22.2: Selective and Differential Media - MacConkey, EMB, MSA

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MacConkey Agar (1)

Purpose: Selective and differential medium; identification of Enterobacteriaceae

Media: Contains bile salts to inhibit most Gram (+) bacteria except Enterococcus and some species of Staphylococcus, peptone, and lactose.

Reagents/Indicators: Contains crystal violet and bile salts, which inhibit Gram (+) bacteria, and neutral red dye, which stains microbes fermenting lactose (and thereby decreasing the pH) a pink color.

Figure \(\PageIndex{1}\): Lactose fermenter(left) and non-lactose fermenter on MacConkey Agar
https://commons.wikimedia.org/wiki/File:MacConkey_agar_with_LF_and_LF_colonies.jpg

Mechanism/reactions: By utilizing the lactose available in the medium, Lac+ bacteria such as Escherichia coli, Enterobacter and Klebsiella will produce acid, which lowers the pH of the agar below 6.8 and results in the appearance of red/pink colonies. Non-Lactose fermenting bacteria such as Salmonella, Proteus species and Shigella cannot utilize lactose, and will use peptone instead. This forms ammonia, which raises the pH of the agar, and leads to the formation of white/colorless colonies.

Directions: Streak agar in a straight line and incubate for 24 – 48 hours.

Interpretation:

(+) = Lactose fermentation, re/pink colonies

(Slow) = Some organisms ferment lactose slowly or weakly, and are sometimes put in their own category – these include Serratia and Citrobacter

(-) = non-lactose fermenters, white/colorless growth

EMB Agar (2)

Purpose: Selective and differential medium; identification of Enterobacteriaceae. Used primarily to distinguish coliform from non-coliform bacteria in water testing.

Media: Eosin, Methylene Blue, lactose, sucrose

Reagents/Indicators: Eosin Y and Methylene Blue

Mechanism/reactions: Selects for Gram Negative bacteria, and differentiates those enterics which ferment lactose (coliforms) from those which do not ferment lactose (non-coliforms). Indicators form a dark purple precipitate at low pH (due to fermentation products) and also inhibit gram positive bacteria. E. coli will often produce a green metallic sheen due to strong fermentation and precipitation of acid and indicator complex.

Figure \(\PageIndex{2}\): Two lactose fermenters growing on EMB. K.C.Burke CC BY-NC SA

Directions: Streak agar in a straight line and incubate for 24 – 48 hours.

Interpretation:

(+) = Lactose fermentation, dark purple colonies with dark center. Weak fermenters will have pink mucoid growth.

Green sheen = vigorous fermentation of lactose

(-) = non-lactose fermenters, colorless (or very faint pink) growth.

Mannitol Salt Agar (3)

(Optional during the unknowns: may be requested with justification for its use.)

Purpose: Selective and differential; identification of pathogenic Staphylococci

Media: Mannitol Salt Agar (MSA) contains mannitol, 7.5% sodium chloride, and phenol red

Reagents/Indicators: Phenol red

Figure \(\PageIndex{3}\): Staphylococcus aureus growing on MSA and exhibiting the typical yellow halo, K.C.Burke CC BY-NC SA

Mechanism/reactions: Salt concentration will inhibit most other organisms so the media is selective for staphylococci. Mannitol is fermented by Staphylococcus aureus and will cause a pH change in the medium (acidic) that is detected by observing phenol red changing to yellow.

Directions: Streak agar in a straight line and incubate for 24 – 48 hours.

Interpretation:

(+) = Growth and yellow halo surrounding it (also record growth/no color)

(-) = No growth, no color change

Materials

Gloves
Student stock organisms
1 plate MAC/GNR
1 plate EMB/GNR
1 plate MSA/ GPC plus Staphycoccus aureus
Staphycoccus aureus

Procedures

Inoculate each GNR onto MAC
Inoculate Each GNR onto EMB
Inoculate each GPC and Staphycoccus aureus onto MSA

Safety

Staphylococcus aureus is a BSL2 organism. Wear glove while handling.

Results

Give the results for the inoculated bacteria. Be careful to not only enter +/-, but also a short description of the colony characteristics on each media.

Bacterium	MAC	EMB	MSA

Post-lab Questions

Compare and contrast MAC and EMB.
Is it normal to use both MAC and EMB when identifying a bacterium? Why or why not?
What similarities and differences did you observe in your results with MAC and EMB?
What is the primary purpose of MSA?
What differences between the different GPCs did you notice on MSA?
Why is MSA optional only during the unknowns?

Conclusion

Explain how MAC, EMB, and MSA are selective and differential media, and predict how this will help you during identification of unknowns.

Resources

Mary E. Allen. 2005. Macconkey agar plates protocols. Web. 1 October 2016 http://www.asmscience.org/content/education/protocol/protocol.2855
Archana Lal, Naowarat Cheeptham. 2007. Eosin-methylene blue agar plates protocol. Web. 1 October 2016 http://www.asmscience.org/content/education/protocol/protocol.2869
Patricia Shields, Anne Y. Tsang. 2006. Mannitol salt agar plates protocols. Web. 1 October 2016 http://www.asmscience.org/content/education/protocol/protocol.3034

Contributors and Attributions

Kelly C. Burke (College of the Canyons)