Cottrell
August, 2010
Sediment direct counts with SYBR Green I
Start at step 6 for samples that have already been fixed with PFA
1- Add 1.5 ml of sediment to 2 mL microfuge tube.
2- Add 75 µl of 20% PFA
3- Vortex
4- Incubate at 4 °C for 4 h.
5- Store at -20°C
6- Thaw at room temperature
7- Transfer a 200 µl aliquot of preserved sediment to 10 ml of 1X PBS in a 15 ml
tube.
8- Vortex
9- Centrifuge at 4,000 rpm for 5 min
10- Remove supernatant
11- Wash with 10 mL of 1X PBS
12- Centrifuge and wash with PBS a second time
13- Remove the final supernatant
14- Add 1 ml of PBS:Ethanol
15- Store at -20 °C
16- Transfer 50 µl to 450 µl of 3% NaCl in a 15 ml tube
17- Mix and incubate at RT for 20 min
18- Add 2 mL of 1 M Tris-HCL:methanol
19- Sonicate at 50% power with 3 sec pulse for 1 min on ice
20- Transfer 1 ml to 2.5 ml of 3% NaCl
21- Filter 1 ml onto a black 0.2 µm filter
22- Add 1 ml of 0.1 M HCl
23- Incubate for 5 min
24- Wash with 5 ml of TE buffer
25- Cut 1/8 to 1/4 filter piece
26- Stain with SYBR Green I in TE buffer with 1% Triton
Cottrell
August, 2010
27- Blot away excess stain
28- Mount with cover slip using Citifluor:Vectashield
Materials
SYBR Green I staining solution
TE buffer (pH 8)
175 µl
SYBR-I
5 µl
1% Triton
20 µl
PBS-ethanol
PBS
90 ml
EtOH 10 ml
Tris-methanol
1 M Tris-HCl (pH 8) 75 ml
Methanol
25 ml
3% NaCl
NaCl
1.5 g
MilliQ H2O
50 ml
0.1 M HCl
1 M HCl
10 ml
H2O
90 ml
Based on:
Lunau, M., A. Lemke, K. Walther, W. Martens-Habbena, and M. Simon. 2005. An
improved method for counting bacteria from sediments and turbid environments by
epifluorescence microscopy. Environ. Microbiol. 7: 961-968.