Selective and Differential Media - Background & Procedure PDF

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Selective and Differential Media Many patient samples contain a mixture of different bacteria. The hospital laboratory often employs selective and/or differential media to aid in the separation and identification of these various species. We will explore 3 types of media: 1.) Phenylethyl Alcohol Agar (PEA) 2.) MacConkey Agar (MAC) 3.) Eosin Methylene Blue Agar (EMB) 4.) Mannitol Salt Agar (MSA) * Selective media = growth or no growth * Differential media = if the selective properties of the media allow growth, additional information about the bacteria can be determined. ****Some media are only selective and some media are both selective and differential. Be sure to understand and note which category each of the media you are using today falls into. Bacterial cultures used: Escherichia coli Proteus vulgaris Staphylococcus aureus Staphylococcus epidermidis

Procedure: 1. Each person obtains four of each of the following agar plates: Phenylethyl Alcohol Agar (PEA), Mannitol Salt Agar (MSA), MacConkey Agar (MAC), and Eosin Methylene Blue Agar (EMB). Label one set of plates for each of the four bacteria being tested with the type of agar and the bacteria you are testing. 2. Using a sterile swab, inoculate the first set of plates with E. coli. Repeat to inoculate the other 3 sets of plates with the remaining three bacteria to be tested. 3. Incubate at 37º C for 48 hours. 4. Observe the plates and record the results of all four bacteria.

Phenylethyl Alcohol Agar (PEA) Phenylethyl Alcohol Agar (PEA) is a selective medium. •

PEA selects for Gram positive organisms and inhibits the growth most other bacteria.

•

Selective ingredient - Phenylethyl Alcohol which functions by interfering with DNA synthesis in Gram negative organisms

Result Poor growth or no growth Good growth

Interpretation

Presumptive ID

Organism inhibited by Phenylethyl Alcohol

Gram negative

Organism not inhibited by Phenylethyl Alcohol

Gram positive

Mannitol Salt Agar (MSA) Mannitol salt agar (MSA) is a selective and differential medium. •

MSA selects for Staphylococcus species and inhibits most other bacteria.

•

Selective ingredient - 7.5% sodium chloride inhibits most bacteria other than Staphylococcus species.

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Pathogenic Staphylococcus aureus can be differentiated from nonpathogenic Staphylococcus species due the ability of Staphylococcus aureus to ferment the mannitol and create acid causing the pH indicator turns yellow.

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Differential ingredient - phenol red; a pH indicator that is yellow below pH 6.8, and red or pink at pH 6.8 and above. Result

Interpretation

Presumptive ID

a. Poor growth or no growth

Organism inhibited by NaCl

Not a Staphylococcus species

Organism not inhibited by NaCl

Is a Staphylococcus sp.

c. The media turns yellow around growth

Organism produces acid from mannitol fermentation

Possible pathogenic S. aureus

d. The media remains red around growth

Organism does not ferment mannitol

Nonpathogenic Staphylococcus

b. Good growth *If this is true then also c. or d.

MacConkey Agar (MAC) MacConkey agar (MAC) is a selective and differential medium. •

MAC selects for Gram negative bacteria.

•

Selective ingredients - bile salts and crystal violet inhibit growth of Gram positive bacteria.

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Organisms that can ferment lactose produce acid and turn red.

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Differential ingredient - neutral red is a pH indicator that is red at a pH less than 6.8 and colorless at a pH greater than 6.8.

Results

Interpretation

Presumptive ID

a. Poor growth or no growth

Organism inhibited by bile salts and/or crystal violet

Gram positive

b. Good growth *If this is true then also c. or d.

Organism not inhibited by bile salts or crystal violet

Gram negative

c. Pink to red growth

Organism produces acid from lactose fermentation

Lactose fermenter

Organism does not ferment lactose

Lactose non-fermenter

d. Colorless growth

Eosin Methylene Blue Agar (EMB) Eosin methylene blue agar (EMB) is a selective and differential medium. •

EMB selects for Gram negative bacteria.

•

Selective ingredient - eosin Y and methylene blue (both are dyes), inhibit growth of Gram positive bacteria.

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Organisms that can ferment lactose and/or sucrose produce acid and turn purple to black sometimes with a green sheen.

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Differential ingredient - under acidic conditions the eosin Y and methylene blue (both are dyes), form a dark purple complex which is sometimes accompanied by a green metallic sheen.

Result

Interpretation

Presumptive ID

a. Poor growth or no growth

Organism inhibited by eosin and/or methylene blue

Gram positive

b. Good growth *If this is true then also c., d. or e.

Organism not inhibited by eosin or methylene blue

Gram negative

c. Growth is pink

Organism ferments lactose and/or sucrose with little acid production

Possible coliform

d. Growth is purple to black, with or without green sheen

Organism ferments lactose and/or sucrose with much acid production

Probable coliform

Organism does not ferment lactose or sucrose

Noncoliform

e. Growth is normal color...