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Dehydrated Culture Media

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DESOXYCHOLATE CITRATE AGAR (HYNES)

Code: CM0227

A selective medium for the isolation of Salmonella and Shigella organisms.

Typical Formula*

gm/litre

`Lab-Lemco’ Powder

5.0

Peptone

5.0

Lactose

10.0

Sodium citrate

8.5

Sodium thiosulphate

5.4

Ferric ammonium citrate

1.0

Sodium desoxycholate

5.0

Neutral red

0.02

Agar

12.0

pH 7.3 ± 0.2 @ 25°C

 
* Adjusted as required to meet performance standards

Directions
Suspend 52g in 1 litre of distilled water. Bring to the boil over gauze and flame, to dissolve completely. Agitate to prevent charring. Cool to approximately 50°C and pour into sterile Petri dishes. Dry the agar surface before use.
THIS MEDIUM IS HEAT SENSITIVE: AVOID EXCESSIVE OR PROLONGED HEATING DURING RECONSTITUTION. DO NOT AUTOCLAVE OR REMELT.

Description
An improved medium, based on the Hynes1 modification of Leifsons medium for the isolation of salmonellae and shigellae.
The improvement gives larger and more numerous colonies of Shigella species which can easily be picked off and emulsified in saline for slide agglutination tests.

Desoxycholate Citrate Agar (Hynes) is more selective than Desoxycholate Citrate Agar CM0035. In particular, Desoxycholate Citrate Agar (Hynes) is more inhibitory to coliforms and Proteus species.

Technique
Inoculate the medium heavily with faeces or rectal swabs, spreading part of the original inoculum in order to obtain well separated colonies on some portion of the plate. Incubate for 18-24 hours at 35°C. If organisms are late developers or if no non-lactose fermenters are observed, incubate for a further 24 hours.

Colonies may be picked directly off the medium for serological and biochemical tests. It should be noted that Escherichia coli survives on the medium even though it does not usually grow - therefore colonial purity should be established by subculture on to a differential but less inhibitory medium, e.g. MacConkey Agar CM0007.

Colonial characteristics
(Following incubation at 35°C.)
The medium is clear and pale pink. Lactose fermenting organisms produce pink colonies and may be surrounded by a zone of precipitated desoxycholic acid, which is due to acid production. The colonies of non-lactose fermenters are colourless, and due to their alkaline reaction they are surrounded by a clear orange-yellow zone of medium.
Escherichia coli - Most strains are inhibited, but the few strains which grow produce pink umbilicated colonies 1-2mm in diameter which may be surrounded by a zone of precipitation.
Shigella sonnei - the colonies are 1mm diameter after 18 hours incubation, and 2mm after 38 hours; they are smooth and initially colourless, becoming pale pink on further incubation due to late lactose fermentation.
Shigella flexneri - colonies are colourless and similar in appearance to those of Shigella sonnei, but often with a narrow plane periphery round a central dome.
Salmonella paratyphi B - from 1mm diameter after 18 hours incubation to 2-4mm on the second day, when they are slightly opaque, dome-shaped, with a central black dot.
Salmonella typhi - 0.25-1mm in diameter after 18 hours and pale pink, a day later they are flat, conical, 2mm in diameter, colourless and slightly opaque, often with a central grey dot.
Other Salmonella colonies - similar to those of Salmonella paratyphi B. Non-pathogenic non-lactose fermenters, such as Proteus and Pseudomonas species, grow on the medium and may produce colonies which closely simulate those of the salmonellae or shigellae. Proteus colonies are often glossy (more translucent than those of the pathogens), with a large central black dot and a `fishy’ odour.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared agar plates at 2-8°C.

Appearance
Dehydrated medium: Straw/pink coloured, free-flowing powder
Prepared medium: Pink coloured gel

Quality control

Positive controls:

Expected results

Salmonella typhimurium ATCC® 14028 *

Good growth; straw coloured colonies with black centres

Shigella sonnei ATCC® 25931 *

Good growth; straw coloured colonies

Salmonella nottingham NCTC 7832 *Good growth; straw coloured colonies with black centres

Negative control:

 
Enterococcus faecalis ATCC® 29212 *No growth
* This organism is available as a Culti-Loop®

Precautions
Observe the precautions about overheating shown under Directions.
The medium is best used freshly prepared.
Stock cultures of Shigella species may become predominantly in the R-phase when subcultured away from DCA media. Such cultures are difficult to use for control purposes without first heavily streaking the cultures on DCA plates and picking off the few S-phase colonies i.e. the macro-colonies on the agar surface, for further subculture.
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 Escherichia coli present as micro-colonies.

Reference
1. Hynes M. (1942) J. Path. Bact. 54. 193-207.

 
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