Desoxycholate Agar

Intended Use

Desoxycholate Agar is a slightly selective and differential plating medium used for isolating and differentiating gramnegative enteric bacilli.
Desoxycholate Citrate Agar is a moderately selective and differential plating medium used for isolating enteric bacilli, particularly Salmonella and many Shigella species.
Desoxycholate Lactose Agar is a slightly selective and differential plating medium used for isolating and differentiating gramnegative enteric bacilli and for enumerating coliforms from water, wastewater, milk and dairy products.

Summary and Explanation

Desoxycholate Agar as formulated by Leifson¹ demonstrated improved recovery of intestinal pathogens from specimens containing normal intestinal flora. The medium was an improvement over other media of the time because the chemicals, citrates and sodium desoxycholate, in specified amounts, worked well as inhibitors. This medium has been used to screen for Salmonella spp. and Shigella spp. from clinical specimens.²
Desoxycholate Citrate Agar is a modification of Desoxycholate Agar formulated by Leifson.¹ 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.¹
Desoxycholate Citrate Agar effectively isolates intestinal pathogens (Salmonella and Shigella species) by inhibiting coliforms and many Proteus species.
Desoxycholate Lactose Agar is a modification of Desoxycholate Agar formulated by Leifson.¹ 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 grampositive bacteria.
Standard methods manuals for dairy² and water³ specified a modification of Desoxycholate Agar to contain less sodium desoxycholate and, accordingly, be less inhibitory to grampositive bacteria. This formulation, known as Desoxycholate Lactose Agar, was used in pour plate procedures for isolation and enumeration of coliforms in milk, water and other specimens. The medium is no longer included in recent editions of these manuals.

Principles of the Procedure

Peptone provides nitrogen and carbon for general growth requirements. Lactose is the fermentable carbohydrate. Sodium chloride and dipotassium phosphate maintain the osmotic balance of the medium. Sodium desoxycholate, ferric citrate and sodium citrate inhibit growth of gram-positive bacteria. Neutral red is a pH indicator. Agar is the solidifying agent.
Differentiation of enteric bacilli is based on fermentation of lactose. Bacteria that ferment lactose produce acid and, in the presence of neutral red, form red colonies. Bacteria that do not ferment lactose form colorless colonies. The majority of normal intestinal bacteria ferment lactose (red colonies), while Salmonella and Shigella species do not ferment lactose (colorless colonies).
Infusion from meat is a source of carbon and nitrogen. This ingredient is used because the inhibition of coliforms produced is greater than when an extract or simple peptone is used.¹ Peptone provides carbon, nitrogen, vitamins and minerals. Lactose is the fermentable carbohydrate. Sodium citrate and sodium desoxycholate inhibit gram-positive bacteria, coliforms and Proteus species. Ferric ammonium citrate aids in the detection of H₂S-producing bacteria. Neutral red is a pH indicator. Agar is the solidifying agent.
In the presence of neutral red, bacteria that ferment lactose produce acid and form red colonies. Bacteria that do not ferment lactose form colorless colonies. If the bacteria produce H₂S, the colonies 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 spp. do not ferment lactose but Salmonella may produce H₂S (colorless colonies with or without black centers). Lactose-fermenting colonies may have a zone of precipitation around them caused by the precipitation of desoxycholate in the presence of acid.

User Quality Control

Identity Specifications
Desoxycholate Agar
Dehydrated Appearance: Pinkish beige, free-flowing, homogeneous.
Solution:                        4.5% solution, soluble in purified water upon
                                     boiling. Solution is reddish-orange, slightly opalescent.
Prepared Appearance:    Orange, slightly opalescent.
Reaction of 4.5%
Solution at 25°C:            pH 7.3 ± 0.2
Cultural Response
Desoxycholate Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-24 hours.

Identity Specifications
Desoxycholate Citrate Agar
Dehydrated Appearance: Pinkish-beige, free-flowing, homogeneous.
Solution:                        7.0% solution, soluble in purified water upon
                                     boiling. Solution is orange-red, very slightly to
                                     slightly opalescent.
Prepared Appearance:     Orange-red, slightly opalescent.
Reaction of 7.0%
Solution at 25°C:            pH 7.5 ± 0.2
Cultural Response
Desoxycholate Citrate Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C

Identity Specifications
Desoxycholate Lactose Agar
Dehydrated Appearance: Pinkish beige, free-flowing, homogeneous.
Solution:                        4.25% solution, soluble in purifed water upon
                                     boiling. Solution is pinkish-red, very slightly to
                                     slightly opalescent.
Prepared Appearance:    Pinkish-red, very slightly to slightly opalescent.
Reaction of 4.25%
Solution at 25°C:           pH 7.1 ± 0.2
Cultural Response
Desoxycholate Lactose Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-24 hours.

Formula

Desoxycholate Agar
Approximate Formula* Per Liter
Peptone..................................................................... 10.0 g
Lactose ..................................................................... 10.0 g
Sodium Desoxycholate.................................................. 1.0 g
Sodium Chloride........................................................... 5.0 g
Dipotassium Phosphate.................................................. 2.0 g
Ferric Ammonium Citrate............................................... 1.0 g
Sodium Citrate.............................................................. 1.0 g
Agar........................................................................... 15.0 g
Neutral Red................................................................. 0.03 g
Desoxycholate Citrate Agar
Approximate Formula* Per Liter
Meat, Infusion from 330 g............................................ 9.5 g
Proteose Peptone No. 3.............................................. 10.0 g
Lactose..................................................................... 10.0 g
Sodium Citrate.......................................................... 20.0 g
Ferric Ammonium Citrate............................................. 2.0 g
Sodium Desoxycholate................................................ 5.0 g
Agar......................................................................... 13.5 g
Neutral Red............................................................... 0.02 g
Desoxycholate Lactose Agar
Approximate Formula* Per Liter
Proteose Peptone....................................................... 10.0 g
Lactose .................................................................... 10.0 g
Sodium Desoxycholate................................................. 0.5 g
Sodium Chloride.......................................................... 5.0 g
Sodium Citrate............................................................ 2.0 g
Agar......................................................................... 15.0 g
Neutral Red............................................................... 0.03 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

Desoxycholate Agar
1. Suspend 45 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. Avoid overheating. DO NOT AUTOCLAVE.
3. Test samples of the finished product for performance using stable, typical control cultures.
Desoxycholate Citrate Agar
1. Suspend 70 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. Avoid overheating. DO NOT AUTOCLAVE.
3. Test samples of the finished product for performance using stable, typical control cultures.
Desoxycholate Lactose Agar
1. Suspend 42.5 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. Avoid overheating. DO NOT AUTOCLAVE.
3. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Desoxycholate Agar
For a complete discussion on the isolation of enteric bacilli, refer to appropriate procedures outlined in the references.^2-4
Desoxycholate Citrate Agar
1. Inoculate specimen directly onto surface of medium.
2. 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.
Desoxycholate Lactose Agar
See appropriate references for specific procedures.^2,3

Expected Results

Desoxycholate Agar
Refer to appropriate references and procedures for results.^2-4
Desoxycholate Citrate Agar
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.
Desoxycholate Lactose Agar
Refer to appropriate references and procedures for results.^2,3

Limitations of the Procedure

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.

*Store at 2-8° C.

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