Phenylalanine Agar

Intended Use

Recommended for the differentiation of Proteus and Providencia group of organisms from other members of Enterobacteriaceae on the basis of their ability to form phenyl pyruvic acid from phenylalanine.

Composition

Final pH (at 25°C): 7.3±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 26.0 grams in 1000 ml purified/distilled water. Heat to boiling to dissolve the medium completely. Dispense in tubes and sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Allow the tubed medium to cool in slanting position.

Principle And Interpretation

The ability of Proteus species to convert phenylalanine to phenylpyruvic acid is an important reaction in the differentiation of Enterobacteriaceae (1,2). Based on this criterion, Buttiaux developed Phenylalanine Agar for differentiation of Proteus and Providencia group from other members of Enterobacteriaceae (3,4) by the ability of organism in the genera within Proteus to deaminate phenylalanine. Phenylalanine Agar is the modification of the medium originally developed by Ewing et al (5). Yeast extract in the medium supports the growth of the organisms. Sodium chloride maintains osmotic equilibrium. The phenylalanine serves as the substrate for enzymes, which are able to deaminate it to form phenylpyruvic acid. A recommended technique is to inoculate the slant surface with plenty of inoculum and incubate it for 12-16 hours. After incubation, add 0.2 ml of 10% ferric chloride solution so that the solution floods all over the growth. The addition of (0.2 ml 3-5 drops) of a 10% aqueous ferric chloride solution (or a 12% aqueous ferric chloride solution acidified with 2.5 ml of concentrated HCl per 100 ml of reagent) to the cultures following incubation results in the appearance of a light to deep green color (positive reaction) or no color change (negative reaction). In a positive reaction, any phenylpyruvic acid present will react with the ferric salt in the reagent to give a green color. Interpret the results within 5 minutes upon addition of reagent as the green colour fades quickly (2,4).

Type of specimen

Isolated Microorganisms

Specimen Collection and Handling

A recommended technique is to inoculate the slant surface with plenty of inoculum and incubate it for 12-16 hours. After incubation, add 0.2 ml of 10% ferric chloride solution so that the solution floods all over the growth. The addition of (0.2 ml 3-5 drops) of a 10% aqueous ferric chloride solution (or a 12% aqueous ferric chloride solution acidified with 2.5 ml of concentrated HCl per 100 ml of reagent) to the cultures following incubation results in the appearance of a light to deep green color (positive reaction) or no color change (negative reaction). In a positive reaction, any phenylpyruvic acid present will react with the ferric salt in the reagent to give a green color. Interpret the results within 5 minutes upon addition of reagent as the green colour fades quickly (2,4). After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

In Vitro diagnostic use only. For professional use only. Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

1. Some organism may show poor growth due to nutritional variation.
2. Other biochemical tests must be carried out in conjunction for confirmation.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance: Cream to yellow homogeneous free flowing powder

Gelling: Firm, comparable with 1.5% Agar gel

Colour and Clarity of prepared medium: Light amber coloured slightly opalescent gel forms in tubes as slants

Reaction: Reaction of 2.6% w/v aqueous solution at 25°C. pH : 7.3±0.2

pH: 7.10-7.50

Cultural Response: Cultural characteristics observed after an incubation at 35-37°C for 12-16 hours

Key : *Corresponding WDCM numbers, ## Formerly known as Proteus vulgaris # Formerly known as Enterobacter aerogenes

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use.

Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with sample must be decontaminated and disposed of in accordance with current laboratory techniques (6,7).

Reference

1. Henrikson S. D., 1950, J. Bacteriol., 60:225.
2. Singer J. and Volcani B. E., 1955, J. Bacteriol., 69:303.
3. Buttiaux R., Osteux R., Fresnoy R. and Moriamez J., 1954, Ann. Inst. Pasteur Lille., 87:375.
4. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams & Wilkins, Baltimore, Md.
5. Ewing W. H., Davis B. R. and Reavis R. W., 1957, Public Health Lab., 15:153.
6. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
7. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.