Fourier-transform infrared microspectroscopy, a novel and rapid tool for identification of yeasts

Abstract

Fourier-transform infrared (FT-IR) microspectroscopy was used in this study to identify yeasts. Cells were grown to microcolonies of 70 to 250 micro m in diameter and transferred from the agar plate by replica stamping to an IR-transparent ZnSe carrier. IR spectra of the replicas on the carrier were recorded using an IR microscope coupled to an IR spectrometer, and identification was performed by comparison to reference spectra. The method was tested by using small model libraries comprising reference spectra of 45 strains from 9 genera and 13 species, recorded with both FT-IR microspectroscopy and FT-IR macrospectroscopy. The results show that identification by FT-IR microspectroscopy is equivalent to that achieved by FT-IR macrospectroscopy but the time-consuming isolation of the organisms prior to identification is not necessary. Therefore, this method also provides a rapid tool to analyze mixed populations. Furthermore, identification of 21 Debaryomyces hansenii and 9 Saccharomyces cerevisiae strains resulted in 92% correct identification at the strain level for S. cerevisiae and 91% for D. hansenii, which demonstrates that the resolution power of FT-IR microspectroscopy may also be used for yeast typing at the strain level.

FIG. 1.

Shapes of microcolonies of different yeast species after replica stamping. (A) Torulaspora delbrueckii. Different apertures are indicated. While recording spectra, the field around the colony has to be covered by an aperture in order to measure only the sample. For this purpose, apertures of different sizes are implemented. The bright central area is left uncovered during measurement. (B) Candida intermedia. (C) Rhodotorula mucilaginosa. (D) R. mucilaginosa and T. delbrueckii growing into each other. (E) Two colonies of R. mucilaginosa growing into each other. (F) Two colonies of D. hansenii.

FIG. 2.

(A) Colony diameter of strains of six different yeast species after transfer to the ZnSe carrier, determined at different growth times. The organisms were incubated at 25°C. (B) Dendrogram of microcolonies of S. cerevisiae recorded at different growth times. Spectral ranges: 3,030 to 2,830 cm⁻¹, 1,350 to 1,200 cm⁻¹, and 900 to 700 cm⁻¹. Average linkage, correlation with normalization to reprolevel (OPUS/Ident Handbook, Bruker Optik GmbH, Karlsruhe, Germany, 1995).

FIG. 3.

External validation of databases containing 45 or 959 average reference spectra recorded with FT-IR microspectroscopy (microscope) and FT-IR macrospectroscopy (samplewheel). If the first hit belonged to an average spectrum of the same species and had an SD of ≤1.0, the result was counted as a correct identification at the species level. If the first hit belonged to a spectrum of the same genus but to a different species, identification was correct at the genus level. Identifications with SD of >1.0 in the first hit were counted as not identified. Results with incorrect identification in the first hit were noted as misidentifications. Abbreviations: 45/45, database consists of 45 average spectra and all 45 spectra were subjected to the external validation; 45/959, database consists of 959 average spectra and 45 spectra were identified against this database.

FIG. 4.

Dendrogram of six different D. hansenii (A to F) and six S. cerevisiae (G to L) strains, each measured three times independently by FT-IR microspectroscopy.