General Guidelines for Handling DNA

Working with bacteria: Good microbiological practice

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Daniel: Alright, Thi, ready to do your first bacterial inoculation?

Thi: Definitely, Daniel! I’m excited to get some hands-on experience.

Daniel: Perfect. I’ll guide you step-by-step and give you some tips along the way to make the process smoother. Let’s start!

Good microbiological techniques will always give you the best yield and quality of plasmid DNA. Follow the steps below to prepare the perfect bacterial culture for your plasmid prep.

  1. Prepare a starter culture by inoculating a single colony from a freshly streaked selective plate into 2–10 mL LB (Luria-Bertani) medium containing the appropriate antibiotic. Grow at 37°C for ~8 hours with vigorous shaking (~300 rpm) (see figure E. coli culture in LB medium).

    Tip: It is often convenient to grow the starter culture during the day so that the larger culture can be grown overnight for harvesting the following morning.

  2. Dilute the starter culture 1/500 to 1/1000 into a larger volume of selective LB medium, as indicated in the appropriate plasmid purification protocol.

    Tip: Use a flask at least 4 times the culture volume to ensure sufficient aeration.

    Tip: Do not use a larger culture volume than recommended in the protocol, as this will result in inefficient lysis and reduce the quality of the preparation.

  3. Grow the culture at 37°C with vigorous shaking (~300 rpm) for 12–16 hours.
  4. Harvest the bacterial culture 12–16 hours after inoculation. This corresponds to the transition from logarithmic into stationary growth phase (see next section) when cell density is high (3–4 x 109 cells per mL) and RNA content of cells is low. Harvesting too early may result in lower than expected yields of plasmid DNA due to a lower cell density. Harvesting too late may result in low plasmid quality and yield due to DNA degradation from over-aging the culture.

    Tip: Growth of cultures is dependent on factors such as host strain, plasmid insert and copy number and culture medium. To determine the optimal harvesting time for a particular system, monitor the cell density and the growth of the culture by measuring the OD600.

  5. Harvest the bacterial culture by centrifugation at 6000 x g for 15 min. Remove all traces of supernatant by inverting the open centrifuge tube until all of the medium has been drained. The cells are now ready for the lysis procedure, as indicated in the appropriate plasmid purification protocol. The procedure may be stopped at this point and continued later by freezing the cell pellets obtained by centrifugation. The frozen cell pellets may be stored at –20°C for several weeks.

The growth curve of an E. coli culture can be divided into several distinct phases. 

Lag phase

Occurs after diluting the starter culture in fresh medium. During this phase, cell division is slow as the bacteria adapt to the fresh medium. 

Logarithmic (log) phase

The bacteria will then start to divide more rapidly (4–5 after dilution), during which the number of cells increases exponentially.

Stationary phase

As the available nutrients in the medium are used up and released metabolites inhibit bacterial growth, the culture becomes saturated and enters stationary phase (~16 hours after dilution), during which cell density remains constant. 

Decline phase

The culture eventually enters the decline phase as cells start to lyse, the number of viable bacteria falls, and DNA becomes partly degraded.

Growth curve of E. coli
You can use different methods for storing E. coli strains depending on the desired storage time. You can use glycerol stocks and stab cultures for long-term storage of bacteria, or agar plates for short-erm storage. Read the preparation instructions and useful tips for each of the methods below.

Glycerol stocks

E. coli strains can be stored for many years at –70°C in 15% glycerol.

Prepare glycerol stocks of bacteria using the following method:

  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 in ethanol–dry ice or liquid nitrogen and store at –70°C.

    Avoid repeated thawing and re-freezing of glycerol stocks as this can reduce the viability of the bacteria.

    Tip: For precious strains, storage of 2 stock vials is recommended.

    Tip: When recovering a stored strain, it is advisable to check the antibiotic markers by streaking the strain onto a selective plate.

Stab cultures

E. coli strains can also be stored for up to 1 year as stabs in soft agar. Stab cultures are used to transport or send bacterial strains to other labs. 

Prepare stab cultures as follows:

  1. Prepare and autoclave LB agar (standard LB medium containing 0.7% agar).
  2. Cool the LB agar to below 50°C (when you can hold it comfortably) and add the appropriate antibiotic(s). While the agar is still liquid, add 1 mL agar to a 2 mL screw-cap vial under sterile conditions, then leave to solidify.
  3. Vials of agar can be prepared in batches and stored at room temperature until required.
  4. Using a sterile straight wire, pick a single colony from a freshly streaked plate and stab it deep down into the soft agar several times (see figure Inoculating a stab culture).
  5. Incubate the vial at 37°C for 8–12 h leaving the cap slightly loose.
  6. Seal the vial tightly and store in the dark, preferably at 4°C.
  7. When recovering a stored strain, it is advisable to check the antibiotic markers by streaking the strain onto a selective plate.
Inoculating a stab culture
Agar plates

Plates of streaked bacteria can be sealed with Parafilm and stored upside-down at 4°C for several weeks. Bacteria should always be streaked onto plates containing the appropriate antibiotic to avoid losing selective markers.

To obtain well-isolated colonies, streak an agar plate as follows:

  1. Flame a wire loop, and cool on a spare sterile agar plate.
  2. Using the wire loop, streak an inoculum of bacteria (from a glycerol stock, stab culture, or single colony on another plate) across one corner of a fresh agar plate, as shown in the figure Streaking bacteria on agar plates.
  3. Flame and cool the wire loop again. Pass it through the first streak and then streak again across a fresh corner of the plate.
  4. Repeat again to form a pattern.
  5. Incubate the plate upside down at 37°C for 12–24 hours until colonies develop.
Streaking bacteria on agar plates
Generating liquid cultures from bacterial stocks

The figure, Essential steps for storing and handling E. coli, shows the sequence of steps you need to take to go from a stored stock of bacteria to a liquid culture for plasmid isolation. You should always streak your bacterial stocks onto selective plates before you use them to check that they will give rise to healthy colonies carrying the appropriate antibiotic resistance. Stocks can potentially contain mutants arising from the cultures used to prepare them or can deteriorate during storage.

It’s always good practice to inoculate your liquid cultures from a healthy, well-isolated colony picked from a freshly streaked selective plate. This will ensure that cells growing in the culture are all descended from a single founder cell and have the same genetic makeup.

Tip: Culture volumes >10 mL should not be inoculated directly from a plate but diluted 1/500 to 1/1000 from a pre-culture of 2–5 mL.

Essential steps for storing and handling E. coli

Tip: High-molecular-weight DNA, such as genomic DNA, should be redissolved very gently to avoid shearing, e.g., at room temperature overnight or at 55°C for 1–2 h with gentle agitation.