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

A five-year-old child presents with four days of bloody diarrhoea, fever, and abdominal cramps. The paediatrician 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 — coliform and Gram-positive suppressor Citrate at the concentration used in DCA inhibits most normal Gram-negative intestinal flora, particularly Escherichia coli and other coliforms. The combination of deoxycholate and citrate creates an environment that is sufficiently hostile for normal flora while being tolerable for enteric pathogens such as Salmonella and Shigella, which have inherent resistance to these compounds.

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-centred 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 colourless 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 diarrhoeal illness, particularly when bacillary dysentery (bloody diarrhoea) 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.

Ingredients	Gm/liter
Peptone	5.0
Lab-Lemco powder	5.0
Lactose	10.0
Sodium citrate	8.5
Sodium thiosulphate	5.4
Ferric ammonium citrate	1.0
Sodium deoxycholate	5.0
Neutral red	0.02
Agar	12.0
pH 7.3 ± 0.2 @ 25°C

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.

Dipotassium phosphate buffers the medium.

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.

The reduction of ferric ammonium citrate to iron sulfide is indicated by the formation of black iron sulfide. If the bacteria produce H2S, the colonies will have 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)

Species	Colony color	H2S (black center)	Key distinguishing feature
Salmonella typhi	Colourless	Black center (variable; may be small dot)	Lactose non-fermenter; H2S positive; confirm with agglutination
Salmonella spp. (non-typhi)	Colourless	Black center (usually prominent)	Lactose non-fermenter; H2S positive; larger black centres than S. typhi
Shigella sonnei	Initially colourless; pale pink after 48 hrs	No black center	Late lactose fermenter — colonies may appear colourless at 24 hrs; pink at 48 hrs; can be confused with Salmonella at early read
Shigella flexneri	Colourless	No black center	Non-fermenter; no H2S; confirmed by serology
Shigella dysenteriae	May be inhibited or show poor growth	No black center	Most inhibited Shigella species on DCA — use MacConkey or blood agar alongside
Escherichia coli	Pink, umbilicated (button-shaped), with deoxycholate precipitate halo	No	Most strains inhibited; those that grow have characteristic pink umbilicated appearance
Enterobacter / Klebsiella spp.	Large, pale mucoid, pink center	No	Mucoid appearance due to capsule; suppressed but not always fully inhibited
Proteus / Providencia spp.	Colourless to tan, large	Variable; large central black dot	Characteristic "fishy" odour; large glossy colonies more translucent than Salmonella; no swarming (unlike on blood agar)
Yersinia enterocolitica	Colourless	No	Small, colourless; confirmed by incubation at 25°C for 48 hrs (enhances growth)
Enterococci	No growth to slight growth	—	Inhibited by deoxycholate

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 colourless (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 colourless 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.

Feature	DCA	SS Agar	XLD Agar
Selectivity level	Moderate	High	High
Shigella dysenteriae recovery	Poor — may be inhibited	Poor — often inhibited	Good
Shigella sonnei/flexneri recovery	Good	Good	Good
Salmonella recovery	Good	Good	Good
H2S indicator	Yes (ferric ammonium citrate)	Yes (ferric citrate + thiosulfate)	Yes (ferric ammonium citrate)
Lactose differentiation	Yes (neutral red — pink vs colourless)	Yes (neutral red)	Yes (phenol red — yellow vs red)
Salmonella colony appearance	Colourless with black center	Colourless with black center	Pink/red with black center
Shigella colony appearance	Colourless	Colourless	Red/pink (distinctive)
E. coli colony appearance	Inhibited or pink umbilicated	Inhibited	Yellow
Do not autoclave	Yes	Yes	Yes
Best for	Standard stool culture; typhoid workup	High-selectivity; reducing coliform breakthrough	Best Shigella recovery; differentiates Salmonella from E. coli by color

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 — two questions:

Question	Answer	Organism to suspect
Is the colony colourless?	Yes	Salmonella or Shigella (NLF) — proceed to confirmatory testing
Is there a black center?	Yes	H2S producer — most likely Salmonella (not Shigella)
Is the colony pink?	Yes	Lactose fermenter — E. coli or Enterobacter/Klebsiella
Is the colony colourless at 24 hrs but pink at 48 hrs?	Yes	Shigella sonnei — late lactose fermenter

The H2S system as a clinical teaching point: Salmonella typhi causes typhoid fever — a systemic illness, not a typical watery diarrhoea. It produces H2S, which means black-centred 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-centred DCA colony.

Memory anchor for the enteric media trio:

• DCA: moderate selectivity, good for most enteric pathogens in standard stool culture
• SS agar: high selectivity, maximum suppression of normal flora, risk of inhibiting Shigella dysenteriae
• XLD agar: best Shigella recovery, color-coded for Salmonella (pink with black center) vs Shigella (red/pink without black center)

References and Further Reading

• Tille, P. M. (2017). Bailey and Scott's Diagnostic Microbiology (14th ed.). Elsevier.
• Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries, Part 2 (2nd ed.). Cambridge University Press.
• Leifson, E. (1935). 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, 40(3), 581–599.
• World Health Organization. (2010). Guidelines for the collection of clinical specimens during field investigation of outbreaks. Geneva: WHO.