Single spore isolation of fungi
ABSTRACT W.H. (1999). Single spore isolation of fungi. Fungal Diversity 3: 29-38. Methods to isolate fungi from single spores are outlined. These methods are specifically designed for mycological laboratories which are not necessarily well funded. Therefore, they involve a simple procedure, are relatively inexpensive, and most importantly effective. Furthermore, only basic equipment is required. By using these methods, most fungi, with the exception of those that do not germinate on artificial medium, can be isolated. Some approaches are suggested to prevent mite infestations and to reduce the risk of bacterial contamination.
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Single spore isolation of fungi
Yuen-Wah Choi*, Kevin D. Hyde and Wellcome W.H. Ho
University of Hong Kong, Pokfulam Road, Hong Kong; * e-mail: email@example.com
for Researchin FungalDiversity, Departmentof Ecologyand Biodiversity,The
Diversity 3: 29-38.
Hyde,K.D. and Ho, W.H. (1999). Singlespore isolationof fungi. Fungal
designed for mycological
to isolate fungi from single spores are outlined.
laboratories which are not necessarily well funded. Therefore, they
procedure, are relativelyinexpensive,
only basic equipment is required. By using these methods, most fungi, with the
that do not germinateon artificial
are suggestedto prevent mite infestations
These methodsare specifically
and most importantlyeffective.
and to reduce the risk of bacterial
can be isolated.Some
Key words:culture collection, fungal isolations
The process of identifying fungi does not rely solely on morphological
features for identification.The use of fungal cultures, obtained from single
spore isolations are fundamental to the identification of many fungi. In terms of
the morphologicalspecies concept, cultures can provide extra characters for
identification, and anamorph-teleomorph
may also be useful and have traditionally been fundamental in the identification
of certaintaxasuchas speciesof Fusarium
morphologicaland molecular characters, in most cases require single spore
cultures (Goh and Hanlin,1997). The biological
tested by mating fungal cultures, thus isolates derived from single spores are
fundamental for these studies. Cultures also allow repeatable testing of results
by other researchersand may also be important in the discovery
metabolites (e.g. Strobel et al., 1996).
The importanceof careful identification
that have been isolated from single spores cannot be over stressed (Smith,
1969). In order to study fungi more successfully, it is important that simplified
keysare developed for identification,
connections. Cultural characteristics
both can of
species concept has been
of fungi and obtaining cultures
and simpleeffective methodsare
Figs. 1-2. Some tools useful in fungal isolation. 1. Extra fine forceps, Pasteur pipette, small
glass container and pipette teat. 2. Glass needle.
perform, even for those who do not have experience in isolation, relatively
inexpensive and effective. There are many methods for isolating and storing
fungi (Hilderbrand,1938; Smith and Onions, 1994), however, some of these
are expensive or difficult to carry out. In this paper, we describe a commonly
used method for single spore isolation, which is inexpensive, easy to carry out,
and effective. Only basic equipment is required and much of the equipment can
be reused,therefore even poorlyfunded
procedures that are outlined.
The most common problems when isolating fungi are contamination
bacteria and yeasts, and the fact that the wrong species may inadvertently be
isolated. To overcome these problems, dilution of spore masses can be used to
in order to isolate fungi. Isolation methods must be simple to
laboratoriescan carryout the
is isolated (Hansen,
cultures stored in a high nutrient medium, such as Potato Dextrose Agar (PDA)
may lose their ability to produce enzymes or metabolites,
functions. Care is therefore needed in selecting a storage medium (Smith and
0.5 g/L, streptomycin,
There are several methods to ensure that the correct single spore
1926) and a commonly used method is reported here.
not all spores will germinate
may be required for successful isolation of these fungi. Fungal
of yeast contaminationand differentantibiotics (e.g.
0.5 g/L) can be used to reduce bacterial
in artificial media, and other
or perform other
For effective isolations to be carried out, an isolation room or clean bench
with as little air movement as possible is desirable. A dissection microscope
with illumination from both below and above, kept permanently
flow cabinet is desirable. A compound microscope can be kept near by to check
spores and to observe germinating spores). This will reduce the likelihood of
contamination.Alcohol (70%) is used to clean the working surface and some
sort of burner is required for sterilizing utensils.
In the methodsrecommended below, water agar (WA) and PDA are
routinely used as inexpensive media. Other media may be required for difficult
fungi, such as V8 juice agar for lower fungi, or corn meal agar (CMA) when
sporulationis requiredin lignicolous
Pasteur pipettes must also be prepared beforehand.
A dissecting microscope and a compound microscope are necessary for
fungal identification.A small glass container or glass slide, sterilised water,
sterilised Pasteur pipettes, and extra fine forceps are desirable for single spore
isolation (Fig. 1). An alcohol lamp, insect needle and a Pasteur pipette are used
to make a glass needle. A clean 25 C incubator is important for the storage of
in a lamina
fungi. Sterilised water and sterilised
Making the glass needle
The narrow end of the pipette is broken so that about 2 cm remains and an
insect needle (metal needle for mounting insects) is placed inside the broken
end and flamed until the glass melts and the needle is firmly fixed (Fig. 2). It is
easier to use the needle if it is less than 1 cm outside the end of the glass
pipette, otherwise it will break easily.
Other kinds of glass needles (Goh, 1999) can be used and are similar to the
one mentioned here. It is more difficult to sterilize glass needles, therefore the
metal one mentioned here is preferred. Special isolation needles, such as the
cleistothecia. 5. Ascomycete with apothecia. 6. Hyphomycete.
3-8. Different typesof fungiand germinated spores.
7,8. Germinated spores.
3,4. Ascomycetes with
Microtool (T5340 Microtool needle and T5344 Microtool Handle, Agar PIano
Scientific Ltd.) can be purchased, but they are very expensive and fragile and
not suitable for beginners.
Addition of antibiotics
Agar is prepared and autoclaved at 121 C for 20 minutes and antibiotics are
added when the temperature of the agar is about 50 C, i.e. it can be held by
hand. If the temperature is too high, the antibiotics will denature and become
ineffective. As a general rule, 0.5 g antibiotics per L of agar will reduce
bacterial contamination significantly,but this figure may need to be varied
dependingon the requirements. If the antibiotics are sterilized, a sterilized
syringe should be used to transfer 2 to 4 ml sterilized water to dissolve the
antibiotics and then suitable amount of antibiotics are added to the agar directly
using the syringe. If the antibiotics are not sterilized, dissolve the antibiotics
into 5 to 10 ml sterilized water and sterilize by passing through a sterilized
membrane filter (0.2 /lm or 0.45 /lm pore size). The agar is poured into 90 mm
or 60 mm Petri dishes inside a laminar flow cabinet (or on a sterilised bench)
and allowed to solidify. They can be dried overnight in a safety cabinet. When
preparing dissolved antibiotics and pouring agar, it is better to use a burner to
keep the surrounding environment aseptic. In order to overcome the problem of
overgrowth by other fast growing fungi, such as Penicillium and Trichoderma,
growth inhibitors can be added to slow down the radial growth of the fungal
colonies. Rose Bengal (0.3 g/L) is commonly used to inhibit growth. This can
be autoclavedwith the media, which is very convenient,
additional likelihood of contamination.
or other inhibitorysubstances
as there is no
Addition of complex materials
In order to maintain the culture in good condition, various substrates can
be used to promote better growth of mycelia or formation of fruiting structures
(Guo et al., 1998). This can include the addition of wood, leaves or cellulose
filter paper to the surface of the media. Material, which is similar to, or the
same as, the substrates on which the fungi originally grew, is desirable. This
material should be autoclaved twice (121 C, 20 minutes), before use and can be
placed on the agar surface aseptically.
Isolationfrom single spores suspensions
There are many different groups of fungi, e.g. ascomycetes, coelomycetes,
basidiomycetesand hyphomycetesand each has different types of fruiting
bodies. The methods to isolate each of them are therefore different.
Fungi with closed fruiting bodies (Figs. 3, 4; e.g. ascomycetes
coelomycetes with pycnidia: Phoma sp.) can be removed from the substrate
surface using fine forceps and broken open in sterilized water in order to
provide a spore suspension. If the fruiting body is submerged in the substrate, a
Figs. 9-12. Fungal cultures. 9. Spore suspension on 16 squares on water agar plate. 10. Five
pieces of agar containing germinated spores on PDA plate. 11. Fungal colonies of about I cm
diam. 12. A pure fungal culture.
razor blade is used to cut the sporocarp open to expose the internal contents.
The contents can then be transferred to a drop of sterilized water on a small
glass container or slide in order to provide a spore suspension.
Fungiwithcup-shaped fruiting bodies(ascomycetes with apothecia:
Rhytisma sp., Fig. 5, and coelomycetes with acervuli: Chaetospermum
be transferreddirectly, by removing the whole fruiting body. This can be
placed in sterilized water and squashed with fine forceps, in order to provide a
suspensioncan be obtained by removing a few segments from the gills and
agitating them in sterilized water. Isolation can also be carried out from sterile
tissue within the cap. A few pieces of sterile tissue can be aseptically torn from
the split fruiting structure and placed on water agar.
Spores of hyphomycetes can be picked up directly from the substrate using
fine forceps or a needle (Fig. 6). It is better to pick up spores only (not
conidiophores),as this reduces the chance of contamination.
placed in sterilized water and agitated in order to provide a spore suspension.
The spores are
Treatment of spore suspensions
A glass container or glass slide is sterilised with ethanol and wiped with a
towel on which ethanol (70%) has been sprayed. A sterilised pipette is then
used to transfer about 6 drops of sterilised water into the glass container or onto
the glass slide. The suspension is then prepared as above. Sixteen squares are
markedon the bottomof the water
homogenous spore suspension is then transferred with a sterilised pipette, onto
the surface of the water agar plate, with a drop placed above each of the drawn
squares. Alternativelyabout six drops of the suspension can be pipetted onto
the centre of the agar plate and this can be carefully shaken to spread the
suspension. If this method is followed it is a good practice to mark the outer
edge of the suspension on the base of the Petri dish. Both of these methods may
help to locate the germinatingspores later. A small drop of the suspension
should be used at this stage to make a permanent slide and to check that the
correct fungus has been selected.
The unsealed plate is incubated at 25 C for 12-24 hours. It is not sealed as
this allows some of the surface water to dry out. If the plate is sealed with
parafilm, water will often accumulate
increase the chance of contamination. The spores are checked within 12 hours
and then every 24 hours to establish
germinated (Figs. 7, 8), a sterilised glass needle is used to pick up a small piece
of agar containinga spore. In order to establish that the spore is the one
desired, and maintain quality control, a slide is prepared and examined under
the compound microscope. If the spores do not germinate after 12 hours, seal
the plate with Parafilm and examine periodically. Ten germinated spores are
transferred and distributed evenly onto two PDA plate (Fig. 10) and incubated
agar plate (Fig.9). The prepared
on the surface of the agar and will
germination.Once the spores have
at 25 C until their colony diam. are about 1 to 2 cm (Fig. 11). A small piece of
mycelium with agar can then be cut and transferred to another PDA plate and
the culture (Fig. 12) is checked after few days, if there is no contamination,
pure culture has been obtained. Cultures can then be stored on the desired
placed on the inside of the top of a Petri dish using Vaseline (Fig. 13). It is then
sealed with parafilm and left horizontally overnight at 25 C. If the basidia are
mature, they will fall on agar surface. In the case of ascomycetes, a small piece
using the spore shooting or drop methods
or a pieceof cap or gill tissue
a piece of cap or gill tissue is cut from the fruiting body and is
e.g. an ascomycete that can ejectits
Infrom a basidiomycete.
of substrate containing a few fresh ascomata, is cut away from the source. Care
is taken to avoid the inclusion of other fungi in order to reduce the chances of
contamination.The substrate is then inserted perpendicularly
that the necks of the ascomata are aimed across the surface of the plate (Fig.
14). The plate is then left on its side overnight at 25 C. If the ascomata are
mature enough, ascospores will be ejected and fall on agar surface below the
The next day, the agar plate surface is checked to see whether spores have
been ejected or dropped. If spores are present on the agar surface, their identity
can be established by preparing a slide of agar containing spores. If the spore is
the correct one, a sterilized glass needle can be used to transfer a piece of agar
containing each spore onto the PDA plate as before.
The PDA plate is incubated in 25 C until their colony diam. are about 1 to
2 cm. A small piece of mycelium with agar is then cut and transferred
another PDA plate and the culture is checked after few days, if there is no
contamination,a pure culture has been obtained.
If there is any contamination in a culture, subcultures may be obtained by
transferringsmall threads of uncontaminated
Although difficult, it is possible to rescue cultures by this method.
into the agar so
hyphae with the glass needle.
Storage of cultures
Once a pure culture has been obtained, it can be maintained on an agar
slant at 4 C or stored as a few pieces of agar with mycelia in cryo vials with
10% glycerol in liquid nitrogen. Alternatively, it can be stored as a few pieces
of agar with mycelia in small bottle with sterilized parafin oil or sterilized
water at 4 C. There are several other methods to maintain fungal cultures and
these can be reviewed in the "Preservation and Maintenance of Living Fungi"
(Smith and Onions, 1994). It is recommended that a mixture of methods are
used so that preservation of a culture is more likely (in case one method fails).
As fungi are an excellent source of food for mites and mites are found
everywhere, fungal cultures in storage may soon become contaminated
mites. If there are any mite infestations in fungal cultures, the mites spread
spores such as Aspergillus
sp., bacteria and yeasts from one plate to another
and if not dealt with quickly can contaminate all of the cultures. There are no
proven methods to eliminate mites, but some precautions can be used to reduce
the risk of mite infestations.
1.The bench and the stage of the microscope where work with cultures is
carried out must be cleaned with 70% ethanol.
2.All stored cultures should be sealed with parafilm, although parafilm will
not keep out mites indefinitely.
The equipment use to remove fungi from the substrate must be sterilized
by flame or in 70% ethanol.
The area for storage of fungal cultures, such as incubator, must be mite-
free. This can be achieved by sterilizing with 70% ethanol every month.
Naphthaleneballs can be stored with fungi and probably slow down the
activity of mites, however, Naphthalene balls may also effect the growth of
If a plastic or perspex container is used for storage, the "feet" of the
container can be immersed in jars with paraffin oil or water, and therefore,
mites cannot climb into container. Once there is mite-contaminated
the collection, it must be removed and sterilised immediately.
Try to avoid leaving plates in the incubator for long periods and transfer
them to storage vessels as soon as possible. This will reduce the risk of
build up of mites.
Mite proof Petri dishes can also be used (e.g. 60 mm air-tight Petri dishes,
Falcon), but they are expensive.
We would like to thank Helen Leung for photographic
following people are thanked for providing imput into the development of the methods we now
routinely use to isolate fungi from single spores: T.K. Goh, E.B. Gareth Jones, Jean Lodge, A.
Nakagiri, R. Peterson and lE. Taylor.
Goh,T.K. and Hanlin,
C1oh,T.K. (1999). Single-spore
Guo, L.O., Hyde, K.O. and Liew, E.C.Y. (I 998). A method to promote sporulation
endophyte fungi. Fungal Diversity 1: 109-113.
Hansen, H.N. (\ 926). A simple method of obtaining single-spore cultures. Science 64: 384.
Hildebrand,E.M. (1938). Techniquesfor the isolation of single microorganisms.
Review 4: 627-664.
Smith, o. and Onions, A.H.S. (\994).Preservation
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