Apr 19, 2024

Public workspacePreparation of Glycerol Stocks from storing microbes from Petri Dishes

 Forked from Preparation of Bacteria Glycerol Stocks
This protocol is a draft, published without a DOI.
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Márcio Fernandes
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Protocol Citation: Stephane Fadanka, Mujar Minette Shalo, Nadine Mowoh 2024. Preparation of Glycerol Stocks from storing microbes from Petri Dishes. protocols.io https://protocols.io/view/preparation-of-glycerol-stocks-from-storing-microb-dced2ta6
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)
  • ReagentGlycerolSigma – 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
ReagentGlycerolMerck MilliporeSigma (Sigma-Aldrich)
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
5m
  • Use a clean measuring cylinder to measure Amount10 mL of distilled water and equal amount Amount10 mL of ReagentGlycerolSigma 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
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
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 Amount500 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 Amount500 µL of 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 (Amount500 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.