Deoxycholate Citrate Agar (DCA): Composition, Principle, Uses, and Colony Characteristics

A five-year-old child presents with four days of bloody diarrhea, fever, and abdominal cramps. The pediatrician suspects bacillary dysentery, either Salmonella or Shigella. A stool specimen is sent to the laboratory. The technician has a specific challenge: the specimen contains millions of normal intestinal flora bacteria: E. coli, enterococci, streptococci, all of which will outgrow any enteric pathogen on a non-selective medium.

The solution is Deoxycholate Citrate Agar. A medium engineered to inhibit all that normal flora while allowing Salmonella and Shigella to grow as distinct, identifiable colonies.

Principle

DCA achieves selectivity and differentiation through three simultaneously acting mechanisms:

1. Deoxycholate (bile salt): primary Gram-positive inhibitor. Sodium deoxycholate at the concentration in DCA (5 g/L) is directly inhibitory to Gram-positive bacteria, disrupting their cell membranes. Most Gram-positive organisms encountered in stool specimens (enterococci, staphylococci, streptococci) are completely suppressed.
2. Sodium citrate, working with deoxycholate to suppress normal flora Citrate at the concentration used in DCA inhibits Gram-positive organisms and helps suppress normal Gram-negative intestinal flora, including coliforms. Citrate and deoxycholate act together rather than separately: their combination creates an environment hostile to normal flora but tolerable for enteric pathogens such as Salmonella and Shigella, which have inherent resistance to bile salts. This is what makes DCA more inhibitory than plain deoxycholate agar.
3. Ferric ammonium citrate + sodium thiosulfate, H₂S indicator system. Bacteria that produce hydrogen sulfide (H₂S) during metabolism (including most Salmonella species) react with ferric ammonium citrate to form black iron sulfide precipitate within and beneath the colony. This produces the characteristic black-centered colonies of Salmonella on DCA, a direct visual indicator of H₂S production that distinguishes most Salmonella from Shigella (which does not produce H₂S).

Differential mechanism: lactose fermentation: Neutral red is the pH indicator. Lactose fermenters produce acid, lowering the pH and turning colonies pink to red. Lactose non-fermenters (Salmonella, Shigella, Yersinia) produce colorless colonies, standing out visually against the pink background created by any residual coliform growth.

Important selectivity limitation: DCA is more inhibitory than MacConkey agar but less inhibitory than SS agar. Shigella sonnei and S. flexneri grow on DCA, but Shigella dysenteriae (the most virulent species, causing epidemic dysentery) may be partially or completely inhibited on DCA. Always use DCA in conjunction with a less inhibitory medium (MacConkey or blood agar) and a more inhibitory medium (SS agar or XLD) to ensure recovery of the full range of enteric pathogens.

Uses of Deoxycholate Citrate Agar

1. Primary isolation of Salmonella and Shigella from stool specimens DCA is a first-line plating medium for acute diarrheal illness, particularly when bacillary dysentery (bloody diarrhea) is suspected. Stool specimens from suspected typhoid fever, non-typhoidal Salmonellosis, and Shigellosis are plated directly onto DCA alongside MacConkey or XLD agar.

2. Subculture from enrichment broth After overnight incubation in selenite F broth or tetrathionate broth (enrichment step for low-count specimens, carriers, or food samples), subculture onto DCA provides selective solid medium for colony isolation.

3. Outbreak investigation In suspected food poisoning or gastroenteritis outbreaks, DCA is used for stool, food, and environmental water samples where Salmonella is the most likely cause.

4. Food microbiology DCA is used for the detection of Salmonella in food products, particularly in poultry, eggs, and dairy products, as part of regulatory food safety testing.

Composition of Deoxycholate Citrate Agar (DCA)

Peptone provides carbon, nitrogen, vitamins, and minerals.

Lab-Lemco is a meat extract made from specially selected raw materials, adjusted to neutrality, and dried to a fine powder. It will enhance the growth of many bacteria and it is incorporated into a wide range of culture media as a solid foundation material as it enhances the growth of many bacteria.

Lactose helps in differentiating enteric bacilli (lactose fermenters produce red/pink colonies while lactose non-fermenters produce colorless colonies). The fermentation of lactose causes acidification and the pH indicator neutral red changes color to red. Lactose-fermenting colonies may have a turbid zone of precipitation around them caused by the precipitation of deoxycholate in the acidic environment.

Organisms that produce H₂S reduce the sodium thiosulfate in the medium to hydrogen sulfide, which then reacts with ferric ammonium citrate to form black iron sulfide. If the bacteria produce H₂S, the colonies develop black centers.

Coliform bacteria and gram-positive bacteria are inhibited or greatly suppressed due to sodium deoxycholate, sodium citrate, and ferric ammonium citrate.

Preparation of Deoxycholate Citrate Agar (DCA)

Prepare the medium as instructed by the manufacturer.

1. Suspend 52g of dehydrated medium (supplied by the manufacturer) in 1 liter of distilled water.
2. Heat with great care to dissolve the medium completely. Do not boil or autoclave the medium. Avoid excessive or prolonged heating. DO NOT AUTOCLAVE. (If autoclaved the agar becomes soft and almost impossible to streak)
3. As soon as the medium has cooled to 50°C -55°C mix well and dispense aseptically in sterile Petri dishes
4. Label the plates “DCA”. Date the medium and give it a batch number. Store the plates at 2°C – 8°C, preferably in sealed plastic bags to prevent loss of moisture.

Storage conditions and Shelf life

• Store the dehydrated medium at 10-30°C and use it before the expiry date on the label.
• Store the prepared agar plates at 2-8°C. Shelf life is up to 6 weeks providing there is no change in the appearance of the medium to suggest contamination or an alteration of pH.

Appearance

• Dehydrated medium: Straw/pink colored, free-flowing powder
• Prepared medium: Pink colored gel

Inoculation of the prepared medium

1. Dry the agar surface before use.
2. 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.
3. Incubate for 18-24 hours at 35°C.
4. If organisms are late developers or if no non-lactose fermenters are observed, incubate for a further 24 hours.

Colony Characteristics on Deoxycholate Citrate Agar (DCA)

Reading DCA plates- practical tips:

1. Always examine plates at both 24 and 48 hours. Shigella sonnei is a late lactose fermenter and can appear falsely colorless (mimicking Salmonella) at 24 hours, becoming pink at 48 hours.
2. Salmonella Typhi often produces a smaller black center than non-typhi Salmonella; do not dismiss a faint black dot as negative.
3. Pick colorless colonies for further testing regardless of whether a black center is present; Shigella does not produce H2S but is still a priority pathogen.

Choosing Your Enteric Pathogen Medium: DCA vs SS Agar vs XLD Agar

Students frequently confuse these three media, which are all used for enteric pathogen isolation. The table below shows the key differences.

Practical recommendation for resource-limited settings: If only one enteric selective agar can be used, XLD agar provides the best balance of selectivity and Shigella recovery. If two media are available, DCA + XLD is the standard combination. SS agar is most useful when high contamination levels demand maximum selectivity, but its inhibitory effect on Shigella dysenteriae means it should not be used as the sole medium.

How to Remember

DCA, the name explains the two key selective agents:

• Deoxycholate: kills Gram-positive organisms
• Citrate: suppresses coliforms
• Agar: the base

Everything else in the medium either supports growth (peptone, Lab-Lemco) or signals differentiation (neutral red for lactose, ferric ammonium citrate + thiosulfate for H₂S).

The colony reading rule:

The H2S system as a clinical teaching point: Salmonella Typhi causes typhoid fever, a systemic illness, not a typical watery diarrhea. It produces H₂S, which means black-centered colonies on DCA. But the black center can be small and easily missed. Always confirm with TSI agar: S. typhi produces an alkaline slant, acid butt, and H₂S on TSI, a pattern as characteristic as the black-centered DCA colony.

Memory anchor for the enteric media trio: DCA is the moderate middle option, SS agar is the most inhibitory (and can miss Shigella dysenteriae), and XLD gives the best Shigella recovery with color-coded Salmonella-versus-Shigella reading. The full comparison is in the table above.

Where Students Get Confused

1. Shigella sonnei can masquerade as Salmonella at 24 hours. S. sonnei is a late lactose fermenter, so at 24 hours it looks colorless like a non-fermenter, the same as Salmonella or other Shigella. By 48 hours it turns pink. Always read plates at both 24 and 48 hours, and do not commit to an identification on the first read alone.

2. A missing black center does not rule out Salmonella. Most Salmonella produce a black center from H₂S, but Salmonella Typhi often makes only a small, faint black dot, and S. Paratyphi A makes none. Pick colorless colonies for confirmatory testing whether or not a black center is present. The black center confirms Salmonella when present; its absence does not exclude it.

3. Black center means H₂S, not necessarily Salmonella. Proteus also produces H₂S and can form colorless colonies with a large black center on DCA, mimicking Salmonella. Do not report Salmonella on colony appearance alone. Proteus is strongly urease positive and Salmonella is urease negative, so a urease test and TSI separate them before serology.

4. DCA is the middle of three media, not a standalone. DCA is more inhibitory than MacConkey but less than SS agar. Shigella dysenteriae, the most virulent Shigella, may be partly or fully inhibited on DCA. Use DCA alongside a less inhibitory medium and, ideally, XLD, so that a suppressed S. dysenteriae is not missed in exactly the dysentery cases where it matters most.

5. Do not autoclave DCA. The citrate and iron hydrolyze the agar when overheated, leaving it soft and impossible to streak. DCA is dissolved by gentle heating and poured; it is never autoclaved. A soft, unstreakable plate usually means the medium was overheated.

References and Further Reading

• Tille PM. Bailey and Scott's Diagnostic Microbiology. 15th ed. St. Louis: Elsevier; 2022.

  Cheesbrough M. District Laboratory Practice in Tropical Countries, Part 2. 2nd ed. Cambridge: Cambridge University Press; 2006.

  Leifson E. New culture media based on sodium desoxycholate for the isolation of intestinal pathogens and for the enumeration of colon bacilli in milk and water. Journal of Pathology and Bacteriology. 1935;40(3):581-599.

  World Health Organization. Guidelines for the Collection of Clinical Specimens During Field Investigation of Outbreaks. Geneva: WHO; 2010.