Simon Holdaway
11-24-2007, 01:45 PM
Lab Management 101 - Glycerol Storage of E.coli bacterial stocks
If you are like us with your planned biology syllabus, then DNA transformation time is just around the corner in your teaching lab. This Lab Management 101 Tip focuses on the bacteria you use in your transformation experiments, and ways to save time and money on next year's lab. In the research laboratory every time a new DNA construct is made and inserted into a plasmid, with subsequent transformation into E.coli, a stock culture is made for the freezer. Stored in 15% glycerol, which acts as a cryoprotectant, the bacterial culture will theoretically last forever at minus 80 degrees, and can be revived at any time. Stored at minus 20 degrees in the type of freezer more commonly found in a teaching laboratory the culture should last 2-5 years.
Glycerol stock cultures are an excellent way to save money by eliminating the need to purchase expensive bacterial strains year after year. The following protocol for glycerol stock cultures was adapted from the Qiagen Bench Guide.
Preparation of glycerol stocks
1. Add 0.15 ml glycerol (100%) to a 2 ml screw-cap vial and sterilize by autoclaving.
Vials of sterilized glycerol can be prepared in batches and stored at room temperature until required.
2. Add 0.85 ml of a logarithmic-phase E. coli culture to the vial of pre-sterilized glycerol.
3. Vortex the vial vigorously to ensure even mixing of the bacterial culture and the glycerol.
4. Freeze at minus 20 degrees. Avoid repeated thawing and re-freezing of glycerol stocks as this can reduce the viability of the bacteria. One way to eliminate this problem in frost-free freezers (which periodically warm up to melt the ice on the inside) is to store your cultures inside a Styrofoam container with an ice pack or two. For precious strains, storage of two stock vials is recommended.
Preparation of stab cultures
An alternative storage technique if you lack a freezer but have a fridge is to prepare stab cultures. E. coli strains can be stored for up to 1 year as stabs in soft agar. Stab cultures can be used to transport or send bacterial strains to other labs.
1. Prepare and autoclave 0.7% LB agar (standard LB medium containing 7 g/liter agar).
2. Cool the LB agar to below 50?C (when you can hold it comfortably) and add the appropriate antibiotic(s). While still liquid, add 1 ml agar to a 2 ml screw-cap vial under sterile conditions, then leave to solidify. Vials of agar can be prepared in batches and stored at room temperature until required.
3. Using a sterile straight wire, pick a single colony from a freshly grown plate and stab it deep down into the soft agar several times.
4. Incubate the vial at 37?C for 8-12 h leaving the cap slightly loose.
5. Seal the vial tightly and store in the dark, preferably at 4?C.
Stab cultures will keep for approximately 12-18 months. Long enough to be revived in time for next year's transformation experiment.
http://www1.qiagen.com/literature/qiagennews/0598/985theqi.pdf
If you are like us with your planned biology syllabus, then DNA transformation time is just around the corner in your teaching lab. This Lab Management 101 Tip focuses on the bacteria you use in your transformation experiments, and ways to save time and money on next year's lab. In the research laboratory every time a new DNA construct is made and inserted into a plasmid, with subsequent transformation into E.coli, a stock culture is made for the freezer. Stored in 15% glycerol, which acts as a cryoprotectant, the bacterial culture will theoretically last forever at minus 80 degrees, and can be revived at any time. Stored at minus 20 degrees in the type of freezer more commonly found in a teaching laboratory the culture should last 2-5 years.
Glycerol stock cultures are an excellent way to save money by eliminating the need to purchase expensive bacterial strains year after year. The following protocol for glycerol stock cultures was adapted from the Qiagen Bench Guide.
Preparation of glycerol stocks
1. Add 0.15 ml glycerol (100%) to a 2 ml screw-cap vial and sterilize by autoclaving.
Vials of sterilized glycerol can be prepared in batches and stored at room temperature until required.
2. Add 0.85 ml of a logarithmic-phase E. coli culture to the vial of pre-sterilized glycerol.
3. Vortex the vial vigorously to ensure even mixing of the bacterial culture and the glycerol.
4. Freeze at minus 20 degrees. Avoid repeated thawing and re-freezing of glycerol stocks as this can reduce the viability of the bacteria. One way to eliminate this problem in frost-free freezers (which periodically warm up to melt the ice on the inside) is to store your cultures inside a Styrofoam container with an ice pack or two. For precious strains, storage of two stock vials is recommended.
Preparation of stab cultures
An alternative storage technique if you lack a freezer but have a fridge is to prepare stab cultures. E. coli strains can be stored for up to 1 year as stabs in soft agar. Stab cultures can be used to transport or send bacterial strains to other labs.
1. Prepare and autoclave 0.7% LB agar (standard LB medium containing 7 g/liter agar).
2. Cool the LB agar to below 50?C (when you can hold it comfortably) and add the appropriate antibiotic(s). While still liquid, add 1 ml agar to a 2 ml screw-cap vial under sterile conditions, then leave to solidify. Vials of agar can be prepared in batches and stored at room temperature until required.
3. Using a sterile straight wire, pick a single colony from a freshly grown plate and stab it deep down into the soft agar several times.
4. Incubate the vial at 37?C for 8-12 h leaving the cap slightly loose.
5. Seal the vial tightly and store in the dark, preferably at 4?C.
Stab cultures will keep for approximately 12-18 months. Long enough to be revived in time for next year's transformation experiment.
http://www1.qiagen.com/literature/qiagennews/0598/985theqi.pdf