UV mutagenesis, photoreactivation and a crowded replica plate
In a college laboratory a student irradiates 10 mL of a log-phase Escherichia coli suspension with a 254 nm UV lamp at a fixed distance for 0, 20, 40 and 60 seconds, stirring the suspension in an open sterile petri dish. The lamp was switched on only one minute before the first exposure. Immediately after irradiation the plates are carried to a neighbouring room with large windows and spread-plated on nutrient agar. The colonies that appear are replica-plated onto minimal medium and onto minimal medium plus amino acids. The student counts the colonies that grow on the minimal plates to estimate the mutation frequency. From the 60-second sample she obtains 3 colonies and concludes that UV does not produce auxotrophs. The 40-second master plate carries 420 colonies and the same velvet cloth is used for all replica plates.
1. Identify the errors in this experiment and state how each one distorts the result.
Read the worked answer
Several errors compound each other.
- Lamp not warmed up. A low-pressure mercury lamp needs several minutes for its output to stabilise; switching on for one minute means the delivered dose is unknown and differs between the samples, so the 'dose series' is not comparable.
- Daylight exposure during plating. The room with large windows is the worst place to handle irradiated cells. Visible light drives photolyase-mediated photoreactivation, which monomerises thymine dimers and repairs the very mutations being counted. Plating must be done under subdued yellow light or in the dark.
- Wrong colonies counted. An auxotroph grows on complete medium but fails on minimal medium. The student counted growth on minimal medium, which is exactly reversed: the mutants are the colonies present on complete but missing on minimal.
- Overcrowded master plate. With 420 colonies the velvet picks up overlapping growth, and neighbouring colonies cross-feed the auxotrophs with leaked amino acids - false negatives.
- Unsterilised, reused velvet. One cloth for all plates carries cells from one master plate onto the next, producing phantom colonies and cross-contamination, and gives no valid negative control.
2. The 40-second master plate has 420 colonies and 9 of them are absent from the minimal plate. Calculate the mutation frequency and explain why the 60-second result must not be used to conclude anything about mutation.
Read the worked answer
Calculation. Mutation frequency per viable cell scored = number of auxotrophs ÷ total colonies replicated = 9 ÷ 420 = 0.021, that is about 2.1 mutants per 100 surviving cells (2.1 × 10⁻²). Because UV is a strong mutagen this figure is plausible for such a dose.
Why the 60-second data are meaningless for this purpose. Killing rises steeply with dose, so at 60 seconds only a tiny surviving fraction remains; 3 colonies is a sample far too small for any statistical statement. More importantly, the survivors at high dose are a selected minority - cells that repair well or are unusually resistant - so the surviving population is not representative. A strain that survives by efficient repair may look mutationally 'quiet' for reasons unrelated to mutagenesis.
The correct design uses the dose that gives a high but manageable survival, so that hundreds of colonies can be screened per plate, and always expresses mutation frequency per survivor, never per cell plated, because the number of survivors changes with dose.
3. A colony from the nine is suspected to be an arginine auxotroph. Describe how you would confirm it and explain why confirmation cannot be done by replica plating alone.
Read the worked answer
Why replica plating is only a screen. It tells you that a colony grows on complete medium and not on minimal medium; it does not tell you which nutrient is missing, and failure to grow can be caused by poor transfer by the velvet, by the colony being at the plate edge, or by cross-feeding on the crowded master plate.
Confirmation.
- Pick the colony from the complete-medium master plate, purify it by streaking for single colonies, and keep a stock.
- Streak the purified culture on: minimal medium (no growth expected), minimal medium plus arginine (growth), and minimal medium plus each of several other single amino acids such as methionine or histidine (no growth).
- Score growth after 24–48 hours at 37 °C and compare with the wild type, which grows on all three.
- Re-streak from the supplemented tube onto minimal medium again to check that growth truly depended on arginine.
Growth only when arginine is supplied is the definition of an arginine auxotroph, and testing single supplements one at a time is what makes the identification specific.