Eleanor Finch
The decarboxylase test is a biochemical test used to differentiate members of the Enterobacteriaceae family based on their ability to produce the enzyme decarboxylase. Amino acids are metabolized differently by gram-negative aerobic and facultatively anaerobic bacteria, as well as gram-positive cocci. These amino acids can be decarboxylated, hydrolysed, or deaminated, depending on the organism and the amino acid in question. Decarboxylase enzymes break the bond holding the carboxylic group to the rest of the amino acid. A positive test result for decarboxylase activity is indicated by a colour change in the broth, with purple indicating a positive result and yellow indicating a negative result.
| Characteristics | Values |
|---|---|
| Colour | Purple |
| pH | Alkaline |
| Turbidity | Turbid |
| Fermentation | No fermentation of glucose |
| Amino acid | Presence of amino acid |
| Broth | Arginine, Lysine, Ornithine |
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What You'll Learn
Purple colouration indicates a positive test
The decarboxylase test involves inoculating each of the three decarboxylase broths (arginine, lysine, and ornithine) and a control broth (without amino acids) with four drops of broth. A layer of sterile mineral oil is added to each tube, and the tubes are incubated at 35-37°C for four days. The tubes are examined at 24, 48, 72, and 96 hours for a colour change.
A positive test result is indicated by a purple colour, which means the media has been decarboxylated and formed amines (alkaline byproducts). This colour change occurs due to the decarboxylation of amino acids, resulting in an alkaline pH. The pH indicators bromocresol and cresol red change from orange to purple in the presence of alkaline byproducts.
Non-glucose-fermenting microorganisms may exhibit weak decarboxylase activity, leading to insufficient amine production and a deeper purple colour in the basal medium compared to an uninoculated tube. This colour change indicates that the organism is positive for the amino acid being tested.
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Differentiating Enterobacteriaceae
Enterobacteriaceae is a large family of Gram-negative rod-shaped bacteria, which includes over 30 genera and more than 100 species. It is commonly found in the intestine of animals, hence the name "enterobacteria" or "enteric bacteria". The classification of this family is still a subject of debate, but one classification places it in the order Enterobacterales of the class Gammaproteobacteria in the phylum Pseudomonadota.
The original classification of species within the family was largely based on 16S rRNA genome sequence analyses, which have low discriminatory power. However, in 2016, the order Enterobacteriales was renamed to Enterobacterales and divided into 7 new families, including the emended Enterobacteriaceae family. This classification was based on the construction of several robust phylogenetic trees using conserved genome sequences.
Many members of the Enterobacteriaceae family are normal members of the gut microbiota in humans and other animals, while others are found in water, soil, or plants, or are parasites. Some well-known human pathogens in this family include E. coli, Klebsiella spp., Salmonella spp., Shigella spp., Proteus spp., Morganella spp., Erwinia spp., Serratia marcescens, Citrobacter spp., and Yersinia spp.
Differentiating between species within the Enterobacteriaceae family can be achieved through various methods. One approach is biochemical testing, such as using systems like API 20E, which identify isolates based on their O (lipopolysaccharide), H (flagellar), and K (capsular) antigens. Another method is molecular diagnosis, which includes techniques such as PCR, DNA homology, and 16s rRNA sequencing. Additionally, siderophore-pattern analysis can differentiate between genera, species, and subspecies within the family. Each strain exhibits a specific pattern, allowing for easy separation between different groups. For example, Morganella, Proteus, Providencia, and Yersinia strains can be easily distinguished from Salmonella, Shigella, Escherichia coli, Enterobacter, Citrobacter, Klebsiella, and Serratia.
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The role of glucose fermentation
The decarboxylase test is a biochemical test used to differentiate members of the Enterobacteriaceae family based on their ability to produce the enzyme decarboxylase. The metabolism of amino acids can vary in aerobic and facultatively anaerobic bacteria, as well as in gram-negative organisms. This metabolism can occur through decarboxylation, hydrolysis, or deamination, depending on the amino acid being metabolised and the organism.
Decarboxylase tests involve the use of decarboxylase broths, which contain glucose as the fermentable carbohydrate. Glucose fermentation occurs within the first 10 to 12 hours of incubation. The acidic environment created by this fermentation is necessary for the production of decarboxylase.
Under aerobic conditions, glucose is oxidised, while under anaerobic conditions, it undergoes fermentation. In the context of the decarboxylase test, the presence of an oil overlay, such as mineral oil, creates an anaerobic environment that allows for the observation of glucose fermentation. The oil layer prevents the release of gases formed during fermentation, ensuring that the glucose is metabolised anaerobically. This is particularly important in differentiating glucose-fermenting organisms, as the fermentation of glucose can cause a distinct colour change in the broth.
In summary, the role of glucose fermentation in the decarboxylase test is to create an environment that facilitates the differentiation of organisms based on their ability to produce decarboxylase. The fermentation process, occurring within the first 10 to 12 hours, produces an acidic environment necessary for decarboxylase production. The colour changes observed during fermentation, in combination with the presence or absence of decarboxylase activity, contribute to the interpretation of positive or negative test results.
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Incubation and observation
During incubation, the tubes are maintained at a temperature of 35-37°C in ambient air for a period of four days. The optimal temperature range is crucial for promoting the growth and metabolic activity of the microorganisms being tested. The tubes are then observed for colour changes at specific time intervals: 24, 48, 72, and 96 hours. These time intervals allow for the detection of any early or delayed responses, ensuring accurate interpretation of the results.
A positive test result for decarboxylase activity is indicated by a distinct colour change in the broth. The media will turn purple, indicating that the amino acid has been decarboxylated and formed amines, which are alkaline byproducts. This colour change is due to the change in pH from the decarboxylation process. The purple colour can range from a turbid purple to a faded-out yellow-purple shade.
On the other hand, a negative test result is characterised by a bright, clear yellow colour or a lack of any noticeable colour change. The yellow colour indicates that glucose fermentation has occurred, but it is not indicative of decarboxylation. It is important to note that the control tube, which does not contain any amino acids, should retain its original colour or turn yellow. Any turbidity or alkaline colour change in the control tube invalidates the test.
In some cases, the broth may exhibit a grey colour, which could suggest a decrease in the indicator's intensity rather than alkaline production. Additionally, non-glucose-fermenting microorganisms may display weak decarboxylase activity, resulting in insufficient production of amines needed to convert the pH indicator. However, some non-fermenters may still produce enough amines to cause a deeper purple colour in the basal medium compared to an uninoculated tube.
It is important to exercise caution when interpreting the results. One should wait for at least 18 to 24 hours of incubation before making conclusions to avoid erroneous results. Glucose fermentation occurs rapidly within the first 10 to 12 hours, creating an acidic environment necessary for decarboxylase production. Therefore, early interpretation might lead to incorrect assumptions about the presence or absence of decarboxylase activity.
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Decarboxylase enzymes
There are three types of decarboxylase enzymes that are routinely tested for: arginine decarboxylase, ornithine decarboxylase, and lysine decarboxylase. These enzymes catalyse the breaking of the bond that binds the carboxylic group to the rest of the amino acid. Arginine decarboxylase is useful for identifying Enterococcus at the species level. For example, Enterococcus faecalis and Enterococcus faecium are arginine-positive, while Enterococcus avium is arginine-negative. Lysine decarboxylase, on the other hand, is used to differentiate between Salmonella (+) and Shigella (-).
The decarboxylase test involves inoculating each of the four decarboxylase broths with four drops of the prepared suspension. A layer of sterile mineral oil is added to each tube to prevent the escape of gases. The tubes are then incubated at 35-37°C for 4 days and observed for colour changes at 24, 48, 72, and 96 hours. A positive test result is indicated by a purple colour, which means the media has been decarboxylated and formed amines (alkaline byproducts). This colour change is not masked by the fermentation of dextrose, which causes an acid colour change. A negative test result is indicated by a bright clear yellow colour, which means the medium has fermented glucose but has not undergone decarboxylation.
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Frequently asked questions
A purple colour indicates a positive test. This indicates that the media has been decarboxylated and formed amines (alkaline byproducts).
A yellow colour indicates a negative test. This means that the broth has fermented glucose, but it is not indicative of decarboxylation.
The test is performed to differentiate members of Enterobacteriaceae based on their ability to produce the enzyme decarboxylase. The procedure involves inoculating each of the four decarboxylase broths with four drops of the prepared suspension. A 4mm layer of sterile mineral oil is then added to each tube before they are incubated for four days at 35-37°C in ambient air. The tubes are observed for colour change at 24, 48, 72, and 96 hours.
