Endo Agar

There are actually two similar Endo agars, Endo agar and LES Endo agar.  They differ mainly in that LES Endo agar has added nutrients that support a wider growth of strains, but otherwise display similar results.  They are both Selective & Differential Media.  The selective and differential aspects are due to sodium sulfte, basic fuchsin, and lactose in each medium.  They are Selective because they encourage some bacteria to grow while inhibiting others.  Sodium sulfte and basic fuchsin generally inhibits Gram positive bacteria from growing (a few will grow) and generally allowing Gram negative organism to grow (some will not grow).

Both media are Differential because if lactose is utilized, it will create the intermediate acetaldehyde which reacts with the sodium sulfte and basic fuchsin will color the colonies pink/red from the acids produced during lactose fermentation.  These plates differentiate between species capable of utilizing  lactose and those unable to metabolize it.

Purpose:  to select for and isolate Gram negative organisms, to check for the presence of coliforms, and to differentiate among the family of Enterobacteriaceae.  Its main use is to detect for fecal contamination especially of water and dairy products.  Both the Gram negative selection and the detection of coliforms is imperfect, a small percentage of strains do not act as expected.

Procedure:  

  1. Appropriately label you plates.  Mark the dish bottom into thirds and label what species will be in each section.  It is best to label the side of the bottom plate or write on the bottom in tiny letters so that you will be able to observe the growth clearly.
  2. Inoculate one third with your unknown streak for isolated colonies if possible.  Isolated colonies work best for this test but it is not essential.
  3. Inoculate one third with E. coli, and the other third with S. epidermidis.  Generally it is best to keep E. coli well away from the other cultures because it may overgrow them.
  4. You will want to compare the growth on these plates to growth inoculated on a general purpose media such as NA. or TSA.
  5. Incubate your plates upside down in the incubator at 35-37 C for 1-2 days.
  6. Slow growing species may require a day or two of additional growth.
Results  

Compare your growth on these plates to growth on a general purpose media.  (If no growth on the general purpose media, discard your results and repeat the test.)  Look for the presence or absence of growth and if there is growth if it is reduced from normal.  It there is growth, check to see if the growth is pink or red in color.  One predicts that if an organism grows well on Endo Agar, it is most likely Gram negative, otherwise it is most likely Gram positive.  And if the growth is good and pink/red, it is most likely a coliform, otherwise it is likely a noncoliform.  Also look if the colonies have a metallic sheen, if they do the organism highly utilizes lactose.  Incidentally, a major difference between a coliform and a noncoliform in the family Enterobacteriaceae is simply if it can metabolize lactose.

References:

Difco Laboratories. 1998.  Difco Manual, 11th ed. Difco Laboratories, Sparks, MD, USA.

Harley J.P. and Prescott L.M. 2002.  Laboratory Exercises in Microbiology, 5th ed. McGraw-Hill Higher Education, New York, NY, USA.

Pommerville J.C.  2011.  Alcamo's Laboratory Fundamentals of Microbiology.  Jones & Bartlett Learning, Sudbury, MA, USA.

Instructor Notes:

Students should confirm their tentative conclusions from this test by performing a Gram stain and testing for lactose utilization.  Other species of bacteria may be used as positive and negative controls.  E. coli will have good pink/red growth with a metallic sheen, Enterbacter aerogenes will have good pink growth, and S. epidermidis will grow poorly if at all.  Instructors may wish to have students test several selective and differential media at the same time and then compare them all to growth on one general purpose media plate to save on the cost.