Christensen Citrate Sulphite Agar

Composition

Final pH (at 25°C): 6.7±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 24.29 grams in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Dispense into test tubes. Sterilize by autoclaving at 12 to15 lbs pressure (118 to 121°C) for 15 minutes. Cool the tubes in slanted position to give slants with generous butts.

Principle And Interpretation

Christensen Citrate Sulphite Agar is a modification of the Christensen Iron Agar (1). This modification was described by Edwards and Ewing (2). Christensen reported that all members of genera Escherichia, Enterobacter, Citrobacter and Salmonella as well as Alkalescens-Dispar were capable of utilizing citrate as a source of energy while Shigella species failed to utilize citrate.

Organisms that metabolize citrate as a sole source of carbon cleave citrate to oxaloacetate and acetate via the citritase enzyme. Another enzyme, oxaloacetate decarboxylase, then converts oxaloacetate to pyruvate and CO2. Further, this CO2 combines with sodium and water to form sodium carbonate, an alkaline compound (3). As a result, the pH of medium rises and the indicator, phenol red changes from orange red to cerise. Presence of the cerise colour indicates a positive finding for citrate utilization. Medium constituent yeast extract provide the necessary nutrients mainly nitrogenous and vitamins for the growth of the organisms.

L-Cysteine hydrochloride is a reducing agent. Dextrose is the fermentable carbohydrate. Sodium citrate is the energy source for citrate utilizing organisms. Care should be taken while inoculating, as, a too heavy inoculum may give a false positive result (4).

The reduction of ferric ammonium citrate to iron sulphide by H2S producing organisms is indicated by blackening of the medium. Sodium thiosulphate enhances H2S production. Strong positive cultures upon prolonged incubation turn the entire butt black. Some members of Salmonella like Salmonella Typhi are weakly positive and require 2-5 days for hydrogen sulphite production.

Quality Control

Appearance: Light yellow to light pink homogeneous free flowing powder

Gelling: Firm, comparable with 1.4% Agar gel.

Colour and Clarity of prepared medium

Orange red coloured, very slightly opalescent gel forms in tubes as slants

Reaction

Reaction of 2.42% w/v aqueous solution at 25°C. pH: 6.7±0.2

pH

6.50-6.90

Cultural Response

M495: Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours.

Storage and Shelf Life

Store below 30°C in tightly cloased container and the prepared medium at 2 - 8°C. Use before expiry date on the label.

Reference

1. Christensen W.B., 1949, Research Bull., Weld County Health Dept., Greenley Co., 1:3.
2. Edwards P.R. and Ewing W. H., 1955 and 1962, Identification of Enterobacteriaceae Minneapolis, Burgess Publishing Co., pg. 179 and 242.
3. Horward B., 1994, Clinical and Pathogenic Microbiology, 2nd ed., Mosby Year Book, Inc.
4. Branson D., 1972, Methods in Clinical Bacteriology, Springfield, III: C. Thomas, 15.