FERRIC CHLORIDE REAGENT, 10%

Cat. no. Z63 Ferric Chloride Reagent, 10% 15ml

INTENDED USE

Hardy Diagnostics Ferric Chloride Reagent, 10% is recommended for use in determining hippurate hydrolysis, phenylalanine deaminase, and tryptophan deaminase reactions in bacteria.

SUMMARY

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.

REAGENT FORMULA*

Ferric Chloride 100.0gm
Hydrochloric Acid, 37% 25.0ml
Deionized Water 975.0ml

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Upon receipt store 2-30ºC. Products should not be used if there are any signs of deterioration, discoloration, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

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.

Hippurate Test: 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 35ºC. 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.(1)

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 35ºC. 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.

INTERPRETATION OF RESULTS

A positive hippurate reaction is denoted by the appearance of a brown precipitate that persists for 10 minutes.

Positive phenylalanine and tryptophan deaminase reactions are denoted by the appearance of a green or brown color, respectively, within five minutes. Positive phenylalanine reactions must be interpreted immediately since the green color fades quickly.

LIMITATIONS

Hippurate hydrolysis, phenylalanine deaminase and tryptophan reactions may be used as aids is the identification of beta-hemolytic streptococci and gram-negative bacilli, respectively. Additional testing, using pure culture, is recommended for complete identification.

In the hippurate test, the final concentration of ferric ion is extremely important. It is recommended that titration of the amount of reagent to be used be performed in order to minimize false-positive reactions.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, Hippurate Broth (Cat. no. Z52), Phenylalanine media (Cat. no. L21), Tryptone Broth (Cat. no. R40), incinerators, incubators, pasteur pipets, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Results
Streptococcus agalactiae
ATCC® 12386
Hippurate Hydrolysis: Positive
Phenylalanine Deaminase: N/A
Tryptophan Deaminase: N/A

Streptococcus pyogenes
ATCC® 19615
Hippurate Hydrolysis: Negative
Phenylalanine Deaminase: N/A
Tryptophan Deaminase: N/A

Proteus mirabilis
ATCC® 12453
Hippurate Hydrolysis: N/A
Phenylalanine Deaminase: Positive
Tryptophan Deaminase: Positive

Escherichia coli
ATCC® 25922
Hippurate Hydrolysis: N/A
Phenylalanine Deaminase: Negative
Tryptophan Deaminase: Negative

User Quality Control

PHYSICAL APPEARANCE

Ferric Chloride Reagent, 10% should appear clear, and yellow in color.

P. mirabilis, positive phenylalanine reaction

Showing posistive phenylalanine deaminase reaction.
Ferric Chloride Reagent, 10% (Cat. no. Z63) added to Phenylalanine slant (Cat. no. L21) with heavy growth of Proteus mirabilis (ATCC® 12453). P. mirabilis was incubated aerobically for 24 hours at 35ºC.

E. coli, negative phenylalanine reaction

Showing negative phenylalanine deaminase reaction.
Ferric Chloride Reagent, 10% (Cat. no. Z63) added to Phenylalanine slant (Cat. no. L21) with heavy growth of Escherichia coli (ATCC® 25922). E. coli was incubated aerobically for 24 hours at 35ºC.

REFERENCES

1. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

2. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.


API is a registered trademark of bioMeriuex, France.
ATCC is a registered trademark of the American Type Culture Collection.

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