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Supplementary Information
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High-resolution imaging of pelagic bacteria by Atomic Force Microscopy and
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implications for carbon cycling
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Francesca Malfatti, Ty J. Samo and Farooq Azam
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Staining Protocol and Mowiol® based antifading medium.
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DAPI staining. The samples were fixed and filtered on polycarbonate or Anodisc filters
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and were mounted on a clean glass slide. A 5 µl drop of DAPI VECTASHIELD solution
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(Vector Laboratories Inc., Burlingame, CA, USA) was spotted on the slide and the filter
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was placed on it face up. A cover slip with a 5 µl drop of DAPI VECTASHIELD was
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placed on the filter (Fuchs et al. 2007).
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SYBRGold® staining. Samples filtered on Anodisc filters were stained and mounted as in
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Nobel and Fuhrman (Noble & Fuhrman 1988, Patel et al. 2007). The wet filter was
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placed on a drop of SYBRGold® (Invitrogen Corp., Carlsbad, CA, USA), 2.5/1000 final
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concentration, for 15 min in the dark at room temperature. Then the dried filter was
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placed, face up, on a slide on a 5 µl drop of the Mowiol® based antifading medium, and a
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cover slip with a drop of SYBRGold® and Mowiol® based antifading medium (1:15 final
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concentration) was then place on the filter (Lunau et al. 2005).
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NanoOrange® staining. Samples were filtered on polycarbonate filters. Once dry, the
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filter was placed on a drop of Mowiol® based antifading medium on a glass slide. A 5 µl
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solution of Mowiol® based antifading medium and NanoOrange® (Invitrogen Corp.,
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Carlsbad, CA, USA), (Grossart et al. 2000) was spotted on a cover slip. The cover slip
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was put on the filter.
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NAO staining. NAO is a vital dye, and it can also stain fixed samples. For live staining,
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the seawater sample was incubated with 10 µl NAO (Sigma-Aldrich, St. Louis, MO,
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USA) per ml of sample (using NAO stock solution 1 mg ml-1 in 100% ethanol) in the
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dark for 5 minutes at room temperature. For fixed samples the NAO solution, final
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concentration 2 µg ml-1, was mixed with the mounting medium (Mowiol® based
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antifading medium or SYBRGold® mounting medium and DAPI VECTASHIELD) and
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subsequently applied to the cover slip as described in the previous staining protocols.
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Mowiol® based antifading medium. 2.4 g of Mowiol® 4-88 (# 81381, Sigma-Aldrich, St.
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Louis, MO, USA) was added to 6 g of glycerol (for fluorescence microscopy) and
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vigorously stirred at room temperature for 30 minutes. Then 6 ml of Milli Q water was
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added to the solution that was stirred for 2 hours. Subsequently 14 ml of 1x TAE buffer
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(pH 7.4) was added and the solution was mixed for 2 hours at 50C to achieve complete
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dissolution of the Mowiol®. The solution was filtered through 0.2 µm pore size filter and
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stored at -20C.
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1% of 1M Ascorbic acid solution (in 1x TAE, pH 7.4) was added to the Mowiol®
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solution. The Mowiol® based antifading solution was kept for 2 weeks at -20C (see
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http://www.hei.org/research/aemi/moviol.htm and (Lunau et al. 2005)).
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EFM imaging. Once stained, the filters were visualized at the epifluorescence
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microscope (Olympus BX51) at 1000x final magnification. DAPI is excited at 345 nm
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and emits at 458 nm. SYBRGold® is excited at 495 nm and emits at 539 nm.
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NanoOrange® is excited at 485 nm and emits at 590 nm. NAO is excited at 494 nm and
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emits at 519 nm.
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Bacterial cultures. The two isolates BBFL7 and SWAT3 were streaked from the -80°C
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stock pure cultures, on ZoBell solid medium (5 g peptone, 1 g yeast extract, 15 g agar in
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1 liter GF/F filtered seawater). After 24 h, a colony was picked and inoculated into liquid
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ZoBell medium overnight. Bacteria cells were centrifuged at 9000x g for 5 min and
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washed twice with 0.02 µm filtered autoclaved seawater (FASW) and resuspended in
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FASW. Bacteria were fixed, washed from the medium and filtered as described above for
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EFM and AFM.
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AFM imaging
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Synechococcus epifluorescence microscopy (EFM)-identification. We used EFM prior to
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AFM imaging in order to make positive identification of Synechococcus. Freshly cleaved
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(0.2 µm thick) mica disc (# 50; Ted Pella Inc.; # 71856-01, Electron Microscopy
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Sciences), reduced in thickness with a clean razor blade, was affixed on a microscopy
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slide using double sided sticky tape. (Note that the tape is strongly autofluorescent, so it
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must be placed at the edges of mica disk in order to prevent excessive background
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fluorescence). Seawater was spotted on mica, dried and washed with HPLC water (W5-4;
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Fisher). On the mica we identified, at 400x, Synechococcus cells based on phycoerythrin
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autofluorescence (filter set 51006; Chroma). We then acquired AFM scans in air of the
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EFM identified Synechococcus cells on mica (see in press: (Malfatti & Azam 2009)).
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AFM image analysis. We recorded trace and retrace of height, amplitude, phase and Z
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sensor channels. Topography images were processed with Planfit and Flatten functions.
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Bacteria were sized with measuring tool part of the Igor Pro 6.03A MFP3D 070111+830
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software. Briefly, a ruler can be drawn on the object of interest to delineate the
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topographic profile and measure all three dimensions.
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Filter effect on volume estimation.
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We tested whether biovolume measurement was affected by filter type at EFM. We
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compared 0.2 µm Anodisc and 0.2 µm Isopore filters. We stained the cell with DAPI
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(n=100). The average volume on Isopore filters was 0.0400.024 µm3 and on Anodisc it
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was 0.0620.033 µm3. The Isopore membrane is made of polycarbonate and only ~1% of
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the surface has pores. The pores are not evenly distributed and there are some double or
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triple pores (Fig. 1a). Anodisc filter is made of a homogenous layer of alumina oxide
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colloids that are compressed and the filter resembles a sand filter in AFM; there are no
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straight-through pores (Fig. 1b). These (and possibly some other) filter properties may
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explain the difference in measurement of cell size distribution on the two different filter
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types. Anodisc might flatten bacteria more than Isopore yielding larger cell area in EFM
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and since our calculation assumed Z=W a greater degree of flattening may explain the
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higher apparent volume on Anodisc filters.
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Supplementary Information Figure 1: Cell volume comparison by AFM and EFM
31.2
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211_10
211_75
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211_220
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197_35
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197_75
197_440
AFM/EFM
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177_10
177_50
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177_300
1-Mar
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10-Mar
14-Mar
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164_7
164_40
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206_15
206_53
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206_100
206_150
1
206_200
20_aug
0
<0.01
0.011-0.03 0.031-0.05 0.051-0.07 0.071-0.09
>0.091
21_aug
23_aug
µm3
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Average biovolume ratio (AFM/EFM). Biovolume calculations assume that W=Z.
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AFM imaging was performed in air on fixed/dried cells. All biovolume size classes are
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presented for coastal, off-shore and Antarctica samples.
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Literature cited
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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
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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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