Enteric Unknowns

Miramar College
Biology 205 Microbiology

Enteric (Greek enteron = intestine) bacteria are comprised of several different genera, but all reside in the
digestive tract of mammals. Because the amount of bacteria in the large intestine particularly numbers in
the billions, care must be taken to quickly eliminate all but the most obvious pathogens for routine
medical care. Typically, enteric pathogens are Gram negative and lack the ability to ferment lactose. There
are many commercially available media which are selective for Gram negative organisms and will
differentiate them based on lactose fermentation. These media contain a dye that is taken up by lactose
fermenters and will pigment these organisms, while lactose non-fermenters remain colorless. Eosin
Methylene Blue (EMB) Agar is a medium in which the dyes eosin and methylene blue inhibit Gram
positive bacteria. In addition, lactose fermenters will lower the pH of the agar, facilitating their uptake of
the dyes, which causes them to display nucleated colonies with dark centers. MacConkey (MAC) Agar
contains bile salts and crystal violet dye to inhibit the growth of most Gram positive bacteria and neutral
red dye which stains lactose fermenters and provides the pigmentation associated with their identification
(Figure 1). The characteristics that distinguish lactose fermenters from lactose non-fermenters are
summarized in Table 1. Once the distinguishing characteristic of lactose fermentation has been determined,
relatively few biochemical tests are necessary to determine the correct bacterial genus (Figure 2 and Figure
3).

Figure 1: Differential and selective media for the growth of Gram negative enterics, MacConkey (MAC)
Agar and Eosin Methylene Blue (EMB) Agar. Media are selective for Gram negatives, by the addition of
various dyes and chemicals and differential for lactose fermentation. Typically, lactose fermenters will
appear pigmented on these so-called enteric media, and lactose non-fermenters will not. On MacConkey
agar, lactose fermenters (left plate, two right side inoculations) appear dark pink. On EMB, lactose
fermenters (right plate, two right side inoculations) appear purple or may have a metallic sheen, depending on
the organism.

Table 1: Various Selective/Differential media for the isolation and characterization of Gram negative enteric bacteria.
Organism Eosin-Methylene Blue (EMB) agar MacConkey (MAC)
agar
Escherichia coli dark center with greenish metallic sheen red or pink
Enterobacter sp. similar to E. coli, but colonies are larger red or pink
Klebsiella large, mucoid, brownish pink
Proteus translucent, colorless transparent, colorless
Pseudomonas translucent, colorless to gold transparent, colorless
Salmonella translucent, colorless to gold translucent, colorless
Shigella translucent, colorless to gold transparent, colorless
 Figure 2: Dichotomous key for the identification of lactose non-fermenting enteric bacteria. Genus named
listed in white will not be assigned to you as an unknown for this particular laboratory exercise. However,
they may be assigned as a Major Unknown organism later in the semester.

Figure 3: Dichotomous key for the identification of lactose fermenting enteric bacteria.

Enterics
For this assignment, you will receive two species in a broth culture. One contains a lactose
fermenting Gram negative bacillus and the other contains a Gram negative bacillus which lacks
the ability to ferment lactose and produce acid and gas. It will be your job to determine the genus
identity of both of these organisms using the biochemical inoculations outlined in the Protocol
section of this Lab Exercise and the Dichotomous Key found in Figure 2. You will first isolate
these organisms by streaking onto Eosin-Methylene Blue Agar (EMB) and MacConkey agar
(MAC). You will be able to identify which is the lactose fermenter. You will then confirm this by
inoculating your organisms into separate Phenol Red fermentation broths of lactose and
glucose. An alternative method is to inoculate each organism into Russell Double Sugar (RDS)
and/or Kligler’s Iron Agar (KIA) slants. Using these multi-test media, you will also be able to
determine glucose fermentation and, in the case of KIA, hydrogen sulfide production.
 Phenol Red broths, RDS and KIA contain the fermentable sugars glucose and lactose and the pH
indicator phenol red. In RDS and KIA the concentration of lactose is 10× the concentration of
glucose, and thus glucose is quickly metabolized if the isolate is capable of fermenting it to
produce an acidic byproduct and/or gas. If an organism ferments glucose only, the entire tube
turns yellow. Because there is a minimal amount of glucose present in the tube, the organism
quickly exhausts it and begins oxidizing amino acids for energy. Ammonia is produced and the
pH rises. Within 24 hours the phenol red indicator reverts to its original red color on the slant.
For this reason, RDS and KIA should not be read prior to 24 hours after incubation. KIA also
contains ferrous sulfate (FeSO4), and if hydrogen sulfide (H2S) is being produced, it will react with
the ferrous sulfate to form ferric sulfide (FeS2) (Figure 4). Because these tests rely on pH changes
in the media, it is important to read the results in a timely manner. As mentioned above, early
observations may erroneously lead to the conclusion that an organism is capable of fermenting
both lactose and glucose. Further, reading results that are older that 48 hours old may lead to the
observation of the phenomenon known as alkaline reversion, whereby the catabolism of amino
acids again leads to a raising of the pH which will give false negative results for both glucose and
lactose fermentation. Once both glucose and lactose fermentation have been noted, you will test
your lactose non-fermenter for motility and urea hydrolysis. Your lactose fermenter will be tested
for indole production (using the multi-test medium SIM), urea hydrolysis and citrate
utilization. As these media were used as part of your Minor Unknown, you should refer to Minor
Unknown Lab for information about these tests and how to interpret them.

Figure 4: Various inoculations onto Kligler's Iron agar slants. Glucose fermentation is noted in the “butt” of
the slant- a red butt indicates a lack of fermentation and a yellow butt indicates positive fermentation.
Lactose fermentation is noted in the “neck” of the slant, with results similar to that of glucose: red necks are
negative for lactose fermentation, yellow necks are positive. Gas production is noted as the slant either splits
or is raised up from the bottom of the test tube. Inoculations which are capable of producing H2S form a
black precipitate.

Protocol:
Enterics
Day One
Individual Supplies
Enteric A & Enteric B mixed broth
EMB agar plate
MacConkey Agar plate
1. Streak your cultures on an EMB and MacConkey plate for isolation. Incubate at 37 degrees for
24 to 48 hours.
 Day Two
Individual Supplies
incubated EMB and MAC plates
2 Phenol Red lactose broth
1 Phenol Red glucose broth
2 Russell Double Sugar agar or NA slants
1. Gram stain each of your isolates and confirm morphology and arrangement indicative of
enteric bacteria.
2. Determine which of the organisms is the lactose non-fermenter.
3. Inoculate both organisms into Phenol Red lactose broths to confirm lactose fermentation.
4. Inoculate the organism you believe is lactose negative into a Phenol Red glucose broth for
glucose fermentation.
5. Inoculate both organisms into NA slants or RDS to confirm lactose non-fermentation and
check for glucose fermentation.
6. Incubate your slants at 37°C for 24 - 48 hours.

Day Three
Individual Supplies
incubated NA or RDS slants
Urea broth
SIM
Simmon’s citrate
1. Confirm the lactose non-fermenter, perform the following inoculations:
a. Identify your organism’s ability to ferment glucose.
b. Inoculate your lactose non-fermenter into urea broth and SIM deep medium.
c. Incubate your media at 37°C for 48 hours. Remember that urea broth is a 3–5 day
incubation and should be left in the incubator if a negative result is seen at the end of
the 48 incubation period.
2. Confirm the lactose fermenter, perform the following inoculations:
a. Inoculate your organism into SIM, Simmon’s citrate and urea broth.
b. Incubate all media at 37°C for 48 hours.

Day Four
1. Record all relevant data.

Protocol scheme diagram

Data Collection and Analysis
Enterics
1. Complete the following table for each of your unknown organisms. If you did not perform a
test, indicate that with “n/a.”
Biochemical Attribute Organism A Organism B
Gram stain
lactose fermentation
glucose fermentation
motility
urea
H 2S production
indole
citrate
1. Identify each of your enteric isolates.

My lactose fermenter is ______________________ _____________________.

My non-lactose fermenter is ______________________ _____________________.

Discussion Questions:
Enterics
1. What additives make EMB and MacConkey agar selective and differential?

2. What can be said of lactose non-fermenters on these types of media?

3. What two characteristics separate Salmonella from Shigella? What media can be used for this
differentiation?

4. How can acid production by glucose and lactose fermentation be differentiated in the same
tube?

5. What is alkaline reversion? Why is it important?

 Media Used in Enteric analysis

Enteric Unknowns

biochemical/
medium enzyme reagent(s) positive & negative results
physiological characteristic

Russell Double multi-medium to test for fermentative lactose [lactose] 10× [glucose] yellow butt indicates glucose fermentation,
Sugar sugar fermentation and/or glucose enzymes phenol red (in medium), yellow neck indicates lactose fermentation,

eosin & methylene blue are selective and inhibit

selective medium inhibits
Eosin- Gram positive bacteria, are taken up by lactose
Gram positive; differentiates eosin, methylene blue (in
Methylene Blue various fermenters resulting in pigmented and
based on lactose medium)
agar sometimes “nucleated” colonies; lactose non-
fermentation
fermenting organisms remain unpigmented
multi-medium to test for fermentative lactose [lactose] 10× [glucose] yellow butt indicates glucose fermentation,
Kligler iron
sugar fermentation and H 2S and/or glucose enzymes, phenol red (in medium), yellow neck indicates lactose fermentation,
agar
production cysteine desulfurase iron salts (in medium) black precipitate indicates H2S production
bile salts & crystal violet are selective and
selective medium inhibits
inhibit Gram positive bacteria, neutral red dye is
MacConkey Gram positive; differentiates bile salts, crystal violet,
various differential and pigments lactose fermenting
agar based on lactose neutral red (in medium)
organisms red; lactose non-fermenters are non
fermentation
pigmented
Differential media to
Acid production turns broth yellow, gas
Phenol Red distinguish glucose
various Phenol Red, glucose production is visible as a bubble in inverted
Glucose Broth fermentation and acid and
Durham tube
gas production
Differential media to
Acid production turns broth yellow, gas
Phenol Red distinguish lactose
Beta galactosidase Phenol Red, lactose production is visible as a bubble in inverted
Lactose Broth fermentation and acid and
Durham tube
gas production
black precipitate indicates H 2S production; red
H 2S Production; indole cysteine desulfurase; iron salts (in medium); color change with Kovac’s reagent indicates
SIM
production; motility trytophanse Kovac’s reagent indole production from tryptophan; cloudy
throughout indicates motility
Simmon citrate citrate as a sole source of bromthymol blue (in
citrase blue color change indicates presence of citrase
agar carbon medium)
color change to hot pink/cerise color indicates
urea broth production of urease urease phenol red
presence of urease