Feb 28, 2025
Bacterial Glycerol Stocks
- 1University of Birmingham
Protocol Citation: David Cleary 2025. Bacterial Glycerol Stocks . protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk9znxv5r/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: February 28, 2025
Last Modified: February 28, 2025
Protocol Integer ID: 123573
Abstract
This protocol describes the basic steps for the preparation of bacterial stocks in glycerol suitable for long-term storage at -20 or -80.
Preparation of o/n bacterial culture
Preparation of o/n bacterial culture
- Prepare liquid culture according to requirements of organism e.g., BHI, TS or LB broth
- Determine required volume (N vials x 500 μL of culture)
- Pick several colonies from a purity plate using a sterile loop and innoculate broth
- Incubate at 37oC o/n if >50mL or 6 h if ~20 mL
Preparation of 50% Glycerol
Preparation of 50% Glycerol
- Measure 10 mL autoclaved glycerol and 10 mL sterile dH2O into falcon tube
- Vortex thoroughly to mix
- Aspirate into 20 mL syringe and pass through a 0.2 μM filter into a sterile falcon tube
Aliquoting and Storage
Aliquoting and Storage
- Add 500 μL glycerol to required number of cryovials
- Add 500 μL of bacterial liquid culture
- Vortex briefly to mix
- Label with ID, preparation date and number (if >1 stock is made)
- Stocks can be stored at -20oC for up to six months or several years at -80oC