Simmons Citrate Agar
Intended Use:
Recommended for differentiation the members of Enterobacteriaceae on the basis of citrate utilization from clinical and
non clinical samples.
Composition**
Ingredients Gms / Litre
Magnesium sulphate 0.200
Ammonium dihydrogen phosphate 1.000
Dipotassium phosphate 1.000
Sodium citrate 2.000
Sodium chloride 5.000
Bromothymol blue 0.080
Agar 15.000
Final pH ( at 25°C) 6.8±0.2
Directions
Suspend 24.28 grams in 1000 ml purified/ distilled water. Heat, to boiling, to dissolve the medium completely. Mix well and
distribute in tubes or flasks. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
Precaution: Before using water, ensure pH of water is 6.5 to 7.0. Initial colour of the medium may deviate from
expected colour, if the above precaution is ignored.
Principle And Interpretation
These media are used for the differentiation between Enterobacteriaceae and the members of aerogenes group on the basis
of citrate utilization as sole carbon source. Initially the citrate medium was developed by Koser (1) containing ammonium salt
as the only nitrogen source and citrate as the only carbon source for differentiating Escherichia coli and Enterobacter
aerogenes by IMViC tests. Later on Simmons (2) modified Kosers formulation by adding agar and bromothymol
blue (3). It is recommended by APHA (4).
Ammonium dihydrogen phosphate and sodium citrate serve as the sole nitrogen and carbon source
respectively. Microorganisms also use inorganic ammonium salts as their sole nitrogen source. Metabolism of these
salts causes the medium to become alkaline, indicated by a change in colour of the pH indicator from green to blue.
Bromothymol blue is the pH indicator. The medium should be freshly prepared because in dry conditions, changes in
colour may appear even before inoculation, especially at the bottom of the slant.
Type of specimen
Isolated microorganism from clinical and non clinical samples.
Specimen Collection and Handling
For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (5).
For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (6,7,8). For
clinical samples follow appropriate techniques for handling specimens as per established guidelines (9,10).
After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions
In Vitro diagnostic use. For professional use only. Read the label before opening the container. Wear
protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices
while handling specimens and culture. Standard precautions as per established guidelines should be followed while
handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.
Limitations
1.Before using water, ensure pH of water is 6.5 to 7.0.Initial colour of the medium may deviate from expected colour, if the
above precaution is ignored.
2.The pH affects the performance of the medium and must be correctly monitored.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at
recommended temperature
Quality Control
Appearance
Cream to yellow homogeneous free flowing powder.
Gelling
Firm, comparable with 1.5% Agar gel
Colour and Clarity of prepared medium
Forest green coloured slightly opalescent gel forms in tubes as slants.
Reaction
Reaction of 2.43% w/v aqueous solution at 25°C. pH : 6.8±0.2.
pH
6.60-7.00
Cultural Response
Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours.
Organism Inoculum Growth
(CFU)
Citrate
utilisation
# Klebsiella aerogenes
ATCC 13048 (00175*)
50-100 good-luxuriant positive
reaction, blue
colour
Escherichia coli ATCC
25922 (00013*)
=10 inhibited 4
Salmonella Typhi ATCC
6539
50-100 fair-good negative
reaction, green
colour
Salmonella Typhimurium
ATCC 14028 (00031*)
50-100
Shigella dysenteriae ATCC
13313
=10 inhibited 4
Salmonella Choleraesuis
ATCC 12011
50-100
50-100 good-luxuriant positive reaction,
blue colour
Salmonella Enteritidis
ATCC 13076 (00030*)
Key: * Corresponding WDCM numbers
# Formerly known as Enterobacter aerogenes
good-luxuriant positive reaction,
blue colour
good-luxuriant positive reaction,
blue colour
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and the prepared medium at 2 - 8°C. Use before expiry date on the label.
On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to
the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated
area protected from extremes of temperature and sources of ignition Seal the container tightly after use. Product performance
is best if used within stated expiry period.
Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow
established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical
sample must be decontaminated and disposed of in accordance with current laboratory techniques (9,10).
