Podcast
Questions and Answers
What is the primary purpose of using an X-gal indicator plate in the experiment?
What is the primary purpose of using an X-gal indicator plate in the experiment?
- To incubate the bacteria at a lower temperature
- To promote antibiotic resistance in the colonies
- To identify colonies that produce β-galactosidase (correct)
- To enhance the growth of all colonies on the LB plate
Which ingredient in the X-gal indicator plates directly leads to the coloring of colonies?
Which ingredient in the X-gal indicator plates directly leads to the coloring of colonies?
- LB agar
- Ampicillin
- Glycerol
- X-gal (correct)
What is the temperature at which the LB/Amp plates should be incubated overnight?
What is the temperature at which the LB/Amp plates should be incubated overnight?
- 20ºC
- 37ºC (correct)
- 40ºC
- 30ºC
After selecting a positive colony from the original LB plate, what is the next step in the process?
After selecting a positive colony from the original LB plate, what is the next step in the process?
What is the purpose of resuspending the cells in 20% glycerol during the preservation process?
What is the purpose of resuspending the cells in 20% glycerol during the preservation process?
What is the purpose of preparing a stock culture?
What is the purpose of preparing a stock culture?
Which method is NOT typically used for the preservation of production cells?
Which method is NOT typically used for the preservation of production cells?
What conditions must be maintained when storing freeze-dried cells?
What conditions must be maintained when storing freeze-dried cells?
What is a critical step when starting a new production run from cryogenic preserved cells?
What is a critical step when starting a new production run from cryogenic preserved cells?
In the microbank method, what is the role of the sterile beads?
In the microbank method, what is the role of the sterile beads?
Why is it advantageous to use 15-50% glycerol in cryogenic preservation?
Why is it advantageous to use 15-50% glycerol in cryogenic preservation?
How can a single microbank vial benefit multiple cultures?
How can a single microbank vial benefit multiple cultures?
What is the significance of sealing ampoules after freeze-drying?
What is the significance of sealing ampoules after freeze-drying?
Flashcards
Stock Culture (Seed Bank)
Stock Culture (Seed Bank)
A collection of production cells stored to ensure consistency between batches.
Freeze-drying
Freeze-drying
A process of preserving production cells by removing water and storing in a vacuum-sealed container.
Cryogenic Preservation
Cryogenic Preservation
Freezing cells in a solution containing glycerol to protect them from damage during freezing.
Microbank
Microbank
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Cryogenic Medium
Cryogenic Medium
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Seed Flask
Seed Flask
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Culture Broth
Culture Broth
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Sterile Transfer
Sterile Transfer
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What are X-gal plates used for?
What are X-gal plates used for?
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What is a 'streak-plate'?
What is a 'streak-plate'?
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What is a 'replica grid'?
What is a 'replica grid'?
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What is beta-galactosidase?
What is beta-galactosidase?
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How do you determine the cell concentration in each vial?
How do you determine the cell concentration in each vial?
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Study Notes
Stock Culture Preparation
- Production cells need consistent starting material for repeated large-scale batches over months or years.
- Preservation methods maintain cell viability, like freeze-drying or cryogenic preservation.
- Freeze-drying allows storage at room temperature under anhydrous conditions in sealed ampoules (e.g., ATCC samples).
- Cryogenic preservation involves freezing cells in a 15-50% glycerol/water buffer solution.
- The cells are then aliquoted into sterile vials and frozen at -70°C or -130°C.
- Microbank method uses cryo-vials with porous beads in cryogenic fluid. This is more space efficient.
- Colonies are transferred directly from plates or liquid cultures to the beads. Excess liquid removed before freezing.
X-gal Plates
- X-gal plates identify colonies producing β-gal.
- The plates contain X-gal (a substrate) and other ingredients (likely a growth media).
- Colonies producing β-gal enzyme cleave X-gal resulting in a blue color change.
Cell Preservation Procedure
- Liquid culture streaked onto LB/Amp plates for initial growth.
- Colonies are replicated onto X-gal plates.
- Confirmation of blue colonies (β-gal producers); selection of positive colonies for stock.
- Positive colonies are transferred to fresh LB broth media for overnight growth at 37°C
- Centrifuge the cells to pellet down the cells; resuspend in 20% glycerol and split into two components .
- Freeze at –20ºC.
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Description
This quiz covers essential techniques in cell culture preparation, including preservation methods like freeze-drying and cryogenic preservation. It also explores the use of X-gal plates for identifying β-gal producing colonies. Test your understanding of these crucial concepts in microbiology and cell biology.
