Day 5 - Selection and Differentiation

Different types of agar plates can encourage the growth of one type of organism, while inhibiting the growth of other types of organisms.  This is called selection media, since it selects a certain type of microorganism to grow on it.  Agar media that produce different characteristics for different types of organisms is called differential media, since it differentiates between different organisms.

Selection and differentiation can be combined in the same plate.  For example, the mannitol salt plate is selective for Staphylococcus spp., but it differentiates S. aureus from other Staphylococcus species.

We used several different selective/differential media in the lab.  Here is what we did:

Blood Agar Plate:
This test selects fastidious bacteria in clinical specimens, and it differentiates among bacteria that lyse red blood cells.  It differentiates between alpha-hemolysis, which is partial degradation of RBC and produces a greenish hue around the colonies; beta-hemolysis, which is complete destruction of RBC and produces a colorless zone around the colonies; and gamma-hemolysis, which is no lysis of RBC.

Materials:
Blood agar plate
Our unknown bacteria in broth culture

Steps:
1.  Label the blood agar plate with our group number. We shared a blood agar plate with group 3, so we divided the plate in half and labeled our half with a "4."
2.  Use aseptic technique to inoculate the blood agar plate from our broth culture.
3.  Incubate the plate upside-down at 35 degrees for 24 hours.
4.  Examine the results.
5.  Discard the plate properly.

Results:

Our bacteria is on the right side of the plate
Positive for growth

Our bacteria is on the left (we flipped the plate over for a different view)

Discussion:  Our bacteria is positive for growth, so it can grow in clinical specimens.  The hue of our colonies is not green, nor is it colorless around the colonies.  Thus, our bacteria must be gamma-hemolytic, meaning that our bacteria does not destroy red blood cells.




Mannitol Salt Agar:
This plate is selective for bacteria that can tolerate salt (7.5% NaCl, to be exact).  These types of bacteria include Staphylococcus, Micrococcus, and Enterococcus.  The mannitol in the mannitol salt agar plate makes it differential between Staphylococcus aureus and other salt-tolerant bacteria.  The mannitol salt media differentiates by turning yellow for S. aureus, because the mannitol fermentation produces an acid that turns the medium yellow.

Materials:
Mannitol salt agar plate
Our unknown bacteria in broth culture

Steps:
1.  Label the manitol salt agar plate with our group number. We shared a mannitol salt agar plate with group 3, so we divided the plate in half and labeled our half with a "4."
2.  Use aseptic technique to inoculate the mannitol salt agar plate from our broth culture.
3.  Incubate the plate upside-down at 35 degrees for 24 hours.
4.  Examine the results.
5.  Discard the plate properly.

Results:

Our bacteria is on the right side of the plate
Positive growth with a very slight amount of mannitol fermentation

Discussion:  Our bacteria grows on this medium, so it can tolerate salt.  Our bacteria can also ferment mannitol, which is a verification of previous test, but it cannot ferment it strongly.  This means that our bacteria is not Staphylococcus aureus. 




MacConkey Agar:
This test is selective for gram-negative enteric bacilli, and it differentiates between them via the bacteria's ability to ferment lactose.

Materials:
MacConkey agar plate
Our unknown bacteria in broth culture

Steps:
1.  Label the MacConkey agar plate with our group number. We shared a MacConkey agar plate with group 3, so we divided the plate in half and labeled our half with a "4."
2.  Use aseptic technique to inoculate the MacConkey agar plate from our broth culture.
3.  Incubate the plate upside-down at 35 degrees for 24 hours.
4.  Examine the results.
5.  Discard the plate properly.

Results:

Our bacteria is on the left side
Positive for growth

Discussion:  Our bacteria grows on this medium, so it is a gram-negative bacillus.  This verifies our results from the staining procedures.  Based on the color, our bacteria can also ferment lactose.  This is another verification of earlier tests.



Phenyl Ethyl Alcohol Agar:
This test is selective for gram-positive bacteria.  This test verifies whether or not our bacteria is gram-positive or gram-negative.

Materials:
Phenyl Ethyl Alcohol agar plate
Pure culture of our unknown bacteria on an agar slant

Steps:
1.  Label the agar plate with our group number and test type
2.  Use aseptic technique to inoculate the phenyl ethyl agar plate with our bacteria
3.  Incubate at 35 degrees for 24 hours
4.  Examine the results
5.  Discard the plate properly

Results:

Note: we are group 4, on the right side of the agar plate.

Discussion:  Our bacteria did not proliferate much in this culture.  This indicates that our bacteria is gram-negative, which verifies the gram stain we did that gave that same result.



Eosin Methylene Blue (EMB):
This test is selective for gram-negative bacteria.  It differentiates between those bacteria that can ferment lactose and/or sucrose, and those that cannot.  Gram-negative bacteria that produce significant acid, via the fermentation of the sugars, produce a green sheen.  Those bacteria that produce less amounts of acid yields a pink color.  Bacteria that cannot produce acid are colorless or the color of the medium. 

Materials:
EMB agar plate
Pure broth culture of our unknown bacteria

Steps:
1.  Label the EMB agar plate with our group number and type of test
2.  Use aseptic technique to inoculate the agar plate.
3.  Incubate at 35 degrees for 24 hours.
4.  Discard the plate properly.

Results: 

Our bacteria is on the right.
It produced pink colonies.

Discussion:  Based on the production of pink colonies, our bacteria is gram-negative and can ferment lactose and sucrose.  However, our bacteria does not produce much acid.