Deoxycholate Citrate Agar (DCA)- Composition, Principle, Preparation, Results, Uses

Deoxycholate Citrate Agar (DCA) is a selective and differential medium for the isolation and differentiation of Gram-negative enteric bacilli from clinical and environmental samples. It was first developed by Leifson in 1935 as a modification of the original formulation by Hektoen and Edwards. DCA is especially useful for the detection of Salmonella and Shigella species, which are the major causes of bacterial gastroenteritis and dysentery. DCA inhibits the growth of most Gram-positive bacteria and some Gram-negative bacteria, such as Proteus, Pseudomonas and Providencia, by the presence of sodium deoxycholate and sodium citrate in the medium. DCA also allows the differentiation of lactose-fermenting and non-lactose-fermenting bacteria by the incorporation of lactose and neutral red in the medium. Lactose-fermenting bacteria produce acid from lactose, which lowers the pH of the medium and turns it red. Non-lactose-fermenting bacteria do not produce acid and remain colorless or light pink. In addition, DCA contains ferric ammonium citrate, which serves as an indicator for hydrogen sulfide (H2S) production by some bacteria. H2S reacts with ferric ions to form black iron sulfide precipitate, which can be seen as black centers or dots on the colonies. DCA is a simple and inexpensive medium that can be used for the preliminary screening of enteric pathogens in routine microbiology laboratories. However, it has some limitations that require further confirmation and identification of the isolated organisms by other methods.

Deoxycholate Citrate Agar (DCA) is a selective and differential medium for the isolation and differentiation of Gram-negative enteric bacilli. It contains the following ingredients:

• HI solids: This is a mixture of peptones and extracts derived from animal tissues. It provides carbon and nitrogen sources for the growth of bacteria. It also inhibits the growth of coliforms and Gram-positive bacteria by lowering the pH of the medium.
• Proteose peptone: This is a partially digested protein that supplies additional carbon, nitrogen, vitamins and minerals for bacterial metabolism.
• Lactose: This is a disaccharide sugar that serves as a fermentable carbohydrate for enteric bacilli. Lactose fermenters produce acid and gas, which change the color of the pH indicator neutral red to red. Lactose non-fermenters do not produce acid or gas, and remain colorless or light pink on the medium.
• Sodium deoxycholate: This is a bile salt that acts as a selective agent by inhibiting or suppressing the growth of coliforms and Gram-positive bacteria. It also precipitates in an acidic environment, forming a turbid zone around lactose fermenters.
• Sodium citrate: This is a salt that acts as a selective agent by inhibiting or suppressing the growth of coliforms and Gram-positive bacteria. It also chelates iron, preventing its reduction by hydrogen sulfide producers.
• Ferric ammonium citrate: This is a source of iron that can be reduced to iron sulfide by hydrogen sulfide producers, such as some Salmonella species. The formation of iron sulfide results in blackening of the colonies or the medium.
• Dipotassium phosphate: This is a buffer that maintains the pH of the medium at 7.5±0.2.
• Neutral red: This is a pH indicator that changes color from yellow to red in an acidic environment. It helps to differentiate lactose fermenters from non-fermenters based on their acid production.
• Agar: This is a solidifying agent that provides a firm surface for bacterial growth.

The final pH of DCA is 7.5±0.2 at 25°C. The medium should not be autoclaved or heated excessively, as this may affect its selectivity and quality. The medium should be cooled to 45-50°C before pouring into sterile Petri plates. The agar surface should be dried before use to prevent spreading of colonies.

Deoxycholate Citrate Agar (DCA) is a selective and differential medium that inhibits the growth of gram-positive bacteria and some gram-negative bacteria, while allowing the growth and differentiation of enteric bacilli based on their ability to ferment lactose and produce hydrogen sulfide.

The main ingredients of DCA are:

• HI solids: This is a source of carbon and nitrogen that helps in the growth of enteric bacilli. It also inhibits the growth of coliforms and other gram-positive bacteria by creating an unfavorable osmotic environment.
• Proteose peptone: This provides additional nutrients such as carbon, nitrogen, vitamins and minerals for the growth of enteric bacilli.
• Sodium deoxycholate: This is a bile salt that acts as a selective agent by disrupting the cell membrane of gram-positive bacteria and some gram-negative bacteria, causing them to lyse and die. It also precipitates in an acidic environment, forming a turbid zone around lactose-fermenting colonies.
• Sodium citrate: This is another selective agent that inhibits the growth of gram-positive bacteria and some gram-negative bacteria by chelating metal ions and interfering with their metabolic processes. It also enhances the activity of sodium deoxycholate by reducing its solubility in water.
• Ferric ammonium citrate: This is a source of iron that serves as an indicator for hydrogen sulfide production. Hydrogen sulfide is produced by some enteric bacilli such as Salmonella and Proteus from the reduction of sulfur-containing amino acids or thiosulfate. Hydrogen sulfide reacts with ferric ions to form black iron sulfide, which appears as a black center or dot in the colonies.
• Lactose: This is a carbohydrate that serves as a substrate for fermentation by enteric bacilli. Lactose-fermenting bacteria produce acid and gas from lactose, which lowers the pH of the medium and changes the color of the pH indicator neutral red from yellow to red. Lactose-fermenting colonies appear as red or pink on DCA. Lactose-nonfermenting bacteria do not produce acid or gas from lactose, and their colonies remain colorless or transparent on DCA.
• Dipotassium phosphate: This is a buffer that maintains the pH of the medium at 7.5, which is optimal for the growth and differentiation of enteric bacilli.
• Neutral red: This is a pH indicator that changes color from yellow to red in an acidic environment. It helps in distinguishing lactose-fermenting colonies from lactose-nonfermenting colonies on DCA.

The principle of DCA is based on the following reactions:

Lactose + Bacteria → Acid + Gas + Energy

Acid + Neutral Red → Red Color

H2S + Fe3+ → FeS + H+

FeS + Colony → Black Center

Deoxycholate + Acid → Precipitate + Turbidity

Deoxycholate Citrate Agar (DCA) is a ready-to-use medium that can be purchased from commercial suppliers or prepared in the laboratory. The preparation and method of use of DCA are as follows:

• To prepare DCA in the laboratory, suspend 70.52 grams of the dehydrated medium in 1000 ml of distilled water. Heat to boiling to dissolve the medium completely. Note: DO NOT AUTOCLAVE. Avoid excessive heating as it is detrimental to the medium. Cool to 45-50°C. Mix well and pour into sterile Petri plates.
• To use DCA, dry the agar surface before inoculation. Inoculate the medium heavily with feces or rectal swabs, spreading part of the original inoculum in order to obtain well-separated colonies on some portion of the plate. Incubate for 18-24 hours at 35°C.
• If organisms are late developers or if no non-lactose fermenters are observed, incubate for a further 24 hours.
• Examine the plates for colony morphology and color. Lactose non-fermenters produce transparent, colorless to light pink or tan-colored colonies with or without black centers. Lactose fermenters produce a red colony with or without a bile precipitate.

On DCA, different types of Gram-negative enteric bacilli can be distinguished by their ability to ferment lactose and produce hydrogen sulfide (H2S).

Lactose fermenters produce red colonies with or without a bile precipitate. The bile precipitate is a turbid zone of deoxycholate that forms around the colonies due to the acidification of the medium by lactose degradation. The pH indicator neutral red changes its color to red in acidic conditions. Examples of lactose fermenters are Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, etc.

Lactose non-fermenters produce transparent, colorless to light pink or tan-colored colonies with or without black centers. The black centers indicate the production of H2S from the reduction of ferric ammonium citrate. Examples of lactose non-fermenters are Salmonella spp., Shigella spp., Proteus spp., etc.

Salmonella and Shigella species do not ferment lactose but Salmonella may produce H2S, forming colorless colonies with or without black centers. Shigella species do not produce H2S and form colorless colonies without black centers. However, some strains of Salmonella may also be non-H2S producers and some strains of Shigella may be weak lactose fermenters, so further biochemical tests are required for confirmation.

The following table summarizes the typical results on DCA for some common enteric bacilli:

Deoxycholate Citrate Agar (DCA) is a selective and differential medium that is used for the isolation and differentiation of Gram-negative enteric bacilli in a laboratory setting. It is especially useful for the detection of organisms that cause bacillary dysentery, such as Shigella and some strains of Salmonella. It can also be used to isolate Salmonella strains that cause food poisoning and Salmonella Paratyphi, which causes paratyphoid fever.

DCA works by inhibiting the growth of most Gram-positive bacteria and some Gram-negative bacteria, such as coliforms, by the action of sodium deoxycholate and sodium citrate. It also allows the differentiation of lactose fermenters and non-fermenters by the presence of lactose and neutral red indicator in the medium. Lactose fermenters produce red colonies with or without a bile precipitate, while lactose non-fermenters produce transparent, colorless to light pink or tan-colored colonies with or without black centers. The black centers are due to the production of hydrogen sulfide from ferric ammonium citrate by some bacteria, such as Salmonella.

DCA is commonly used in conjunction with other media, such as MacConkey Agar, Bismuth Sulphite Agar, Xylose Lysine Deoxycholate (XLD) Agar, etc., for the routine examination of stool and urine specimens. DCA can provide a preliminary identification of enteric pathogens based on their colony morphology and biochemical reactions. However, further confirmation tests are required to verify the species and serotypes of the isolated organisms.

DCA is a valuable tool for the diagnosis and epidemiology of enteric infections caused by Gram-negative bacilli. It can help to identify the causative agents of dysentery, food poisoning, paratyphoid fever, and other gastrointestinal diseases. It can also help to monitor the prevalence and distribution of these pathogens in different regions and populations.

• DCA is a highly selective and inhibitory medium that may suppress the growth of some strains of Salmonella and Shigella. Therefore, suspected pathogens must be subcultured on a less inhibitory medium prior to identification.
• DCA is not specific for Salmonella and Shigella, as many other bacteria can also produce colorless or black colonies on this medium. For example, Proteus, Citrobacter, Edwardsiella and some coliforms can also produce H2S and mimic Salmonella. It is widely recommended that more selective agars are used for the identification of Salmonella, such as xylose lysine deoxycholate (XLD) agar or Hektoen enteric agar.
• DCA is a heat-sensitive medium that should be prepared and poured as soon as possible after the addition of the deoxycholate, otherwise it tends to become very soft and difficult to handle. Excessive heating or prolonged storage may also affect the performance of the medium.
• DCA is not suitable for the routine examination of stool and urine specimens, as it may miss some enteric pathogens that do not grow well on this medium. It is suggested that other media such as MacConkey agar, Eosin methylene blue agar or Bismuth sulfite agar be used in conjunction with this medium for better isolation and differentiation of Gram-negative enteric bacilli.
• DCA does not provide sufficient information for the confirmation of species. Further biochemical and serological tests are required to identify the isolates to the species level. For example, indole test, urease test, motility test, triple sugar iron agar test, lysine decarboxylase test and slide agglutination test with specific antisera are some of the commonly used methods for confirming Salmonella and Shigella species.

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