What is Deoxycholate Citrate Agar (DCA)?

• Deoxycholate Citrate Agar (DCA) is a specialized plating medium used for the isolation of enteric bacilli, specifically Salmonella and various Shigella species. It is a modification of Leifson’s formula, designed to provide moderate selectivity for enteric pathogens by increasing the concentrations of citrate and deoxycholate salts.
• The composition of DCA includes sodium deoxycholate, which at a pH of 7.3 to 7.5, inhibits the growth of gram-positive bacteria. Additionally, the citrate salts present in the medium inhibit the growth of gram-positive bacteria and most other normal intestinal organisms. These properties make DCA a selective medium, allowing for the isolation of enteric pathogens while suppressing the growth of unwanted bacteria.
• DCA is prepared using a ready-to-use dehydrated powder, readily available from culture media suppliers. The medium is formulated based on Leifson’s modified formula, ensuring optimal recovery of intestinal pathogens from food samples. However, if the primary goal is to isolate Shigella, it is recommended to use a less inhibitory medium alongside DCA.
• When using DCA for the routine examination of stool and urine specimens, it is suggested to combine it with other media such as MacConkey Agar or Bismuth Sulphite Agar. This combination helps improve the detection of specific pathogens.
• DCA shares similarities with deoxycholate agar but offers a slightly higher selectivity for enteric pathogens due to the increased concentrations of citrate and deoxycholate salts. The medium’s components, such as HI solids, proteose peptone, sodium deoxycholate, sodium citrate, and ferric ammonium citrate, contribute to inhibiting or suppressing the growth of coliform bacteria and gram-positive bacteria.
• Lactose is included in DCA to differentiate between lactose fermenters and non-fermenters. Lactose fermenters produce red colonies, while non-fermenters produce colorless colonies. The fermentation of lactose causes acidification of the medium, leading to a change in color of the pH indicator (neutral red) to red. Colonies surrounded by a turbid zone of precipitated deoxycholic acid indicate acidification of the medium. In an acidic environment, sodium deoxycholate combines with neutral red, causing the dye to precipitate as deoxycholate.
• The reduction of ferric ammonium citrate to iron sulfide results in the formation of black iron sulfide, indicating the presence of certain pathogens. Salmonella and Shigella species, although non-lactose fermenters, may produce hydrogen sulfide (H2S), leading to colorless colonies with or without black centers.
• One important consideration when working with DCA is that the combination of citrate and iron has a hydrolyzing effect on agar when the medium is heated. This can cause the agar to become soft and unelastic, making streaking difficult. Autoclaving the agar can further exacerbate this issue, resulting in soft agar that is almost impossible to streak. Salmonella Gallinarum can be inhibited by increasing the concentration of sodium deoxycholate to 0.1% or greater.
• It is worth noting that surface colonies of non-lactose fermenters may absorb a small amount of color from the medium, appearing pinkish, which could lead to confusion with coliforms.
• In summary, Deoxycholate Citrate Agar (DCA) is a selective and differential medium used for the isolation of enteric pathogens, specifically Salmonella and Shigella species. It provides moderate selectivity due to increased concentrations of citrate and deoxycholate salts, inhibiting the growth of gram-positive bacteria and most normal intestinal organisms. DCA’s composition and indicators aid in the differentiation of lactose fermenters and non-fermenters, as well as the detection of specific pathogenic characteristics.

Principle of Deoxycholate Citrate Agar (DCA)

The principle of Deoxycholate Citrate Agar (DCA) is based on several key components and reactions within the medium.

The HI solids present in DCA serve as a source of carbon and nitrogen while inhibiting the growth of coliform bacteria. Proteose peptone provides additional carbon, nitrogen, vitamins, and minerals necessary for bacterial growth. The combination of sodium deoxycholate, sodium citrate, and ferric ammonium citrate in the medium inhibits or greatly suppresses the growth of both coliform bacteria and gram-positive bacteria.

Dipotassium phosphate acts as a buffer in the medium, maintaining a stable pH. Lactose, a fermentable carbohydrate, plays a crucial role in differentiating enteric bacilli. Bacteria that ferment lactose produce acid, leading to the formation of red colonies. On the other hand, lactose non-fermenters produce colorless colonies.

Coliform bacteria, if present, form pink colonies on DCA. The degradation of lactose by lactose-fermenting bacteria causes acidification of the medium surrounding the colonies. This acidification results in a color change of the pH indicator neutral red, turning it red. Additionally, these colonies are often surrounded by a turbid zone of precipitated deoxycholic acid due to the acidification process.

In an acidic environment, sodium deoxycholate interacts with neutral red, causing the dye to precipitate out of the solution along with the deoxycholate. The reduction of ferric ammonium citrate to iron sulfide is indicated by the formation of black iron sulfide, which aids in the detection of certain bacteria, particularly those producing hydrogen sulfide (H2S).

Salmonella and Shigella species, although unable to ferment lactose, may produce H2S. Therefore, they form colorless colonies on DCA, with or without black centers.

It is important to note that the combination of citrate and iron has a hydrolyzing effect on agar when the medium is heated. This hydrolysis leads to the production of a soft and unelastic agar, which can pose challenges during streaking or manipulation of the medium.

Overall, the principle of DCA revolves around the utilization of specific components and reactions to differentiate lactose fermenters from non-fermenters, inhibit the growth of certain bacterial species, and detect the production of H2S by select pathogens.

Composition of Deoxycholate Citrate Agar (DCA)

Ingredients	Gms/liter
HI solids	10.00
Proteose peptone	10.00
Lactose	10.00
Sodium deoxycholate	5.000
Neutral red	0.020
Sodium citrate	20.00
Ferric ammonium citrate	2.000
Agar	13.50

Final pH (at 25°C) 7.5±0.2

Deoxycholate Citrate Agar w/ 1.5% Agar

Ingredients	Gms / Litre
Beef extract	5.000
Peptic digest of animal tissue	5.000
Lactose	10.000
Sodium deoxycholate	2.500
Sodium citrate	5.000
Sodium thiosulphate	5.000
Ferric citrate	1.000
Neutral red	0.025
Agar	15.000
Final pH (at 25°C)	7.0±0.2

Preparation of Deoxycholate Citrate Agar (DCA)

Here are the steps for the preparation and method of use of Deoxycholate Citrate Agar (DCA):

1. Suspend 70.52 grams of DCA powder in 1000 ml of distilled water.
2. Heat the suspension to boiling to ensure complete dissolution of the medium. Avoid excessive heating, as it can be detrimental to the agar. Note that autoclaving is not recommended for DCA.
3. Allow the medium to cool down to a temperature of 45-50°C.
4. Mix the agar suspension well to ensure homogeneity.
5. Pour the DCA mixture into sterile Petri plates, ensuring even distribution.
6. Allow the agar surface to dry completely before use. This can be achieved by leaving the plates uncovered in a sterile environment or using a laminar flow hood.
7. Inoculate the medium heavily with feces or rectal swabs, ensuring a significant amount of the original inoculum is spread on the plate. This helps in obtaining well-separated colonies on certain areas of the plate.
8. Incubate the plates at a temperature of 35°C for 18-24 hours.
9. After the initial incubation period, check for colony development. If certain organisms are late developers or if no non-lactose fermenters are observed, continue incubating for an additional 24 hours.
10. After the appropriate incubation period, observe and analyze the colonies for color, morphology, and other distinguishing characteristics.

Note: It is important to follow good laboratory practices and maintain proper sterility throughout the preparation and use of DCA.

Inoculation of the prepared Deoxycholate Citrate Agar (DCA)

The process of inoculating the prepared Deoxycholate Citrate Agar (DCA) medium is as follows:

1. Dry the Agar Surface: Before use, ensure that the surface of the DCA agar has completely dried. This can be achieved by allowing the plates to sit uncovered in a sterile environment or using a laminar flow hood.
2. Heavy Inoculation: Inoculate the DCA medium heavily with feces or rectal swabs, ensuring a significant amount of the original inoculum is spread on the plate. This is done to promote the growth of the target bacteria and obtain well-separated colonies on certain areas of the plate. The heavy inoculation helps in ensuring that the desired pathogens are adequately represented and can be differentiated from other bacterial species.
3. Spreading the Inoculum: To obtain well-separated colonies, it is recommended to spread part of the original inoculum on the plate. This helps to distribute the bacteria evenly and prevent overcrowding of colonies. It also aids in the visual differentiation of individual colonies, which is important for further analysis.
4. Incubation: After inoculation, incubate the DCA plates for 18-24 hours at a temperature of 35°C. This temperature provides an optimal growth environment for many enteric pathogens and allows for the detection of characteristic reactions and colony appearances.
5. Extended Incubation (if necessary): If certain organisms are late developers or if no non-lactose fermenters are observed within the initial incubation period, it may be necessary to incubate the plates for an additional 24 hours. This extended incubation period allows for the detection of slower-growing or delayed-reacting bacterial species.

By following these steps, the prepared DCA medium can be effectively inoculated, allowing for the growth and differentiation of enteric pathogens. The incubation period is crucial for the development of characteristic colony appearances and reactions, which aid in the identification and analysis of the targeted bacteria.

Result Interpretation on Deoxycholate Citrate Agar (DCA)

The interpretation of results on Deoxycholate Citrate Agar (DCA) is as follows:

1. Lactose Non-Fermenters:
   • Appearance: Transparent, colorless to light pink or tan-colored colonies.
   • Black Centers: May or may not be present.
   • Examples: Escherichia coli.
   • Growth: Poor growth.
   • Bile Precipitate: Negative reaction.
   • Hydrogen Sulfide (H2S): Negative reaction.
2. Lactose Fermenters:
   • Appearance: Red colonies.
   • Bile Precipitate: May or may not be present.
   • Examples: Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Abony.
   • Growth: Good-luxuriant growth.
   • Hydrogen Sulfide (H2S): Positive reaction.
   • Black Centers: Present in colonies of Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Abony.
3. Shigella flexneri:
   • Appearance: Good growth.
   • Color: Colorless.
4. Shigella sonnei:
   • Appearance: Colonies are initially smooth and colorless, but become pale pink with further incubation due to late lactose fermentation.
5. Enterobacter/Klebsiella spp.:
   • Appearance: Large, pale mucoid colonies with a pink center.

It is important to note that these interpretations are based on the characteristics observed on Deoxycholate Citrate Agar (DCA). Additional confirmatory tests are usually required for accurate identification of specific bacterial species.

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

Species	Colony Morphology
Escherichia coli	Most strains are inhibited, but the few strains which grow produce pink umbilicated colonies, encircled by a zone of precipitate.
Shigella sonnei	Colonies are smooth and initially colorless, becoming pale pink on further incubation due to late lactose fermentation
Shigella flexneri	Colonies are colorless and similar in appearance to those of Shigella sonnei
Salmonella spp	Salmonellae produce non-lactose fermenting colorless colonies with (for H2S producing salmonellae) or without black centers
Enterobacter/Klebsiella spp.	Large, pale mucoid colonies with a pink center.
Proteus/Providencia spp.	Large, colorless to tan, with or without a black center. Proteus colonies are often glossy (more translucent than those of the pathogens), with a large central black dot and a `fishy’ odor.
Yersinia enterocolitica	Colorless
Enterococci	No growth to slight growth

Quality control Organisms

Quality control organisms are essential in ensuring the reliability and accuracy of microbiological tests, including those performed using Deoxycholate Citrate Agar (DCA). These control organisms help verify the performance of the medium and ensure that it functions as expected. Here are some examples of quality control organisms and their expected results when using DCA:

Positive Controls:

1. Salmonella typhimurium ATCC® 14028:
   • Expected Result: Good growth; straw-colored colonies.
2. Shigella sonnei ATCC® 25931:
   • Expected Result: Good growth; straw-colored colonies.
3. Salmonella poona NCTC 4840:
   • Expected Result: Good growth; straw-colored colonies.

Negative Control:

1. Enterococcus faecalis ATCC® 29212:
   • Expected Result: No growth.

The positive control organisms are chosen because they represent common pathogens that can be isolated and identified using DCA. These organisms are expected to exhibit good growth on the medium and form colonies with straw-colored appearances.

The negative control organism, Enterococcus faecalis, is a non-enteric bacterium that should not grow on DCA. Its inclusion as a negative control helps ensure that the medium is selective and specific for enteric pathogens, and that it does not support the growth of unrelated organisms.

By including these quality control organisms in routine testing, laboratories can verify the proper performance of DCA and ensure that it consistently provides accurate and reliable results. It is important to follow established guidelines and standards for the selection and use of quality control organisms in microbiological testing.

Precautions

When working with Deoxycholate Citrate Agar (DCA), it is important to observe certain precautions to ensure accurate results and maintain the integrity of the medium. Here are some precautions to consider:

1. Overheating: Avoid excessive heating of the medium during preparation, as it can be detrimental to the agar. Follow the directions provided and be cautious about overheating, as it can lead to soft agar that is difficult to handle.
2. Fresh Preparation: DCA is best used when freshly prepared. It is recommended to prepare the medium as needed and avoid storing it for extended periods. Freshly prepared medium ensures optimal performance and reliable results.
3. Stock Culture of Shigella Species: When working with stock cultures of Shigella species, be aware that subculturing them away from DCA media can cause a shift to the R-phase, making them difficult to use for control purposes. To mitigate this, heavily streak the cultures on DCA plates and select the few S-phase colonies (macro-colonies on the agar surface) for further subculture.
4. Purity of Biochemical Tests: When performing biochemical tests on colonies picked from the surface of DCA plates, it is important to carry out purity subcultures. This is because the colony may be contaminated with Escherichia coli, which can be present as micro-colonies. Purity subcultures help ensure that the selected colony is free from contamination, allowing for accurate biochemical testing.

By following these precautions, you can minimize potential errors and ensure the reliability of your results when working with Deoxycholate Citrate Agar. It is also important to adhere to good laboratory practices and follow any specific guidelines or recommendations provided by relevant authorities or manufacturers.

Uses of Deoxycholate Citrate Agar (DCA)

Deoxycholate Citrate Agar (DCA) finds several uses in the laboratory setting for the isolation and differentiation of Gram-negative enteric bacilli. Some of the specific uses of DCA are as follows:

• Isolation of Enteric Bacilli: DCA is primarily employed for the isolation of Gram-negative enteric bacilli. It provides a selective environment that inhibits the growth of Gram-positive bacteria and many other normal flora present in the intestines. This selectivity allows for the isolation of target enteric pathogens.
• Differentiation of Pathogens: DCA aids in the differentiation of various Gram-negative enteric bacilli based on their ability to ferment lactose and produce other characteristic reactions. Lactose fermenters and non-fermenters exhibit distinct colony appearances, enabling differentiation between different bacterial species.
• Identification of Bacillary Dysentery: DCA is particularly useful in the isolation of organisms that cause bacillary dysentery, such as Shigella species. The medium’s composition and selective properties provide an optimal environment for the growth and differentiation of these pathogens.
• Isolation of Salmonella Strains: DCA is employed for the isolation of Salmonella strains that cause food poisoning, including Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis. These pathogens can be effectively recovered and differentiated on DCA plates based on their characteristic growth and reactions.
• Detection of Salmonella Paratyphi: DCA is also used for the isolation of Salmonella enterica serovar Paratyphi, a pathogen responsible for paratyphoid fever. The medium’s selective properties facilitate the growth and differentiation of this particular Salmonella serovar.

By utilizing DCA, microbiologists can isolate and differentiate various Gram-negative enteric bacilli, including those causing dysentery, food poisoning, and specific Salmonella serovars. The medium’s selectivity and differential properties play a crucial role in the accurate identification and characterization of these pathogenic bacteria.

Limitations of Deoxycholate Citrate Agar (DCA)

Deoxycholate Citrate Agar (DCA) has certain limitations that should be taken into consideration. These limitations include:

1. Subculturing on Less Inhibitory Medium: DCA is a moderately selective medium, but for the accurate identification of suspected pathogens, it is recommended to subculture them on a less inhibitory medium before performing further identification tests. This is because there are other bacteria that may exhibit similar growth characteristics to Salmonella on DCA.
2. Limited Selectivity for Salmonella Identification: While DCA can be used for the isolation of Salmonella, it is not the most selective medium for their identification. For more reliable identification of Salmonella, the use of more specific agar media like xylose lysine deoxycholate (XLD) agar is widely recommended.
3. Heat Sensitivity: DCA is sensitive to excessive heat exposure. To maintain its integrity and usability, it should be poured and cooled as soon as possible after adding deoxycholate. Prolonged heating can result in a soft agar that becomes difficult to handle.
4. Complementary Use with Other Media: For routine examination of stool and urine specimens, it is suggested to use DCA in conjunction with other media such as MacConkey Agar, Bismuth Sulphite Agar, or other suitable culture media. This helps to enhance the detection of a broader range of pathogens and provides a more comprehensive analysis.
5. Biochemical Confirmation Required: While DCA can provide initial differentiation and identification of certain pathogens, further biochemical tests are needed for confirmation of species. This is particularly important to ensure accurate identification and differentiation of similar-looking colonies.
6. Variation in Growth: Some organisms may show poor growth on DCA due to nutritional variations. The medium may not support optimal growth for all bacterial species, leading to potential limitations in detecting certain pathogens.

Considering these limitations, it is important to use DCA in conjunction with other suitable media and perform additional confirmatory tests to ensure accurate identification and characterization of suspected pathogens.

FAQ

References

• https://exodocientifica.com.br/_technical-data/M1639.pdf
• http://exodocientifica.com.br/_technical-data/M065.pdf
• https://microbeonline.com/deoxycholate-citrate-agar-dca-preparation-uses-colony/#google_vignette
• https://universe84a.com/deoxycholate-citrate-agar-dca/
• https://legacy.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/227410.pdf
• https://www.onlinebiologynotes.com/deoxycholate-citrate-agar-dcacompositionpreparation-and-use/
• https://www.neogen.com/globalassets/pim/assets/original/10000/7186_pi.pdf
• https://www.thomassci.com/Laboratory-Supplies/Microbiological-Media/_/Deoxycholate-Citrate-Agar-Modified-Hynes
• http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=cm0035&c=UK&lang=EN