Pure Cultures and Streak Plating: Isolation of Single Bacterial Colonies from a Mixed Sample

1. Set up

1. All microbes should be treated as if they are hazardous. Always wear a lab coat and gloves, tie back long hair, and ensure that any wounds are particularly well protected.
2. Ready the work space by sterilizing it using 70% ethanol.
3. Ensure that agar plates, sample solution(s) and either a box of pre-sterilized plastic inoculation loops or a metal loop plus a Bunsen flame, are close at hand. Disposable, plastic loops are typically pre-sterilized. Metal loops should be dipped in 70% ethanol, and then held near the blue area of a Bunsen flame and heated until fiery hot. Allow the wire to cool down by raising the lid of the plate (only slightly to prevent contamination) and tapping it against the solidified medium.
4. Finish each procedure with a repeated sterilization of the work space and a thorough wash/sterilization of hands and wrists.

2. Protocol

1. Preparation of media
   1. Identify and prepare a solid medium (typically containing 1.5% (w/v) agar) that will sustain the utilized bacterial species/strain. Mix the medium in a bottle able to hold twice the final volume to avoid overflow when autoclaving.
   2. Sterilize the media by placing the bottle, with a semi-tightened cap, in an autoclave set to 121°C for 20 min.
   3. Close the cap properly as soon as the bottle is removed from the autoclave. If the media is to be used shortly, place the bottle in a water-bath set to 45°C to preserve it in a liquid state. The agar will otherwise solidify at anything less than 32-40°C, and can later be re-heated (typically using a microwave) to a melting point at 85°C.
2. Preparation of culture plate(s)
   1. Mark the base of sterile Petri dishes (typically 100 x 15 mm) on either the side or the bottom with the experimenter's name, the date, and media type.
   2. Pour 20-25 mL of 45°C agar culture medium (previously prepared) into each of the labelled plates. Should foam appear along the edges, this should be swiftly removed using a regular pipette and a sterile tip.
   3. Immediately place all lids back onto the dishes to prevent contamination.
   4. Allow the agar to solidify for approximately 2 h at room temperature or overnight at 4°C. Once set, bacterial culture plates should subsequently be stored upside-down at 4°C to minimize condensation on the medium surface.
3. Streak plating
   1. Submerge a sterile loop into the desired inoculum and immediately disperse the collected sample onto the first quadrant of the plate using a zig-zag motion (Figure 1, I).
   2. Close the lid and re-sterilize the inoculation loop or collect a new sterile disposable loop.
   3. Make 3-4 strokes radiating from the first quadrant (containing a relatively dense bacterial population) towards the second quadrant of the plate (Figure 1, II).
   4. Close the lid and re-sterilize the inoculation loop or discard the disposable loop and collect a new sterile one.
   5. Repeat this streaking of 3-4 strokes from the second into the third quadrant, and then from the third to the fourth quadrant, using a sterile loop each time (Figure 1, III - IV).
   6. Using a sterile loop, make one final stroke in a zig-zag pattern from the fourth quadrant towards the middle of the plate (Figure 1, V). The bacterial prevalence will be lower in this area, ideally allowing individual colonies to be established from a single, viable mother-cell.
   7. Close the lid and (if required by the bacterial species) seal with parafilm to prevent air flow.
   8. Depending on the bacterial species/strain, place the culture plate up-side-down in a suitable environment and incubate until bacterial colonies are visible (segregated colonies can appear in either area of the plate as the initial concentration may vary).
   9. To generate a clonal bacterial population, streak out another plate, exchanging the inoculum plated onto the original plate for cells isolated from a single colony of the original plate.