Phenylethyl Alcohol Agar: Composition, Preparation, Uses

Last updated on June 21st, 2021

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.

IngredientsAmounts (gm/L)
Pancreatic digest of casein15.0 g
Papic digest of soybean meal5.0 g
Sodium chloride5.0 g
Agar15.0 g
Sterile defibrinated sheep blood50.0 ml
β-Phenylethyl alcohol2.5 g
Distilled water1 liter

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.

  1. Suspend the ingredients in one liter of distilled water and mix thoroughly
  2. Heat with frequent agitation and boil for 1 minute to dissolve completely.
  3. Autoclave for 15 min at 121°C and 15 lb/in^2,
  4. Cool to about 45°C
  5. Aseptically add sterile 5% sterile defibrinated sheep blood, and mix well.
  6. Pour the melted medium into sterilized Petri plates (approximately 20 to 30 ml per plate) and let it solidify.

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

Note:

CharacteristicsPEA without sheep bloodPEA with 5% sheep blood
Color of the prepared mediaclear to slightly hazy and pale yellow.firm, opaque, and red in color.
Shelf liveprepared plates can be stored in the refrigerator for up to 4 weeks before use.prepared plates could be stored in the refrigerator up to 1 week before use.

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.

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

OrganismGram reactionGrowth response
Escherichia coliGram-negativeInhibited
Enterobacter aerogenesGram-negativeInhibited
Proteus mirabilisGram-negativeMarkedly inhibited. Swarming inhibition.
Pseudomonas aeruginosaGram-negativePartially inhibited
Salmonella enteritidisGram-negativeInhibited
Bacillus sp.Gram-positiveGood
Clostridium perfringensGram-positivePartially inhibited
Enterococcus faecalisGram-positiveGood
Micrococcus luteusGram-positiveGood
Staphylococcus aureusGram-positiveGood
Streptococcus pneumoniaeGram-positiveGood
Streptococcus pyogenesGram-positiveGood
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 in
    mixed 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.

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.
  • Pesudomonas 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.

References and further readings

  • Archana Lal, Naowarat Cheeptham. 2011. Phenylethyl alcohol agar protocol. American Society for Microbiology
  • Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology. https://doi.org/10.1128/9781555818814
About Acharya Tankeshwar 473 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

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