- Explain what the citrate test indicates about a bacteria species' genes, enzymes, and metabolism.
- Tell what citrate permease is and what it does for bacterial species that have it.
- Tell that the citrate test is useful for bacterial identification and characterization.
- Successfully conduct a citrate test and interpret the results.
All living things need carbon to survive. The carbon-containing molecules that bacteria can utilize as a carbon source differs based on the bacterial species and is dependent on their genes. Their genes dictate what enzymes the bacterial species can produce. Since enzymes are necessary for a cell's metabolic reactions, the genes of a bacterial species dictate their abilities to use different carbon sources.
Some bacterial species, but not all bacterial species, can utilize citrate as a source of carbon. Organisms that can survive using citrate as the sole source of carbon have a citrate permease enzyme that can transport citrate molecules into the cell. The citrate is then made into pyruvate, which can be converted into a variety of different products in the cell.
Figure 1: Bacteria with the citrate permease gene can produce the protein citrate permease by gene expression (left). Citrate permease is a membrane protein that enables the transport of citrate into the cell. (Right) When a bacterial cell produces citrate permease, citrate molecules are transported into the bacterial cell. This enables the bacteria to utilize citrate as a source of carbon.
Figure 2: Bacteria without the citrate permease gene cannot produce the protein citrate permease (left). Without the citrate permease protein, citrate is unable to enter the bacterial cell (right). As a result, the bacteria cannot use citrate as a source of carbon.
Simmons' citrate is a chemically defined microbiological medium that contains sodium citrate as the sole carbon source. A pH indicator, bromothymol blue, is also included in Simmons' citrate medium. Bacteria that can grow on this medium (i.e., that can survive on citrate as the sole source of carbon) produce alkaline byproducts that will change the medium color from green (neutral pH) to blue (alkaline pH).
Figure 3: Results of the citrate test. When the medium stays green (no color change), the test indicates the bacterial species is citrate negative (left). When the medium changes color to blue, the test indicates the bacterial species is citrate positive (right).
Bacterial species that are capable of metabolizing citrate are considered citrate positive and will cause Simmons' citrate agar to change from green to blue. Bacterial species that are incapable of metabolizing citrate are considered citrate negative and will result in Simmons' citrate agar remaining green (no color change). Since only some bacterial species can metabolize citrate, the citrate test is useful for bacterial identification and characterization.
- Obtain two test tubes with Simmons' citrate agar slants.
- Use tape to label the test tubes with your group name, bacteria name, and the type of test medium.
- Using a sterile inoculating loop, aseptically obtain a small amount of bacteria and streak the entire slant of the citrate agar. Repeat for both species.
- Incubate the inoculated tube at 37 °C until next lab session.
- Observe the color of the Simmons’s citrate slant.
- bright blue is citrate positive
- no color change (agar is still green is citrate negative
Results & Questions
|bacterial species has citrate permease (+/-)||bacterial species has citrate permease gene (+/-)|
- Complete the table above with the results observed.
- How does the citrate test relate to understanding the metabolism of a bacterial species?
- What does the color change from green to blue mean about the pH of the culture?
- What does the color change from green to blue mean about the bacterial species in the culture?
- Can the citrate test be used for bacterial identification and characterization? Explain your answer.
- Chapter Image: Citrate Simmons DSCN0604.jpg by Jnjoffin is licensed under CC BY-SA 4.0
- Laboratory Exercises in Microbiology: Discovering the Unseen World Through Hands-On Investigation by Susan McLaughlin and Joan Petersen is licensed under CC BY-NC
- PDB 1pv7 EBI.jpg by Jawahar Swaminathan and MSD staff at the European Bioinformatics Institute is in the public domain
- Red Mountain Microbiology by Jill Raymond Ph.D.; Graham Boorse, Ph.D.; Anne Mason M.S. is licensed under CC BY-NC 4.0
- Sodium citrate.png by Velandur, updated by User:Rifleman_82 is in the public domain