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Dehydrated Culture Media

Sector: Clinical

SIMMONS CITRATE AGAR

Code: CM0155

An agar medium for the differentiation of Enterobacteriaceae based on the utilisation of citrate as the sole source of carbon.

Typical Formula*

gm/litre

Magnesium sulphate

0.2

Ammonium dihydrogen phosphate

0.2

Sodium ammonium phosphate

0.8

Sodium citrate, tribasic

2.0

Sodium chloride

5.0

Bromothymol blue

0.08

Agar

15.0

pH 7.0 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards

Directions
Suspend 23g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes.

Description
Simmons Citrate Agar is recommended (Ewing and Edwards1 ) for the differentiation of the family Enterobacteriaceae based on whether or not citrate is utilised as the sole source of carbon.

The medium is virtually a solidified form of Koser citrate medium which, in its original form, suffered from the disadvantage that false appearance of growth occurred when large inocula were employed. The addition of bromothymol blue indicator to the medium was a distinct improvement.

Simmons Citrate Agar complies with the recommendations of the APHA2.

Technique
The medium may be used either as slopes in test tubes or as a plate medium in petri dishes. In both cases the surface of the medium is lightly inoculated by streaking and, where slopes are used, the butt of medium is inoculated by stabbing. Incubation for 48 hours at 35°C is recommended.

Positive growth (i.e. citrate utilisation) produces an alkaline reaction and changes the colour of the medium from green to bright blue, whilst in a negative test (i.e. no citrate utilisation) the colour of the medium remains unchanged.

Escherichia coli including serotypes from epidemic infantile enteritis, as well as Shigella, Yersinia and Edwardsiella species do not grow on the medium. Serratia and the majority of the Enterobacter, Citrobacter, Klebsiella, Proteus and Providencia species, except Morganella morganii and Klebsiella rhinoscleromatis utilise citrate and produce the characteristic blue coloration3.

Simmons Citrate Agar may be used to differentiate citrate-positive Salmonella enteritidis and members of Salmonella subgenus II, III and IV from the citrate-negative Salmonella typhi, Salmonella paratyphi A, Salmonella pullorum and Salmonella gallinarum.

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 medium at 2-8°C.

Appearance
Dehydrated medium: Yellow coloured, free-flowing powder
Prepared medium: Dark blue-green coloured gel        

Quality control

Positive control:Expected results
Klebsiella pneumoniae ATCC® 13883*Growth; colour change of medium to blue  
Negative control:  
Escherichia coli ATCC® 25922*Inhibited or no growth; no colour change of medium
* This organism is available as a Culti-Loop®

Precautions
It is important not to carry over any nutrients into the citrate medium because this will result in false positive tests. Dilute the inoculum in saline before inoculating the citrate medium to avoid a carry-over of other carbon sources4.

References
1. Ewing W. H. and Edwards P. R. (1960) Bull. Bact. Nomen. and Taxon. 10. 1-12.
2. American Public Health Association (1998) Standard Methods for the Examination of Water and Wastewater. 20th Edn. APHA Inc. Washington DC.
3. Kauffman F. (1954) `Enterobacteriaceae’ 2nd ed., Munksgaard, Copenhagen.
4. Matsen J. M. and Sherris J. C. (1969) Appl. Microbiol. 18. 452-454.

 
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