Question

A strain of Salmonella typhimurium has a base pair substitution which makes it His \(^{-}\). This strain's rate of spontaneous reversion to His \({ }^{+}\) is \(5 \times 10^{-8}\). Chemical \(\mathrm{X}\) is added to the growth medium of a culture of these cells. Solutions are diluted and plated onto minimal medium (a \(10^{-2}\) dilution) and onto nutrient rich medium (a \(10^{-3}\) dilution). The plates are incubated for 48 hours. The colonies are counted: 50 colonies on the minimal medium and 1,000 colonies on the nutrient rich medium. What can you conclude from this experiment?

Short answer

The effect of chemical X on the mutation can be determined by comparing the reversion rates. The spontaneous reversion rate is given as \(5 \times 10^{-8}\), while the calculated reversion rate from the experiment is \(5 \times 10^{-3}\). This indicates that chemical X significantly enhances the reversion rate in the culture, suggesting its potential role in causing the His- to His+ reversion.

Step-by-step solution

Understanding the Colony Counts

First, it's crucial to understand that the minimal medium plate only allows growth of His+ bacteria (i.e., revertants), while the nutrient rich medium plate allows all bacteria (both His- and His+) to grow. Also, since these are dilutions, the actual colony count will need to be adjusted by the dilution factor in later steps.

Calculating the Total Number of Bacteria

The next step is to calculate the total number of bacteria in the culture; this can be obtained from the nutrient-rich medium where both His- and His+ can grow. The original sample had \(1000 \times 10^{3}=10^{6}\) colonies.

Calculating the Number of Revertants

The revertant colonies are those that managed to grow on the minimal medium. The undiluted culture thus contained \(50 \times 10^{2} = 5 \times 10^{3}\) revertants.

Determining the Reversion Rate

The reversion rate can be determined by dividing the total number of revertants by the total number of bacteria. This would equate to: \(\frac{5 \times 10^{3}}{10^{6}}= 5 \times 10^{-3}\).

Interpreting the Results

Now it's time to compare the spontaneous reversion rate (given as \(5 \times 10^{-8}\)) with our calculated reversion rate. The calculated reversion rate from the experiment is \(5 \times 10^{-3}\). This indicates that chemical X drastically enhances the reversion rate in this culture by several orders of magnitude, suggesting its potential role in causing the His- to His+ reversion.

Key concepts

Understanding Genetic Mutation

A genetic mutation is a change in the nucleotide sequence of the genetic material of an organism. Mutations can range from a single base pair change, known as a point mutation, to large-scale changes involving chunks of the chromosome. These alterations can lead to changes in the proteins produced by genes, potentially affecting an organism's phenotype.

When discussing Salmonella typhimurium, which is the bacterium in our exercise, mutations in certain genes can result in the inability to synthesize histidine, indicated as His^-. Of particular importance are spontaneous mutations, which occur without external influence and can affect the fitness of bacteria. In the context of the exercise, the focus is on the spontaneous reversion rate, which measures how often a mutated gene spontaneously changes back to its original, functional form.

The Role of Salmonella typhimurium

Salmonella typhimurium is a species of bacteria that can cause infections in humans and animals. In genetics research, it is frequently used in mutation studies due to its ability to grow on simple growth mediums and its relatively well-understood genetics.

In laboratory experiments, the growth characteristics of Salmonella typhimurium can be explored by introducing specific mutations. The His^- strain utilized in the textbook exercise contains a mutation that results in it being unable to grow without the amino acid histidine. This characteristic makes it an excellent candidate for experiments that aim to explore genetic mutations and the effects of various substances, like chemical X, on mutation rates.

His+ Revertants Explained

His⁺ revertants are individual Salmonella typhimurium cells that have undergone a spontaneous genetic change that rectifies a previously mutated histidine synthesis gene. Consequently, these revertants regain the ability to grow in the absence of external histidine inputs, as they can once again synthesize this essential amino acid internally.

This reversion is crucial for the exercise at hand as the number of His⁺ revertants directly correlates to the mutation reversion rate. By cultivating these bacteria on a minimal medium, we can select for His⁺ revertants and then count these colonies to quantify the rate at which the spontaneous mutation reverses.

Colony Counting Techniques

Colony counting is a fundamental microbiology technique used to estimate the number of viable bacteria in a sample. In this exercise, counting colonies allows for the quantification of revertant bacteria. Here's the process:

• After incubation, visible colonies are counted.
• These counts are factored by the dilution used to ensure accurate estimates of the original sample's bacterial population.

It is through this colony counting that researchers can deduce not only the total number of bacteria in a culture but also calculate the proportion of His⁺ revertants.

Minimal Medium: A Selective Growth Environment

A minimal medium is a type of growth environment for bacteria that only contains the essential nutrients required for the survival of those which are able to synthesize all necessary cellular components from these basics. It typically consists of a carbon source, like glucose, and salts. Importantly, it lacks amino acids like histidine, necessitating that bacteria must be able to produce these compounds to grow on such a medium.

In the context of our exercise, the minimal medium selects for His⁺ revertants because only those revertants can synthesize the amino acid histidine required for their growth.

Nutrient Rich Medium: An Unrestricted Environment

In contrast to minimal medium, a nutrient rich medium, or complete medium, contains a variety of nutrients, including amino acids, nucleotide precursors, and other growth factors. This type of medium supports the growth of a broader range of bacterial types, not just those with specific genetic capabilities.

When Salmonella typhimurium is grown on this medium, both His^- and His⁺ strains can grow because the medium supplies the necessary histidine. This allows for the determination of the total population of bacteria in the culture, a key step in our exercise for calculating the reversion rate.