CSIRO PUBLISHING
Australasian Plant Pathology, 2009, 38, 132–134
A simple and effective method for the elimination
of bacteria from fungal cultures
N. J. Cother A,B and M. J. Priest A
A
New South Wales Department of Primary Industries, ASCU, Orange Agricultural Institute,
Forest Road, Orange, NSW 2800, Australia.
B
Corresponding author. Email: norma.cother@dpi.nsw.gov.au
Abstract. A quick and effective method to remove bacteria from fungal cultures without the requirement for antibiotics
or complex manipulations is described.
Introduction
Despite best laboratory practice, contamination of fungal
cultures by bacteria is still a frequent occurrence, hindering the
preparation of axenic cultures and resulting in such cultures
usually being discarded. In many laboratories, antibiotics are
used to overcome the contamination. However, treatment using
antibiotics will not necessarily eliminate all of the bacteria,
and may only mask their presence. In mycological culture
collections where long-term storage is a high priority this
can be a problem as the bacteria often reappear when cultures
are revived.
Constantinescu (1990) noted that purifying fungal cultures by
using agar medium was not a new practice. While this assertion is
correct and several papers have been published on methods to
eliminate bacterial contamination using only agar media, all
methods have their disadvantages. Brown (1924) was the first
author to describe such a method of eliminating bacteria, by
inverting a segment of agar culture, previously inoculated with the
contaminated fungus, and taking inoculum from the under-
surface. This involves double handling of the contaminated
fungus and works only if the fungus penetrates well into the
agar to avoid accidentally picking up some of the contaminant.
Machacek (1934) eliminated bacterial contamination by placing a
small portion of contaminated agar culture into molten cooled
agar and then pressing a sterile coverslip onto the agar to seal the
top of the culture, preventing the movement of the bacterial
contaminant and enabling culturing from uncontaminated
subsurface hyphal growth. This requires transfer at the right
moment of agar cooling combined with careful sealing of the
agar surface with the coverslip and is unsuitable if several
different cultures are being processed at the same time. It is
also not suitable for thermolabile fungi. Sleeth (1945) used a
defined nutrient agar at pH 5.0–5.5, which, after pouring into
Petri dishes to form a thick layer, was cut into quadrants and
inoculated so that each quadrant could be turned over and the
pure fungus removed similar to that described by Brown (1924).
This method has the same limitations as Brown (1924) with
the additional requirement of the preparation of a special agar.
Schmitthenner and Hilty (1962) described a method that relied
Ó Australasian Plant Pathology Society 2009
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on a fungal culture producing spores and added additional
water, which would encourage the appearance of any bacterial
contaminant. Ko et al. (2001) described two methods for the
removal of bacteria and mites from contaminated cultures of
Phytophthora, Pythium and Botryosphaeria, in both glass and
plastic Petri dishes. For plastic dishes, a heated blade was
used to remove a section of the bottom of a plastic Petri dish
containing the mites or contaminant, scraping the exposed agar
and transferring to fresh agar similar to the method of Brown
(1924). This is not easy to do without melting the agar while
cutting the bottom of the Petri dish. The second method involved
removing the entire agar layer by inversion onto clean paper and
removing small pieces of agar from the exposed surface in a
manner similar to that described by Brown (1924). This second
method was the one chosen by the authors.
Other non-antibiotic methods which have been employed
include the use of van Teighem rings (Harrower 1989) or
Raper’s rings (Raper 1937), on the principle that most
filamentous fungi will grow down into the agar within the ring
and emerge into clear agar outside it, leaving any bacterial
contaminant behind. Both Raper’s and van Tieghem rings
normally use only one ring per culture (although several may
be used), which needs to stay in place until the mycelium has
grown down, underneath the ring and back to the surface again.
The rings also require sterilisation between uses. Raper’s
rings need to be inserted into the agar while it is being
poured whereas van Tieghem rings require being heated and
then placed onto pre-poured agar to become embedded
and sealed into the agar. Heating the ring sufficiently without
melting through the agar and into the base of the Petri dish or
the agar cracking and not sealing if the ring is too cool
requires some practice. Neither method is suitable for slow-
growing fungi.
We describe a quick and effective method to remove bacteria
from fungal cultures that can be used in any laboratory without the
requirement for antibiotics or special media. This method is used
routinely in the culture collection quality assurance and
maintenance program of Herbarium DAR for both our bottle
and freeze-dried collection.
10.1071/AP08096 0815-3191/09/020132
Bacterial-free fungal cultures
Method
A very small amount of mycelium, or agar with mycelium, is
removed from the contaminated plate and placed in the centre of
the base of a sterile, plastic Petri dish (Fig. 1). A large rectangular
piece of agar is cut aseptically from a fresh agar plate and
inverted onto the top of the mycelium using a spatula (Figs 2
and 3). By inverting the piece of agar onto the contaminated
piece of culture there is no risk of the spatula spreading
contamination. The edges of the rectangle are smoothed
down onto the Petri dish to seal the contaminated piece and
remove most of the air (Fig. 4). From the remaining agar of the
fresh plate, a second small plug is cut (Fig. 5) and placed on top
of the rectangle of agar above the contaminated mycelium
(Figs 6 and 7). Mycelium is allowed to grow up through the
rectangle and onto the agar plug. A binocular microscope can be
1 2
3 4
5 6
Figs 1–6. Procedure for the removal of bacterial contaminants from fungal cultures. Details in the text.
Australasian Plant Pathology 133
used to check that the clean mycelium has grown into the plug. As
soon as the mycelium has emerged, the plug is transferred onto a
new agar plate and incubated (Fig. 8).
Discussion
No specialised equipment is required with our method. It is
possible to perform many cultures at a time without the
predetermined need for molten agar, sterile rings or coverslips.
The agar can be standard laboratory agar of normal thickness and
strength, or a recipe can be selected to optimise the growth of the
fungus. The plug on the top of the large rectangle is easily
removed with a scalpel without disturbing the large rectangle
of agar. If the mycelium has attached the plug to the rectangle, a
portion can be readily removed using a scalpel.
134 Australasian Plant Pathology
Agar
Agar plug
Contaminated
mycelium
Fig. 7. Cross section of the Petri dish.
Fig. 8. Removal of the agar plug.
Some of the species this technique has been used successfully
on are – Ascochyta rabeii, Botryosphaeria dothidea, Botrytis
cinerea, Colletotrichum acutatum, Cylindrocarpon sp., Eutypa
lata, Fusarium spp., Guignardia mangiferae, Leptosphaeria
maculans, Marssonina rosae, Monilinia fructicola, Phoma
caricae-papayae, Phomopsis leptostromiformis, Phyllosticta
telopeae, Phytophthora cinnamomi, P. fallax, P. medicaginis,
http://www.publish.csiro.au/journals/app
N. J. Cother and M. J. Priest
Pleiochaeta setosa, Pythium spp., Sarocladium attenuatum,
Sclerotinia minor, S. sclerotiorum, Sphaceloma sp.,
Trichoderma spp., and Verticillium spp.
This method has also been used to successfully remove light
infestations of culture mites from cultures. The seal between the
Petri dish and the rectangular agar piece acts as a barrier to mite
movement.
The only cultures that proved to be difficult were slow-
growing fungi such as Phialophora, Postia and Phellinus, or
those infected with endophytic bacteria. The slow-growing fungi
have eventually grown through the covering agar to be
successfully retrieved. Cultures with endophytic bacteria are
unlikely to be successfully decontaminated using this method.
References
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Constantinescu O (1990) Purifying fungal cultures by using the agar method
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Harrower KM (1989) A simple technique for purifying mycelial cultures of
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Ko SS, Kunimoto RK, Ko WH (2001) A simple technique for purifying
fungal cultures contaminated with bacteria and mites. Journal of
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Machacek JE (1934) A simple method of obtaining Pythium cultures free from
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Raper JR (1937) A method of freeing fungi from bacterial contamination.
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bacteria. Phytopathology 35, 1030–1031.
Manuscript received 20 August 2008, accepted 6 November 2008