The Aspergillus and Penicillium website

Aspergillus Reference Cultures

M. A. Klich
U.S. Department of Agriculture. Agricultural Research Service, Southern Regional Research Center, New Orleans, Louisiana, USA

R.A. Samson
CBS Fungal Biodiversity Centre, Utrecht, The Netherlands.

and members of the International Commission on Penicillium and Aspergillus (ICPA).

Introduction

The purpose of this culture set is to provide interested individuals with basic information on the genus in order to facilitate research and identification. The isolates were selected to represent a wide variety of morphological and physiological characteristics of the genus. Using these living cultures, individuals can observe these characteristics under their own lab conditions. This is extremely important because some of the characteristics of the species are influenced by minor variations in media and incubation conditions. Based on a comparison of their own results to those obtained by ICPA members, users may find it necessary to adjust their own media or incubation conditions, or to interpolate the identification keys. We also foresee this culture set being useful in teaching mycology laboratories. The culture set will be available from the major collections listed at the end of this booklet.

The International Commission on Penicillium and Aspergillus (ICPA) is a commission of the International Union of Microbiological Societies Mycology Division. One of the aims of ICPA is improvement of the accuracy of identification of these two genera. This project was undertaken to address this goal. For this study, members of ICPA were asked to make detailed morphological observations of two strains of each species and to select the strain they considered to be most typical. The data sets were used to write the species descriptions herein. Nine members of ICPA participated including, Katsuhiko Ando (Japan), Ellen Hoekstra (Netherlands), Zofia Kozakiewicz (United Kingdom), Toru Okuda (Japan), Stephen Peterson (USA), John Pitt (Australia), Keith Seifert (Canada), and the authors.

A number of identification systems for the genus and sections thereof are available (see References). The choice of the system used should be based on the nature of the research being conducted. One of the goals of ICPA is to stabilize nomenclature of Aspergillus and Penicillium, therefore, we do recommend that "Names in Current Use" (Pitt and Samson 1993) be used when publishing papers to assure the current name of the organism is correct.

Species included in the set are Aspergillus candidus, A. clavatus, A. flavus, A. niger, A. ochraceus, A. penicillioides, A. terreus, A. versicolor and two species with Aspergillus anamorphs, Emericella nidulans and Eurotium amstelodami. Aspergillus fumigatus is available upon request to users familiar with the handling of potential human pathogens.

Media and Incubation

All ICPA participants were asked to make observations using Czapek Yeast Extract Agar with trace metals (CYA; Pitt 1973, Smith 1994 ), incubated at 25°C and 37°C; Malt-Extract Agar (MEA; Blakeslee 1915) incubated at 25°C; and Czapek Yeast Agar with 20% Sucrose (CY20S; Pitt and Hocking 1985) CYA with 2OOg sucrose instead of 30 g) incubated at 25°C. Most members also contributed data sets on Czapek Agar (CZ, see Raper and Fennell 1965).

Cultures were incubated in the dark for seven days. Plates with teleomorphic states requiring more than one week for asci to mature were placed back in the appropriate incubators and observed regularly until asci matured. Macroscopic observations were made on all media, but only colony diameter was measured for CYA 37°C. The medium used for microscopic observations was left to the discretion of the individual contributor. Most contributors chose to use MEA.

All of the identification systems use standard size Petri dishes, with three inoculation points - equidistant from the edge of the plate and from one another. The inoculum should be fresh, ideally from slants one to two weeks old. Some laboratories use a needle to take conidia from the slant directly onto the petri plate. This method is not practical in areas with low humidity because the spores tend to scatter. Plates can be inoculated from spore suspensions made by taking a needle/loop of spores from the slant and mixing them in small vials containing 0.2-0.5 ml of a sterile mixture of 0.2% agar and 0.05% detergent (such as Tween 80).

CYA- Czapek Yeast Agar

Czapek Agar

Czapek concentrate

MEA- Malt Extract Agar

Important Morphological Characteristics of the Genus

Aspergillus is an anamorphic genus that produces asexual spores (conidia, conidiospores) on a specialized structure characteristic of the genus called an aspergillum. The aspergillum is composed of the swollen apical section (vesicle) of the fruiting structure bearing either a single palisade of cells (phialides, primary sterigmata) that give rise to conidia, or with two palisades of cells, with those arising from the vesicle (metulae, secondary sterigmata) each bearing one to ten phialides (FigurEach palisade of cells is formed synchronously on the surface of the vesicle. Aspergilli with only phialides are referred to as uniseriate, those with both metulae and phialides are biseriate. The shape of thee 1). vesicle varies from almost spherical (globose) to club-shaped (clavate), with many intermediate forms including pear-shaped (pyriform) and spoon-shaped (spathulate). The vesicle is borne on a long stipe (stalk) the basal end of which curves or forms a "T" where it joins the vegetative hyphae. This basal portion is referred to as a "foot cell" even though it is part of the same cell as the stipe. The aspergillum, stipe and foot cell together are referred to as the conidiophore.

Macroscopic characters are important in subgeneric classification in the genus Aspergillus. Conidial colors as observed on Petri plates serve as a major subgeneric classification characters. Colors vary from black to white to yellow to green to blue. A color guide is useful for determining this character. We have used Kornerup and Wanscher (1978). Colonies may also produce droplets of liquid (exudate) on the surface of the colony. Colored pigment may be produced in the agar on the reverse of the colony (reverse color). Some of these may be soluble enough to extend beyond the edge of the colony (soluble pigment).

Colony diameter is sometimes a useful characters, as species in the genus vary in ability to grow at different water potentials and at different temperatures. Colony diameter is measured by holding the plate up to the light and measuring from one edge of mycelial growth to the opposite edge.

Some members of the genus Aspergillus also reproduce sexually, with the teleomorphic states belonging to the Ascomycete family Trichocomaceae. Two of these, Eurotium amstelodami and Emericella nidulans are included in this reference set. Other structures found in this genus include sclerotia that are asexually formed firm masses of hyphae and, Hülle cells, which are thick walled cells frequently associated with cleistothecia.

The species selected for this Reference Culture set were chosen to demonstrate the variability of morphological features of the genus. For quick reference, many of these are listed in Table 1. The species descriptions were compiled from the observations made by ICPA members for the isolate indicated.

Safety

Care should be taken to avoid inhalation of spores and other fungal products such as volatiles. These fungi may produce toxins or cause allergic reactions. They may also be human pathogens, especially in immunosuppressed populations.

Consistently Observed Morphological Characters of Reference Isolates

Legend

clav=Aspergillus clavatus, pen=Aspergillus penicilliodes, vers=Aspergillus versicolor, terr=Aspergillus terreus, cand=Aspergillus candidus, niger=Aspergillus niger, ochr=Aspergillus ochraceus, flav=Aspergillus flavus, amst=Eurotium amstelodami, nid=Emericella nidulans, fum=Aspergillus fumigatus

Isolate Numbers of Reference Cultures

	CBS	IMI	ATCC	CCFC
Emericella nidulans	288.95	370083	96921	222012
Eurotium amstelodami	518.65	229971	16464	222011
Aspergillus fumigatus	286.95	370079	96918	222005
A. terreus	601.65	17294	1012	222009
A. versicolor	583.65	229970	96920	222010
A. flavus	282.95	370082	22546	222004
A. ochraceus	280.95	370080	96919	222007
A. niger	554.65	50566	16888	222006
A. clavatus	514.65	321306	10058	222003
A. candidus	283.95	78734	13686	222002
A. penicillioides	285.95	370081	42692	222008

Addresses of collections from which the Reference Cultures are available

CBS

Centraalbureau voor Schimmelcultures
Oosterstraat 1
3742 SK BAARN
The Netherlands
Fax:+ 31 35 541 6142
email: SALES

IMI

International Mycological Institute
Bakeham Lane
EDHAM, Surrey TW20 9TY
United Kingdom
fax:+44 1784 470909
email: z.lawrence@cabi.org

ATCC

American Type Culture Collection
12301 Parklawn Drive
Rockville, MD 20852 USA
fax: +1 301 231 5826
e-mail: sales@atcc.org

FRR

FRR Culture Collection
Dr. Ailsa Hocking
CSIRO Division of Food Science and Technology
P.O. Box 52
North Ryde, NSW
Australia
Fax:+612 887 3107
E-mail: Ailsa.Hocking@foodadmin.dfp.csiro.au (klopt mailadres? was "raar")

CCFC

Canadian Collection of Fungal Cultures
Eastern Cereal and Oilseed Research Centre
Agriculture and Agri-Food Canada
Ottawa, Ontario
K1A 0C6
fax: +1 613 7591924
e-mail: babcockc@em.agr.ca

ICPA HOMEPAGE