Influence of cell geometry and number of replicas in the reproducibility of whole cell FTIR analysis

Abstract

Fourier Transform InfraRed (FTIR) spectroscopy is an increasingly used technique in biology, especially for whole cell metabolomic fingerprint. The reproducibility of this technique is influenced by a large number of factors such as the physiological state of cells, sample manipulation and growth conditions. Evidence exists suggesting that the cell shape and dimension can be further elements to consider in whole cell FTIR analysis. In this study we aimed to address the effect of cell geometry on the FTIR spectra and to define the extent of variability occurring between machine and biological replicas with a standardized protocol. The yeast species Saccharomyces cerevisiae (large oval-shaped cells) and Debaryomyces hansenii (small round shaped cells) were employed for their different morphology. Thirty machine replicas of each were analyzed separately and after averaging in groups of three, showing a three to four-fold reduction of the variability. Similarly, a two-fold reduction of variability was observed when thirty biological replicas of the two yeast species were analyzed. The optimal number of replicas to average was then estimated with a bootstrap-like procedure in which biological and machine replicas were randomly resampled 2000 times and averaged in groups spanning from 2 to 12 replicas. This simulation has shown that little if any advantage can be obtained by increasing the number of replicas over five and that the variability exhibited by the small regular cells of D. hansenii was always roughly half of that displayed by the large S. cerevisiae cells, confirming the results obtained with standard non-bootstrapped averages.

Assessing the Potential of FT-IR to Identify Clinical Strains of Candida spp.

“…Recently however, Roscini, L. et al [1] give way to a more consistent approach with the selection of the most variable sections of IR spectra combined with a minimum amount of 5 replicas for consistency. Since our goal of using this method routinely is to determine rapidly the relatedness of strains when confronted with a possible outbreak more than for identification itself, we will carry on using this method following Roscini, L. et al's results [1].…”

“…Since our goal of using this method routinely is to determine rapidly the relatedness of strains when confronted with a possible outbreak more than for identification itself, we will carry on using this method following Roscini, L. et al's results [1].…”

“…Many phenotypic characteristics are unstable and too dependent on growth conditions. However, Roscini et al demonstrated that the analysis of an average of 5 replicas of each spectrum for a strain is sufficient to eliminate most of the variations observed individually, thus reducing the background noise when comparing strains [1]. Also the region of the infra-red spectra to use can be manipulated in order to enhance the differences between strains by choosing regions related mainly to RNA and Proteins -RNA (ca 1080cm 1) and proteins (amide II ca 1540cm 1) [2].…”

“…Infra-red (IR) spectroscopy is based on the excitation of molecular bonds. Each bond absorbs a specific wave-length within the IR spectra, revealing a specific fingerprint for each molecule generated by the sum of the absorption of all atom-atom bonds [1]. Many advanced spectrophotometers scan ranges IR radiation, through the means of an interferometer use together with Fourrier mathematical analysis of the raw data.…”

“…35 microliters of a suspension of each strain in distilled water (OD500=3) were deposited on a well of the FT-IR plate, followed by drying at 44+/-0.5ºC for 45+/-5 minutes. Scans were made in a Bruker ® FT-IR spectrophotometer (in the HTS-XT module), of the region of 500 to 2000 cm -1 and with the aid of Ward's algorithm and base-line corrections, dendrograms of the second derivatives of the spectrophotometer's output were built by Opus (Bruker ® ) as described in Roscini, L. et al [1]. Seven replicates of each sample were run but only green results were used for the building the dendrogram and subsequent comparison of identification with biochemical identification Table 1 reveals the biochemical identification and the number of green wells (quality approved by the software of the equipment) out of seven obtained in one run of FT-IR analysis for each strain.…”