Lysine decarboxylase test – Principle, Procedure, Result

What is Lysine decarboxylase test?

• Lysine decarboxylase test relies on the capability of certain bacteria to degrade L-lysine into caverine after the release of carbon dioxide.
• Decarboxylase mediums were first described in the work of Moeller (2-4) to detect lysine and ornithine decarboxylase and arginine dihydrolase. Falkow created a decarboxylase lysine medium to detect and distinction from Salmonella as well as Shigella.
• Taylor modified the medium by removing peptone from the formulation in order to prevent false positives due to Citrobacter freundii as well as its paracolons. This modified method has been suggested by the ISO committee.
• Lysine Decarboxylase Broth is generally used for studying the decarboxylase reactions that occur in the members of Enterobacteriaceae.
• This enzyme Lysine decarboxylase converts Lysine into caverine.
• Lysine decarboxylase (LDC) is a crucial enzyme in maintaining pH equilibrium and biosynthesis of cadaverine.
• The majority of bacteria use acid stress-induced lysine-decarboxylase to aid in the response to environment’s acidity. At extremely low pH LDC is stimulated and it increases acidity of medium for growth by eating one proton during the reaction of enzymatic.
• Additionally, LDC plays a crucial role in the creation of cadaverine, a vital industry-grade platform chemical. Cadaverine can be used for many different applications like the creation of polyamides and polyurethanes as well as additives and chelating agents. Lysine Decarboxylase Test is a rapid test to prove that lysine has been decarboxylated that can be used to confirm the identification of enterobacteria. The tubes contain desiccated fluid that is suitable for this purpose.
• The lysine decarboxylase is an enzyme that targets the carboxylic component in the amino acid, lysine and results in the formation of amine cadaverine.
• The sugar inside the medium gets fermented by enterobacteria and results in a color change of the indicator system, from purple to yellow.
• The acid medium facilitates the decarboxylation of amino acids reaction by forming the molecule cadaverine. The production of amines alkalizes the medium, causing the indicator to change color between yellow and purple. The negative reaction is evident through the yellow hue within the tube, while it is positive represented by a purple hue.

Principle of Lysine decarboxylase test

• The medium used is lysine descarboxylase broth. It is nutritional broth in which 0.5 percent lysine has been added.
• The most important ingredient in the media is a small amount of glucose, which is required to allow the process to continue.
• The pH indicator , bromocresol, is purple at neutral pH. It is alkaline or basic pH. However, it becomes yellow at pH <5.2.
• In the first stages of incubation, after the inoculation, the fermentation of dextrose by the microorganisms causes acid production. This causes an eventual change in colour of the indicator bromocresol from purple to yellow.
• The acidic reaction that results triggers decarboxylase activation, which results in the decarboxylation of Lysine to the cadavarine.
• The alkaline effects through cadaverine production trigger the indicator of bromocresol (changed into yellow) to return to the purple colour.
• If the organisms don’t produce decarboxylase enzymes The colour of the medium is remain yellow.
• Non-utilizers of Dextrose will not exhibit any changes in the medium color. Utilize light inoculas and do not take the results until after 24 hours incubation time, since some organisms require a longer time of incubation as long as 4 days.

Lysine decarboxylase test purpose

• The aim is to find out whether the microbe is able to utilize an amino acid called lysine to serve as an energy source and carbon energy to grow. Lysine utilization is achieved by the enzyme lysine-decarboxylase.
• To differentiate Salmonella Arizonae from the Bethesda Ballerup group of Enterobacteriaceae.

Requirement

• Media: lysine decarboxylase broth medium.
• Organism: Inoculum of selected organism.
• Other: Test Tubes, Autoclave

Composition of decarboxylase broth medium

Preparation of decarboxylase broth medium

1. Take a suspension of 14.02 grams in 1000 ml of distilled water.
2. If necessary, heat to melt your medium to the point of complete dissolution.
3. Inject 5 ml into test tubes with screw caps.
4. Sterilize using autoclaving at 15lbs tension (121degC) during 15 minutes.

Lysine decarboxylase test procedure

Inoculation of Medium 

1. Choose the lysine decarboxylase broth medium.
2. Start your Bunsen burner.
3. Choose the inoculating loop tool.
4. Inoculating loops can be flambé in order to make it sterilizable.
5. Caps off the tubes you are testing.
6. Flaming into the tubes’ mouths.
7. Utilize the inoculating instrument that is sterile to obtain an inoculum in the culture tube of the unknown bacteria.
8. The inoculum should be immediately transferred directly into the clean sterilized medium.
9. Flame The openings of your tube.
10. Replace the caps that are on test tubes.
11. Re-flame the inoculation tool. 

Incubation of the Inoculated Medium

1. Place the tube with the inoculated strain within the 35-37 C incubator.
2. Click on the New Day button to move ahead 24 hours.  

Determination of Test Results

1. Incubate for the proper amount of time. To conduct testing this method, the hue should be observed at 24 – and 48-hour intervals to determine the outcome. (i.e. it is necessary to repeat steps 12-16 two times to finish your test).
2. Remove desired incubated cultures from the incubator.
3. Check the results of the test. In the event that test results were conducted according to the above procedure it is likely that the culture changed color to yellow when exposed to acids , or remain purple when pH is neutral or when there are bases or alkali. To conduct this test, observing the color after 24 hours is essential for a an accurate interpretation of results however, an additional 24 hours of incubation is required to establish the result of the test that has been completed. Once the tube’s color is determined, place the tube inside the incubator for one additional period of time (perform steps 12-16 over again).
4. Record test results.
5. Get rid of the culture.

Lysine decarboxylase test results

• Positive lysine decarboxylase test: The alkaline conditions that are triggered by cadaverine production causes the indicator bromocresol to turn purple (changed into yellow) to return to its purple. This indicates that the organism can produce decarboxylase enzymes.
• Lysine decarboxylase Negative test:  If the organisms fail to produce decarboxylase enzymes, then the medium’s color remains yellow.

Lysine decarboxylase test positive organisms

Uses of Lysine decarboxylase test

• Lysine Decarboxylase Broth is used for differentiating the Salmonella Arizonae group from Bethesda Ballerup group of Enterobacteriaceae.

Limitations

• The test cannot determine the amount of intracellular enzymes and will only be able to detect it when it is enough to trigger a change in pH within the media.
• Changes in the growth conditions (i.e. the levels of glucose, lysine and amino acids that are not the amino acid lysine) can significantly affect decarboxylase activity of lysine in coliforms.
• The dextrose fermentation in the medium results in an acid-colored change. But, it will not cover the color change that is caused by a positive decarboxylation process.

References

• Phirke PM. Application of the rapid lysine decarboxylase test for early isolation and detection of salmonellae in sewage and other wastewaters. Appl Environ Microbiol. 1977 Oct;34(4):453-5. doi: 10.1128/aem.34.4.453-455.1977. PMID: 335973; PMCID: PMC242676.
• Bunsupa S, Katayama K, Ikeura E, Oikawa A, Toyooka K, Saito K, Yamazaki M. Lysine decarboxylase catalyzes the first step of quinolizidine alkaloid biosynthesis and coevolved with alkaloid production in leguminosae. Plant Cell. 2012 Mar;24(3):1202-16. doi: 10.1105/tpc.112.095885. Epub 2012 Mar 13. PMID: 22415272; PMCID: PMC3336119.
• KOTT, Y. Lysine Decarboxylase Activity as a Simple Test in Differentiation of Enterobacteriaceae. Nature 196, 90–91 (1962). https://doi.org/10.1038/196090b0
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