To rehydrate E. coli, you typically introduce a small volume of sterile liquid to a dried or lyophilized bacterial sample, allowing the cells to become metabolically active again.
The Basic Rehydration Process
A common method for rehydrating E. coli involves adding 250 microliters (µL) of sterile water directly to the dried bacterial culture. This sterile water will dissolve the dried pellet or film, allowing the E. coli cells to reabsorb water and begin their metabolic processes.
Why Rehydrate E. coli?
Microorganisms like E. coli are often stored in a dried or freeze-dried (lyophilized) state to maintain viability over long periods without refrigeration and for easy transport. This method prevents metabolic activity, preserving the genetic integrity and characteristics of the strain. Rehydration is the necessary step to revive these dormant cells for laboratory use, research, or further culturing.
Materials You'll Need
Before starting, ensure you have all necessary materials and maintain aseptic conditions to prevent contamination.
- Dried E. coli culture: This could be a lyophilized pellet in a vial, a dried film, or a sample on a filter paper.
- Sterile rehydration liquid:
- Sterile water: As per the reference, 250 µL is a suitable starting volume. Ensure it's molecular-grade or distilled and autoclaved.
- Sterile Luria-Bertani (LB) broth or Tryptic Soy Broth (TSB): These nutrient-rich media can be used instead of or after sterile water to provide immediate nutrients for growth.
- Sterile micropipette and tips: For accurate liquid transfer.
- Sterile culture tube or vial: To transfer the rehydrated culture.
- Sterile growth medium (e.g., LB agar plates or broth): For subsequent culturing.
- Incubator: Set to the appropriate temperature for E. coli growth (typically 37°C).
- Bunsen burner or alcohol lamp (optional): For sterilizing tools if working in a non-laminar flow hood environment.
- Ethanol (70%): For surface sterilization.
Step-by-Step Rehydration Procedure
Follow these steps carefully to ensure successful rehydration and minimal contamination:
- Prepare your workspace: Clean your laminar flow hood or workspace with 70% ethanol. Ensure all materials are sterile and within reach.
- Open the E. coli container: Carefully open the vial or container holding the dried E. coli culture. If it's a lyophilized pellet, ensure it's at the bottom of the vial.
- Add sterile rehydration liquid:
- Using a sterile micropipette, draw 250 µL of sterile water (or sterile LB broth).
- Gently transfer this liquid directly onto the dried E. coli pellet or film.
- If using a very small pellet, you can slightly swirl or tap the vial to help the pellet dissolve. Avoid vigorous shaking that might aerosolize cells.
- Allow rehydration: Let the mixture sit for 5-15 minutes at room temperature to allow the cells to fully rehydrate. You should see the dried material dissolve into a milky suspension.
- Transfer to growth medium:
- Once rehydrated, the E. coli suspension is ready for culturing.
- Pipette the entire rehydrated suspension into a sterile culture tube containing fresh liquid growth medium (e.g., 3-5 mL of LB broth).
- Alternatively, streak a loopful of the rehydrated suspension onto a sterile agar plate (e.g., LB agar).
- Incubate:
- Place the liquid culture tube or agar plate into an incubator set to 37°C.
- Incubate liquid cultures with shaking (e.g., 200-250 rpm) for 4-8 hours, or until visible turbidity.
- Incubate agar plates inverted overnight (12-18 hours) until colonies appear.
Important Considerations for Successful Rehydration
- Sterility is paramount: Always use sterile equipment and work in an aseptic environment to prevent contamination, which can lead to false results or loss of your culture.
- Gentle handling: Dried bacteria can be fragile. Handle vials and pipettes gently to avoid damaging the cells.
- Rehydration medium: While sterile water is effective for rehydration, using a nutrient-rich broth like LB or TSB can sometimes promote faster recovery and growth for sensitive strains.
- Incubation: Adequate incubation time and temperature are crucial for the revived cells to multiply and form a viable culture.
Rehydration Medium Options
| Rehydration Medium | Advantages | Disadvantages | Common Use |
|---|---|---|---|
| Sterile Water | Simple, readily available, minimal osmotic shock | No immediate nutrients for growth | Initial rehydration of robust strains |
| LB Broth | Provides essential nutrients for immediate growth | Contains components that can be contaminated easily | Preferred for most E. coli rehydrations |
| TSB Broth | Richer medium, good for general bacterial growth | Can sometimes be too rich for specific applications | General-purpose rehydration for diverse bacteria |
By following these guidelines, you can effectively rehydrate your E. coli cultures for further experimental work and propagation.
