Freeze drying bacteria is a useful method for long-term preservation especially after cloning and inserting a new gene. How can BL21 Competent E. coli be Lyophilization? Thank you
Hi,
You are bacteria growth in general media (Nutrient broth) at 32-37C for 16-18h than centrifuging and taking biomass to Freeze drying tool.
Dear Abdol
According some sources freeze drying bacteria is a useful method for long-term preservation. Freeze drying bacteria has been done a multistep process which involves culturing the microbes, suspending them in a lyophilization medium/buffer, subjecting them to the freeze drying process, and then subsequently storing them properly.
Theresa Philips describe the lyophilization in 10 steps
1-First, grow an overnight culture, or lawn, of the microorganism on LB or other appropriate nutrient agar plate.
2-Next, prepare sterile crimp-cap vials by autoclaving ahead of time, with the caps (rubber stoppers) placed loosely on top. Place paper labels printed with the culture's identification inside the tubes prior to autoclaving. Alternatively, use tubes with caps designed for sterility.
3-Add 4 mL lyophilization buffer to the plate. If necessary, the cells can be suspended using a sterile glass rod.
4-Quickly transfer the culture suspension to the sterilized vials. Add approximately 1.5 mL per vial. Seal with the rubber cap.
5-Freeze the culture suspension inside the vials by placing the vials in a -20 degrees Celcius freezer.
6-Once the cultures are frozen, prepare the freeze-dryer by turning it on and allowing time for the appropriate temperature and vacuum conditions to stabilize. Do this according to the manufacturer's instructions for the particular brand of freeze-dryer you're using.
7-Carefully, and aseptically, place the vial caps loosely on top of the vials, so that moisture can escape during the freeze-drying process, and place the vials into a freeze-dryer chamber. Apply the vacuum to the chamber according to the manufacturer's instructions.
8-Allow the culture time to completely lyophilize (dry out). This may take from a few hours to overnight depending on the volume of each sample and how many samples you have.
9-Remove the samples from the freeze-dryer chamber according to the manufacturer's instructions and immediately seal the vials with the rubber caps, then the crimp tops.
10-Store the lyophilized culture collection at room temperature.
Also you can check the following links.
Hoping to be helpful
https://www.thebalance.com/how-to-freeze-dry-a-bacterial-culture-lyophilization-375685
http://opsdiagnostics.com/notes/ranpri/rpbacteriafdprotocol.htm
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC545105/
Marius has given a detailed and simplified answer which should be adequate. However you have to experimentally determine the optimum pressure and temperature conditions to give you the best product suitable for your purpose. It is a function of the material being freeze dried, freeze drying buffer/medium (especially cryoprotectants) used. It is important to note that freeze dried material be sealed under vacuum to avoid exposure to moisture and oxygen. Test your material periodically to measure its viability.
SP Scientific. Basic Principles of Freeze Drying. http://www.spscientific.com/freeze-drying-lyophilization-basics/
https://opsdiagnostics.com/notes/ranpri/rpbacteriafdprotocol.htm
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