Jan 02, 2025
Bacterial SYBR-Cell Staining and Counting with the Attune Flow Cytometer
Forked from a deleted protocol
- Maggi Mars1
- 1usf
Protocol Citation: Maggi Mars 2025. Bacterial SYBR-Cell Staining and Counting with the Attune Flow Cytometer . protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4w3nzvo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 30, 2024
Last Modified: January 02, 2025
Protocol Integer ID: 117417
Keywords: bacterial sybr, counting with the attune flow cytometer, cell staining, sybr cell, attune flow cytometer, cell
Abstract
SYBR Cell staining in MICO lab
Troubleshooting
Prep
Clean culture hood with 70% ethanol and use UV light to sterilize the hood (about 15 minutes)
Pre-heat mini-incubator to 37ºC.
Take foil-wrapped diluted SYBR stock out from fridge door and allow it to come to room temperature (about 5-10 minutes), while keeping it protected from light.
After warmed, vortex the SYBR stock and make sure it is well mixed
Take out samples for the day (in fridge, labeled with station, depth, date, and "BACT") and gently vortex each sample.
Note: cruise 3 has BACT 1 and 2 so that we have enough volume for the imaging flow cyto as well
Staining
put a fresh 96-well plate, mixed samples, and mixed SYBR stock into the cleaned culture hood
Add 1 (one) µl of SYBR stock to each well you intend to use in the assay. Make sure to add the 1 µl to the bottom center of the well so that you can see it.
- do not use the furthest column to the right
add 99 (ninety-nine) µl of water to well 1A - this will be a blank
add 99 (ninety-nine) µl of sample to each of the other wells and mix 5-10x so that SYBR and samples are mixed.
This can be printed and used to write in sample names
Record sample-well locations
Either print template, use the postits, or record in protocols (makes sure you save run):
| 1 | 2 | 3 | 4 | 5 | |
| A | Blank | ||||
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| C | |||||
| D | |||||
| E | |||||
| F | |||||
| G | |||||
| H |
| 6 | 7 | 8 | 9 | 10 | |
| A | |||||
| B | |||||
| C | |||||
| D | |||||
| E | |||||
| F | |||||
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| H |
| 11 | 12 | |
| A | ||
| B | ||
| C | ||
| D | ||
| E | ||
| F | ||
| G | ||
| H |
Incubate
put sample plate into the mini-incubator and incubate at 37ºC for 10 minutes
Edit Attune Template
Open proper experiment from template and edit sample names to match actual sample names
Run Experiment on Attune
use bacterial template