Ames Test Activity
Rodríguez E, Piccini C, Sosa V, Zunino P. The use of the ames test as a tool for addressing problem-based learning in the microbiology lab. J Microbiol Biol Educ. 2012 Dec 3;13(2):175-7. doi: 10.1128/jmbe.v13i2.421. PMID: 23653807; PMCID: PMC3577329.
- One week before the activity, minimal medium plates were prepared. This medium consists of the following solutions: an agar solution (15 g of agar in 600 ml of water), a saline solution (0.2 g of MgSO4, 1 g of sodium citrate, 2 g of (NH4)2 SO4, 6 g of KH2PO4 and 14 g of K2HPO4 in 200 ml of water) and a glucose solution (5 g of glucose in 200 ml of water). All three solutions were autoclaved separately. Once cooled, they were combined and plated.
- The day prior to the activity 150 μl of a biotin solution was spread on the plates.
- A fresh culture of S. typhimurium strain TA100, grown on Mueller-Hinton agar, was used.
- A suspension of this strain (equivalent to 108 cfu/ml) was made in sterile saline solution.
- A sterile swab was dipped in this bacterial suspension, taking care to press the swab against the inner wall of the tube to eliminate excess liquid upon removal.
- This swab was used to spread the bacterial suspension on one of the previously prepared agar plates, covering its entire surface with inoculum.
- The plate was allowed to dry before further manipulation (approx. 10 mins).
- Fourteen plates were inoculated in this way; twelve were used for assaying the substances brought by the students (the assay was done in duplicate for each of the six substances), one was used for the positive control and one for the negative control.
- To carry out the test, forceps were dipped in ethanol, and then flamed. The sterilized forceps were used to take a disk of sterile filter paper (12 mm diameter) and place it in the center of each of the inoculated agar plates.
- A drop (70 μl) of each substance to be analyzed was deposited on the corresponding disk thus placed on each of the experimental plates.
- For the negative control, 70 μl of sterile water was placed on the corresponding disk of filter paper.
- For the positive control, 70 μl of a solution of sodium azide was placed on the corresponding disk of filter paper.
- Plates were incubated at 37°C for 48 to 72 hours.
- Results were analyzed after this incubation period.