Download Enteric unknown

ENTERIC UNKNOWNS
The term “enteric” means intestinal (usually the large intestine, known as the
colon). Enteric organisms cause many diseases, so they are clinically
important. Some Enterobacteraceae are normally found in the soil. Those
Enterobacteraceae that are only found in the colons of warm-blooded animals
are called “coliforms”. Coliforms are considered fecal indicators. In other
words, if there are coliforms found on lettuce in the grocery store, that is
evidence of fecal contamination.
Your goal with the enteric unknown is to determine the genus of the organism you
have.
REVIEW OF ENTERIC TEST RESULTS
The purpose of this test strip is to test for 20 different enzymes to get a profile of
organisms to help identify them. The profile is based on the fact that different genes
produce different enzymes for these organisms.
One such enzyme is β-galactosidase, which breaks lactose (a milk sugar) into glucose and
galactose. If you don’t have this enzyme and you drink milk, you feed the bacteria in
your colon, and they break the lactose down and produce CO2 gas, and you also get
diarrhea.
CHARACTERISTICS OF ALL ENTEROBACTERACEAE
- Gram negative rods
- Oxidase negative
- Catalase positive
- Fermentation of glucose (positive)
- reduce nitrate (NO3; an inorganic substrate in the API tube. Reduction = positive)
- facultative anaerobes
- If they are motile, they have peritrichous flagella, so they can run and tumble.
There is a flow chart that you follow to determine which genus of Enterobacteraceae you
have. The first question in the flow chart asks you what your results were for your IMViC
test.
IMViC stands for the following (the “i” is just added to make pronunciation easier):
Indole
Methyl red
Voges-Proskauer
Citrate
Methyl Red (MR) is a pH indicator that starts out yellow and becomes red if acid is
present. If the organism has the ability to ferment sugars, the end products of the
fermentation process are acids. Therefore, if the MR tube turns red, the organism was
able to ferment sugars.
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EXPLANATION OF THE ENTEROTUBE
NOTE: Know which wells have what color of a positive test: Brown, Orange or Red,
Blue, Yellow, Diffused black pigment, Pink ring on top, etc.
LACTASE: this tests for an enzyme that breaks down milk sugar (lactose) into glucose
and galactose. If the bacteria have this enzyme, the test is positive (yellow), but the
organism is not pathogenic. Only pathogens are missing this enzyme (negative is clear).
ADH: the substrate is arginine. The indicator is phenol red. If the organism has the
enzyme arginine dehydrogenase , it can break down arginine (an amino acid), test is
positive (red). Negative is yellow. (argentine dehydrolase) breaks down argentine, which
is an amino acid. All amino acids have a carboxyl group (with a free radical attached to
the carbon) and an amino group. When ADH breaks down argentine, it becomes
ornithine, which is more basic. A pH indicator (phenol red) is present in the well. The pH
of phenol red is 6.8, which is close to neutral. If the pH goes down, it is acid (yellow),
and if the pH goes up, it is basic (red). Phenol red, in the presence of ornithine, gets
redder, although in the first 24 hours it may still be just orange. The substrate of the ADH
test is arginine, because that is what is broken down. The product is ornithine. Since
ornithine is more basic, the phenol red turns red.
LDC: Lysine decarboxylase. The substrate is lysine, another amino acid. If the organism
has the enzyme lysine decarboxylase, it will take lysine and break it down into
cadaverine (rotten flesh smell). Red is positive and yellow is negative.A decarboxylase
breaks off the carboxyl group. The result in this case is cadaverine. When an animal dies,
there is a lot of protein breakdown, called putrefaction, and give that stink of “death”.
Cadaverine contributes to that smell. Lysine breaks down into cadaverine, which is more
basic, so phenol red becomes redder.
ODC: Ornithine decarboxylase. This tests for decarboxylation of ornithine (an amino
acid) into putricine (putrid smell). Red is positive and yellow is negative. Ornithine was
the PRODUCT of the ADH well, but in the ODC well, it is the SUBSTRATE. The
product here is putricine, which is also a product of putrefaction and has that death smell.
It is more alkaline (basic), so phenol red turns red. Therefore, in tubes 2, 3, and 4, we are
looking for red as a positive result.
CIT: Citrate is the sole carbon source in this medium. If the organism can use citrate as
its only carbon source, the medium will become basic. The pH indicator is brom thymo
blue. Acid = green or yellow (negative) and base = blue (positive). Citrate (a salt of citric
acid). Citric acid is a part of the Krebs’s cycle. Citrate is the only API well with one
carbon source: citrate. The break down of citric acid produces a more basic medium
because an acid is breaking down. In this case, the pH indicator is brom thymol blue.
Blue color after the test indicates a basic pH, which is a positive result. Yellow indicates
acid, and green is slightly acid.
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H2S: Hydrogen sulfide gas. This tests for the production of sulfide. Black is positive.This
is the result of the reduction of thiosulfate (the substrate). Thiosulfate breaks down into
hydrogen sulfide gas. Iron is added to the medium, and if positive, it precipitates out and
turns the medium black. The enzyme involved is thiosulfate reductase (therefore, it is a
reduction reaction). The gas has to react with iron to detect it. This reaction can also
occur in eggs, which have the amino acid cysteine in them. The bacteria break down the
amino acid into H2S, and you get a rotten egg smell from the sulfur in the H2S. The
substrate is sodium thiosulfate. You look for the black color in the TSI slant. Remember,
iron is needed for this test.
UREA: This tests checks for the production of certain proteases (urease), which is an
enzyme that breaks urea down into ammonia and carbon dioxide. The indicator is phenol
red. Positive is red, negative is yellow.Urea is broken down into ammonia by the enzyme
urease. Urea is made when proteins are broken down. The kidneys are supposed to filter
the urea and put it into the urine for elimination. If the kidneys malfunction, urea can
build up in the blood and be very toxic. The smell of OLD urine is from ammonia.
Bacteria break down urea into ammonia (a base). Phenol red turns red for a positive test.
Urea broth can also be used. The most basic tube in this photo is pink (positive test), and
the least basic is yellow (acid). Only a few of your unknowns were positive.
TDA: Tryptophan deaminase; tests for the presence of pyruvic acid. A dark brownish red
is positive and yellow is negative. The substrate is tryptophan. tryptophan deaminase
(breaks off an amine group). The substrate is tryptophan (an amino acid; also found in
turkey. This amino acid might encourage sleep, which is why you might feel sleepy after
a Thanksgiving turkey meal. The TDA tests for the product: endopyruvic acid. Reagent
(ferric chloride: FeCl3) is added to the tube. A positive result turns dark brown with a
reddish hue. The same people who had a positive urea test will have a positive TDA test.
INDOLE: Indole test; Kovac’s reagent is added. If indole is present, it will form a red
ring if positive, and be yellow if negative. The substrate is tryptophan again.This test also
uses tryptophan. It breaks down more than the amino group, and you end up with indole,
which contributes to the smell of feces. Many of your unknowns are positive for this test,
including E. coli. To test for indole, add Kovac’s reagent, which has alcohol in it.
Alcohol is lighter than water, so when the test is positive and turns red, the red ring floats
to the top of the tube (fig 5-66 and 5-88). TDA and indole have the same substrate.
VP: Voges-Proskauer test; It will initially turn acidic, but it will further break down to
neutral end products. This test checks for the production of acetoin, a product of
fermentation. If acetoin is present, it turns red (positive) and colorless if negative. A
positive test means the organism uses a fermentation pathway. Glycolysis forms pyruvic
acid, which undergoes fermentation, which produces acetoin, which then ends in the
product 2, 3 butandiol. We don’t have a test for the end product, but we can test for
acetoin. This test uses Barret’s reagent A (alpha napthol, a carcinogen!) and Barret’s
reagent B (KOH, a very caustic base, found in draino). When reagent A reacts with
acetoin in a basic environment (takes 10 minutes), it turns red if positive and brown if
negative.
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GEL: This tests for an enzyme called protease that breaks down geletan. If the whole medium
turns black, the test is positive. Gelatin is broken down into amino acids in the presence of
gelatinase (fig 5-53). Gelatin comes from the hooves of horses, pigs, and cows. If the enzyme
is present, it breaks the gelatin down and liquefies it (fig 5-54). It is seen on the API strip (fig
5-88). If the charcoal stays clumped, the test is negative. If it diffuses, it is positive. The same
people who had a positive TDA and urease will have a positive GEL test. This organism makes
several protein-breaking enzymes, and gets its name from these proteonacious enzymes. It is
the only organism that breaks down gelatin.
SUGAR WELL TESTS
GLU, MAN, INO, SOR, RHA, SAC, MEL, AMY, ARA
These are all sugars that are yellow (acid) throughout if positive and blue or green (basic) if negative.
NOTE: You are looking for yellow throughout the medium, not just at the top. Since you all have an
organism in the Family Enterobacteraceae, they will all ferment glucose, so all of your glucose cups
should be yellow within one day. These wells contain different carbohydrates and the same
indicator: brom thymol blue. They all start blue, and we are looking for the acid products
(yellow) of fermentation. Fermentation is anaerobic, so look for the color change at the bottom
of the tube. Yellow is positive. All Enterobacteraceae should be positive for glucose. None
should be positive for all of the sugar wells. If you see green, that is a weak acid, and is still
positive.
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TSI (Triple Sugar Iron Agar)
This medium contains three sugars (below), iron (Fe ++), thiosulfate (oxidized sulfur),
and phenol red (indicator where acid is yellow and basic is red).
10x Sucrose (1%) (sucrose is a plant sugar made of glucose and fructose)
10x Lactose (1%)
1x Glucose (0.1%)
Note that there is 10x more Sucrose and Lactose than there is glucose.
NOTE: When protein is digested, the pH becomes basic because the byproducts are
amino acids, which break down into CO2 and ammonia. When sugars are fermented, the
pH turns acidic because the byproducts are acids.
Know how to record the results of this test, and know what each of the following looks
like: A/A,G, K/A,G K/A,G, H2S K/A, H2S A/A, H2S
A/A,G, H2S
Test results are recorded for the slant and the butt. “A” is for acid, and “K” is for base.
A TSI slant that is all yellow is recorded as A/A
A TSI slant that is red at the slant and yellow at the butt is recorded as K/A
If either sucrose or lactose is fermented, slant will be A/A because there is so much acid
present, it overwhelms the little bit of ammonia that was there.
If just glucose is fermented, more protein was degraded, so the ammonia will be stronger
and show up basic, K/A
If the tube has black in it, the iron was reduced and H2S formed.
If the tube has produced enough gas to separate the agar, it is positive for gas.
An A/A tube with black is recorded as A/A + H2S.
The same tube that also has gas is recorded as A/A + H2S + G
Problems in preparing TSI:
You need the ferrous sulfate or you will be unable to detect hydrogen sulfide gas.
If you forget to slant the media or if you make a very small butt, you will not be able to
differentiate glucose from lactose or sucrose fermentation.
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METHYL RED TEST
Add 5 drops of methyl red to the MRVP broth. If it turns red, it is positive.
If it is yellow or orange, it is negative.
LITMUS MILK
NOTE: Do not shake these tubes to mix them.
Know how to record the results of this test, and know what each of the following looks
like: AR, ACR, AGCR.
Litmus is a pH indicator that turns color only when oxidized. Reduced litmus is colorless. If litmus
is reduced, it will be colorless, so the white milk shows through. This is recorded as “R”.
If it turned pink, it fermented lactose and is acidic. Record this as “A”
If it is bluish, the pH is basic, record it as “K”
If it formed an acid curd (tilt the tube and the milk doesn’t spill), record it as “C”
If you see tracks of gas in a curd, also record “G”.
If the casein protein in the milk was broken down and it is clear on the top and brown in
the tube, that is proteolysis, recorded as “P”.
Read your results and put the tubes back in the racks for further incubation.
NITRATE REDUCTION
This test will be done in the glucose well.
Add one drop of Nitrate A and then one drop of Nitrate B. Color change is positive.
Nitrate is reduced to nitrite, which can be further reduced to nitrogen. All of you should
have had a positive test. Add two reagents. If it turns red, the test is positive for nitrate. If
it does not turn red, add zinc powder to detect for nitrogen, since reduces nitrate to nitrite.
This time, if there is a color change, it means there was nitrate present, so the organism
did not reduce it, and the test is negative. If there is NO color change, the organism
already reduced the nitrate, so the test is positive for nitrate reduction.
NO3 (nitrate)  NO2 (nitrite) N2 (nitrogen gas)
To test for nitrite production, Zinc powder is added with a toothpick to the glucose cup to make NO3 
NO2. If there is no color change, there was no NO3 present.
CATALASE TEST
This is an enzyme that breaks down H2O2 into H20 and O2. Hydrogen peroxide (H2O2) is added to 3 of
the wells. If it bubbles, O2 was released, and catalase is present.
Add one drop of H2O2 to three sugar wells. If it bubbles, test is positive.
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Summary of enteric tests:
Methyl Red starts out yellow and becomes orange or red when acid is present.
Phenol red starts out red and becomes yellow when acid is present.
Phenol red is used for media with a lower pH than an MR.
What color would urea broth be if it produced ammonia? pink
What color would urea broth be if it produced acid? yellow
In the test for nitrate reduction, the presence of nitrite causes the tube to turn red. This is a
positive test. If there is no color change, the test is not necessarily negative. We need to
add zinc powder to the tube to detect the presence of molecular nitrogen. If there is a
color change after adding zinc, the test is negative. If there is no color change this time,
the test is positive.
Know what gases are present:
H2S: FeS gas
Bubbles due to nitrate reduction: Nitrogen gas
Bubbles after the addition of hydrogen peroxide: Oxygen gas
The glucose broth after fermentation: Carbon dioxide gas
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