DHL Agar: Introduction, Principle, Composition, Preparation, Procedure

DHL Agar: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

Introduction of DHL Agar

DHL Agar is the short name for Deoxycholate Hydrogen Sulfide Lactose Agar. It is a modification of Deoxycholate Agar and it was proposed by Sakazaki et. al. DHL agar supplies contain a rich nitrogen base to permit the growth of fastidious strains of Salmonella and Shigella species. The colonies formed are considerably larger than those found on similar selective media (MacConkey agar, S-S agar, etc.).

Principle of DHL Agar

Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue are the sources of nitrogen and vitamin sources in DHL Agar. Carbohydrate, sucrose permits differentiation of weakly lactose-positive, lactose-negative, or sucrose-positive species from sucrose and lactose-negative Enterobacteriaceae. H₂S production is indicated by a blackening of the colonies due to the formation of iron sulfide. Sodium Deoxycholate, Sodium Thiosulfate, Ammonium Iron Citrate, and Sodium Citrate are inhibitory agents of Gram-positive bacteria. Neutral red is a pH indicator. Sodium chloride maintains osmotic balance while agar is the solidifying agent.

Composition of DHL Agar

Typical Formula gm/liter

Enzymatic Digest of Casein: 5.0
Enzymatic Digest of Animal Tissue: 5.0
Lactose: 10.0
Sodium Deoxycholate: 1.0
Sodium Chloride: 5.0
Dipotassium Phosphate: 2.0
Ferric Citrate: 1..0
Sodium Citrate:1.0
Neutral Red:0.03
Agar: 16.0
Final pH: 7.2 ± 0.2 at 25°C

Preparation of DHL Agar

Suspend 46.0 grams in 1 liter of purified/distilled or deionized water.
Heat with frequent agitation and boil for one minute to completely dissolve the medium.
Leave for cooling to 45-50°C.
Pour medium into each plate and leave plates on the sterile surface until the agar has solidified.
Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life DHL Agar

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), or contamination.
The product is light and temperature sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements

Test specimens ( Clinical sample or growth of bacteria)
Inoculating loop
Bunsen burner
CO₂Incubator
Control strains (Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922)
Culture Media

Procedure of DHL Agar

Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
Inoculate and streak the specimen as soon as possible after collection.
If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
Streak for isolation with a sterile loop.
Incubate plates aerobically at 35-37ºC for 18-24 hours.
Examine colonial characteristics.

Colony Morphology of DHL Agar

Cultural responses of bacteria on DHL Agar are as follows-

Staphylococcus aureus ATCC 25923: No growth
Escherichia coli ATCC 25922:r Red colonies
Enterococcus faecalis: No growth or partial growth-colorless colonies
Klebsiella pneumoniae: Pink colonies
Proteus mirabilis: Colorless ± black centers
Proteus vulgaris: Pink colonies
Salmonella Typhi: Ccolorless ± black centers
Salmonella Typhimurium: Colorless colonies w/ black centers
Shigella flexneri: Colorless colonies

Result-Interpretation

Enteric bacilli can be differentiated on the basis of lactose fermentation. An organism that ferments lactose produces acid and, in the presence of a neutral red indicator, makes pink to red colonies. Non-lactose fermenting bacteria colonies remain colorless. The majority of normal intestinal bacteria ferment the lactose (red colonies) while Salmonella and Shigella species do not ferment lactose and thus remain colorless colonies

Use of DHL Agar

It is used for the detection and isolation of pathogenic Enterobacteriaceae.

Keynotes on DHL Agar

It is not autoclavable medium.
Other than DHL not autoclave media are Wilson & Blair’s, TCBS, Salmonella-Shigella agar,XLD agar and Chromagar.
DHL agar is also called Sakazaki DHL agar.

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