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Simmons Citrate Agar biochemistry test for Enterobacteriaceae

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Simmons Citrate Agar
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Simmons Citrate Agar is an agar medium used for the differentiation of Enterobacteriaceae based on the utilization of citrate as the sole source of carbon. 



Ingredients per liter of deionized water:


Principle of Simmons Citrate Agar


Uses of Simmons Citrate Agar
1.    It is used for the differentiation of Gram-negative bacteria on the basis of citrate utilization.
2.    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.
3.    Simmons Citrate Agar is primarily used to aid in the identification of Enterobacteriaceae. Uses
include:
  • Escherichia coli (usually -) and Shigella spp. (-) from other commonly encountered
    Enterobacteriaceae (variable +).
  • Edwardsiella spp. (-) from Salmonella spp. (usually +)
  • Serratia proteamaculans (+) from Yersinia pseudotuberculosis (-) and Yersinia enterocolitica
    (usually -)
  • Klebsiella-Enterobacter groups (usually +) from E. coli (usually -)
  • Proteus rettgeri (+) from Morganella morganii biogroups 1 and 2 (-)
  • Yokenella regensburgei (+) from Hafnia alvei (-)
  • Leminorella grimontii (+) from L. richardii (-)
  • Acidovorax delafieldii (+) from A. facilis (-) and  A. temperans (-)

Preparation 
1.    Dissolve above salts in deionized water.
2.    Adjust pH to 6.9.
3.    Add agar and Bromothymol blue.
4.    Gently heat, with mixing, to boiling until agar is dissolved.
5.    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.
6.    For tubes, dispense 4.0 to 5.0 ml into 16-mm tubes.
7.    Autoclave at 121 degree C under 15 psi pressure for 15 minutes.
8.    Cool in slanted position (long slant, shallow butt).
9.    Tubes should be stored in a refrigerator to ensure a shelf life of 6 to 8 weeks.
10. The uninoculated medium will be a deep forest green due to the pH of the sample and the bromothymol blue.

 Culture medium 18-24h 
subculture on simmons citrate agar tube and streaking an agar slant with the loop.Then take in incubation 37c 18-24h
Image result for simmons citrate agar streaking
Result Interpretation


 positive growth (i.e. citrate utilisation) produces an alkaline reaction and changes the colour of the medium from green to bright blue.

Negative test (i.e. no citrate utilisation) the colour of the medium remains unchanged.

Limitations of Simmons Citrate Agar
1.    It is important not to carry over any nutrients into the citrate medium because this will result in false positive tests.
2.    Some organisms are capable of growth on citrate and do not produce a color change. Growth is considered a positive citrate utilization test, even in the absence of a color change.
3.    Tests with equivocal results should be repeated.
4.    The reactions of this medium alone are not sufficient for identification to the species level.
5.    The inoculum should be from a pure, overnight culture grown on a solid medium and not from a broth suspension.
Quality Control on Simmons Citrate Agar
Positive control: 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
List of Bacteria which gives positive citrate utilization test
  1. Klebsiella pneumoniae
  2. Enterobacter species (minority of strains gives negative result)
  3. Citrobacter freundii
  4. Salmonella other than Typhi and Paratyphi A
  5. Serratia marcescens
  6. Proteus mirabilis (minority of strains gives negative result)
  7. Providencia
Citrate Test: variable (different strains give different results)
  1. Proteus vulgaris
  2. Vibrio cholerae
  3. Vibrio parahaemolyticus
Citrate test: negative
  1. Escherichia coli
  2. Shigella spp
  3. Salmonella Typhi
  4. Salmonella Paratyphi A
  5. Morganella morganii
  6. Yersinia enterocolitica
Although uncommon, natural E. coli variants that are citrate positive have been isolated.  Citrate-negative strains of E. aerogenes have also been found.
References
www.time2026end.com

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