Jan 26, 2026
Version 2
Culturing of E.coli stocks for Whole Genome Sequencing V.2
- Lindsay Newbold1,
- Susheel Bhanu Busi1,
- Manasa Suresh1,
- Denise Pallett1,
- Wiebke Schmidt2,
- Martin Spurr2
- 1UK Centre for Ecology & Hydrology;
- 2The Environment Agency
- Lindsay Newbold
Protocol Citation: Lindsay Newbold, Susheel Bhanu Busi, Manasa Suresh, Denise Pallett, Wiebke Schmidt, Martin Spurr 2026. Culturing of E.coli stocks for Whole Genome Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl88p6rl2w/v2Version created by Lindsay Kate Newbold
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: January 22, 2026
Last Modified: January 26, 2026
Protocol Integer ID: 239134
Keywords: culturing methodology, Whole Genome Sequencing, whole genome sequencing, culturing methodology
Funders Acknowledgements:
The Environment Agency
Grant ID: SC250010
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Abstract
This protocol outlines a simple culturing methodology for the growth of E.coli prior to Whole Genome Sequencing.
Safety warnings
Ensure good laboratory practice is followed throughout waste is suitably disposed of either through treatment with Rely+ Virkon (liquid waste) or via autoclave (solid waste).
Before start
All culture vessels and plates should be labeled prior to use. Luria Bertani (LB) broth (Lennox) should be pre-prepared following manufacturers instructions. All reagents and plasticware should be sterile prior to use.
Reviving culture from stock
Take stock sample out of the -80oC Freezer and transfer to upright -80oC Freezer in microbiology lab.
Alternatively keep stock on ice whilst in use to minimize thawing.
Take sub-set of samples from box, place sample and plate in laminar flow cabinet and use a sterile loop to take small amount of glycerol stock. Return to -80oC
Lift plate to 45 degree angle and swipe loop over pre-prepared Tryptone Bile X-Glucuronide (TBX) agar plate using streak plating method.
Figure 1: Streak plate method for revival of E.coli isolates.
Incubate plates at 37oC overnight.
Colony culture
Remove individual colony from overnight culture using sterile loop.
Note: E.coli on TBX will be blue, only select blue colonies.
Add loop to 5ml LB (Lennox) Broth in 15ml centrifuge tube, and swirl to remove colony. Dispose of loop.
Seal centrifuge tube with lid, then slighty release to allow some airflow. Place in tube rack, and incubate overnight in a shaking incubator pre-set to 37oC and 200 rpm.
Figure 2: Colony culture of E.coli in LB (Lennox) Broth.
Culture pellet and preparation for sequencing
Remove overnight culture from shaking incubator.
Tighten tube lid and place in centrifuge capable of holding rotor with 15ml tube adapter. Such as Beckman Coulter Avanti J-E, and JS 5.3 Rotor with 15ml adapters. Centrifuge at 4500 rpm for 20 minutes.
Remove tubes from centrifuge, and pour supernatant into waste bottle. For later treatment with Rely+ Virkon (one tablet per 500mls).
Suspend resultant pellet in Zymo DNA/RNA shield preservation buffer, in 2ml screw top tubes provided by microbes ng.
Add pellet suspension, back into 2ml tube (provided by microbes ng).
Secure preparation with 2ml tube lid and store in the fridge until dispatch.