SIMMONS CITRATE AGAR

Cat. no. L80 Simmons Citrate Agar, 16x100mm Tube, 5.5ml Slant 20 tubes/box

INTENDED USE

Hardy Diagnostics Simmons Citrate Agar is recommended for use in the differentiation of gram-negative enteric bacilli based on citrate utilization.

SUMMARY

In the early 1920s, Koser developed a liquid medium formulation for the differentiation of fecal coliforms from the coliform group. (5) Simmons later modified this formulation to produce a solid medium that eliminated potential errors when interpreting growth. (9)

Simmons Citrate Agar contains ammonium dihydrogen phosphate, which supplies the only source of nitrogen, and sodium citrate, which serves as the sole source of carbon. Organisms capable of utilizing ammonium dihydrogen phosphate and citrate will grow unrestricted on this medium. Bromothymol blue acts as a pH indicator, causing the medium to change from green (neutral) to blue (alkaline) with increasing pH. Citrate utilization produces an alkaline carbonate, resulting in a deep blue color change within the agar. The medium will remain green if organisms are not able to metabolize sodium citrate.

FORMULA

Ingredients per liter of deionized water:*

Sodium Chloride 5.0gm
Sodium Citrate 2.0gm
Ammonium Dihydrogen Phosphate 1.0gm
Dipotassium Phosphate 1.0gm
Magnesium Sulfate 0.2gm
Bromothymol Blue 0.08gm
Agar 15.0gm

Final pH 6.9 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-30ºC. Products should not be used if there are any signs of contamination, deterioration, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Consult listed references for information on specimen collection. (2-4,6,7) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport medium and refrigerated until inoculation.

Refer to the keywords "Specimen Collection and Transport" on the Hardy Diagnostics Technical Document website for more information.

Method of use: This medium is not intended to be used for primary isolation. Using a light inoculum, inoculate medium with growth from a pure culture. Inoculate the slant by streaking the surface in a serpentine manner. Replace caps loosely on the tube(s). Incubate aerobically for 24-96 hours at 35 degrees C. Examine daily for growth and color change.

INTERPRETATION OF RESULTS

A positive reaction is indicated by growth with development of a deep blue color reaction within the medium. A negative reaction is evidenced by no growth or growth with the medium remaining green in color.

LIMITATIONS

Failure to incubate tubes with loose caps may result in false-negative results.

Use a light inoculum to streak the slant; a heavy inoculum may result in false-positive results.

When inoculating multiple biochemicals from the same culture, inoculate this medium first, or flame inoculating needle prior to streaking this medium. Carryover of glucose or other nutrients onto this medium may result in false-positive results.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Salmonella enterica
ATCC ® 14028
E 24-48hr 35°C Aerobic Growth; deep blue color in medium
Klebsiella pneumoniae
ATCC ® 13883
E 24-48hr 35°C Aerobic Growth; deep blue color in medium
Escherichia coli
ATCC ® 25922
E 24-48hr 35°C Aerobic Inhibited; no color change to blue

User Quality Control

PHYSICAL APPEARANCE

Simmons Citrate Agar should appear slightly opalescent with possible slight precipitate, and dark green in color.

S. typhimurium growing on Simmons Citrate Agar

Salmonella enterica (ATCC ® 14028) colonies growing on Simmons Citrate Agar (Cat. no. L80). The blue color change is indicative of citrate utilization. Incubated aerobically for 24 hours at 35ºC.

E. coli inhibited on Simmons Citrate Agar

Escherichia coli (ATCC ® 25922) growth inhibited on Simmons Citrate Agar (Cat. no. L80). Incubated aerobically for 24 hours at 35ºC.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

4. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

5. Koser, S.A. 1923. Utilization of the salts of organic acids by the colon-aerogenes group. J. Bacteriol., 8:493.

6. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

7. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

8. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

9. Simmons, J.S. 1926. A culture medium for differentiating organisms of typhoid-colon aerogenes groups and for isolation of certain fungi. J. Infect. Dis., 39:209.


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041116gr