Question

You wish to isolate temperature-sensitive mutants (e.g., those able to grow at \(30^{\circ} \mathrm{C}\) but not at \(37^{\circ} \mathrm{C}\) ). Describe experiments to isolate such a cell.

Short answer

Prepare culture medium, inoculate, incubate at 30°C, replica plate, incubate plates at both 30°C and 37°C, identify non-growing mutants at 37°C, and isolate them.

Step-by-step solution

Prepare the Culture Medium

Start by preparing a nutrient-rich culture medium suitable for the organism you are working with. Make sure the medium can support the growth of the organism at both temperatures, 30°C and 37°C.

Inoculate the Organism

Inoculate the culture medium with the organism you wish to isolate temperature-sensitive mutants from. Ensure that the inoculum is thoroughly mixed into the medium.

Incubate at 30°C

Incubate the inoculated culture at 30°C for an adequate amount of time to allow for growth. This step ensures that any potential mutants capable of growing at 30°C will proliferate.

Replica Plating

Replica plate the colonies from the 30°C culture onto two separate plates, ensuring identical colony placement. One plate will be incubated at 30°C and the other at 37°C.

Incubate at Both Temperatures

Incubate one of the replica plates at 30°C and the other at 37°C. Allow sufficient time for growth, typically overnight or as per the organism's growth rate.

Identify Temperature-Sensitive Mutants

After incubation, compare the growth on both plates. Identify colonies that grew on the 30°C plate but failed to grow on the 37°C plate. These colonies are temperature-sensitive mutants.

Isolate and Purify Mutants

Select the identified temperature-sensitive mutants from the 30°C plate and re-streak them onto fresh plates to purify the clones. Incubate again at 30°C to ensure pure cultures.

Key concepts

Culture Medium Preparation

The foundation of isolating temperature-sensitive mutants begins with preparing an appropriate culture medium. This medium provides essential nutrients for the organism you are studying.
Ensure that the medium is rich and can support growth at both target temperatures, such as 30°C and 37°C. A common choice might be LB agar for bacteria, but the specific medium will depend on your organism.
The process involves:

• Sterilizing the medium to prevent contamination.
• Allowing the medium to cool before pouring it into Petri dishes.
• Ensuring even distribution to facilitate consistent growth.

Proper medium preparation is crucial for accurate results as it ensures that growth differences are due to temperature sensitivity and not nutritional deficiencies.

Inoculation

Once your culture medium is ready, the next step is inoculation, where you introduce the organism into the medium. To do this effectively:

• Start with a pure culture of your organism to avoid contamination.
• Use a sterile loop or pipette to transfer a small amount of organism to the prepared plates.
• Spread the organism evenly to ensure single colonies form.

Mixing the inoculum thoroughly is crucial as it helps in the even distribution of cells, making it easier to identify individual colonies that may show temperature sensitivity later on. After inoculation, let the plate sit at room temperature for a few minutes to allow the organism to adhere to the medium before incubation.

Replica Plating

Replica plating is a technique used to transfer the same pattern of colonies from one plate to several other plates. This allows you to test the same colonies under different conditions.
First, let your inoculated plates grow at 30°C until visible colonies appear. Use a sterile cloth or velvet to gently touch the surface of the colonies to pick them up. Then, press this onto two fresh plates prepared earlier. These new plates will have the same colony pattern as the original.
Immediately label one plate for incubation at 30°C and the other at 37°C. This step is vital for pinpointing the temperature-sensitive mutants as it ensures identical colony placement, allowing a direct comparison of growth at different temperatures.

Incubation Temperatures

Incubation is a critical phase to differentiate between normal colonies and temperature-sensitive mutants. Label and place your replica plates in different incubators set at 30°C and 37°C, respectively.
The idea here is to monitor growth at both temperatures. Typically, an overnight incubation is sufficient, but this can vary based on your organism's growth rate.
It's essential to:

• Ensure accurate temperature control in the incubators.
• Prevent disturbance during incubation to maintain consistent conditions.

Proper incubation allows temperature-sensitive mutants to be easily identified, as they will grow at the permissive temperature (30°C) but not at the restrictive temperature (37°C).

Mutant Identification

After incubation, it's time to identify your temperature-sensitive mutants. Carefully compare the growth patterns on both plates. Look for colonies that appear on the 30°C plate but are absent on the 37°C plate.
These absent colonies are your temperature-sensitive mutants, as they cannot survive or grow at the higher temperature.
To ensure accuracy:

• Use a marker to circle suspected colonies on the 30°C plate.
• Re-streak these colonies on new plates to isolate and purify them further.
• Repeat incubation at 30°C to obtain pure mutant cultures.

This process ensures you have isolated true temperature-sensitive mutants, which can now be studied further for their unique properties.