A selective medium called phenylethyl alcohol (PEA), is used to cultivate Gram positive organisms, especially cocci, in a sample containing pathogens. Phenylethyl alcohol is the active ingredient that inhibits or significantly reduces growth of Gram-negative organisms. It interferes with DNA synthesis. Staphylococcus aureus is a Gram positive organism that grows on PEA, while Serratia marcescens is a Gram negative organism.

Principle of Phenylethyl Alcohol Agar

A selective medium, Phenylethyl alcohol (PEA), allows the growth of gram positive cocci and inhibits most gram negative organisms. PEA changes the membrane permeability of Gram-negative bacteria, allowing for the influx of other molecules. This causes large amounts of cellular potassium to leak into the cells, which ultimately leads to disruption or inhibition in DNA synthesis in Gram-negative bacteria.

Composition of PEA

Suppliers can purchase PEA agar medium as a premixed product. To prepare the plates, you should follow the manufacturer’s instructions. Premade agar plates can also be made from this media.

Ingredients	Amounts (gm/L)
Pancreatic digest of casein	15.0 g
Papic digest of soybean meal	5.0 g
Sodium chloride	5.0 g
Agar	15.0 g
Sterile defibrinated sheep blood	50.0 ml
β-Phenylethyl alcohol	2.5 g
Distilled water	1 liter

Final pH at 25°C 7.3 ± 0.2.

Preparation of the Media

You can make phenylethyl alcohol (PEA) with or without 5% sheep’s blood. To enhance the growth anaerobic bacteria, five percent sheep blood can be added to the base medium.

1. Mix the ingredients in 1 liter of distilled Water.
2. To dissolve the mixture completely, heat with constant stirring.
3. Autoclave for 15 minutes at 121°C and 15 lb/in2.
4. Keep it at 45 degrees Celsius
5. Mix well.
6. Allow the melted medium to cool in sterilized Petri dishes.

Step 5. is required when preparing PEA with 5% sheepblood supplementation.

Note:

Characteristics	PEA without sheep blood	PEA with 5% sheep blood
Color of the prepared media	clear to slightly hazy and pale yellow.	firm, opaque, and red in color.
Shelf live	prepared 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 Technique

Inoculation

Before inoculation, store media should be at room temperature.

Aseptically transfer potential mixed cultures onto the surface using a spreading or streaking technique depending on the purpose of the study.

Incubation

Plates should be incubated for between 24 and 48 hours at 35degC +-2 degC in an appropriate environment. According to the purpose of the study, PEA blood-agar plates can either be incubated under anaerobic or aerobic conditions. High CO2 conditions allow bacteria to be detected and can also promote the growth of most other pathogens.

Interpretation of results

The colonies will form on the agar plates after proper incubation. While Gram-positive bacteria can grow well, most gram-negative bacteria cannot or are only partially inhibited.

Growth Response

Growth response to some gram positive and gram negative bacteria on PEA agar

Organism	Gram reaction	Growth response
Escherichia coli	Gram-negative	Inhibited
Enterobacter aerogenes	Gram-negative	Inhibited
Proteus mirabilis	Gram-negative	Markedly inhibited. Swarming inhibition.
Pseudomonas aeruginosa	Gram-negative	Partially inhibited
Salmonella enteritidis	Gram-negative	Inhibited
Bacillus sp.	Gram-positive	Good
Clostridium perfringens	Gram-positive	Partially inhibited
Enterococcus faecalis	Gram-positive	Good
Micrococcus luteus	Gram-positive	Good
Staphylococcus aureus	Gram-positive	Good
Streptococcus pneumoniae	Gram-positive	Good
Streptococcus pyogenes	Gram-positive	Good

Quality Control 

After checking for correct pH, colour, depth, and sterility, the following organisms are used to determine the growth performance of the completed medium. 

Organism	Expected Result
Streptococcus pyogenes ATCC 19615	Growth
Staphylococcus aureus ATCC 25923	Growth
Proteus mirabilis ATCC 12453	Partial Inhibition

Uses of Phenylethyl Alcohol Agar (PEA)

• If the source of the samples contains multiple pathogens, such as peritoneal fluid or gastrointestinal contents, PEA agar can be used. Gram staining indicates that the culture has Gram-negative rods. In specimens with mixed bacterial flora, phenylethyl alcohol-agar inhibits Gram-negative bacteria, particularly Proteus species. It is used to selectively grow Streptococcus and Staphylococcus in mixed cultures.
• PEA agar containing 5% sheep blood can be used to isolate most gram positive and gram negative anaerobes from enteric specimens. It prevents facultative gram negative rods from growing, which is important for Enterobacteriaceae to avoid overgrowing the anaerobes. Also, it inhibits the swarming by Clostridium septicum and Proteus.

Limitations

• Some gram-positive cocci can be inhibited slightly by PEA. Many require incubation for up to 48 hours to allow sufficient growth to be visible.
• This medium does not inhibit Pesudomonas Aeruginosa, a gram-negative bacteria. While some gram-negatives may produce tiny colonies, they are usually limited to the first quadrant of a streak plate.