Supplementary Information
High-resolution imaging of pelagic bacteria by Atomic Force Microscopy and implications for carbon cycling
Francesca Malfatti, Ty J. Samo and Farooq Azam
Staining Protocol and Mowiol® based antifading medium.
DAPI staining. The samples were fixed and filtered on polycarbonate or Anodisc filters and were mounted on a clean glass slide. A 5 µl drop of DAPI VECTASHIELD solution (Vector Laboratories Inc., Burlingame, CA, USA) was spotted on the slide and the filter was placed on it face up. A cover slip with a 5 µl drop of DAPI VECTASHIELD was placed on the filter (Fuchs et al. 2007).
SYBRGold® staining. Samples filtered on Anodisc filters were stained and mounted as in Nobel and Fuhrman (Noble & Fuhrman 1988, Patel et al. 2007). The wet filter was placed on a drop of SYBRGold® (Invitrogen Corp., Carlsbad, CA, USA), 2.5/1000 final concentration, for 15 min in the dark at room temperature. Then the dried filter was placed, face up, on a slide on a 5 µl drop of the Mowiol® based antifading medium, and a cover slip with a drop of SYBRGold® and Mowiol® based antifading medium (1:15 final concentration) was then place on the filter (Lunau et al. 2005).
NanoOrange® staining. Samples were filtered on polycarbonate filters. Once dry, the filter was placed on a drop of Mowiol® based antifading medium on a glass slide. A 5 µl solution of Mowiol® based antifading medium and NanoOrange® (Invitrogen Corp., Carlsbad, CA, USA), (Grossart et al. 2000) was spotted on a cover slip. The cover slip was put on the filter.
NAO staining. NAO is a vital dye, and it can also stain fixed samples. For live staining, the seawater sample was incubated with 10 µl NAO (Sigma-Aldrich, St. Louis, MO, USA)per ml of sample (using NAO stock solution 1 mg ml-1 in 100% ethanol) in the dark for 5 minutes at room temperature. For fixed samples the NAO solution, final concentration 2 µg ml-1, was mixed with the mounting medium (Mowiol® based antifading medium or SYBRGold® mounting medium and DAPI VECTASHIELD) and subsequently applied to the cover slip as described in the previous staining protocols.
Mowiol® based antifading medium. 2.4 g of Mowiol® 4-88 (# 81381, Sigma-Aldrich, St. Louis, MO, USA) was added to 6 g of glycerol (for fluorescence microscopy) and vigorously stirred at room temperature for 30 minutes. Then 6 ml of Milli Q water was added to the solution that was stirred for 2 hours. Subsequently 14 ml of 1x TAE buffer (pH 7.4) was added and the solution was mixed for 2 hours at 50C to achieve complete dissolution of the Mowiol®. The solution was filtered through 0.2 µm pore size filter and stored at -20C.
1% of 1M Ascorbic acid solution (in 1x TAE, pH 7.4) was added to the Mowiol® solution. The Mowiol® based antifading solution was kept for 2 weeks at -20C (see and (Lunau et al. 2005)).
EFM imaging. Once stained, the filters were visualized at the epifluorescence microscope (Olympus BX51) at 1000x final magnification. DAPI is excited at 345 nm and emits at 458 nm. SYBRGold® is excited at 495 nm and emits at 539 nm. NanoOrange® is excited at 485 nm and emits at 590 nm. NAO is excited at 494 nm and emits at 519 nm.
Bacterial cultures. The two isolates BBFL7 and SWAT3 were streaked from the -80°C stock pure cultures, on ZoBell solid medium (5 g peptone, 1 g yeast extract, 15 g agar in 1 liter GF/F filtered seawater). After 24 h, a colony was picked and inoculated into liquid ZoBell medium overnight. Bacteria cells were centrifuged at 9000x g for 5 min and washed twice with 0.02 µm filtered autoclaved seawater (FASW) and resuspended in FASW. Bacteria were fixed, washed from the medium and filtered as described above for EFM and AFM.
AFM imaging
Synechococcus epifluorescence microscopy (EFM)-identification.We used EFM prior to AFM imaging in order to make positive identification of Synechococcus. Freshly cleaved (0.2 µm thick) mica disc (# 50; Ted Pella Inc.; # 71856-01, Electron Microscopy Sciences), reduced in thickness with a clean razor blade, was affixed on a microscopy slide using double sided sticky tape. (Note that the tape is strongly autofluorescent, so it must be placed at the edges of mica disk in order to prevent excessive background fluorescence). Seawater was spotted on mica, dried and washed with HPLC water (W5-4; Fisher). On the mica we identified, at 400x, Synechococcus cells based on phycoerythrin autofluorescence (filter set 51006; Chroma). We then acquired AFM scans in air of the EFM identified Synechococcus cells on mica (see in press: (Malfatti & Azam 2009)).
AFM image analysis. We recorded trace and retrace of height, amplitude, phase and Z sensor channels. Topography images were processed with Planfit and Flatten functions. Bacteria were sized with measuring tool part of the Igor Pro 6.03A MFP3D 070111+830 software. Briefly, a ruler can be drawn on the object of interest to delineate the topographic profile and measure all three dimensions.
Filter effect on volume estimation.
We tested whether biovolume measurement was affected by filter type at EFM. We compared 0.2 µm Anodisc and 0.2 µm Isopore filters. We stained the cell with DAPI (n=100). The average volume on Isopore filters was 0.0400.024 µm3 and on Anodisc it was 0.0620.033 µm3. The Isopore membrane is made of polycarbonate and only ~1% of the surface has pores. The pores are not evenly distributed and there are some double or triple pores (Fig. 1a). Anodisc filter is made of a homogenous layer of alumina oxide colloids that are compressed and the filter resembles a sand filter in AFM; there are no straight-through pores (Fig. 1b). These (and possibly some other) filter properties may explain the difference in measurement of cell size distribution on the two different filter types. Anodisc might flatten bacteria more than Isopore yielding larger cell area in EFM and since our calculation assumed Z=W a greater degree of flattening may explain the higher apparent volume on Anodisc filters.
Supplementary Information Figure 1: Cell volumecomparison by AFM and EFM
Average biovolume ratio(AFM/EFM). Biovolume calculations assume that W=Z. AFM imaging was performed in air on fixed/dried cells. All biovolume size classes are presented for coastal, off-shore and Antarctica samples.
Literature cited
Fuchs BM, Pernthaler J, Amann R (2007) Single cell identification by fluorescence in situ hybridization. In: Reddy CA, Beveridge TJ, Breznak JA, Marzluf G, Schmidt TM, Snyder LR (eds) Methods for general and molecular microbiology. ASM Press, Washington, D.C., p 886-896
Grossart H-P, Steward GF, Martinez J, Azam F (2000) A simple, rapid method for demonstrating bacterial flagella. Appl. Environ. Microbiol. 66:3632-3636
Lunau M, Lemke A, Walther K, Martnes-Habbena W, Simon M (2005) An improved method for counting bacteria from sediments and turbid environments by epifluorescence microscopy. Environmental Microbiology 7:961-968
Malfatti F, Azam F (2009) Atomic Force Microscopy reveals microscale networks and possible symbioses among pelagic marine bacteria. Aquatic Microbial Ecology
Noble TR, Fuhrman JA (1988) Use of SYBR Green I for rapids epifluorescence counts of marine viruses and bacteria. Aquatic Microbial Ecology 14:113-118
Patel A, Noble TR, Steele JA, Schwalbach MA, Hewson I, Fuhrman JA (2007) Virus and prokaryote enumeration from planktonic aquatic environments by epifluourescence microscopy with SYBR Green I. Nature Protocols 2:269-276
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