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Selective and Differential Media

Selective media contains certain compounds or chemicals that favor the growth of certain species of bacteria.  Selective media is often used when one is trying to isolate a specimen of interest from a mixed culture.  Let us say that the specimen of interest is one cell in a million.  It is unlikely that anyone could isolate such a rare specimen from a mixed sample.  Yet, if one enriches for the specimen of interest with a selective media, isolation of the organism of interest may become much more likely if not probable.  Selective media is often used when trying to find the rare pathogen from a patient's numerous mixed normal flora.  As some organisms inhibit or grow faster than others, using selective media may be required.  (MSA, MAC, and PEA are all selective media.)

Differential media contains certain indicators (compounds) that allow one to differentiate between different species of bacteria.  The test is often positive or negative or has a color change or does not, allowing one to differentiate between two species.  For example, Staphylococcus aureus when grown on MSA ferments the sugar mannitol, but Staphylococcus epidermidis does not.  (MSA is both a differential media and a selective media.  MAC is also selective and differential.)

It is recommended that students streak their unknown culture, E. coli, and S. epidermidis onto the following selective and differential media, but other species may be used.  E. coli is positive for growth and lactose metabolism on MAC and is negative for growth on PEA and MSA.  S. epidermidis is negative for growth on MAC, is positive for growth on PEA and MSA.

Purpose:  To test your unknown bacteria on several differential and selective media.  You will need to compare this growth to growth on a control TSA or NA plate.

I.  Inoculate a TSA or NA Plate as a Control

TSA (Trypticase Soy Agar) and NA (Nutrient Agar) are General Purpose Media that are not selective or differential, their purpose is simply to grow many types of bacteria cheaply.  Here we will use a general purpose media to compare the growth on the selective and differential media.  If you obtain growth on TSA or NA, but not on a selective or differential media, your results are most likely valid.  Yet, if you do not get growth on TSA or NA, then your results on the other media are in question.

Purpose:  to compare your growth observed on this "control media" to your growth on the selective and differential media.

Procedure:  

  1. Mark the dish bottom into thirds and label what each third will be, adding the date and your name.  
  2. 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.
  3. Inoculate one third with your unknown using a single quick streak.  Generally, a W or Z pattern works best.
  4. 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.  ( Streak only one species/third, don't mix the bacteria!  Do not isolate for colonies!  See below for how to do this quickly.)
  5. Incubate your plates upside down in the incubator at 35-37 C for 1-2 days.
Results -You are looking for positive growth for all species.  Compare this growth to growth in your other plates.

II.   Inoculate a MacConkey Agar (MAC) plate

MacConkey Agar (or MAC) is a Selective & Differential Medium.  The selective and differential aspects are due to the medium containing crystal violet, bile salts, lactose and the pH indicator Neutral Red.  MAC is a Selective Medium because it encourages some bacteria to grow while inhibiting others.  Crystal violet and bile salts apply the selection, generally inhibiting Gram positive bacteria (though a few will grow) and generally allowing growth of Gram negative organism (though a few will not grow).

Mac is a Differential Medium because it contains the sugar lactose and a pH indicator that expose differences between species.  These plates are used for distinguishing enteric coliforms (diarrheal disease, stool samples, etc.) from enteric noncoliforms.  MAC differentiates between species capable of utilizing  lactose and those unable to metabolize it.  If an organism metabolizes lactose, acids are produced which will change the color of Neutral Red to pink (Neutral Red is colorless above pH 6.8 and pink below pH 6.8).

Purpose:   to compare your growth observed on this media to your "control media," and for isolating Gram negative organisms from a mixed culture.  Also used to differentiate among the Gram negatives, especially among the enterics.

Procedure:  

  1. Mark the dish bottom into thirds and label what each third will be, including the date and your name.  
  2. 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.
  3. Inoculate one third with your unknown using a single quick streak.  Generally, a W or Z pattern works best so that you can observe in between the streak.
  4. 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.  (Streak only one species/third, don't mix the bacteria!  There is no need to isolate for colonies!  See later for how to do this quickly.)
  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  

Look for presence or absence of growth and if there is growth if the growth is bright pink or red.  Compare your growth on the MAC plate to growth on either TSA or NA.  Generally, MAC plates are pretty good about inhibiting the growth of Gram positive cells.  Usually the growth is absent not just inhibited a little.  One predicts that if an organism grows on MAC it is most likely Gram negative, but this has to be confirmed with a Gram stain, as a few Gram positive organisms will grow on Mac plates.  Incidentally, a major difference between a coliform and a noncoliform is simply whether the enteric can metabolize lactose.

III.  Inoculate a Phenylethyl Alcohol (PEA) plate

Phenylethyl Alcohol  is a Selective Medium.  It is Selective because phenylethyl alcohol is in the medium and inhibits the growth of most Gram negative organisms (but not all of them) and encourages the growth of Gram positive organisms (but not all of them).  Generally, if one has a culture that grows well on PEA, it probably is a Gram positive species.  If the growth is slowed or absent, it is probably a Gram negative species.  Phenylethyl alcohol interferes with DNA synthesis in Gram negative species, but the growth may only be slower, not absent.  PEA & other selective media are often used because clinical cultures of mixed organisms frequently have Gram negative organisms inhibiting the Gram positive organisms. When one wants to study the Gram positive species in the mixed culture (such as staphylococci or streptococci), use a medium such as PEA to select for the Gram positive bacteria.

Purpose:  to compare your growth observed on this media to your "control media," and for isolating Gram positive cocci from a mixed culture.  Also used to isolate anaerobic bacteria from mixed clinical samples.

Procedure:  

  1. Mark the dish bottom into thirds and label what each third will be, including the date and your name.  
  2. 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.
  3. Inoculate one third with your unknown using a single quick streak.  Generally, a W or Z pattern works best so that you can observe in between the streak.
  4. 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.  (Streak only one species/third, don't mix the bacteria!  Do not isolate for colonies!  See below for how to do this quickly.)
  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  

Look for reduction or absence of growth on PEA compared to growth on an TSA or NA plate.  If growth is reduced or absent on PEA, then you most likely have a Gram negative species.

IV.  Inoculate Mannitol Salt Agar (MSA) plate

MSA is a Selective and Differential Medium that favors the growth of salt loving organisms (halophiles).  MSA is a Selective Medium because of its  high (7.5%) sodium chloride concentration that inhibits the growth of most organisms.  MSA is a Differential Medium because of the presence of the sugar mannitol and the pH indicator Phenol Red.  If an organism ferments mannitol, an acid will be produced turning the indicator yellow.  If one is testing among the Staphylococcus species, pathogenic Staphylococcus aureus ferments mannitol, nonpathogenic Staphylococcus species do not.

Purpose:  to compare your growth observed on this media to that of your "control media."  Also for isolating halophiles and for isolating and differentiating among the staphlococci.

Procedure:  

  1. Mark the dish bottom into thirds and label what each third will be, including the date and your name.  
  2. 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.
  3. Inoculate one third with your unknown using a single quick streak.  Generally, a W or Z pattern works best so that you can observe in between the streak.
  4. 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.  (Streak only one species/third, don't mix the bacteria!  Do not isolate for colonies!  See below for how to do this quickly.)
  5. Incubate your plates upside down in the incubator at 35-37 C for 1-2 days.
  6. Slow growing species may require two days or so of additional growth.
Results  

Look for the presence or absence of growth and if there is growth, whether it has a yellow hallo or not.

V.  How to Plate the Above Tests Quickly

Performing each test one plate at a time is not the optimal way to perform these tests.  Instead use the procedure below to plate them quickly.

  1. Get all the plates you need, being careful to keep track of NA (or TSA) and PEA as they look alike.
    • Label the plates.  Divide the plate bottoms in thirds and label the organisms on each third.
    • Organize your plates into neat piles.
  2. First streak your unknown onto one plate, using only a single streak.  If you keep the loop sterile, there is no need to reflame it!
  3. Get another swab of your unknown and streak the second plate, repeat till all plates are done.
  4. Now reflame your loop (important!) and cool it keeping it sterile.  Obtain another culture.
  5. Then streak out the second bacteria species on all the plates, repeating the above procedure using sterile technique.
  6. Reflame your loop and do the same for the third bacteria species until all plates are plated with all the bacteria.
  7. Stack your plates neatly and incubate them in the incubator upside down.

References:

Atlas R.M. 1997.  Handbook of Microbiological Media, 2nd ed.  CRC Press, Boca Raton, FL, USA.

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. (Not PEA)

MacFaddin J.F. 1985. Media for the Isolation-Cultivation-Identification-Maintenance of Medical Bacteria. Williams & Wilkins, Baltimore MD, USA.

Pommerville J.C.  2011.  Alcamo's Laboratory Fundamentals of Microbiology.  Jones & Bartlett Learning, Sudbury, MA, USA. (Not PEA)

Instructor Notes:

S. epidermidis does not ferment mannitol on MSA.  If this is desired, S. saprophiticus may be used instead.  Other strains may be used for controls.  It is recommended that these tests be done before the Gram stain and the KOH tests.  Bile salts may be precipitated in the Mac plates by some organisms, but this is generally not used to identify species.

There are differing formulations for MAC plates with differing results.  For example, MAC w/o Crystal Violet is sometimes used to differentiate among Gram positive cocci.  The standard formulation is provided here.

The selection by PEA is often incomplete.  A few Gram positives are inhibited partially, a number of Gram negatives are partially inhibited (instead of fully inhibited), and Pseudomonas aeruginosa (Gram negative) is not inhibited at all.