2 IDENTIF I C AT I O N O F M O U L D S

The identification of filamentous fungi is based on the examination of their macroscopic

(colonial) and microscopic characteristics. In the seventeen years since the first edition of
this manual, there has been an explosion of new species names, many based more heavily
on nucleic acid sequencing methodology than on the traditional morphological approach.
It is thus important to recognise that definitive identification of, for example, atypical,
unusual or non-sporing moulds, will often require molecular analysis to fully support
morphological identification.
One of the consequences of molecular analysis of moulds has been the recognition that
many medically important species are in fact complexes that are composed of a number of
genetically distinct, but morphologically identical species. Nonetheless, as an introduction
to the major groups of pathogens for clinical diagnosis, microscopic morphology remains
the standard approach. Wherever possible we have indicated where a species complex has
been deliberately simplified for primary identification.
Macroscopic features such as colonial form, surface colour and production of pigments
are often helpful in identification. The growth rate of mould colonies depends on the
culture medium and temperature of incubation but provided conditions are standardised,
these characteristics can be taken into consideration in the process of identification.
Morphological examination of microscopic structures such as spores and spore-bearing
cells is an essential part of mould identification. Moulds that fail to sporulate are often
impossible to speciate and it is therefore important to select culture conditions which
favour sporulation.
Many clinical laboratories today employ DNA sequencing as part of their routine proto-
col for fungal identification. In circumstances where morphology-based identification is
not helpful, an isolate may be a candidate for DNA-based identification. This approach
may be useful when an isolate displays atypical morphology, fails to sporulate, requires
lengthy incubation or incubation on specialized media in order to sporulate, or if the phe-
notypic results are nonspecific or confusing.

Media
The texture and colour of mould colonies often depend on the age of the culture and agar
medium on which the organism is grown. Nevertheless, these characteristics are useful in
identification. Owing to the almost universal use of Sabouraud’s glucose peptone agar,
the descriptions in this manual are based on cultures prepared on it. However, there are

Identification of Pathogenic Fungi, Second Edition. Colin K. Campbell, Elizabeth M. Johnson,

and David W. Warnock.
© 2013 Health Protection Agency. Published 2013 by Blackwell Publishing Ltd.

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I D E N T I F I C AT I O N OF MOULDS

numerous formulations of that medium, both with and without antibiotics, and it is advis-
able to confine supplies to one manufacturer as the morphological appearance of moulds,
and pigmentation in particular can differ from one formulation to another. Moulds often
grow best on rich media, such as glucose peptone agar, but over-production of mycelium
often results in loss of sporulation. If a mould isolate fails to produce spores or other rec-
ognisable structures after two weeks, it should be subcultured to a less-rich medium to
encourage sporulation and permit identification. The composition of a number of useful
media is given at the end of this chapter.

Methods of Slide Preparation

Microscopic examination of slide preparations is the most important part of the identifica-
tion of a mould culture. If well prepared, these will often give sufficient information on
the form and arrangement of spores and other structures for an identification of the fungus
to be made. The usual method is to remove some of the surface growth from a culture
plate with a sharp rigid needle and place it in a drop of mounting fluid (such as lacto-
fuchsin or lactophenol cotton blue) on a clean microscope slide. The material is then teased
apart with two sharp needles and a cover slip applied. Gentle pressure is used to spread
out the preparation before it is examined under a microscope using ×10 and ×40 objective
lenses.
There are several other methods for the preparation of slides for microscopic examination
of fungi. One of the most helpful is to use clear adhesive tape. A small flag of tape (about
20mm long) is cut with scissors and placed on the end of a rigid needle. The tape is pressed,
adhesive side downwards, on to the surface of the culture using a second needle applied
to the back of the tape. The coated tape is then placed, adhesive side upwards, in a small
drop of mounting fluid on a microscope slide. A second small drop of mounting fluid is
placed on the preparation and a cover slip is applied.
If a slide preparation shows no spores, it is often helpful to try nearer the centre of the
colonies, where the mould is older and has had more time to sporulate. If there are too
many spores and the sporing structures cannot be discerned, it is useful to try nearer the
edge of the colonies. If no spores are found in slide preparations, it is sometimes worthwhile
to remove the lid from the culture plate and examine the colonies for evidence of sporula-
tion under the low-power objective of a microscope.
Both the ‘needle’ and the ‘tape’ methods give suitable preparations for microscopic
examination but each has its drawbacks with certain forms of fungal growth. Needle
preparations dislodge chains and wet masses of spores, and these features are best seen
with tape preparations. On the other hand, structures such as pycnidia and spores hidden
deep in the mycelium are not picked up on tape and require dissection with a needle. The

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IDENTIFICATION OF MOULDS

needle also allows sub-agar growth to be studied. Mounting fluids such as lactophenol and
lactofuchsin attack some types of adhesive tape and render it unsuitable for preparation of
permanent mounts. Needle preparations can be sealed around the edge with DPX for long-
term storage.

Slide Culture
The slide culture technique is useful for observing the intact arrangement of spores or
spore-bearing structures. A thin, square block of a suitable nutrient agar (smaller than a
cover slip) is placed on a sterile microscope slide supported on a bent glass rod in a petri
dish. The four sides of the agar block are then inoculated with portions of mycelium of the
fungus to be identified. The block is then covered with a sterile cover slip, sterile distilled
water added to the base of the petri dish, the lid replaced and the plate incubated at 30oC.
Once adequate sporulation has occurred, the cover slip is removed from the agar and
placed on a drop of mounting fluid on a clean glass slide with the adherent mycelium
downwards. The agar block is then removed and discarded, leaving adherent mycelium
on the slide. Mounting fluid is added and a clean cover slip applied. The preparations can
be sealed for long term preservation.

Conversion of Dimorphic Pathogens from Mould to Yeast Phase

Commercial kits are available for the rapid identification of the dimorphic fungi Histoplas­
ma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Sporothrix schenckii.
These include the AccuProbe test (Gen-Probe Inc., San Diego, California, USA) and the
exoantigen test (ImmunoMycologics Inc., Norman, Oklahoma, USA). In addition, the iden-
tification may be further supported by inducing conversion from the mycelial to the yeast
phase. Conversion of Coccidioides spp. to the pathogenic (spherule) phase is problematic
and less commonly attempted. Note that all these fungi, with the exception of S. schenckii
are hazardous pathogens and should only be handled under safe laboratory conditions
(UK Hazard Group 3 or equivalent).
Conversion to yeast phase usually requires special media and a temperature above 35°C.
An extensive body of literature has been devoted to conversion conditions as they apply
to the various species, and specialist laboratories will have their favourite procedures.
Bacteriological blood agar, heat-treated blood (‘chocolate’) agar, brain-heart infusion agar
or even Sabouraud’s glucose peptone agar may achieve conversion. Whatever medium is
used, conversion often leads to a colony of yeast cells mixed with hyphae, and serial sub-
cultures may be needed to fully convert to the yeast phase. It is also helpful to keep the
agar surface from becoming too dry.

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MEDIA FO R M O U L D I D E N T I F I C AT I O N

Cornmeal Agar Dermatophyte Test Agar

This medium is useful for stimulating This medium turns red in colour with
ascocarp and pycnidium production in dermatophytes and is useful for
some moulds. distinguishing those species from other
moulds. It is important to remember that
cornmeal extract 2g some non-dermatophyte moulds can also
produce that colour change.
agar 15 g
distilled water 1L
glucose 40 g
mycological peptone 10 g
Heat to dissolve. Autoclave at 121°C for
15 min. phenol red 0.2 g
agar 12 g
distilled water 1L
Czapek-Dox Agar
This defined medium is recommended for Heat to dissolve. Autoclave at 121°C for
the identification of Aspergillus and 15 min.
Penicillium spp. It is also useful for
stimulating sporangium production in Malt Extract Agar
mucoraceous moulds.
This rich medium is recommended as an
alternative to Sabouraud’s glucose
sucrose 30 g
peptone agar for stimulating sporulation
sodium nitrate 2g
in a wide range of moulds, including the
potassium chloride 0.5 g dermatophytes.
magnesium glycerophosphate 0.5 g
potassium sulphate 0.35 g malt extract 30 g
ferrous sulphate 0.01 g mycological peptone 5g
agar 12 g agar 15 g
distilled water 1L distilled water 1L

Heat to dissolve. Autoclave at 121°C for Heat to dissolve. Autoclave at 115°C for
15 min. 10 min.

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IDENTIFICATION OF MOULDS

Philpot’s Urea Agar glucose 20 g

This medium turns red in colour if the potato extract 4g
fungus produces urease enzyme. It is used agar 15 g
to distinguish Trichophyton rubrum distilled water 1L
(usually urease negative) from
T. interdigitale (urease positive). It is
Heat to dissolve. Autoclave at 121°C for
important to remember that the granular
15 min.
form of T. rubrum gives a positive result,
as will most dermatophytes.
Sabouraud’s Glucose Peptone
Agar
glucose 5g
mycological peptone 1g This medium is recommended for the
sodium chloride 5g isolation and cultivation of dermatophytes
and other moulds requiring a rich
potassium-dihydrogen 2g
substrate with a high content of organic
orthophosphate
nitrogen. Antibacterial antibiotics (in
phenol red 0.012 g
particular chloramphenicol) can be added
agar 15 g
to control bacterial contamination.
distilled water 1L

glucose 40 g
Heat to dissolve. Autoclave at 115°C for mycological peptone 10 g
20 min. Cool to 50°C and add 50 ml of
agar 15 g
sterile 40% urea solution.
distilled water 1L

Potato Dextrose Agar

Heat to dissolve. Autoclave at 121°C for
This is a good general purpose medium 15 min.
which stimulates sporulation in many
moulds. It stimulates pigment production
in some dermatophytes.

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100 mL

100 mL
0.075 g

0.1 g
Store away from direct sunlight.

Store away from direct sunlight.

Lactophenol cotton blue

Lactofuchsin
acid fuchsin
lactophenol
cotton blue

lactic acid

16
Heat gently to dissolve. Store away from
20 mL
40 mL
20 mL
20 g
MOUNTIN G F L U I D S

phenol crystals

distilled water

direct sunlight.
Lactophenol

lactic acid
glycerol