Data on preparation of psychrotolerant bacterium Shewanella olleyana sp. nov. cells for transmission electron microscopy

This data article contains transmission electron microscopy (TEM) images of psychrotolerant bacterium Shewanella olleyana sp. nov. Cells of S. olleyana were grown following an optimized culture conditions in liquid medium. Procedure for the preparation of cells suitable for TEM is described in detail.

To our knowledge, this data article is first to report the ultrastructure of S. olleyana. The data presented here confirmed the presence of high levels of iron and/or iron sulfide in the broth culture precipitates of S. olleyana. Researchers using this psychrotolerant bacterium can take these findings into consideration for its culture propagation and other purposes (e.g. microscopy).

Data
The data presented in this article show the morphological and ultrastructural characteristics of psychrotolerant bacterium Shewanella olleyana sp. nov. obtained by TEM. Cells were grown and prepared following an optimized procedure. S. olleyana cells were grown and propagated in solid medium at temperatures between 4 and 10°C for 24-48 h. Complete sedimentation of suspended S. olleyana cells (grown in solid medium) was achieved following centrifugation at 4000 x g for 10 min at 4°C. The presence of residual or contaminating reduced form of iron and/or iron sulphide was detected when cells are grown in liquid medium.
For TEM analysis, 24-48 h old S. olleyana cells were harvested from agar plates. Cells were flooded with 4 ml of sterile cold physiological saline (0.85% NaCl) and scraped using a glass rod to detach the cells. The cell suspension was centrifuged at 4000 g for 10 min at 4°C. The resulting pellet was washed with cold physiological saline. Cells were resuspended in MH buffer and stored at À 86°C until use. Growth of S. olleyana cells in marine broth medium was also observed.

X-ray fluorescence (XRF) spectroscopy analysis of S. olleyana precipitates from broth cultures
To verify the occurrence of iron reduction in the broth medium by S. olleyana cells, XRF analysis of the recovered precipitates was done. The PANalytical Epsilon5 EDXRF spectrometer was used for the multi-elemental analysis of the sample. The instrument is equipped with 600 W-anode x-ray tube and 100 kV generator, up to 15 secondary targets and a high resolution PAN-32 detector. To identify the elemental constituents of the sample, it was analyzed qualitatively using the spectrum generated by the EDXRF. Cells were grown on 10 ml marine broth [1] incubated at 4-10°C for 3-5 days. Formation of black precipitates during incubation was observed (Fig. 2). Precipitates were recovered and dried free of moisture at 110°C for 14 min in an aluminum dish. The precipitate that adhered on the aluminum dish was placed on top of an "XRF insert", covered with X-ray thin film sample support and inserted in stainless steel cap. The sample assembly was then put inside the EDXRF spectrometer and analyzed using the PANalytical Epsilon Software for elemental analysis. Fig. 3 shows the result of XRF analysis of the black precipitates. The precipitates contain high levels of elemental iron (17%), confirming that this could be a mixture of reduced form of iron (Fe 2 þ ) and/or iron sulphide precipitates produced by S. olleyana.

TEM and negative staining of S. olleyana
S. olleyana cells were fixed in buffered 2.5% glutaraldehyde and 4% paraformaldehyde. Cells were washed three times with physiological saline to remove excess fixative and were fixed in unbuffered 1% osmium tetroxide and washed with physiological saline. It was then dehydrated in a graded series of acetone solutions and gradually impregnated in Epon resin with heat polymerization. Semi-thin survey sections were sliced with glass knives, stained with toluidine blue and used to orient sections. Ultra-thin sections were mounted on uncoated copper grids and stained with uranyl acetate and lead citrate. Sections were examined and viewed in a JEOL 1010 TEM. Negative staining of S. olleyana cells was done following a previously published method [2]. Samples were viewed using a JEOL 1200 EX electron microscope (Figs. 4-6).

Statistical analysis
Each data point represents the mean þSD of two trials. GraphPad InStat software was used to determine the differences among the means. Data were compared using one-way analysis of variance (ANOVA) with post-test. Dunnett's test was used to compare treatment means against the control mean. Statistical significance was determined at p o0.05. Table 1 Growth of S. olleyana in Zobell's marine agar medium incubated at different temperatures. Growth was grouped into Aþ ¼excellent growth (4300 cfu/plate); A¼ good growth (250-300 cfu/plate); B ¼ fair growth (1-100 cfu/plate); and NG ¼ no growth (0 CFU/plate).