Citrate is a tricarboxylic acid trianion that is produced by the deprotonation of the three carboxyl groups of citric acid. The main reason behind performing the citrate test is to ascertain the capacity of an organism to make use of sodium citrate. Often, the citrate test is part of a battery of tests useful for identifying gram-negative pathogens and environmental isolates.

Negative Citrate Test Meaning

After performing the citrate test, the outcome of the test can be positive or negative. An organism is considered citrate negative when no trace is visible. A bacteria is citrate negative if there is no change in colour and as a result, the medium will continue to exist deep forest green colour of the uninoculated agar.

Procedure to perform a Citrate Test

1. To perform a citrate test, a fresh pure culture needs to be taken as an inoculation source. 
2. For this, collect a single isolated colony, and lightly streak the surface of the slant. To limit the amount of cell material transferred to the agar slant, a needle is mostly preferred. (Note: avoid using liquid cultures as the inoculum source.)
3. Oxygen is used for citrate utilization, thus, if screw caps are used, they should be placed loosely on the tube. 
4. Incubate at 35oC (+/- 2oC) for 18 to 48 hours and observe a colour change from green to blue along the slant. 
5. Few organisms may require up to seven days of incubation because of their limited rate of growth in citrate medium.

List of bacteria giving negative citrate test

The following bacteria give negative citrate tests:

• Escherichia coli: Escherichia Coli, also identified as E. Coli, is a large and diverse group of bacteria, usually found in the environment, foods, and intestines of people and animals. Although most strains of E. coli are not likely to cause harm, some strains can cause diseases like diarrhoea, urinary tract infections, respiratory illness and pneumonia. 
• Yersinia enterocolitica: This bacteria is an enteropathogenic species that is associated with the genus Yersinia. It is broadly distributed in nature and affects both humans and animals. This type of infection has symptoms such as acute diarrhoea, mesenteric adenitis, pseudo appendicitis and even sepsis. Often, children are more susceptible to the infection than adults, and it is more common in the winter. 
• Salmonella Typhi: Salmonella Typhi is a gram-negative bacterium that is responsible for typhoid fever. This bacterium infects the intestinal tract and the blood. This bacteria can reach humans through various sources like uncooked or raw food, and the consumption of non-vegetarian foods.
• Edwardsiella Tarda: This bacterium is a motile, facultatively anaerobic, gram-negative rod that is categorised as a member of the family Enterobacteriaceae. It causes the Haemorrhagic septicaemia disease in fish and gastro and extraintestinal infections in humans. The genus Edwardsiella is mainly composed of three species: E. Tarda, E. ictaluri and E. Hoshinae. The E. Hoshinae usually infects birds and reptiles, whereas E. Ictaluri or E. Tarda are reported in fish.
• Vibrio Hollisae: It is a small, non-cholera gram-negative bacterium. This bacterium is a halophilic species that was reclassified as Grimontia Hollisae. This type of organism is known to cause moderate to severe cases of gastroenteritis.

Limitations of Citrate Tests

Here are a few limitations of the citrate test:

1. In a few cases where the growth of organisms on the slant of the tube may not be in excess, it may not show colour change. On further incubation for a longer time, the colour change may be observed. If not, the test should be repeated.
2. Inoculation from broth culture should be avoided to eliminate the mixing of different media.
3. Citrate tests may not always give accurate results. They may give false positive or false negative results, hence other confirmatory tests are advisable.

Conclusion

While performing the citrate test, it is crucial to not add any other nutrients to the medium during inoculation. This is because any extra nutrients can lead to incorrect outcomes. Another limitation of citrate tests is that certain citrate-positive bacteria may take several days to turn the medium blue. Thus, complementary tests must be performed to identify the species of the strain isolated, and pure, fresh cultures must always be used to obtain interpretable results.