Preparation of Bacteria Glycerol Stocks

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.

Before start

Make sure all materials and reagents needed for this protocol are available

Steps

Preparing liquid culture of the bacteria to be stored

Prepare LB following this protocol depending on the desired amount of LB and subsequent number of glycerol stock tubes needed.

Using an inoculating wire loop, pick up some bacteria colonies from a culture plate and inoculate in an Erlenmeyer flask containing the LB (with the right antibiotic if applicable).
Grow the cells by incubating in an incubator at 37°C for 3h 0m 0sto obtain maximum cell growth.

Diluting Pure Glycerol to 50% with Distilled water

Use a clean measuring cylinder to measure 10mLof distilled water and equal amount 10mL of into a 50 mL falcon tube.

Cork the tube and shake thoroughly until the liquids are evenly mixed

Filtering the 50% glycerol

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

Aliquoting the bacterial culture into 50% glycerol and storing

To make 1mL of Bacteria glycerol stocks, aliquot 500µLof 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 (500µL) 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.