Patent GB-1172290-A (GB)
Title: Paper Strip Test for Citrate Utilization
IPC: C12Q1/04
Filed: 1967-11-20
Abstract: 1,172,290. Micro-biological testing. WARNER-LAMBERT PHARMACEUTICAL CO. Nov. 20, 1967 [April 24, 1967], No.52758/67. Heading C6F.  A diagnostic preparation for the rapid and positive detection of citrate utilization by microorganisms comprises a bibulous material containing four zones (A, B, C and D): zone A being impregnated with a reagent medium consisting essentially of sodium citrate, monobasic potassium phosphate and dibasic potassium phosphate in distilled water, the reagent medium being adjusted to a pH of about 6À4 with an acid or base which does not interfere with the micro-biological reaction, zone C being impregnated with an indicator system consisting essentially of Bromthymol Blue, methyl, ethyl, propyl or butyl alcohol, water-soluble Phenol Red and distilled water, the indicator system being adjusted to a pH of about 6À4 with an acid or base which does not interfere with the micro-biological reaction, and zones B and D being impregnated with a micro-biologically inert hydrophobic barrier composition; and zone A is contiguous only to zone B, zone B is contiguous only to zones A and C, zone C is contiguous only to zones B and D, and zone D is contiguous only to zone C and the untreated portion of the bibulous material. Reagent medium; 8-12g of sodium citrate, about 2À5g of monobasic potassium phosphate and about 0À5g of dibasic potassium phosphate in 100 ml distilled water and adjusted to pH 6À4 by IN hydrochloric, nitric or phosphoric acid, or sodium, potassium or ammonium hydroxide, respectively. Indicator system: 0À18-0À22g of Bromthymol Blue, 22À5-27À5 ml methyl, ethyl, propyl or butyl alcohol, 0À18-0À22g of water-soluble Phenol Red and a sufficient amount of distilled water to bring the volume to 100 ml. The pH is adjusted in two stages by the addition of 0À1N sodium hydroxide to a solution of Bromthymol Blue in the alcohol, and to which part of the distilled water has been added, for obtaining a measured pH of 6À4 and subsequently, after the addition of the Phenol Red and sufficient distilled water to bring the volume to 100 ml, by the addition of 0À1N hydrochloric acid to obtain a pH of 6À4 for the final indicator system. Barrier composition: waxes, lacquers or plastics, preferably 75-100ml of a colourless polymerized methyl methacrylate coating composition in a toluene vehicle and 0-25ml. ethyl alcohol. Bibulous materials: filter paper, felt, porous ceramic strips, and woven or matted glass fibre. A sheet of paper 82À4mm. by 157À5mm. is used as the stock for making 25 strips 6À3mm. wide. Five bands are marked across each strip viz. reagent zone (14mm.), barrier zone (12À7mm.), indicator zone (14mm.), barrier zone (12À7mm.), and blank zone (29mm.).  The barrier composition is first applied to the barrier zones and then, after drying, the reagent mixture and indicator system are applied to their respective zones. The diagnostic strips are packed in a test tube containing a desiccant. In operation, the reagent zone is allowed to come into contact with a saline suspension of an unknown culture in a test tube which is incubated for four hours at 37‹C., after this the test tube is momentarily tilted in order to wet the indicator zone with the suspension of  organisms and 30 minutes are allowed to elapse for the colour to develop as an indication of the utilization, whether positive (purple) or negative (brown). The test enables Salmonella and Escherichia coli to be differentiated from Shigella and Aerobacter-Klebsiella, respectively.