Ames Test - Section The Ames test is commonly used as an efficient and inexpensi

Question

Ames Test - Section The Ames test is commonly used as an efficient and inexpensive test for the mutagenic property of substances. Bacteria that are deficient in histidine biosynthesis (His-) are spread on solid agar containing inorganic salts and sugars (minimal medium). Minimal medium does not contain the amino acid histidine or any other amino acid supplements. On minimal medium, the his- bacteria will not grow unless they undergo a reversion mutation. In the Ames test, His- bacteria are mixed with a substance and spread on minimal medium (plated). If the substance is mutagenic, some of the bacteria will undergo a reversion mutation and be able to grow in the absence of histidine. If the chemical is not mutagenic then the bacteria will not grow. When a bacterial strain deficient in histidine biosynthesis (His-), is plated for confluent growth (about 1 X1 0 6) on minimal medium (containing only inorganic salts and sugar), it does not grow (See below). However, the bacteria grow when plated coated with a mystery substance on minimal medium (substances #1 and #2.)

a) Why did we plate on rich medium and minimal medium with no additions?What are these called?

b) What are two possibilities for the identities of the mystery substances?
Indicate which type of mystery substance corresponds with the growth on each plate.

c) If the mystery substance #1 is a mutagen, how would that enable the bacteria to grow in the absence of histidine?

d) If you examine many control plates (lacking histidine and without the addition of a drug – minimal plates) you find the occasional colony with both strains A and B. Explain the presence of these colonies.

Explanation / Answer

Answer: (a) We plated on rich medium to see if the bacterial culture used for experiments is growing and in healthy condition. This is called positive control.

Similarly, we plated on minimal medium without any addition to see if the bacterial cultural has some mutants generated by random reversions or it has 100 % His- cells. This is called negative control.

(b): One of the mystery substance could be histidine or any compund containing histidine. If histidine is added, then all of the bacteria that has been plated should grow, which is shown by #2.

Second chemical compound could be a mutagen. If mutagen is added, then only a few bacteria will grow, in which a reversion mutation has occurred, shown by #1

(c): A mutagen can cause a new mutation in the DNA molecule of an already mutant allele of the gene that encodes the enzyme required for histidine biosynthesis. The mutation caused by mutagen could either restore the wild-type DNA sequence or it will make a compensating change (that includes addition of the basepairs to restore reading frame, or change a basepair to restore the amino acid sequence). Fixing gene automatically fixes the protein product and restore the final phenotype to wildtype.

(d): The occasional colony with both strain A and B could be because of spontaneous reversion mutations that occurs at a very low frequency.

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