Medical Mycology December 2004, 42, 499 /504
Evaluation of Microsporum canis in different methods of
storage
R. S. N. BRILHANTE*$, C. S. P. CAVALCANTE*, F. A. SOARES-JÚNIOR*, A. J. MONTEIRO%, E. H. S. BRITO*,
R. A. CORDEIRO$, J. J. C. SIDRIM$ & M. F. G. ROCHA*$
*School of Veterinary Medicine, Post-Graduation Program in Veterinary Science, State University of Ceará, Fortaleza;
$Department of Pathology and Legal Medicine, School of Medicine, Medical Mycology Specialized Center and %Department
of Statistics and Applied Mathematics, Federal University of Ceará, Fortaleza, Brazil
The main objective of this investigation was to evaluate different methods of
storage for Microsporum canis based on materials and equipment that are readily
available in developing countries. We tested 32 strains of M. canis at 208C in
potato dextrose agar (PDA) in its plain condition, or amended with 10% dimethyl
sulfoxide or with 10% glycerol. In addition, we tested 258C storage of isolates in
plain saline (0.9% NaCl) and in saline covered with a mineral-oil layer. After 9
months of storage, none of the M. canis strains frozen in PDA supplemented with
glycerol survived, while only 16 and 6%, respectively, of the isolates in plain and
DMSO medium lost viability. Nine month’s storage in saline with or without
mineral oil increased the amount of pleomorphic development of sterile hyphae;
this phenomenon occurred at a significantly higher level than was seen in isolates
stored at 208C. The physiological characteristics of M. canis were not affected
by the different storage tests. The results suggest that, in order to ensure optimal
viability, purity and pristine isolate condition, each M. canis isolate maintained
should be held in at least two methods of storage, namely, PDA at 208C and
saline with a mineral-oil layer at 258C.
/
/
/
Keywords
cats, dogs, Microsporum canis, storage
Introduction
The preservation of fungal strains is a very important
activity in a mycological laboratory. Strains may be
preserved as diagnostic reference stocks, or for comparative studies, or for use in training [1]. Some fungi
are difficult to maintain in good condition, however,
and some dermatophytes in particular mutate rapidly
to produce morphological variants quite unlike the
parental strain. Changes in physiological, biochemistry,
pathogenicity and genetic characteristics may
also occur, often without obvious morphological
changes [2].
Received 31 January 2003; Accepted 10 October 2003
Correspondence: R. S. N. Brilhante, Rua Barão de Canindé 210;
Montese, CEP 60.425-540, Fortaleza CE, Brazil. Tel: /55 085 214
2853; Fax: /55 085 295 1736; E-mail: [email protected]
– 2004 ISHAM
Various methods have been proposed for the high
quality preservation of fungal cultures; for example,
good results have been shown for storage in sterile soil
[3], storage in sterile distilled water [1,4,5], freezing at
/708C [6], freeze-drying (lyophilization) [7], maintenance under paraffin oil overlays [8] and immersion in
vessels held in liquid nitrogen (cryopreservation) [9].
It is recommended that in order to minimize the
probability of strains being lost, each strain should be
maintained by at least two different procedures, whenever practical. At least one of these, where possible,
should be lyophilization or storage in cryopreservation,
as for most strains these are the best methods for
minimizing the risk of genetic change [7,9,10]. In many
parts of the world, however, such methods are not
available and alternative techniques must be found.
The purpose of the present investigation was to assay
which widely available, inexpensive methods would be
suitable for preservation of the macroscopic and
DOI: 10.1080/13693780410001712052
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Brilhante et al.
microscopic features of Microsporum canis isolates in
a Brazilian laboratory lacking routine access to lyophilization, ultra-low-temperature freezing and liquid
nitrogen storage.
Between November 2000 and August 2001, 32 strains
of M. canis were obtained from 22 dogs and 10 cats
examined in the Medical Mycology Specialized Center,
Faculty of Medicine, Federal University of Ceará,
Brazil. The samples were obtained in collaboration
with five veterinary clinics located in the city of
Fortaleza, Ceará, Brazil.
further analysis was done. For strains producing
macroconidia, 10 nonoverlapping /40 microscope
fields were examined. Macroconidial numbers in the
combined fields was noted as 0 /10, 11/50 or /50.
Quantification of microconidia was done similarly.
After quantification, macroconidia were checked to
determine if they had typical morphology (fusiform,
with thick and roughened walls). Thereafter, macroconidia were randomly selected and measured with a
reticule for length and width. These features were
observed at /40.
Physiological characteristics of the revived isolates
were evaluated, as recommended by Sidrim and
Moreira [11], with nutritional tests (thiamine, nicotinic
acid, inositol and histidine) and the urease test. In
addition, the in-vitro hair perforation test was done.
Storage strains
Statistical analysis
Definitively identified M. canis isolates were maintained in saline (0.9% NaCl) at room temperature
(258C). This procedure was intended to establish a
common zero time for all strains. Inoculum from the
saline stocks was then used to inoculate potato dextrose
agar (PDA; Difco, Detroit, MI), which was incubated
for 15 days at room temperature. The strains of M.
canis were then subcultured into the following storage
media for storage at /208C: (i) PDA, (ii) PDA with
10% dimethyl sulfoxide (DMSO) and (iii) PDA with
10% glycerol. In addition, each strain was stored at
258C in saline (7-ml vol. of 0.9% NaCl) with and
without a 2-ml covering of sterile mineral oil.
A test for homogeneity, the Fischer’s exact test, was
used to compare numbers of surviving M. canis strains
in different methods of storage.
Materials and methods
Isolates
Fungal viability verification for the different storage methods
At intervals of 3, 6 and 9 months, frozen strains were
thawed and material was inoculated into Sabouraud
agar and PDA tubes. Strains stored in saline and saline
with mineral oil were manually agitated and a portion
of each suspension was transferred to growth media as
used for the frozen samples.
Microsporum canis colonies in Sabouraud and PDA
media were analyzed after 15 days, and the colony
surface texture and reverse pigmentation were noted.
Micromorphology of the revived isolates was
observed in rice agar, lactrimel agar, Sabouraud agar
and PDA as recommended by Sidrim and Moreira [11].
Slides were made in lactophenol cotton blue, and for
each culture, 10 microscope fields were scanned at
/40.
The quantification of macroconidial production was
as follows. Initially, we noted whether each test strain
produced macroconidia or just sterile mycelium on the
test media. If the strain did not sporulate initially, no
Results
In pre-storage micromorphological analysis, Sabouraud agar was observed not to favor conidiogenesis: 63%
of the M. canis strains grown on this medium formed
only sterile hyphae (Fig. 1A). Of the 37% strains that
produced macroconidia, only 13% produced typical
macroconidia. PDA, rice agar and lactrimel agar were
equivalent for macroconidia production; the difference
among these media was not significant (P /0.4190).
However, the lactrimel agar medium appeared in
general to be more favorable for the observation of
conidium formation, since of all media it induced the
lowest production of sterile mycelium, a problem
affecting only a single one of the 32 cultures grown
on it prior to preservation (Fig. 1B).
When preserved isolates were examined for sterile
mycelium production, it was seen that storage in saline,
with or without mineral oil, increased the amount of
sterile mycelium seen (Fig. 2). This difference is
significant (P /0.0015), when compared to M. canis
isolates preserved frozen.
Lactrimel agar stimulated macroconidium production in 31 of the 32 test strains in baseline checks prior
to storage (Fig. 3). In addition, 75% of the strains
grown on this medium produced typical macroconidia.
The macroconidial sizes, ranging from 50 to 85 mm in
length, and 7.5 to 20 mm in width, remained stable
during the 9 months of preservation irrespective of the
method used.
– 2004 ISHAM, Medical Mycology, 42, 499 /504
M. canis and different storage methods
501
Fig. 1 Frequency of sterile mycelial proliferation
in Microsporum canis isolates maintained under
different preservation methods and then grown
for analysis on different media. (A) Sabouraud
agar results; (B) lactrimel agar.
Isolates stored 9 months in both saline treatments
and in plain PDA and PDA with DMSO lost their
capacity to produce microconidia. These results were
analyzed using lactrimel agar, which stimulated microconidial production in all but one of the strains prior to
preservation (Fig. 4).
Only 9% of pre-storage strains grown on Sabouraud
agar had low cottony colonies with fimbriate margins
and canary yellow pigment, even though these characteristics are considered typical for M. canis. However, after storage in PDA with DMSO and saline with
mineral oil, 28% of M. canis strains grew on Sabour-
Fig. 2 Isolates of Microsporum canis showing
proliferation of sterile hyphae after 9 months of
storage in plain saline and in saline overlaid with
mineral oil.
– 2004 ISHAM, Medical Mycology, 42, 499 /504
aud as typical colonies (Table 1). Cottony, unpigmented
colonies made up the largest morphological category,
accounting for 41% of strains pre-storage. These strains
showed further attenuation of morphological differentiation during the preservation period, independent of
the method evaluated. Only a single M. canis strain
preserved 6 months in either saline treatment showed a
typical fringed, yellow colony. After 9 months frozen
on PDA with DMSO, three of the 32 test colonies
showed these characters.
The tested physiological characteristics of M. canis,
as outlined above, were not changed by the different
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Brilhante et al.
Fig. 3 Number of Micorsporum canis isolates
showing macroconidial production after preservation by different methods, as analyzed by subsequent growth in lactrimel agar.
preservation methods. All results were typical for the
species.
Death of stored M. canis strains was observed in the
three techniques involving freezing, but was not seen
either saline storage method at 258C. All M. canis
strains stored in PDA with added glycerol were dead
after 9 months. Two strains (6%) stored in stored in
PDA with DMSO died, while 16% of strains in plain
PDA failed to survive (Fig. 5). This difference between
plain PDA and agar with DMSO was marginally
significant (P /0.04258).
Discussion
The storage of microorganisms is one of the major
problems at research centers, because few laboratories
possess the technological support for storage of strains
by lyophilization or liquid nitrogen. Thus many mycologists seek to store their microorganisms with cheaper
and more accessible methodologies. Each technique
used requires evaluation, as storage can induce morphological changes and alterations in cell wall components as well as a loss of virulence in some species
[12,13].
Determination of the correct preservation method
for each fungal species requires periodic monitoring
with special attention directed to morphology, pathogenicity and genetic stability [14].
Sabouraud agar, despite being the classic medium
used in medical mycology, was found not to be
adequate for micromorphological studies in M. canis,
failing to elicit macroconidia that appeared in 97% of
strains on lactrimel agar. This finding is likely related to
the high nutrient levels in Sabouraud medium. Marchisio et al. [15] have already shown that lactrimel
medium is capable of stimulating macroconidial production in sterile isolates of various dermatophyte
species.
Our finding that the two types of saline storage
tested increased the proliferation of sterile hyphae over
the 9 months of storage may reflect the microaerophilic
conditions found in the stored saline solutions, as well
as low nutrient levels and an accumulation of toxic
metabolites. In 1984, Barnes [16] reported that the
longevity of microorganisms stored in mineral oil can
vary with species as well as with storage temperature.
Factors that should be taken into account also include
the age of the inoculum used to initiate the storage
Fig. 4 Number of Microsporum canis isolates
showing microconidial production after preservation by different methods, as analyzed by subsequent growth in lactrimel agar.
– 2004 ISHAM, Medical Mycology, 42, 499 /504
M. canis and different storage methods
Table 1 Time of preservation and storage technique versus changes
in Microsporum canis colony macromorphology
Culture media
Type* 3 months 6 months 9 months
Saline covered
with mineral oil
A
B
C
A
/
21.8%
/
9.0%
9.0%
56.3%
3.0%
12.5%
28.0%
31.3%
/
9.0%
B
C
A
18.7%
/
3.1%
34.3%
3.0%
3.1%
28.0%
3.0%
28.0%
Potato dextrose
B
agar with 10% DMSO C
25.0%
/
18.7%
/
15.6%
9.0%
Saline
*Colony types: A, low, fringed, cottony colony with canary yellow
reverse; B, deeply cottony colony without pigment; C, fringed,
farinaceous colony with canary yellow reverse. Initial tests on
Sabouraud agar showed A colonies at 9%, B at 41% and C and
others combined at 50%. Other colony types observed were fringed,
low cottony unpigmented colony; deeply cottony colony with yellow
pigment and fringed, farinaceous, unpigmented colony. These types
make up the balance of 100% of the colonies in each of the time
periods tested for each storage condition.
procedure, as well as the concentration and quality of
the mineral oil [17].
Cryopreservation, frequently used in several areas,
was tested in the present study with two cryoprotectants in PDA, DMSO and glycerol. These compounds
are expected to protect the cell surface against production of intracellular ice crystals. However, numerous
investigators suggest that such additives can alter the
chemical and mechanical nature of the cell during
freezing, making it necessary to stabilize the kinetics of
cell permeation by balancing the changes in osmotic
concentration brought about by such cryoprotectants
[18]. We found that DMSO was associated with
viability in 30 (94%) of the stored strains, while on
plain PDA, 27 strains survived (84%). This difference
was insignificant (P /0.4258).
Fig. 5 Number of deaths in Micorpsorum canis
strains stored by different methods.
– 2004 ISHAM, Medical Mycology, 42, 499 /504
503
Glycerol appeared to be lethal. We used the protocol
recommended by Stockdale et al. [19], involving
covering cultures with 10% glycerol (or 10% dimethyl
sulfoxide) before transferring to the /208C freezer.
None of our strains survived this protocol with
glycerol. The toxicity of 10% glycerol to some fungi
has also been observed in stored strains of Madurella
mycetomatis [20]. As Kim et al. [21] have stated, the
cryoprotectants, despite being undeniably successful at
the initial time of freezing, can become cytotoxic during
prolonged periods at temperatures below 08C. On the
other hand, toxicity is not always observed. For
example, 10% glycerol was an excellent cryoprotectant
in strains of Malassezia spp. at /808C [22], a
temperature at which molecular mobility in glycerol
solutions is negligible. It is not clear whether or not
these differences between M. canis and Malassezia
spp. are organism-specific or derive, at least in part,
from the difference in temperatures used. We limited
our study to /208C freezing because this is widely
accessible at our institution. Certainly, when M. canis
is stored at /208C, 10% glycerol should not be used.
Macromorphologically, the Sabouraud agar medium
was recognized as ideal for macromorphological
studies, as it consistently showed greater uniformity in
the characteristics observed than was seen on other
media. However, what was unexpected was that the
classic M. canis variant (which, as described by Evans
and Richardson [2] has a fimbriate margin, a low
cottony texture and canary yellow pigmentation) was
the phenotype least commonly isolated from our
test animals. A supposedly atypical primary isolate
type, deeply cottony and unpigmented was most
common. Though Marchisio et al. [15], indicate that
only two macromorphological strain variants of M.
canis can be found, one with low mycelium and the
other with deeply cottony mycelium, we also found a
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Brilhante et al.
third variant. This variant, seen after 6 months
of storage, had a cinnamon-colored, minutely scalylooking, farinaceous colony with or without canary
yellow reverse pigmentation.
Strains stored in saline tended to form cottony
colonies with yellow reverse when subcultured in
Sabouraud agar, possibly due an effect of the stress of
diminished metabolism in the saline, as well as the low
availability of nutrients. Saline treatments and PDA
with DMSO cryoprotectant, evaluated at 6 months,
favored the production of farinaceous strains with or
without pigment. These isolates completely lost the
appearance of being M . canis isolates, possibly due to
the stress of readapting to the nutrient-rich environment of fresh growth medium. We speculate that these
isolates might regenerate their characteristics after
successive subcultures on fresh medium. Similar phenotypic changes can be observed in other stored
dermatophytes, such as in Trichophyton tonsurans
isolated obtained in various regions of the world.
Kim et al. [23] using random amplification of polymorphic DNA, demonstrated the genetic similarity of
these strains, independent of phenotypic polymorphisms. Raven et al. [24] suggest that the adaptive
processes of species are associated with instability of
their habitat, whether in biotic or in abiotic factors.
Preservation and revival can be expected to elicit the
types of changes brought about by instability.
We know no precedent for our observation that
microconidia were formed in 97% of strains initially,
but disappeared after storage irrespective of the
method used. Unfortunately, we can suggest no
explanation for this phenomenon.
Our conclusion is that, in order to ensure optimal
viability, purity and morphological stability, each strain
of M. canis should be maintained in at least two
methods of storage, namely, PDA at /208C and saline
with a mineral-oil layer at 258C.
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