USE OF SELECTIVE AND

DIFFERENTIAL MEDIA

Materials

Working in groups of two, obtain a 24-48 hour mixed culture containing Staphylococcus epidermidis, Proteus vulgaris and Escherichia coli, and one set of 3 plating media:  EMB agar, Mannitol Salt agar, and McConkey agar.

Media

Eosin and methylene blue agar (EMB agar) contains the dyes eosin and methylene blue.  They inhibit Gram-positive organisms.  Such a medium is selective for Gram-negative species.  Lactose-fermenting organisms such as E. coli produce a black precipitate on EMB.  Their colonies will be either black or possess dark centers with transparent, colorless peripheries.  Non-lactose fermenters such as Proteus sp., Salmonella sp., or Shigella sp. appear pink or uncolored.  Thus, the medium is considered differential with respect to lactose fermentation.

MacConkey agar similar to EMB agar in that it is also selective for Gram-negative species and differential with respect to lactose fermentation. MacConkey agar is used for the detection of coliforms and enteric pathogens based on their ability to ferment lactose.  Lactose-fermenting bacteria appear red to pink while non-lactose fermenting bacteria appear as colorless or transparent colonies. 

Mannitol salt agar (7.5% NaCl) is a medium selective  for staphylococci and differential with respect to pathogenicity.  Growth of most bacteria other than staphylococci, which are halophilic, is inhibited by the high concentration of salt in the medium.  Pathogenic staphylococci fermenting the mannitol sugar turn the phenol red indicator bright yellow.  Non-pathogenic staphylococci produce small colonies surrounded by red or purple zones.

Student Instructions

DAY 1

1.  Prepare gram stains of mixed culture, observe and report findings.

2.  Streak mixed culture on each medium, incubate until next lab.

DAY 2

1.  Pick and stain representative colonies from each plate, record and observe findings.

2.  Discuss implications