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

Deoxycholate Citrate Agar (DCA) is a  recommended medium for the isolation of enteric pathogens particularly Salmonella and Shigella species from clinical  as well food samples. DCA is a modification of Desoxycholate Agar formulated by Leifson. His original medium demonstrated improved recovery of intestinal pathogens from specimens containing normal intestinal flora by using citrates and sodium desoxycholate in specified amounts as inhibitors to gram-positive bacteria. Leifson modified his original medium by increasing the concentration of sodium citrate and sodium desoxycholate and found Desoxycholate Citrate Agar reliable for isolating many Salmonella and Shigella species. DCA  effectively isolates intestinal pathogens (Salmonella and Shigella species) by inhibiting coliforms and many Proteus species.

Introduction of Deoxycholate Citrate Agar (DCA)

Deoxycholate Citrate Agar (DCA) is a  recommended medium for the isolation of enteric pathogens particularly Salmonella and Shigella species from clinical  as well food samples. DCA is a modification of Desoxycholate Agar formulated by Leifson. His original medium demonstrated improved recovery of intestinal pathogens from specimens containing normal intestinal flora by using citrates and sodium desoxycholate in specified amounts as inhibitors to gram-positive bacteria. Leifson modified his original medium by increasing the concentration of sodium citrate and sodium desoxycholate and found Desoxycholate Citrate Agar reliable for isolating many Salmonella and Shigella species. DCA  effectively isolates intestinal pathogens (Salmonella and Shigella species) by inhibiting coliforms and many Proteus species.

Principle of Deoxycholate Citrate Agar (DCA)

Deoxycholate Citrate Agar (DCA) contains meat, peptone ,lactose, sodium citrate, ferric ammonium citrate, sodium desoxycholate, reutral red indicator  and agar. Meat peptone provides carbon, nitrogen, vitamins and minerals. Lactose is the fermentable carbohydrate. Sodium citrate and sodium deoxycholate inhibit Gram-positive bacteria, coliforms (Escherichia, Klebsiella, Enterobacter, Citrobacter and Hafnia) and Proteus species. Ferric ammonium citrate aids in the detection of  hydrogen sulphide (H2S)-producing bacteria. Neutral red is a pH indicator. Bacteria that ferment lactose produce acid and form red  colonies. Bacteria that do not ferment lactose form colourless colonies. Bacteria producing H2S will have black centers. The majority of normal intestinal bacteria ferment lactose and do not produce H2S (red colonies without black centers). Salmonella and Shigella species do not ferment lactose but Salmonella may produce H2S (colourless colonies with or without black centers). Lactose fermenting colonies may have a zone of precipitation around them caused by the precipitation of deoxycholate in the presence of acid.

Composition of Deoxycholate Citrate Agar (DCA)

           ( Difco™ )

Deoxycholate Citrate Agar (DCA) ingredients and their amounts are as follows-

Ingredients                        Gms / Litre

Meat, Infusion from 330 g:  9.5
Proteose Peptone No. 3: 10.0
Lactose: 10.0
Sodium Citrate:  20.0
Ferric Ammonium Citrate: 2.0
Sodium Desoxycholate:  5.0
Agar : 13.5
Neutral Red: 0.02

Distilled water (D/W): 1000 ml

Final pH 7.5 ± 0.2

Preparation of Deoxycholate Citrate Agar (DCA)

  1. Suspend 70 grams of the powder of Deoxycholate Citrate Agar (DCA)   in 1 liter purified / distilled or deionized water.
  2. Mix thoroughly and heat with frequent agitation and boil for 1 minute to
    completely dissolve the powder.
  3. Avoid overheating and do not autoclave.
  4.   Leave for cooling to 45-50°C.
  5. Mix well before dispensing.
  6. Pour into each plate  and leave plates on the sterile surface until the agar has solidified.
  7. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Deoxycholate Citrate Agar (DCA)

  • Store at 2-8ºC  and away from direct light.
  • Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination.
  • Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

Test Requirements for DCA test Procedure

  • Test specimens
  • Inoculating loop
  • Bunsen burner
  • Incubator
  • Control strains ( For negative control-Enterococcus faecalis ATCC 29212,  and Escherichia coli ATCC 25922  while positive control-Salmonella enterica subsp. enterica serotype Typhimurium ATCC 14028 , Shigella flexneri 12022 ATCC )

Test procedure of Deoxycholate Citrate Agar (DCA)

  1. Allow the plates to warm at room temperature, and the agar surface to dry before inoculating.
  2. Take DCA plates.
  3. Inoculate the specimen directly onto the medium.
  4. Incubate plates at 35 ± 2°C for 18-24 hours. Plates can be incubated for an additional 24 hours if no lactose fermenters are observed.

Result Interpretation  of DCA

Lactose nonfermenters 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.

Control strains: Enterococcus faecalisNo growth, Escherichia coli  Poor growth; pink with bile precipitate negative reaction for H2S, Salmonella enterica subsp. enterica serotype Typhimurium -Good-luxuriant growth; colorless; positive reaction for H2S, black centered colonies and Shigella flexneri-good growth with colorless colonies.

Colony Characteristics of various organisms in Deoxycholate Citrate Agar (DCA)

Organisms                              Growth

Escherichia coli:  Poor growth; pink with bile precipitate negative reaction for H2S

Enterobacter/Klebsiella species:  Theses bacteria show Poor growth, Large, pale mucoid colonies with the pink center.

Salmonella Typhimurium:  Good-luxuriant growth; colorless; positive reaction for H2S, black centered colonies as shown above picture

Salmonella enteritidis:    Good-luxuriant growth; colorless; positive reaction for H2S, black centered colonies and predominant cause of the food-borne salmonellosis in humans

Salmonella abony : Good-luxuriant growth; colorless; positive reaction for  H2S, black centered colonies.

Shigella flexneri: Good growth; colorless

Shigella sonnei: Colonies are smooth and initially colorless, becoming pale pink on further incubation due to late lactose fermentation as shown above picture.

Uses of Deoxycholate Citrate Agar (DCA)

  1. DCA  uses  for the isolation and differentiation of enteric gram-negative bacilli from clinical (feces, blood)  as well as food samples.
  2. It is particularly useful for the isolation of organisms that cause bacillary dysentery (Shigella) , Salmonella strains that cause food poisoning, typhoid fever and Salmonella Paratyphi causes paratyphoid fever.

Key Notes 

  1. Complete inhibition of enteric gram negative bacilli may not be guaranteed which also depend on microbial load in specimens.
  2. DCA is less selective as xylose lysine deoxycholate ( XLD) for Salmonella.
  3. Amount of Deoxycholate Citrate Agar powder varies manufacturer to manufacture for preparation. e.g. Microxpress  69.02 gm , Difco™ 70 gm where as Himedia 70.52  gm.

Limitations of Deoxycholate Citrate Agar (DCA)

  1. Coliform strains may be encountered that will grow on this medium, making it difficult to detect pathogens.
  2. Heavy inocula should be distributed over the entire surface of the medium to prevent complete masking of pathogens by coliform organisms.
  3. The medium is best used freshly prepared.
  4. Stock cultures of Shigella species may become predominantly in the R-phase when subcultured away from deoxycholate citrate agar. Such cultures are difficult to use for control purposes without first heavily streaking the cultures on deoxycholate citrate agar plates and picking off the few S-phase colonies i.e. the macro-colonies on the agar surface, for further subculture.
  5. As there are many bacteria that also look like Salmonella on deoxycholate citrate agar, it is widely recommended that more selective media are used for the identification of Salmonella species , namely XLD agar.
  6. When making biochemical tests on colonies picked from the surface of DCA plates, purity subcultures should be carried out because the colony may be contaminated with E. coli present as microcolonies.
  7. Suspected pathogens must be subcultured on a less inhibitory medium prior to identification.
  8. This growth medium is heat-sensitive and should be poured and cooled as soon as possible otherwise, it tends to become very soft and difficult to handle.
  9. Further tests need colonies from pure culture for complete identification in species level like biochemical, immunological, molecular, or mass spectrometry.
  10. Due to nutritional variations some organisms may show poor growth in this medium.

References

  1. https://www.sigmaaldrich.com/catalog/product/sial/d7809
  2. http://www.tulipgroup.com/MicroExpress/Accumix/PackInsert/Dehydrated%20Culture%20Media/D%20Files/Deoxycholate_Citrate_Agar_Agar_Medium_J_EP.pdf
  3. https://legacy.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/227410.pdf
  4. http://himedialabs.com/TD/M065.pdf
  5. http://www.medical-labs.net/desoxycholate-citrate-agar
  6. https://microdok.com/desoxycholate-citrate-agar-dca/
  7. https://www.eolabs.com/product/km0050-deoxycholate-citrate-agar-hynes/
  8. https://www.neogen.com/en-gb/categories/microbiology/acumedia deoxycholate-citrate-agar/
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