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: April 19, 2024
Last Modified: April 19, 2024
Protocol Integer ID: 98469
Keywords: Glycerol stocks, Bacteria stocks, Bacteria glycerol stocks, Store bacteria
Abstract
This protocol is meant to provide researchers with a step by step procedure on how to prepare glycerol stocks in order to preserve and store bacteria for long term.
Bacterial glycerol stocks are important for long-term storage of plasmids. The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years and -20°C for several months.
Glycerol reduces the harmful effects of ice crystals on bacteria which cause dehydration and damage cells through a localised increase in salt concentration leading to the denaturation of proteins.
Guidelines
This protocol describes the steps in preparing bacterial glycerol stocks. This protocol can be performed by anyone with basic molecular biology skills.
Materials
Reagents
LB Broth (with or without antibiotic)
GlycerolSigma – Aldrich
Sterile Distilled water
Bacteria(E. coli) strain of interest (from a LB Agar plate)
Antibiotic stock of choice (if required)
Equipment and glassware
Refrigerator
Incubator
Timer
Sterile 1.5 mL Eppendorf tubes or cryo-tubes
P-1000 micropipette
Sterile 1000 uL pipette tips
50 mL Erlenmeyer flask
Inoculation wire loop
Sterile 0.2 µM micro filter
Sterile 20 mL syringe
Sterile 3 mL plastic dropper
Sterile 50 mL falcon tube
Protocol materials
GlycerolMerck MilliporeSigma (Sigma-Aldrich)
Step 1
Safety warnings
Endeavor to segregate the waste generated and discard appropriately.
Before start
Make sure all materials and reagents needed for this protocol are available
Diluting Pure Glycerol to 50% with Distilled water
Diluting Pure Glycerol to 50% with Distilled water
5m
Use a clean measuring cylinder to measure 10 mLof distilled water and equal amount 10 mL of GlycerolSigma Aldrich into a 50 mL falcon tube.
Cork the tube and shake thoroughly until the liquids are evenly mixed
Filtering the 50% glycerol
Filtering the 50% glycerol
2m
Use a 20 mL sterile syringe to aspirate the 50% glycerol from the falcon tube
Plug in a 0.2 µM micro filter and filter out the glycerol into a sterile falcon tube
From the plate to a PBS eppendorf
From the plate to a PBS eppendorf
3m
If you have colonies already growing in a Petri dish you can simply collect them using an inoculating wire loop and transfer to a eppendorf tube with 500 µL of 1x PBS
Once you have collect enough pure colonies you can go to the next step.
3m
Aliquoting the bacterial culture into 50% glycerol and storing
Aliquoting the bacterial culture into 50% glycerol and storing
To make 1mL of Bacteria glycerol stocks, aliquot 500 µLof the filtered 50% glycerol into separate 1.5 mL Eppendorf tubes (triplicates or more depending on the quantity of glycerol stocks needed.
Use sterile micropipette and tips to measure out equal volumes (500 µL) of the bacterial culture from the Erlenmeyer flask into the tubes containing the 50% glycerol (We now have equal proportion of bacteria and 50% glycerol).
Keep your thumb pressed firmly against the lid of the Eppendorf tube and shake vigorously to make sure the 2 liquids mix completely.
Use a marker pen to label the tubes with the name of the bacterial strain and date of preparation of the glycerol stock
Store the vials in the freezer at -20°C until they are used.
Note
After pipetting the bacteria culture and 50% Glycerol, shake several times to ensure it mixes completely and uniformly.
Bacteria glycerol stocks prepared and stored in this manner are stable for up to year.
Avoid frequent freeze-thaw of the glycerol stocks.