Name_______________________________
Section______________________________
Introductory Microbiology Pre-Lab Assignment
Intestinal Pathogens
Note: ALWAYS write scientific names correctly!
1. (1pt) Using your textbook or other resources, describe the difference between
commensal and mutualistic organisms.
2. (1pt) How can the fecal-oral cycle of disease be broken?
3. (1pt) List the two genera of enteric pathogens of prime medical concern used in this
experiment.
4. (1pt) List two examples given in this lab of coliforms.
5. (1pt) Which of the above (questions 3 & 4) are…
a. Non-lactose fermenters:
b. Lactose fermenters:
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10/07/15
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Lab Exercise: Intestinal Pathogens
In this exercise you will examine the morphological and biochemical features of intestinal
pathogens. The microbial populations of the large intestine are enormous. It is estimated that
there are approximately one TRILLION bacterial cells per gram of feces (that’s about the weight
of one paperclip). Most of the intestinal organisms (enterics) are commensals, and many are in
mutualistic relationships with their human hosts. Some intestinal bacteria synthesize useful
vitamins, such as folic acid and vitamin K. In addition, the normal intestinal microbiota prevents
colonization of pathogenic species by producing antimicrobial substances and by competition
(mutual exclusion).
The enteric pathogens of prime medical concern are the genera Salmonella and Shigella. They
cause enteric fevers, food poisoning, and bacillary dysentery. Salmonella typhi, which causes
typhoid fever, is by far the most significant pathogen of the Salmonella group. Shigella species
are the prime cause of human dysentery. Most diseases of the gastrointestinal system result from
the ingestion of food or water contaminated with pathogenic microorganisms. Good sanitation
practices, modern methods of sewage treatment, and the disinfection of drinking water help
break the fecal-oral cycle of disease.
Since a patient’s feces usually contain many genera of bacteria, not just the etiologic agents of
disease, it is necessary to use media that are selective and differential to favor the growth of the
pathogens. For example, media has been developed to differentiate between lactose fermenting
enterics and non-lactose fermenting enterics. The lactose fermenters are called coliforms and
with just a few exceptions, such as some strains of Escherichia coli, are generally not
pathogenic. The non-lactose fermenting group includes Salmonella and Shigella.
You will be given samples of two unknown intestinal organisms. One will be a lactose
fermenting coliform (such as Escherichia or Enterobacter), and the other will be a non-lactose
fermenter (either Salmonella or Shigella). These organisms will be of less dangerous strains, but
their presence will demand caution in handling. Your problem will be to make a genus
identification of both. A separation outline is included (next page) listing a series of tests you
will perform that differentiate the lactose and non-lactose fermenters and will allow you to
determine which organisms in each group you are working with.
In actual practice, a fecal sample would initially be placed in an enrichment broth. Enrichment
media serves two purposes. It is selective- it inhibits the growth of nonpathogens commonly
found in the intestinal tract and favors (or enriches) the growth of pathogenic enterics. Since we
are not using stool samples in this exercise, this enrichment procedure is omitted. Instead, you
will inoculate the media used in the first test, Kligler’s iron agar, directly from the cultures
provided to you.
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Separation
Outline
Lactose
Lactose +
Lactose -
Indole +
Citrate +
Indole -
Citrate -
Urea
+
Glucose +
Urea
Motile
H2S+
-
Urea+
Indole+/-
Citrobacter
Escherichia
Klebsiella
Enterobacter
Proteus
Glucose -
Nonmotile
H2S Indole Urea -
Urea Indole -
Salmonella
Shigella
Pseudomonas
alcaligenes
-
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Day 1: Differential Media, Fermentation, and Production of Identifying Metabolites





Many biochemical tests utilize a bacterial species’ ability to synthesize specific enzymes for
identification purposes.
Since enzymes catalyze chemical reactions, the products of those reactions can sometimes be
detected. For example, if a chemical reaction produces acid, a pH indicator in the medium
will change color in the presence of that acid. This would indicate that the bacterium carried
out that reaction.
Since many bacteria have established enzymes, we can identify these bacterial species by
testing a collection of reactions that a specific bacterium is known to undergo.
The types of chemical reactions tested often include fermentation reactions or reactions
requiring or producing specific metabolites. Metabolites are small molecules produced
during or are necessary for metabolism.
The media used contain the reactants of the reaction in question and often an indicator
molecule that detects the products.
Materials: per group of 2 students
1 unknown culture on a MacConkey agar plate with a label from 1 to 10
1 unknown culture on a MacConkey agar plate with a label from 11-20
2 Kligler iron agar slants
inoculating wire & loop
Bunsen burner and striker
Gloves, safety glasses
1. MacConkey agar morphology (outward appearance) observations. There are several
differential media that have been developed for the isolation of intestinal pathogens.
MacConkey agar, observed today, contains lactose and a dye so that if an organism is a
lactose fermenter, its colony will take on the reddish color of the dye. Since coliforms
ferment lactose, they produce reddish colonies that may take on a mucoid appearance, or
dark centered colonies. Non-lactose fermenters, such as Salmonella and Shigella (the socalled SS enterics), produce smooth, colorless colonies that are not reddish/pink.

Obtain 1 unknown culture on a MacConkey agar plate with a label from 1 to 10

Obtain another unknown culture on a MacConkey agar plate with a label from 11-20
–Procedure: Observe the morphology of both of your unknowns’ colonies on the MacConkey
agar plates. Look for isolated colonies. Determine which plate contains colonies
characteristic for Salmonella or Shigella organisms (smooth, colorless colonies) and which
plate contains colonies characteristic of coliforms (reddish, mucoid, or dark centered
colonies). Using the morphology results of your unknowns, make a preliminary
determination of which of your unknowns is a lactose fermenter and which is not.
Record your results in the lab report now. Be sure to write your unknowns’ numbers in
the lab report results table (#4 in lab report).
5
2. Kligler iron agar test. You will inoculate Kligler iron agar slants for further
differentiation. Kligler iron agar contains two sugars, glucose and lactose. Therefore, you
will be able to confirm your lactose fermentation results from the MacConkey agar. In
addition, iron also is included in order to detect H2S production. A pH indicator in the media
will cause it to turn from red to yellow if each sugar is fermented while a black precipitate
will indicate if the organism is a producer of H2S.

Obtain 2 Kligler iron agar slants. Label the Kligler iron agar tubes with the ‘unknown
numbers’ found on the MacConkey agar plates, along with your lab section, your initials,
the date, and media type. Do NOT mix up your numbers!
-Procedure: With much care and aseptic technique, use a straight inoculating wire to streakstab each Kligler iron agar slant with the appropriate bacterial culture (move the wire over
the entire surface of the slant in a zig-zag motion before stabbing).
-Incubate the slants at 370C.
-Refrigerate the unknown MacConkey agar plates for use on Day 2.
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Day 2
First, you will determine the fermentation and H2S results of both of your unknowns from the
Kligler iron agar slants and compare the Kligler results with your determination from the
MacConkey plates. Then you will begin several more tests to finalize the identity (the genus) of
your coliform and your non-lactose fermenter.
Materials: per group of 2 students:
Your MacConkey agar plates from previous lab
2 Kligler iron agar slants inoculated during the previous lab
2 Urea broth tubes
2 SIM tubes
2 Simmons citrate agar slants-Slants should be room temperature at the time students
inoculate them.
Inoculating wire and loop
Bunsen burner, Striker, Gloves, Safety glasses

Obtain your Kligler iron agar slants from the previous lab (these may be in the
refrigerator instead of the incubator)
1. Kligler iron agar results. Examine the Kligler agar slants from the previous period. You
will observe the slant area of the tube and the butt area of the tube.
a. Lactose fermentation. The slant area of the Kligler iron agar slant contains
lactose. If the slant is yellow, the organism ferments lactose. Sometimes a poor or
false lactose fermentation result may occur. If the organism strongly fermented
glucose in the butt, some of the resulting acid could leak up into the slant.
Compare your lactose results in the Kligler slant to your MacConkey plate results.
Do they match? They should.
b. Glucose fermentation. The butt of the Kligler iron agar tube contains glucose.
Tubes that have a yellow butt ferment glucose. Note: Occasionally a poor glucose
result may occur (yellow not present or poorly discernable in butt, even though
the organism ferments glucose). All of the organisms used for student unknowns
in this lab DO ferment glucose, so a poor result may occur due to low levels of
glucose fermentation or coverage of most of the yellow with the production of the
black H2S.
c. A black precipitate in the medium will indicate that the organism is an H2S
producer.
d. Use the Interpretation on the page 10 to determine your results. Record all results
in the lab report (#4).
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
Obtain 2 of each of the following (one of each for each unknown):
Urea broth, SIM media, Simmons citrate slants
Label each of the new tubes with your unknowns’ numbers, your lab section, the
media type, and your initials
2. Urea broth. Urea broth is a differential medium that tests the ability of an organism to
produce the exoenzyme urease that hydrolyzes urea to ammonia and carbon dioxide. The
broth contains urea, a small amount of nutrients for the bacteria, and the pH indicator
phenol red. Phenol red turns yellow in an acidic environment and fuchsia in an alkaline
environment. If the urea in the broth is degraded and ammonia is produced, an alkaline
environment is created, and the media turns pink.
-Procedure: With a loop, inoculate appropriate urea broth tubes using a colony from each
Kligler slant (or MacConkey agar plate, if needed).
3. SIM media. You will inoculate tubes of SIM (sulfur-indole-motility) media to
determine 1. motility, 2. hydrogen sulfide production, and 3. indole production. The
indole test is a biochemical test performed to determine the ability of the organism to
split indole from the amino acid tryptophan. This division is performed by a series of
different intracellular enzymes, a system generally referred to as "tryptophanase."
-Procedure: With a straight wire, stab a colony from each of the unknowns into
appropriate SIM tubes. Stab in the center to 2/3 of the depth of the medium. Pull the
straight wire out of the same hole you stabbed it in.
4. Simmons citrate agar slants. Citrate, a Krebs cycle intermediate, is generated by many
bacteria; however, utilization of external citrate in the environment requires the presence
of citrate transport proteins (permeases). In the citrate test, citrate is the sole source of
carbon in the Simmons citrate medium while inorganic ammonium salt (NH4H2PO4) is
the sole fixed nitrogen source. When an organic acid such as citrate is used as a carbon
and energy source, usually alkaline carbonates and bicarbonates are produced.
The visible presence of growth on the medium or visible growth with a change in the pH
indicator color due to the increased pH are the signs that an organism can import citrate
and use it as a sole carbon and energy source; such organisms are considered to be citrate
positive.
-Procedure: Slants should be room temperature at the time students inoculate them.
With a straight wire, lightly streak each of your unknowns on the surface (from bottom to
top with a straight streak) of a different Simmons citrate agar slant. Oxygen is needed for
citrate utilization (it is used in the Krebs cycle) so place the caps on these tubes
LOOSELY; do not screw them shut tightly.
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Incubate all tubes at 370C
Day 3: Final Evaluation
Today, the tubes of SIM medium, urea broth, and Simmons citrate agar slants will be evaluated.
You will determine which of your unknowns was a coliform, which was a non-lactose fermenter,
and both genus identifications.
Materials: per group of 2 students:
Tubes of SIM medium, urea broth, and Simmons citrate agar from previous period
Kovacs’reagent
Use the Interpretation on the following pages to determine your results. Record all results in the
lab report (#4).
1. SIM medium.
a. First, check for motility. If you see cloudiness spreading away from the stab line, the
organism is motile.
b. Second, check for H2S production. A black precipitate will be evidence of H2S
production.
c. Third, test for indole production by adding a few drops of Kovacs’ reagent into the SIM
medium. A pink to deep red or violet color in the surface alcohol layer of the broth will
develop if indole is produced. A negative result appears yellow. A variable result can also
occur, showing an orange color as a result. This is due to the presence of skatole, another
possible product of tryptophan degradation.
2. Urea broth. Examine the urea broth tubes. If the medium has changed from yellow to red or
deep pink, the organism is urease positive.
3. Simmons citrate slant. If the medium has turned from green to an intense Prussian blue, the
organism is citrate.
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Selective and Differential Test Interpretation
MacConkey Agar Colony morphology
Result
Interpretation
Symbol
smooth, colorless colonies
non-lactose fermenter
lactose -
reddish, mucoid, or dark centered
lactose-fermenter
lactose +
Kligler’s Iron
http://www.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/211317.pdf
Result
Interpretation
Symbol
red slant/red butt
no fermentation
lactose & glucose -
red slant/yellow butt
glucose fermentation
glucose+, lactose-
yellow slant/yellow butt
glucose and lactose fermentation
glucose+, lactose+
black in agar
H2S production
H2S +
SIM Medium
http://iws2.collin.edu/dcain/CCCCD%20Micro/sim_deep.htm
Sulfur Reduction Results
Result
Interpretation
Symbol
black in the medium
H2S production
+
no black in the medium
sulfur is not reduced
-
Motility Results
Result
Interpretation
Symbol
growth has spread from stab line
motility
+
little spreading of growth
nonmotile
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Indole Production Results
Result
Interpretation
Symbol
red after addition of Kovacs’ reagent
tryptophane converted to indole
+
no color change after Kovacs’ reagent
tryptophane not converted to indole
-
http://en.wikipedia.org/wiki/Indole_test
Simmons citrate medium
Result
Interpretation
Symbol
growth is visible or
growth with blue color
blue color
organism can import citrate and use it as a
sole carbon and energy source
+
no growth and media
remains green
organism cannot import citrate and use it as a
sole carbon and energy source
-
http://www.bing.com/images/search?q=simmons+citrate&qpvt=simmons+citrate&FORM=IGRE
#view=detail&id=E8229A40467F5A71C11C444A32D33C2C349312EE&selectedIndex=4
To determine the genera of your unknowns, use the separation
outline near the beginning of the lab. START with lactose
fermentation and work your way down the tests in the outline.
11
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Name__________________________
Lab Section:
Intestinal Pathogens Lab Report
1. (1pt) What is the fermentation characteristic that separates the SS (Salmonella/Shigella)
pathogens from the coliforms (such as E. coli and Enterobacter)?
SS pathogens:_______________________________________________
Coliforms:
2. (1pt) Describe the expected appearance of each of the following organisms on MacConkey
agar. Look in the lab’s description of what the coliform and the SS bacterial morphologies
should look like on this medium.
Coliforms________________________________________________________
SS pathogens________________________________________________________
3. (5pt) Using the Separation Outline, fill in the table below with the expected test results (+or-)
for these organisms. Expected results not listed in the Separation Outline can be found in
Bergey’s Manual of Determinative Bacteriology (Found in the lab- look up the organisms in the
index and go to the bolded pages first).
Test
Salmonella
Lactose
Glucose
Motility
H2S
production
Indole
Urea
Citrate
Shigella
Proteus
E. coli
Enterobacter
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4. (2pt) Record all of the actual results of your unknowns’ in the table below.
Medium
MacConkey Agar
Unknown #__________
Write the colony morphology:
Unknown #__________
Write the colony morphology:
Circle one: Is this morphology on
MacConkey agar indicative of a
lactose fermenter or a non-lactose
fermenter?
Circle one: Is this morphology on
MacConkey agar indicative of a
lactose fermenter or a non-lactose
fermenter?
Lactose fermentation_____
Lactose fermentation_____
Does this result match with the lactose
fermentation results from the
MacConkey agar?
Does this result match with the lactose
fermentation results from the
MacConkey agar?
Glucose fermentation_____
Glucose fermentation_____
H2S _____
Sulfur (H2S)_____
H2S_____
Sulfur (H2S)_____
Motility_____
Motility _____
Indole_____
Indole _____
See your answer to
question #1.
Kligler Iron agar
Indicate + or –
SIM MediumIndicate + or -
Urea + or Citrate + or -
5. (1pt) What is the genus of each of your two unknowns?
Coliform Or Non-lactose fermenter?
Unknown #
Genus
Unknown #
Genus
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