Hardy Diagnostics Ferric Chloride Reagent, 10% is recommended for use in determining hippurate hydrolysis, phenylalanine deaminase, and tryptophan deaminase reactions in bacteria.
The hippurate test has been recommended for use in the presumptive identification and differentiation of group B streptococci from other beta-hemolytic streptococci. In the hippurate test, hippurate is hydrolyzed to glycine and benzoic acid. The addition of acidified ferric chloride combined with benzoic acid form a brown precipitate of ferric benzoate.Phenylalanine and tryptophan deaminase reactions are used in the identification and differentiation of gram-negative bacilli. Oxidative deamination of these amino acids yields keto acids phenylpyruvate and indolepyruvate respectively, and ammonia. The addition of acidified Ferric Chloride Reagent to the keto acids produces green or brown colored compounds, respectively.
Specimen Collection: This product is not intended for primary isolation of patient specimens. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism.
Heavily inoculate 5ml of Sodium Hippurate Broth (Cat. no. Z52) with test organism. Concurrent isolation of positive and negative control organisms is also recommended. Incubate 24-48 hours at 35 degrees C. Centrifuge the medium to pack the cells and transfer 0.8ml of the clear supernatant to a small test tube. Add Ferric Chloride Reagent.Note: It is recommended that the amount of Ferric Chloride Reagent to be used in the assay be titrated to insure optimal performance.
Ferric Chloride Reagent Titration:
To 0.8ml amounts of uninoculated Hippurate Broth (Cat. no. Z52) control tubes, quickly add 4, 6, 8, and 10 drops of Ferric Chloride Reagent. Mix thoroughly. Let stand for 10 minutes with occasional shaking. The smallest quantity of acidified Ferric Chloride Reagent giving a clear solution is the quantity to be used in the assay. Examine for a precipitate which persists for longer that 10 minutes.
Phenylalanine or Tryptophan Deaminase Test:
Heavily inoculate Phenylalanine (Cat. no. L21) or Tryptophan (Tryptone Broth, Cat. no. R40) media with the test organism. Incubate 18-24 hours at 35 degrees C. Add 4 to 5 drops of Ferric Chloride Reagent directly to the culture. Examine for the appearance of a green color when using Phenylalanine media or a brown color when using Tryptophan (Tryptone Broth) media.If ferric chloride is to be used for a commercial identification test strip, such as API®, consult the manufacturer's literature
Cat. no. HDZ63