Download Supportive Selective and Differential Media

Unit 7
Unit 7: Supportive, Selective and Differential Media and
Streak Isolation Practice
By Karen Bentz, Patricia G. Wilber and Heather Fitzgerald.
Copyright Central New Mexico Community College, 2015
Introduction
Like all living organisms, bacteria require nutrients in order to grow. Basic media contains
ingredients such as partially digested milk, soy, yeast extract, or beef broth, which provide
nutrients for the growth of many bacteria. T-soy, which you used in solid, liquid, and plate form
for your initial inoculations, is an example of a basic medium.
Supportive media contain additional ingredients, such as red blood cells, which support the
growth of more fastidious (picky) bacteria. Red blood cells are an excellent source of iron and
amino acids as well as required bacterial growth factors such as NAD(factor V) and hemin(factor
X). In Chocolate agar, a type of supportive medium, the RBCs have been lysed (broken open) to
make their contents more readily available to bacteria for growth.
A third class of media, selective and/or differential media, are used to enhance growth of
some bacterial types and inhibit growth of other bacterial types. These media will also
differentiate the bacteria that do grow, according to specific metabolic processes they may
have. TSA-blood is an example of a differential medium, and MacConkey’s is an example of a
selective and differential medium.
TSA-blood can be used to differentiate between bacteria based on the bacteria’s ability to
produce hemolysins, enzymes that lyse the red blood cells. Bacteria that can hemolyze blood
utilize the nutrients and iron in the RBCs for growth. Hemolysin production is associated with
pathogenicity.
Different amounts of hemolysin production are described using the following terms:
a. Beta hemolysis: Complete lysis of the red blood cells resulting in a clear halo in the red
medium underneath the bacterial growth. The bacteria produce a high level of
hemolysins.
b. Alpha hemolysis: Partial digestion of the red blood cells. The hemoglobin is reduced to
methemoglobin, which results in an olive green halo in the red medium underneath the
bacterial growth. The bacteria produce some hemolysins.
c. Gamma hemolysis: Growth of the bacteria, but no lysis or digestion of the red blood
cells underneath the bacterial growth. The bacterial growth is often a whitish color on
the surface of the red medium. The bacteria do not produce hemolysins.
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Unit 7
MacConkey’s agar contains crystal violet and bile salts that inhibit the growth of Gram(+)
bacteria but allow the growth (selection) of Gram(-) bacteria. In MacConkey’s medium, the
disaccharide lactose and the pH indicator neutral red also permit differentiation of the
previously selected Gram(-) bacteria based on the bacteria’s ability to produce the enzyme
lactase. Those bacteria that produce lactase are able to ferment the lactose sugar in the
medium. This will cause a drop in the pH (to less than 7) of the medium, which causes the
bacteria to absorb the neutral red and produce a bright fuchsia color. Bacteria that do produce
lactase and can ferment lactose generally live in the intestines and are not pathogenic. Normal
gut organisms are also called coliforms and are generally not pathogenic (unless they get into
the water supply!). Bacteria that do not produce lactase and thus do not ferment lactose are
more likely to be pathogenic.
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Unit 7
Table 7-1: Characteristics of Chocolate Agar, TSA-blood and MacConkey
Medium
Supportive
Mechanism of Support
Ingredient
Chocolate
Agar
lysed sheep
red blood cells
Medium
Differential
Ingredient
TSA-blood
whole sheep
red blood cells
Medium
MacConkey
Selective
Ingredients
crystal
violet
and
bile salts
The lysed blood provides the bacterial growth factors NAD(factor V) and hemin(factor X), which are
inside red blood cells. The agar is named for the color and contains no actual chocolate.
Mechanism of Differentiation
Bacteria can be differentiated according to their ability to produce enzymes called hemolysins that
digest the sheep blood in the medium.
Bacterial Enzyme: Hemolysin
Beta hemolysis: complete digestion of the blood, the blood has been completely digested and the
medium under the bacteria is clear. Bacteria produce a high level of hemolysins. The bacteria is a likely
pathogen.
Alpha hemolysis: partial digestion of the blood hemoglobin, the medium has an olive-green color. The
bacteria produce some hemolysin and is a possible pathogen.
Gamma hemolysis: no hemolysis. Bacteria do grow on top of the medium, and this growth is often
white, but the blood in the medium underneath the cells retains a red color. The bacteria do not
produce hemolysins, and are probably not pathogenic.
Mechanism of Selection
Growth indicates a
Gram(-) organism.
Gram (+) organisms will
not grow because the
crystal violet and bile
salts interfere with the
function of the
peptidoglycan layer.
Differential
Ingredient
lactose
Mechanism of
Differentiation
If the bacteria can
ferment lactose, the
acid waste they
produce will cause
the pH indicator to
turn fuchsia color.
Required Bacterial
Enzyme: Lactase
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pH indicator
neutral red
Neutral red is a
red color at
neutral pH, and
turns fuchsia if
the pH is acidic.
Of Special Note
The bacterial cells that ferment
lactose become permeable to
the pH indicator and absorb it,
turning the cells as well as the
medium fuchsia.
Pink (lactose fermentation) =
coliform = generally non
pathogen;
Not pink (no lactose
fermentation) = non-coliform =
possible pathogen
Unit 7
DAY 1:
Video Link
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Chocolate Agar https://www.youtube.com/watch?v=6H1Uz3IU4nY
TSA with Blood https://www.youtube.com/watch?v=-ysnHBMToBo
MacConkey’s https://www.youtube.com/watch?v=e9iFdY0ncck
Video by Corrie Andries
Materials
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Metal Inoculating loop
Microincinerator
Black Sharpie-style marker
Appropriate personal protective gear (lab coats, gloves, face shield, hair ties)
Media
o 1 MacConkey, TSA-Blood, Chocolate per pair of students
o 1 T-soy agar petri plate per person
Bacteria cultures, from which to inoculate new media (Note: substitutions may be made
as needed)
o Escherichia coli (Ec), Gram(-)
o Haemophilus haemolyticus (Hh) Gram(-)
o Proteus vulgaris, (Pv) Gram(-)
o Streptococcus mitis, (Stmi) Gram(+)
o Streptococcus pyogenes, (Spy) Gram(+)
Procedures
A. Inoculating Chocolate, Blood and MacConkey Agar
1. Work with a partner for these inoculations
2. Use a black marker to divide the bottom of your plates into thirds.
3. Using the marker, write your initials and the initials of the bacteria on the bottom of
your plates. Also include the date and the type of medium you are working with.
4. Inoculate your labeled plate with the bacteria, following the pattern for bacteria shown
in the diagrams below.
5. Use a sterile loop to pick up a small amount of bacteria from a stock plate.
6. Flame and cool your loop before and after transferring each type of bacteria.
7. When finished inoculating all of your plate media, place them upside down in the
appropriate rack at the front of the lab. (See special note for blood plates.)
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Unit 7
Chocolate Agar Inoculation
1 Chocolate agar plate inoculated with:
Streptococcus pyogenes (Spy)
Spy
Hh
Escherichia coli (Ec)
Haemophilus haemolyticus (Hh)
Ec
MacConkey’s Inoculation
1 MacConkey plate inoculated with:
Escherichia coli (Ec)
Ec
Sm
mm
m
Proteus vulgaris (Pv)
Streptococcus mitis(Sm)
Pv
TSA-blood **
1 Blood plate inoculated with:
Streptococcus mitis (Sm)
Sm
Streptococcus pyogenes (Sp)
Spy
Hh
Haemophilus haemolyticus (Hh)
** TSA-blood plates should be placed upside down in a candle jar for incubation. The candle
jar provides a low oxygen environment that is required for proper function of the bacterial
blood hemolysins.
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B. Streak Isolation Practice
1. Each student should practice the streak isolation technique on a T-soy plate.
2. Use very little bacteria!!! Overlap less. Don’t forget to flame!
3. Choose Ec or Pv for your streak isolation.
4. Refer back to Unit 3 for a refresher on the streak isolation procedure.
Figure 7-1: Pattern for Streak Isolation Procedure
B. 10 streaks through A
A. 1 cm smear
C.
E.
D.
Image created by Patricia G. Wilber, 2015
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Unit 7
Day 2:
Results
1. Collect the plates that you inoculated in the previous lab.
2. Take a picture of each type of media and place your photo in the appropriate area below.
3. Look at the growth of the bacteria on your plate, and note any changes to the color of the
medium surrounding the bacteria.
I. Chocolate Agar (Supportive)
This medium is supportive because the RBCs in the medium have been partially lysed.
Fastidious (picky) bacteria that will not grow on other media may grow on Chocolate agar.
Figure 7-2: Sputum Sample Streak-isolated on Chocolate Agar.
How many species (=colony types) do you see on this plate? ________________________
Accessed 8/31/2015 from http://www.microbelibrary.org/library/2-associated-figure-resource/2263-sputum-chocolate-agarfour-quadrant-streak-enlarged-view but licensed for use by the American Society for Microbiology, Creative Commons
Attribution – Noncommercial – No Derivatives 4.0 International license.
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Unit 7
Insert Photo of Your Chocolate Agar Plate Here:
Name of Bacteria
Supportive Feature:
Did it Grow on
Chocolate Agar?
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Describe Bacterial Growth
Unit 7
II. TSA-blood (Differential)
Figure 7-3. Bacterial Growth showing Alpha (α), Beta (β) and Gamma (γ) hemolysis.
Accessed 7/29/2015 from https://www.studyblue.com/notes/note/n/block-5-study-guide-2013-14hamill/deck/10254665, but licensed for use by the American Society for Microbiology, Creative Commons
Attribution – Noncommercial – NoDerivatives 4.0 International license
Ability to digest red blood cells (RBCs):
Complete RBC Digestion: medium is clear underneath the bacterial growth; beta
hemolysis, organism is a likely pathogen.
Partial RBC Digestion: medium is olive-green under the bacterial growth; alpha
hemolysis; organism is a possible pathogen.
No RBC Digestion: bacteria grows, but medium under the growth stays red; gamma
hemolysis; organism not a likely pathogen.
No Growth: indicates the bacteria may be fastidious (picky)
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Insert Your TSA-blood Photo Here:
Name of Bacteria
Differential Feature:
Did it Grow on the
TSA-blood?
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If the Bacteria Grew, What Does
the Medium Under the Bacterial
Growth Look Like?
(clear= beta, olive-green=alpha,
red=gamma)
Unit 7
III. MacConkey (Selective and Differential)
Figure 7-4: Growth of Bacteria on MacConkey Media. The bacteria on the left grew, meaning it is
Gram(-) but the colonies are clear, meaning the bacteria does not ferment lactose. The bacteria on the
right grew, meaning it is Gram(-), and the dark pruple color means the bacteria ferment lactose.
Accessed 8/31/15 from http://www.microbelibrary.org/library/laboratory-test/2927-lactose, but licensed for use by the
American Society for Microbiology, Creative Commons Attribution – Noncommercial – No Derivatives 4.0 International license.
How many species (=colony TYPES) do you see on each plate? ______________
A. Type of Cell Wall (selective feature):
Positive test for Gram(-) organisms: growth on medium
Negative test for other types of organisms: no growth on medium
B. Ability to Ferment Lactose: (differential feature):
Positive test for lactose fermentation: bacteria and surrounding medium turn fuchsia;
produces lactase, normal intestinal flora.
Negative test for lactose fermentation: bacteria shows clear growth, medium remains
purple; bacteria does not produce lactase, not normal intestinal flora, possible
pathogen.
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Insert Photo of Your MacConkey Plate Here:
Selective Feature:
Did it Grow on
MacConkey?
(Write yes or no)
Name of Bacteria
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Differential Feature:
If Bacteria Grew, Did It
Turn a Fuchsia Color?
(Write yes or no)
Unit 7
IV. Streak Isolation Practice
In the space below, draw or insert a photograph of the results of your streak isolation. The goal
is to have eight or more isolated colonies on your plate.
How many species do you expect to see on your plate? ___________________
How many species do you actually see on your plate? ____________________
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Unit 7
Interpretation
Based on all of your results, explain what you know about the metabolism, cell wall structure,
and enzymes of each bacterial species that you tested. As an example, interpretation has been
completed for Pseudomonas aeruginosa, a species you did not test.
Example for a species you did not test:
Bacterial Species: Pseudomonas aeruginosa
Based on Your Test Results, What Do You Know
About This Bacteria’s Cell Wall, Metabolism, and
Enzymes?
Chocolate agar:
1. The bacteria grew; chocolate agar is a supportive
medium and many species of bacteria will grow on
it. The colonies were whitish.
What is the Evidence For or Against
this Organism Being a Likely
Pathogen?
These test results gave no information
about pathogenicity.
MacConkey’s agar:
1. The bacteria grew, so it is a Gram(-) organism.
2. The bacterial growth was clear to slightly
pink/purple. It was not bright fuchsia. This means
bacteria cannot ferment lactose.
3. This species lacks the enzyme lactase.
Based on this test, Pseudomonas
aeruginosa is a possible pathogen
because it does not ferment lactose as
shown by the clear to slightly
pink/purple color of the colonies on
the MacConkey. Lack of lactose
fermentation indicates a possible
pathogen.
The complete lysis of the blood (Beta
hemolysis) indicates pathogenicity.
TSA-blood:
1. The bacteria grew.
2. The bacteria showed Beta hemolysis, which
means the blood cells were completely lysed.
3. The bacteria produced the enzyme hemolysin.
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Unit 7
You tested five species on one or two plates each. Based on all of your results, explain what you
know about the metabolism, cell wall structure, and enzymes of each bacterial species that
you tested.
Bacterial Species: ______________________________
Based on Your Test Results, What Do You Know About This
Bacteria’s Cell Wall, Metabolism, and Enzymes?
What is the Evidence For
or Against this Organism
Being a Likely Pathogen?
Bacterial Species: ______________________________
Based on Your Test Results, What Do You Know About This
Bacteria’s Cell Wall, Metabolism, and Enzymes?
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What is the Evidence For
or Against this Organism
Being a Likely Pathogen?
Unit 7
Bacterial Species: ______________________________
Based on Your Test Results, What Do You Know About This
Bacteria’s Cell Wall, Metabolism, and Enzymes?
What is the Evidence For
or Against this Organism
Being a Likely Pathogen?
Bacterial Species: ______________________________
Based on Your Test Results, What Do You Know About This
Bacteria’s Cell Wall, Metabolism, and Enzymes?
What is the Evidence For
or Against this Organism
Being a Likely Pathogen?
Bacterial Species: ______________________________
Based on Your Test Results, What Do You Know About This
Bacteria’s Cell Wall, Metabolism, and Enzymes?
Unit 7 Page 16
What is the Evidence For
or Against this Organism
Being a Likely Pathogen?
Unit 7
Post Lab Questions
1. Fill in the blanks in the table below.
Media
Selective
Ingredient(s)
Differential Ingredient
pH Indicator
MacConkey
TSA-blood
Chocolate Agar
none
none
none
none
none
2. Using your results and interpretation information from this lab, give the name of a
bacteria with the following characteristics:
*Be sure to write the names of your bacteria using proper scientific nomenclature. All of
the following are acceptable:
Staphylococcus aureus (underline genus and species names)
STAPHYLOCOCCUS AUREUS (all capital letters)
Staphylococcus aureus (italicized genus and species)
A. Gram(-), lactose fermenter:
What is your evidence for this choice?
B. Requires lysed RBCs to grow:
What is your evidence for this choice?
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Unit 7
C. Beta hemolytic:
What is your evidence for this choice?
D. Gram(-), does not ferment lactose:
What is your evidence for this choice?
E. Alpha hemolytic:
What is your evidence for this choice?
3. Create a set of study cards for the three types of media that you used in this lab. An
example of a study card for the MacConkey medium has been done for you.
No growth means not
a Gram(-) bacteria
Growth of clear to
slightly pink bacteria
means Gram(-) and does
not ferment lactose;
potential pathogen
Growth of fuchsia
bacteria means Gram(-)
and ferments lactose,
normal flora
The authors of this lab unit would like to thank Andrea Peterson and Deyanna Decatur for testing new media and organisms,
our associate dean Linda Martin for many kinds of aid, Michael Jillson and Alex Silage for IT support, and our dean John Cornish.
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