How to Grow Escherichia coli

E. coli's ease of cultivation, rapid growth, and genetic tractability make it an indispensable tool in biological research. By understanding and following proper growth and maintenance procedures, researchers can effectively utilize this versatile organism in a wide range of applications, from basic molecular biology to advanced biotechnology.

I. What is Escherichia coli?

Escherichia coli, commonly known as E. coli, is a Gram-negative, rod-shaped bacterium belonging to the Enterobacteriaceae family. First discovered and isolated by Theodor Escherich in 1885 from fecal samples of healthy individuals, E. coli has since become a model organism in both research and teaching laboratories. Most laboratory strains of E. coli are derived from either the K-12 or B strains, which are considered non-pathogenic and safe for use in research settings. E. coli's genetic tractability, rapid growth rate, and ease of cultivation have made it an invaluable tool in various fields of biology and biotechnology.

II. Why is E. coli important in laboratory settings?

E. coli's importance in laboratory settings stems from its versatility and well-characterized nature. Its use as a model organism dates back to the early 1940s, with pioneering work by researchers like Max Delbruck and Salvador Luria demonstrating its genetic tractability. The discovery of bacterial conjugation by Joshua Lederberg and Edward Tatum in 1946 further solidified E. coli's position as a key model organism. Today, E. coli is widely used in molecular biology, genetics, and biotechnology for applications such as gene cloning, protein expression, and studying fundamental biological processes.

III. What are the optimal growth conditions for E. coli?

E. coli grows optimally at 37°C under aerobic conditions, although it is a facultative anaerobe and can grow in the absence of oxygen. Some strains have been reported to grow at temperatures up to 53°C, but this is not typical for common laboratory strains. E. coli can survive at 4°C for extended periods (up to 3 months) on solid media, though prolonged storage at low temperatures may decrease viability. The bacterium grows best at a neutral pH of 7.0 but can tolerate a range of pH conditions. Under optimal conditions in rich media like Luria-Bertani (LB) broth, E. coli has a doubling time of approximately 20 minutes and can reach cell densities of over 10^9 colony-forming units (CFU) per milliliter in overnight cultures.

IV. What media are commonly used for growing E. coli?

E. coli can grow on a variety of carbon sources, but it is typically cultured in rich media such as Luria-Bertani (LB) broth or on LB agar plates. These media provide a complex mixture of nutrients that support rapid growth. The composition of LB broth typically includes tryptone, yeast extract, and sodium chloride. For specific experimental needs, the media can be modified or supplemented with additional components such as antibiotics or chromogenic agents like X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) for blue-white colony screening in molecular cloning experiments.

V. How do you initiate E. coli growth from frozen stocks?

To initiate E. coli growth from frozen stocks, it's essential to follow proper aseptic technique to avoid contamination and maintain stock viability. Using a sterile wooden applicator stick, remove a small ice chip from the frozen stock vial without allowing it to thaw completely. Streak this ice chip onto an LB agar plate (with appropriate antibiotics if necessary) to obtain single, well-isolated colonies. Incubate the plate overnight (14-18 hours) at 37°C. It's crucial to minimize the thawing of the stock during this process, as repeated freeze-thaw cycles can significantly decrease the viability of the culture.

VI. What is the procedure for growing E. coli on solid media?

Growing E. coli on solid media is often necessary for enumerating bacteria or isolating single colonies. To do this, prepare serial dilutions of an overnight liquid culture (typically from 10^-1 to 10^-10). Apply a small volume (20-100 μL) of the appropriate dilutions to labeled LB agar plates. Use a sterile glass spreader to evenly distribute the culture across the surface of the plate. Incubate the plates overnight at 37°C. For quantification purposes, plates with 30-300 colonies are ideal for accurate counting.

VII. How do you grow E. coli in liquid media?

To grow E. coli in liquid media, start by aseptically transferring 5 mL of LB broth (with appropriate antibiotics if needed) into a sterile glass culture tube. Using a sterile wooden applicator stick or inoculating loop, pick a single, well-isolated colony from a streak plate and inoculate it into the liquid medium. Incubate the culture at 37°C with aeration (shaking or rotating) for the desired amount of time. For larger volumes, Erlenmeyer flasks can be used instead of test tubes.

VIII. What are the methods for long-term storage of E. coli?

For long-term storage, E. coli can be preserved in glycerol stocks at -80°C. To prepare these stocks, mix an overnight culture with sterile 50% glycerol to achieve a final glycerol concentration of 30%. Aliquot this mixture into cryovials and immediately freeze at -80°C. It's recommended to prepare two identical vials for each strain, using one as the working stock and keeping the other as a backup. Alternatively, E. coli can be stored in agar stabs at room temperature, which is particularly useful for shipping strains or when -80°C storage is not available.

IX. How do you prepare E. coli agar stabs?

To prepare an agar stab, use a sterile inoculating needle or toothpick to pick a single colony from a plate or a loopful of liquid culture. Stab the needle straight down into a vial containing LB agar, then pull it straight up. Incubate the vial at 37°C overnight with the cap slightly loosened. After incubation, tighten the cap and store the vial in a cool (15-22°C), dark place.

X. What precautions should be taken when working with E. coli?

Although most laboratory strains of E. coli are considered non-pathogenic, it's crucial to follow proper biosafety protocols. E. coli is typically classified as a Biosafety Level 2 (BSL-2) organism, requiring appropriate safety measures for handling and disposal. Always follow your institution's guidelines for working with microorganisms. When subculturing or passaging E. coli, be aware that repeated transfers can increase the chance of mutations, potentially leading to unintended genetic changes. To maintain strain integrity, it's best to start fresh cultures from frozen stocks for critical experiments.