Phenylethyl Alcohol (PEA) Agar: Composition, Principle, Uses, and Applications

A peritoneal fluid specimen from a patient with abdominal sepsis is plated on blood agar, MacConkey agar, and phenylethyl alcohol blood agar. MacConkey grows several enteric gram-negatives. PEA blood agar, incubated anaerobically, grows Bacteroides fragilis — the clinically most important anaerobe in intra-abdominal infection — that was completely obscured on blood agar by the swarming Proteus mirabilis growing in the same specimen.

This is PEA agar's most important clinical role: suppressing the swarming of Proteus and Clostridium septicum to prevent them from overgrowing slow-growing anaerobes and gram-positive organisms in mixed anaerobic cultures.

Phenylethyl alcohol agar (PEA) is a selective medium used to cultivate Gram-positive organisms, particularly cocci, from a sample containing a mixture of pathogens. The active ingredient, phenylethyl alcohol, inhibits or markedly reduces the growth of Gram-negative organisms by interfering with DNA synthesis. Staphylococcus aureus, a Gram-positive organism, grows on PEA while Serratia marcescens, a Gram-negative organism, does not.

Principle

Phenylethyl alcohol agar (PEA) is a selective medium that permits the growth of gram-positive cocci while inhibiting most gram-negative organisms. PEA alters the membrane permeability, of Gram-negative bacteria allowing influx of otherwise blocked molecules. This results in leakage of large amounts of cellular potassium that ultimately results in disruption or inhibition of DNA synthesis of Gram-negative bacteria.

Composition of PEA

PEA agar medium can be purchased as a premixed powder from suppliers. The manufacturer’s instructions should be followed to prepare the plates. This media can also be purchased as premade agar plates.

Final pH at 25°C 7.3 ± 0.2.

Preparation of the Media

Phenylethyl alcohol agar (PEA) may be prepared with and without 5% sheep blood supplement. Five percent sheep blood is added to the base medium to enhance the growth of anaerobic bacteria.

Step no. 5 is required while preparing PEA with 5% sheep blood supplementation.

Note:

Culture

Inoculation

Stored media should be brought to room temperature before inoculation.

Aseptically transfer potentially mixed cultures onto the surface of the agar using a streaking or spreading technique, depending on the objectives of the study.

Incubation

Incubate plates for 24 to 48 hours at 35°C ± 2 °C in an appropriate atmosphere. Depending on the objectives of the study, PEA blood agar plates can be incubated under aerobic, anaerobic, and 5% CO2 atmosphere. Incubation in a high CO2 atmosphere allows the detection of bacteria that require an increased CO2 concentration and also results in better growth of almost all of the other pathogens.

Interpretation of results

After proper incubation growth of bacteria will appear as colonies on the surface of agar plates. Gram-positive bacteria demonstrate good growth while most gram-negative bacteria do not grow or are partially inhibited.

Figure: PEA agar plates with 5% sheep blood incubated at 48 hours at 35°C Left: Escherichia coli. Right: Staphylococcus aureus Image source: Cheeptham and Farday, ASM MicrobeLibrary

Growth Response

Growth response of some gram-positive and gram-negative bacteria on PEA agar

Figure: PEA agar plates without blood incubated at 48 hours at 35°C Left: Escherichia coli. Right: Enterococcus faecalis Image source: Cheeptham and Farday, ASM MicrobeLibrary

Uses

• PEA agar is used if the sample source contains a mixture of pathogens (e.g., gastrointestinal content or peritoneal fluid) or Gram stain indicates that the culture contains Gram-negative rods. Phenylethyl alcohol agar inhibits gram-negative bacteria, specifically Proteus species, in specimens containing mixed bacterial flora. It is used for the selective growth of Staphylococcus and Streptococcus inmixed cultures.
• PEA agar with 5% sheep blood is used to isolate most gram-positive and gram-negative anaerobes from enteric samples. It inhibits facultative gram-negative rods, preventing Enterobacteriaceae from overgrowing the anaerobes and inhibiting the swarming of Proteus and Clostridium septicum.

PEA vs Columbia CNA Agar — Which to Choose?

Both PEA and Columbia CNA select for gram-positive organisms, but they use different mechanisms and have different strengths:

Key rule: Use PEA for anaerobic cultures and specimens with swarming Proteus. Use Columbia CNA for aerobic gram-positive isolation from mixed aerobic specimens.

Limitations

• Some gram-positive cocci may be slightly inhibited by PEA and many require incubation up to 48 hours for sufficient growth to be visible.
• Pseudomonas aeruginosa (a gram-negative bacteria) is not inhibited on this medium. Other gram-negatives sometimes may give tiny observable colonies but they are often confined to the first quadrant on a streak plate.

Key Exam Facts in One Table

References and further readings

1. Lal A, Cheeptham N. Phenylethyl alcohol agar protocol. American Society for Microbiology; 2011.
2. Garcia LS. Clinical Microbiology Procedures Handbook. 4th ed. ASM Press; 2016.
3. Forbes BA, Sahm DF, Weissfeld AS. Bailey & Scott's Diagnostic Microbiology. 14th ed. Elsevier; 2023.
4. Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology. 9th ed. Elsevier; 2020.