Background: Aspergillus spp. is an important cause of food degradation and biodeterioration, in addition to being pathogenic to animals. Moreover, some species are used in biotechnology and can produce mycotoxins. This paper reports the use of CREA compounds combined with three alternative pH indicators for the Aspergillus systematics. CREA was added to the basal medium with different pH indicator combinations (bromocresol green and bromothymol blue).
Results: Among the 16 Aspergillus isolates, no difference was observed in the three conditions tested in CREA medium, using different pH indicators. A. ostianus, A. sydowii and Aspergillus sp showed good growth and sporulation. Aspergillus niger 1 and A. niger 2 reacted within three days of growth. As for A. carbonariuns and A. nidulans, they reacted in 5 days. Additionally, Aspergillus sp showed a delayed reaction on the tenth day. When considering the intensity relation of the reaction among the five positive isolates, A. niger 1 and A. niger 2 presented a larger and distinct zone around the colony.
Conclusion: Bromocresol green and bromothymol blue are easy to prepare, and they are recommended for diagnostic purposes in Aspergillus, specially the Nigri section.
The genus Aspergillus contains approximately 250 species9. Some of these species have been used for many years in the fermentation industry for the production of organic acids and, more recently, for the production of a variety of enzymes and other secondary metabolites. On the other hand, some Aspergillus species produce mycotoxins that cause adverse effects in animal and human health8. Further descriptions of new species have been published, raising their total number to approximately 2503. Fungal identification has been performed mostly according to the morphological criteria. Even though several physiological tests have been proposed for filamentous fungi, they are still used mostly in the classification and identification of bacteria and yeast. However, creatine sucrose agar (CREA), containing a pH indicator, was developed as a differential medium for Penicillium subgenus Penicillium12, and modified for use in the Aspergillus and Penicillium taxonomy2.
The reactions observed in the medium are consistent1 and presented great results as another tool for the black Aspergillus taxonomy9. pH indicators are frequently weak acids or bases. When added to a solution, pH indicators bind to H+ or OH–. This association causes a change in the electronic configuration of indicators, thus changing their color. This paper reports the use of CREA compounds with three alternative pH indicators for Aspergillus systematics.
Materials and Methods
All fungal strains were obtained based on the literature and research available at the Laboratório de Micologia of Núcleo de Pesquisa Micológica e Micotoxicológica do Departamento de Microbiología e Imunología Veterinária, Projeto Sanidade Animal/Embrapa/UFRRJ. The Aspergillus spp. identification was based on the morphological taxonomy, performed according to the colony diameter, conidial color, exudates, mycelium texture, soluble pigment, excretions, shape and size of conidiophores, vesicles, metulae, phialides, conidia, in addition to their texture and color, observing the identification system for the Aspergillus genus4569.
The creatine sucrose agar consisted of bromocresol purple, 0.05 g L-1; agar, 15 g; mineral solution, 10 mL L-1; trace metal solution, 1 mL L-1. The mineral solution consisted of (g/100 mL): KCl, 5; MgSO4.7H2O, 5; FeSO4.7H2O, 0.1. The trace metal solution consisted of (g/100 mL): ZnSO4.7H2O, 1; CuSO4.5H2O, 0.5; the final pH was 8.0, adjusted after the medium was autoclaved24.
A different combination of pH indicator (bromocresol green and bromothymol blue) was added to the basal medium. Twelve isolates of Aspergillus were tested in the CREA compound containing three different pH indicators, and then they were compared to all media mentioned above. A hemocytometer was used to count mould spores. Spore suspensions were adjusted by serial dilutions of NaCl (0.85 g/100 mL) plus Tween 80 (0.1 %), containing approximately 106 spores/mL. The culture duplicates consisted of three inoculated points, incubated at 25oC in the dark, being examined after 3, 5 and 10 days.
Results and Discussion
In this study, the growth abilities of Aspergillus strains were tested in CREA medium (Table 1). CREA is useful in the classification of various fungal cultures7. Colonial growth characteristics, acid production (medium color change from purple to yellow) and base production can be used as diagnostic features in CREA9.
Among 16 Aspergillus isolates, no difference was observed in the three conditions tested in CREA medium, containing different pH indicators. The reaction time, in days, was identical for those with positive Aspergillus, as well as the intention of reaction. Among the 16 isolates, A. flavipes developed a very limited profile, displaying other fungi such as Penicillium and Aspergillus.
According to Frisvad1, this characteristic is due to the presence of N-sources in the medium, since A. ostianus, A. sydowii and Aspergillus sp showed good growth and sporulation. Aspergillus niger 1 and A. niger 2 presented reactions within three days of growth. A. carbonariuns and A. nidulans presented reactions within 5 days. Aspergillus sp showed an overall delayed reaction on the tenth day. When considering the intensity relation of the reaction among the five positive isolates, A. niger 1 and A. niger 2 presented a larger and distinct zone around the colony (Table 1), and this feature was cited by Frisvad2.
For diagnostic purposes, both pH indicators (bromocresol green and bromothymol blue) can be used to replace bromocresol purple. Bromocresol green and bromothymol blue are easy to prepare, and they are recommended for diagnostic purposes in Aspergillus, specially the Nigri section.
- Frisvad JC. 1985. Creatine sucrose agar, a differential medium for mycotoxins producing terverticillate Penicillium species. Letters in Applied Microbiolgy, 1:109-113.
- Frisvad JC. 1993. Modifications on media based on creatine for use in Penicillium and Aspergillus taxonomy. Letters in Applied Microbiolgy, 16:154-157.
- Geiser DM, Klich MA, Frisvad JC, Peterson SW, Varga J, Samson RA. 2007. The current status of species recognition and identification in Aspergillus. Studies in Mycology, 59:1-10.
- Klich MA. 2002. Identification of common Aspergillus species. Utrecht, Netherlands: CBS, Fungal Biodiversity Centre.
- Klich MA, Pitt JI. 1988. A laboratory guide to common Aspergillus species and their teleomorphs. North Ryde, Australia: CSIRO Division of Food Processing.
- Raper KB, Fennell DI. 1973. The Genus Aspergillus. New York: Robert E. Krieger.
- Samson RA, Hoekstra ES, Frisvad JC. 2004. Introduction to food and airbone fungi. Utrecht, Netherlands: Centraalbureau voor Schimmelcultures.
- Samson RA, Pitt JI. 2000. Integration of modern taxonomic methods for Penicillium and Aspergillus classification. Amsterdam: Ed. HAP.
- Samson RA, Varga J. 2007. Aspergillus systematics in the genomic era. Utrecht, Netherlands: CBS Fungal Biodiversity Centre.