Phenolphthalein Starch Medium for Rapid Isolation of V. Cholerae

Abstract

Among the various media that have been advocated for rapid isolation of V. cholerae, the most widely used is the alkaline peptone enrichment medium which, however, is by no means perfect. During a cholera epidemic in Peiping in the summer of 1932, we felt that a more efficient medium was desirable. To meet this demand the following medium (phenolphthalein-starch solution) was devised, utilizing the unique property of rapid fermentation of starch by V. cholerae 1 in a highly alkaline solution in combination with an indicator as an index for its growth. As the cholera epidemic was over when the technique of preparation of this medium had been perfected, its efficiency was tested out on stools seeded with V. cholerae and gave satisfactory results.

The medium is prepared by dissolving peptone (Witte) 2 gm., maltose 1 gm., potassium nitrate 0.5 gm., sodium carbonate (crystallized) 0.5 gm., sodium chloride 10 gm., and magnesium chloride (crystallized) 0.5 gm. in 900 cc. of distilled water. The solution is boiled for 3 minutes and filtered. 100 cc. of a 5% solution of soluble starch previously boiled for 2 minutes is added to 900 cc. of the above solution and mixed thoroughly. The mixture is filtered through cotton and then through asbestos filter, yielding a clear filtrate which is first measured and then sterilized by boiling for 3 minutes. (The solution cannot be autoclaved in the ordinary way as the starch is decomposed by prolonged heating). The sterilized filtrate is kept in ice chest. Just before use the reaction of the solution is adjusted to pH 9.0-9.2 in the following way: First 5 cc. of the indicator phenolphthalein (saturated solution in 50% alcohol) is added to each litre of the filtrate and the solution is carefully brought to decolorization point (pH 8.3) by addition of necessary amount of N/20 HCl or N/20 NaOH and finally to each litre of the decolorized solution is added exactly 1.5 cc. of N/I NaOH which brings the solution to pH 9.0-9.2 (pink colored).