This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Counting marine microbes with Guava Easy-Cyte 96 well plate reading flow cytometer
H. James Tripp
Oregon State University
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Figure 1
The Guava Easy-Cyte flow cytometer accepts 96 well plates containing stained cells in liquid media and reports the cells/ml for each well in Excel format (csv file). Marine microbes stained with SYBR-Grn I in natural seawater can be counted within approximately 5% error at densities of 1.0E4 to 5.5E5 cells per ml. With exceptional care, cell cultures can be reliably detected but not accurately counted down to 2E3 cells per ml. At densities above 5.5E5, cells must be diluted for accurate counting. There are a few critical adaptations to the manufacturer's operating instructions that must be made relating to use of seawater media for extremely small SAR11 cells. Neutral pH detergent must be used for cleaning or salts will precipitate and foul the fluid pump of the cytometer. Stain concentration must be higher than recommended and made fresh daily, voltage must be higher than recommended, and rinse tubes must be changed daily. Also, freeze/thaw cycles of SYBR-Grn I stock should be kept to a minimum, so it is wise to aloquot stocks upon purchase and receipt.
SYBR-Grn I Nucleic Acid Stain
TE Buffer
Guava Easy-Cyte Plate Reading Flow Cytometer
Approved 96-well microtiter plate
Repeating pipette
Sterile hood
- Prepare SYBR-Grn I stain by completely thawing an aliquot of stock and diluting 1:20 with clean TE buffer.
- Transfer 198 microliters of cells from culturing containers into a manufacturer-approved 96-well microtiter plate in a sterile hood.
- Pipette 2 microliters of 1:20 SYBR-Grn I diluted stock into each well of the microtiter plate.
- Cover the microtiter plate and let stain for 60 min. in the dark.
- Make a worklist for acquiring data on the Guava, as described by the manufacturer.
- Start the worklist and load the microtiter plate into the Guava, taking care that the plate is properly located in the tray.
- Adjust settings on the green channel to 716 volts with a threshold of 8-10 and gating from just over 10 to just over 100.
- Acquire data, consult log, and review the results.
- Regate or dilute and recount any wells that were too concentrated, improperly stained, or dirty.
- Transfer csv file from run to jump drive and copy to PC.
2 hours
Step 7 is critical. All aspects of the procedure come together here. Properly stained cells can only be accurately counted with the correct settings in a clean machine.
Step 1. Insufficient stain will require long stain times; excess stain will result in too much noise.
Step 2. A non-approved microtiter plate will cause broken flow cell tips at over $100.
Step 3. Stain that has been thawed more than 5-7 times will give poor results.
Step 4. Insufficient stain time will result in insufficient signal.
Step 5. Don't forget to put in the dilution factor for a well if the cells were diluted, or the reported cell density will be off by the dilution factor.
Step 6. If the microtiter plate is improperly located, the flow cell tip will break.
Step 7. If signal cannot be separated from noise, cleanliness, stain time, voltage, and thresholds must all be double-checked and compensated for.
See Figure 1.
Ssemakalu Cornelius
commented on 07 June, 2009
Wonderful and straight forward however is the SYBR green the one supplied by invitrogen? In which case is the working concentration 5x? Thank you
Kevin Vergin
commented on 24 September, 2009
true, we use Sybr Green from Invitrogen. We use a 1:20 or 1:30 dilution of the stock and stain cells at a 1:100 dilution so it is a final dilution of 1:2000 or 3000. It is possible that the 1:3000 dilution gives less background noise. We stain for at least one hour before counting.
Ssemakalu Cornelius
commented on 07 June, 2009
Wonderful and straight forward however is the SYBR green the one supplied by invitrogen? In which case is the working concentration 5x? Thank you
Kevin Vergin
commented on 24 September, 2009
true, we use Sybr Green from Invitrogen. We use a 1:20 or 1:30 dilution of the stock and stain cells at a 1:100 dilution so it is a final dilution of 1:2000 or 3000. It is possible that the 1:3000 dilution gives less background noise. We stain for at least one hour before counting.
Version History
CURRENT STATUS: Posted
Version 1
Posted 18 Mar, 2008
Metrics
Comments: 4
HTML Views: ...
Subject Areas
Microbiology
Associated Publications
SAR11 marine bacteria require exogenous reduced sulphur for growth
by H. James Tripp, Joshua B. Kitner, Michael S. Schwalbach, +2
Nature (31 January, 2008)
Method Article
Counting marine microbes with Guava Easy-Cyte 96 well plate reading flow cytometer
H. James Tripp
Oregon State University
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 18 Mar, 2008
Metrics
Comments: 4
HTML Views: ...
Subject Areas
Microbiology
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Figure 1
The Guava Easy-Cyte flow cytometer accepts 96 well plates containing stained cells in liquid media and reports the cells/ml for each well in Excel format (csv file). Marine microbes stained with SYBR-Grn I in natural seawater can be counted within approximately 5% error at densities of 1.0E4 to 5.5E5 cells per ml. With exceptional care, cell cultures can be reliably detected but not accurately counted down to 2E3 cells per ml. At densities above 5.5E5, cells must be diluted for accurate counting. There are a few critical adaptations to the manufacturer's operating instructions that must be made relating to use of seawater media for extremely small SAR11 cells. Neutral pH detergent must be used for cleaning or salts will precipitate and foul the fluid pump of the cytometer. Stain concentration must be higher than recommended and made fresh daily, voltage must be higher than recommended, and rinse tubes must be changed daily. Also, freeze/thaw cycles of SYBR-Grn I stock should be kept to a minimum, so it is wise to aloquot stocks upon purchase and receipt.
SYBR-Grn I Nucleic Acid Stain
TE Buffer
Guava Easy-Cyte Plate Reading Flow Cytometer
Approved 96-well microtiter plate
Repeating pipette
Sterile hood
- Prepare SYBR-Grn I stain by completely thawing an aliquot of stock and diluting 1:20 with clean TE buffer.
- Transfer 198 microliters of cells from culturing containers into a manufacturer-approved 96-well microtiter plate in a sterile hood.
- Pipette 2 microliters of 1:20 SYBR-Grn I diluted stock into each well of the microtiter plate.
- Cover the microtiter plate and let stain for 60 min. in the dark.
- Make a worklist for acquiring data on the Guava, as described by the manufacturer.
- Start the worklist and load the microtiter plate into the Guava, taking care that the plate is properly located in the tray.
- Adjust settings on the green channel to 716 volts with a threshold of 8-10 and gating from just over 10 to just over 100.
- Acquire data, consult log, and review the results.
- Regate or dilute and recount any wells that were too concentrated, improperly stained, or dirty.
- Transfer csv file from run to jump drive and copy to PC.
2 hours
Step 7 is critical. All aspects of the procedure come together here. Properly stained cells can only be accurately counted with the correct settings in a clean machine.
Step 1. Insufficient stain will require long stain times; excess stain will result in too much noise.
Step 2. A non-approved microtiter plate will cause broken flow cell tips at over $100.
Step 3. Stain that has been thawed more than 5-7 times will give poor results.
Step 4. Insufficient stain time will result in insufficient signal.
Step 5. Don't forget to put in the dilution factor for a well if the cells were diluted, or the reported cell density will be off by the dilution factor.
Step 6. If the microtiter plate is improperly located, the flow cell tip will break.
Step 7. If signal cannot be separated from noise, cleanliness, stain time, voltage, and thresholds must all be double-checked and compensated for.
See Figure 1.
Ssemakalu Cornelius
commented on 07 June, 2009
Wonderful and straight forward however is the SYBR green the one supplied by invitrogen? In which case is the working concentration 5x? Thank you
Kevin Vergin
commented on 24 September, 2009
true, we use Sybr Green from Invitrogen. We use a 1:20 or 1:30 dilution of the stock and stain cells at a 1:100 dilution so it is a final dilution of 1:2000 or 3000. It is possible that the 1:3000 dilution gives less background noise. We stain for at least one hour before counting.
Ssemakalu Cornelius
commented on 07 June, 2009
Wonderful and straight forward however is the SYBR green the one supplied by invitrogen? In which case is the working concentration 5x? Thank you
Kevin Vergin
commented on 24 September, 2009
true, we use Sybr Green from Invitrogen. We use a 1:20 or 1:30 dilution of the stock and stain cells at a 1:100 dilution so it is a final dilution of 1:2000 or 3000. It is possible that the 1:3000 dilution gives less background noise. We stain for at least one hour before counting.
Associated Publications
SAR11 marine bacteria require exogenous reduced sulphur for growth
by H. James Tripp, Joshua B. Kitner, Michael S. Schwalbach, +2
Nature (31 January, 2008)