
uous, short term subculturing.
BENEDEK'S suggestion for establishment of a "mycotheca" pre-
served in chlorallactophenol on the analogy of the "herbarium" of
phanerogamists, m a y fill a long felt gap in mycological laboratories.

Zusammenfassung
CASTELLANI'S "Wasserkultur-methode'" ftir mikroskopische Pilze
wurde einer Kontrolluntersuchung unterzogen. Sie konnte in all
ihren Einzelheiten best~tigt werden. Sie ist eine ideale Methode,
wenigstens ffir kleine Kultursammlungen, um eine fortgesetzte,
kurz-fristige Uberimpfung zu vermeiden.
BENEDEK'S Vorschlag ftir die Einrichtung einer "Mykotheka" in
Chlorallactophenol in Analogie mit dem "Herbarium" ffir Bltiten-
pflanzen, mag eine lang geffihlte Lficke im mykologischen Labora-
torinm beseitigen.

R6sum6
La m6thode de CASTELLANI,la "culture en d'eau" des champi-
gnons microscopiques, a 6t6 rd-examinde. Elle pouvait 6tre confirm6e
en tousles d6tails. C'est une m6thode id6ale, au moins, pour la collec-
tion de cultures de petite dimension, pour dviter les transferts conti-
nus et d'intervalle courte.
La suggestion de BENEDEK pour dtablir une mycoth&que en
chlorallactoph6nol, par analogie avec 1' "herbier" pour les phandro-
games peut remplir une lacune, existante pour long temps, aux
laboratoires mycologiques.

Bibliography
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CASTELLA~II, A. 1960. A brief note on the viability of some pathogenic fungi in
sterile distilled water. Imprensa M6dica, 24. June.
17"
260 T. BENEDEK

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