Gram Negative Rods
Enterobacteriaceae (Coliforms)
Tests Used to ID
Unknown Organisms
HOW TO ID AN UNKNOWN
ORGANISM
• Your goal with the enteric unknown is to determine the genus of the
organism you have.
• Individual tests can be performed in separate tubes (conventional
method) or all the tests can be performed in one specially designed
Enterotube (rapid identification method). Comparisons between
rapid identification methods and conventional methods show that
they are equally accurate.
• NOTE: Be able to match tests and media to their reagents, pH
indicators, substrates, products, enzymes, and results, as indicated
in the table at the end of the lecture transcript and flashcards.
• Complete
chart is on
flashcards
GETTING STARTED
• Perform a Gram stain. Since the longest step is air drying, make 34 slides and allow them to air dry at the same time, but only use one
to perform your Gram stain. That way, if your culture is not
decolorized properly, you have several slides ready to go so you can
perform another stain quickly. When you observe your organism
under the microscope, check to make sure your culture is pure.
Sometimes, the Gram stain becomes contaminated or your culture
may be contaminated. If you see more than one organism, you need
to go back to the original pure culture and start again. If you only see
one organism, the next step is to do a Negative stain with India ink,
which is the best way to see the arrangements of the organism.
Record the shape (rods or cocci?) and arrangement (singles,
clusters, or chains?).
METHOD OVERVIEW
• Determine the morphological characteristics of your unknown
organism by performing a Gram stain, motility stab, capsule stain,
and negative stain. If you have a Gram positive rod, you will also
need to do a spore stain, since only Gram positive rods make
endospores (only certain species).
• Next, determine the cultural characteristics of your organism by
observing the growth patterns in broth and on agar slants and
plates. Determine the optimal temperature and oxygen
requirements, and determine what type of hemolysis your organism
displays.
• Next, determine the physiological characteristics of your
organism. This will require about 18-20 individual tests to find out
what enzymes your organism makes, its fermentation pathway, etc.
MORPHOLOGICAL CHARACTERISTICS:
Gram stain, size determination, motility, capsule stain, spore stain
• SIZE DETERMINATION
• If you have a Gram + organism, mix a
loopful of it with a loopful of a Gram neg
organism whose size is known. If you
have a large organism, pick a large
organism to compare it with. If you have a
small organism, pick a small organism.
Estimate the size of yours compared to a
known organism.
Controls
• When performing tests on unknown organisms, control organisms
are also used.
• A “control” is a test media that is inoculated with a pure culture of a
known organism.
• One tube is inoculated with an organism that is known to be positive
for that test, another tube is inoculated with an organism that is
known to be negative for that test, and another tube is inoculated
with the unknown organism.
• When you see the results of the unknown organism, it is compared
to the positive and negative control tubes to determine if the
unknown is positive or negative for that test.
• If the positive control or negative control did not turn the expected
color, we know something is wrong with the media.
MOTILITY TEST
• Inoculate a motility stab. Use a needle to obtain the inoculum. Stab
the needle into the motility medium, almost all the way to the bottom,
then pull the needle back out in a straight line, backing the needle
out of the same stab line you made going in. Remember, these need
to be incubated at room temperature (25°C). If they are placed at
room temperature, the flagella will detach, giving a false negative
result for motile organisms. Also remember that motility media uses
TTC as a terminal electron acceptor. If the organism can use it, the
media will turn red, meaning the TTC has been reduced. If there is
no red color at all, you will need to do a wet mount or hanging drop
to observe the organism directly to determine if it is motile. NOTE:
an Enterotube can test for MRVP, citrate, indole, carbohydrate and
protein catabolism, but a motility test cannot be performed in an
Enterotube.
MOTILITY TEST
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HANGING DROP PREPARATION
Supplies: Toothpick, petroleum jelly (Vaseline), cover slip, depression slide, gloves
Spank the bottom of the nutrient broth to mix it.
Remove one loop of broth and touch it lightly to the center of the coverslip, which is
sitting on a paper towel. Try to get the smallest drop possible.
Use the toothpick to apply one small dab of Vaseline to each corner of the coverslip.
This keeps the coverslip secure when it is upside down.
Clean the depression slide and press the open well down gently onto the coverslip.
Gently flip the slide over so the drop hangs in the depression well.
Since we will look under the microscope at live cells, there will be no contrast, so turn
the iris down to increase the contrast.
Observe under scanning (40x); position the slide so that the edge of the drop is in the
center of the field. Focus on the edge of the drop.
Increase to LP then HP, but do NOT go to 1000x because the depth of field at 1000x
is too small to observe the depth of the droplet.
Look for tiny specks that are moving and record what type of motility is present.
Motility Test
Positive
Motility Test
• Hanging Drop Method
• http://www.youtube.com/watch?v=ujzSms
mg7ok
MOTILITY TEST
• Problems with hanging drop slides:
• False positive results: the organism appears to move, but it
cannot. This can happen if you are observing it too long under the
hot light, and the liquid starts to evaporate and recede.
• False negative results: the organism is capable of motility, but it
does not appear to be moving.
• No motility could be observed if the drop falls into the well and you
don’t realize it, or if the iris diaphragm is opened too wide. Motility
could be observed without false results if the culture is too
concentrated.
MOTILITY TYPES
• TUMBLES: the bacterium seems to be rolling over itself
like a rolling stone.
• RUNS: the bacterium moves from point A to point B
• JIGGLES: the bacterium jiggles like it is in an
earthquake, but it does not move from one part of the
slide to another. THIS IS BROWNIAN MOTION, WHICH
IS NOT TRUE MOTILITY.
• BROWNIAN MOTION is caused by water molecules
hitting a cell with low mass. It causes movement of low
mass cells by the inertia created by molecular
bombardment of water molecules.
NOTE:
• After a person has identified if their
organism is Gram positive, the next test is
to do a spore stain. If it has no spores,
then you need to do an Acid Fast stain to
see if it is Mycobacteria.
• After a person has identified if their
organism is Gram negative, the next test
to do is to use a thioglycollate broth to
determine oxygen requirements.
CULTURAL CHARACTERISTICS:
Growth patterns, temperature, hemolysis, and oxygen requirements
• GROWTH PATTERNS
• Use your working stock and reserve stock to observe the
growth patterns of your organisms and record that
information in your journal. The terminology to use is in
your lab manual. When you have recorded the
morphology on your reserve stock, it will be kept in the
refrigerator. You will not use it except in emergency. Also
use your TSB tubes from your optimum temperature
experiment to determine the pattern of grown in broth.
OPTIMUM TEMPERATURE
• Inoculate one loop-full of your organism into 3 TSB
tubes. Label one tube 25 °C, one tube 30 °C, and one
tube 38°C. Make sure your name is on the tube. These
tubes will be used to determine the optimal temperature
for your organism. Use the spectrophotometer to
calculate their optical density at the next lab period. The
tube with the most growth (highest OD) is the
temperature they prefer. Organisms that grow well in
room temperature as well as body temperature might be
opportunistic pathogens. These tubes can also be used
to determine their pattern of growth in broth.
SODIUM THIOGLYCOLATE TUBES (OXYGEN REQUIREMENT)
• This medium has an oxygen gradient, which means that most of the
oxygen is at the top of the tube, and the least amount of oxygen is at
the bottom of the tube. To prepare this medium, a reducing agent
called Sodium thioglycolate was added, which removes the free
oxygen by chemically binding with it. Therefore, thioglycolate broth
is called a REDUCING MEDIUM. It gets rid of the oxygen. There is
also a pink indicator dye called rasazarin that shows you where
the oxygen is. Notice that the pink color is only at the top of the tube.
We have to be careful not to shake the tube, or we will aerate it (add
more oxygen). We need the oxygen gradient to be maintained for a
successful test. The results of this test determine what oxygen
requirements your organism has.
SODIUM THIOGLYCOLATE TUBES (OXYGEN REQUIREMENT)
• Procedure:
• Hold the thioglycolate tube carefully, taking care to move it gently
without shaking, jiggling, or stirring them (which introduces oxygen
into the medium).
• Label the tube with your name, the date, the organism, and “Thio”
for Thioglycolate.
• Put some of your unknown bacteria on a sterile loop and gently push
the loop straight down to almost the bottom of your tube. Do not
touch the bottom as this may ruin the loop, and do not introduce air
by stirring or shaking the tube!
• Gently pull the loop straight out of the tube and sterilize it.
SODIUM THIOGLYCOLATE TUBES (OXYGEN REQUIREMENT)
• STRICT AEROBES require oxygen to grow. There will only be
growth on the surface of the thio broth tube (pseudomonas and
Bacillus megatarium)
• STRICT ANAEROBES require the absence of all oxygen. There will
only be growth at the butt (bottom) of the tube (clostridium).
• FACULTATIVE ANAEROBES grow best aerobically but do not
require it. Growth is throughout the tube, but is best at the top and
decreases as one descends. (E.coli, staph aureus)
Thio Broth: Determines oxygen
requirements
This test is done to see if the organism
is aerobic (growth only at the top),
anaerobic (growth only at the butt), or
facultative (growth throughout). A
facultative organism can live with or
without air, so that is considered a
virulence factor.
Thio Broth
Not shown:
anaerobic; growth
only at the butt
Aerobic
Facultative
CHARACTERISTICS OF ALL ENTEROBACTERACEAE (Gram neg rods in colon)
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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.
Amino Acids
• All proteins are made of amino acids.
• There are only about 22 different types of
amino acids in our bodies.
• Each amino acid has an amine group (NH2)
and a carboxyl (acid) attached to it.
Amine group:
NH2
Carboxyl group:
COOH
“R” refers to the rest of the molecule
that makes it a particular amino acid.
Amino Acids
• The amino acids we will be working with in lab are these:
• Tryptophan (breaks down into ammonia, pyruvic acid,
and indole; causes feces smell)
• Arginine (breaks down into ornithine)
• Lysine (breaks down into cadaverine)
• Ornithine (breaks down into putrescine)
• Phenylalanine (breaks down into ammonia, pyruvic
acid, and hydrogen sulfide)
Enzymes
• An enzyme is a protein that cuts a
substance into smaller parts.
• Enzymes that cleave proteins are called
protease enzymes.
• Enzymes that cut an NH2 group off are
called deaminases.
• Enzymes that cut the carboxyl group off
are called decarboxylases.
Enzymes
• The substrate is the item that is cut by
the enzyme.
• The products are the pieces that remain
after the enzyme cuts the substrate.
• The pH indicator is what changes color to
show us the presence of the products
created by enzymes.
• You need to know the substrates,
products, enzymes, and pH indicators
used in the tests we will discuss in lab.
PHYSIOLOGICAL TESTS FOR
GRAM NEGATIVE RODS
• NOTE: Know which tests 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). The medium is
MaConkey’s agar or a lactose fermentation tube.
Lactase Test
• MacConkey’s agar
Lactase Test
• Fermentation Broths with Durham tubes
FERMENTATION BROTHS
• If an organism has the ability to ferment sugars, the end
products of the fermentation process are acids. We are
looking for fermentation with acid (A) or acid + gas (AG).
If there is fermentation, it will be yellow. If there is gas,
the inverted miniature tube inside the media will fill with a
gas bubble. If there is no fermentation, it is red, so
record it as no change (NC) or Alk (protein digestion).
• The medium has a Durham tube (a miniature tube that
is inverted on the inside of the test tube). If gas is
produced, it will form a bubble inside the inverted tube. It
also has phenol red as an indicator. Phenol red turns
yellow if acid is present, and red if bases are present.
FERMENTATION BROTHS
• Inoculate one each of the following tubes: glucose, lactose, and
sucrose.
• These are different carbohydrates. After 24 hours, if the inoculated
medium is yellow, it fermented the sugar in that tube. It may or may
not have produced gas. Gas is produced during sugar fermentation,
so when gas is present, fermentation is present as well, but not all
organisms ferment with gas. If it is yellow, record it as (A). If it has
gas in the Durham tube (a bubble that take up 10% of the tube, not
a little bubble), record it as (AG). If it did not turn yellow (stays red),
you have to look at it again in another 24 hours. After 48 hours, if the
media is still red, the organism is negative for fermentation of that
sugar. These tubes must be read in 24 hours, because in 48
hours, any change in color will revert to the original color.
FERMENTATION BROTHS
• This is what happens:
• Some organisms that ferment sugars can also
digest proteins. When these organisms begin to
ferment a sugar, the media becomes acidic
(yellow in 24 hours), which enables them to
begin digestion of the proteins which are in the
media. When proteins are digested, the media
becomes alkaline, and the media will turn back
to red. If you want to know if it fermented the
sugar, you need to read the tube in 24 hours.
FERMENTATION BROTHS
• Suppose a student did not observe their tube
right away, and then they see that it is red but it
has gas. Since the gas is present, that indicates
that it probably fermented the glucose (turned
yellow at 24 hours, but he missed it), and then
the organism proceeded to digest the protein,
turning the media alkaline (back to red again).
That would explain why it was red, but has gas
(gas is produced during the fermentation
process).
If Lactose Negative
• Perform an Indole test (using SIM media
or IMViC test):
– Positive
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Proteus vulgaris (H2S negative)
Providencia stuartii (H2S positive)
E. coli (Citrate negative)
Citrobacter freundii (H2S positive, Citrate positive)
– Negative
• Perform a urease test (all should be negative)
• Perform the MR-VP test
IMViC
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This stands for a series of tests:
1) Indole (this test is also used in SIM Media)
2) Methyl Red
3) Voges-Proskauer
4) Citrate
The small “i” does not stand for anything; it
just makes pronunciation easier.
IMViC
• The IMViC tests were developed as a means of
separating members of the Enterobacteriaceae,
particularly the coliforms, to determine if drinking water is
contaminated with sewage. A coliform is a gram
negative, aerobic or facultative anaerobic rod which
produces gas from lactose within 48 hours. The
presence of some coliforms indicates fecal
contamination. Coliforms are only found in the GI tract of
warm-blooded animals. We will perform the indole test
as part of the SIM media. We performed the citrate test
in the Simmon’s Citrate media. Now we need to perform
the MR-VP test to complete the IMViC series.
Indole Test
• The enzyme tryptophan deaminase breaks an amine
group off of tryptophan (an amino acid) down into
indole (which contributes to the smell of feces), pyruvic
acid, and ammonia. If tryptophan deaminase is present,
the indole end product reacts with Kovac’s reagent. If a
red ring forms at the top of the tube, it is positive for
indole, so the organism makes tryptophan deaminase.
Kovac’s reagent 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. (Control = E. coli). 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.
Tryptophan Deaminase
(Indole) Test
Tryptophan
deaminase is
an enzyme
that slices
tryptophan into
indole and
pyruvic acid.
Add Kovac’s
reagent. Red
ring is positive.
MR-VP TEST (Methyl
Red/Voges-Proskauer)
• We do two tests with this medium: The MR
test and VP test. We will inoculate one
MR-VP tube today, let the culture grow
until the next lab period, and then add 5
drops of Methyl Red to perform the MR
test. In the next lab period, we will
inoculate a new MR-VP tube, let the
culture grow, and then add alpha-naphthol
and potassium hydroxide reagents to
perform the VP test.
MR-VP TEST
• Anaerobic respiration is called fermentation. We are
looking for the ability of the organism to perform glucose
fermentation. Bacteria convert glucose to pyruvate using
different metabolic pathways. One pathway produces
unstable acidic products which quickly convert to neutral
compounds. Another pathway (the butylene glycol
pathway) produces neutral end products, including
acetoin and 2,3-butanediol. A third pathway is the mixed
acid pathway, which produces stable acidic end products
which remain acidic.
MR-VP TEST
• If an organism produces a lot of acid from the
fermentation of sugars, it can override the buffer in the
test media. If this happens, the amber media will turn
red. MR-VP broth differentiates organisms that are single
acid fermenters from organisms that are mixed acid
fermenters because it contains over-riding buffers that
affect organisms that are single acid fermenters.
MR-VP TEST
• An organism that produces only one type of acid after
sugar fermentation will not produce much acid, so the
buffer blocks the media from changing color. But if the
organism produces many different kinds of acids, it
overrides the buffer and causes the color to change.
MR = METHYL RED TEST
• A positive MRVP broth will be red.
• Methyl Red is a yellow colored pH indicator which turns
red if the organism uses the mixed acid fermentation
pathway, which is that pathway that produces stable
acidic end-products. If positive, the enzyme present is
formic hydrogenylase. The acids will overcome the
buffers in the medium and produce an acidic pH. When
methyl red is added, it will go from yellow to red, which is
positive for an organism that uses the mixed acid
fermentation pathway.
MR Test: pH indicator
Negative
Positive
(acid present)
MR = METHYL RED TEST
• NOTE: Methyl red differs from Phenol red
• Methyl Red: starts off yellow, turns red
when acids are present (indicating
glucose fermentation). Used in MR-VP
test (the first part of the test) for mixed
acid fermentation
• Phenol Red: starts off red, turns yellow
when acids are present (indicating
glucose fermentation). Used in Urea broth
and in the Fermentation broths
VP (Voges-Proskauer) TEST
• The VP test is an indirect method of testing for an
organism that ferments glucose using the butylene
glycol pathway. Glycolysis forms pyruvic acid, which
undergoes fermentation and produces acetoin, which
can then be taken into one of several different
fermentation pathways, depending on the organism. In
the butylene glycol pathway, the end product is 2, 3
butanediol. We cannot test for that, but we can test for
acetoin. If acetoin is present, it turns red (positive) and
colorless if negative. A positive test means the organism
uses that particular pathway for fermentation.
VP Test
Negative
Positive
VP (Voges-Proskauer) TEST
• The VP reagents are called Barritts’s A
(alpha napthol, a carcinogen!) and
Barrett’s B (potassium hydroxide; KOH,
a very caustic base, found in draino). If
acetoin is present, it will turn a rust or
red color (Gram negatives tend to do
this). Therefore, red is a positive result,
colorless or brown is negative.
IMViC (+ + - -)
E. coli
Indole
Positive
MR
Positive
VP
Negative
Citrate
Negative
IMViC (- - + +)
Enterobacter
Indole
Negative
MR
Negative
VP
Positive
Citrate
Positive
IMViC (+ - - -)
Indole
Positive
MR
Negative
VP
Negative
Citrate
Negative
MR-VP Tests
+/Citrobacter freundii
-/+
Klebsiella pneumoniae (non-motile)
Enterobacter aerogenes (motile)
CITRATE TEST
• Citrate is a salt of citric acid. It is a part of the Kreb’s
cycle. In this medium, citrate is the sole carbon
source. If the organism can use citrate as its only
carbon source, the medium will become basic (blue)
because an acid (citric acid) is breaking down. The
medium starts out green and turns blue (basic pH) if it
is a positive test. The pH indicator is Bromthymol blue,
which is green when acids are present. It may only be
blue at the top, which is still positive. Acid = green
(negative) and base = blue (positive). A negative tube
will also show no growth.
Citrate
Positive
Negative
Amylase Test with Starch Agar
• This media has starch. Some molecules, such as starch, cannot be
taken into a bacterial cell because the molecules are so large. The
organism can only use starch if it has an enzyme, called amylase,
which can hydrolyze (break down) the starch into simple sugars
that can be absorbed into the cell. We will flood the plate with iodine,
which reacts with starch and turns it black. If the organism has the
enzyme, there will be no more starch left, so there is nothing for the
iodine to react with. Therefore, the presence of amylase will show
up as a halo (area of clearing) around the organism (positive test).
If the organism could not use the starch, the starch forms a
complex with iodine to give a black precipitate around the
organism. That means the organism is negative for amylase.
NOTE: the black color only lasts a few minutes, so you have to read
the test right away before the color disappears.
Amylase Test with Starch Agar
•
After incubation, iodine is poured on the plate, which turns starch black. If
there is a clear zone around the inoculation, the bacteria already digested
the starch. That means it has the enzyme called Amylase.
Oxidative Fermentation (O-F) Test
• We are looking for the ability to ferment or oxidize
glucose. The pH indicator is Bromthymol blue, which
is yellow when acid is present. You will STAB two O-F
tubes of glucose. One tube will need a layer of sterile oil
to create an anaerobic environment so we can check for
fermentation. The other tube will not have oil, so we can
have an aerobic environment to check for oxidation. Next
time, you will see if it turns yellow. If it is yellow, record it
as “A “ (acid present). If there is gas in the tube, also
record “G”. If there was no change (stayed green), write
“NC”.
Oxidative Fermentation (O-F) Test
• Yellow means it
fermented
glucose. If it can
only do that in the
tube exposed to
air, it is a positive
O-F test.
Oxidative Fermentation (O-F) Test
Urease Test
• When protein is digested, the pH becomes a base. This
test checks for the enzyme called urease, which breaks
urea down into ammonium (a base) and carbon
dioxide (water is not a product of this reaction). The
ammonium will increase the pH. The medium has a pH
indicator called phenol red. When pH goes up, it will
turn bright pink (positive), negative is yellow. 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.
Urease Test
Positive
Negative
Negative
Urease Test
• Pink is
positive
If Urease Negative
• Perform a motility test
– Positive
• Salmonella enterica (H2S positive)
• Serratia marcescens (H2S negative)
– Negative
• Shigella somnei
Gelatinase
• Some organisms produce an enzyme
called gelatinase, which breaks down
gelatin. If the gelatin is broken down, it
becomes liquefied, and can no longer
solidify, even when cooled in the
refrigerator. Gelatin is a protein, so
gelatinase is a protease. Gelatin is the
only thing making the media solid. If it
remains liquid, even after refrigeration, it is
positive. Solid is negative.
Gelatinase
• Being able
to liquefy
gelatin is
positive.
Phenylalanine Deaminase Test
• We are looking for the enzyme,
phenylalanine deaminase, which
removes an NH2 group from the amino
acid, phenylalanine, to produce pyruvic
acid, ammonia, and hydrogen sulfide.
When 5 drops of ferric chloride is added
to this, it will turn green, indicating a
positive test. A negative test stays yellow.
Don’t get this mixed up with the SIM
media, where ferrous sulfate turns the
media black.
Phenylalanine Deaminase Test
• When 5 drops
of ferric
chloride are
added, green
color is
positive.
SIM Media
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•
•
•
•
Check for three things on this one tube.
H2S PRODUCTION (Hydrogen sulfide): Some organisms use sulfur as their
terminal electron acceptor in the electron transport chain. These bacteria have the
enzyme thiosulfate reductase, which reduces thiosulfate (the substrate in the
medium) and produces H2S gas (a rotten egg smell). When the H2S reacts with
iron in the medium, a dark precipitate of iron sulfide is produced and the media will
turn black (positive for H2S production). (Control = Proteus vulgaris). Don’t get this
mixed up with the phenylalanine test, where the addition of ferric chloride turns the
media green.
INDOLE PRODUCTION: The enzyme tryptophan deaminase breaks an amine
group off of tryptophan (an amino acid) down into indole (which contributes to
the smell of feces), pyruvic acid, and ammonia. If tryptophan deaminase is
present, the indole end product reacts with Kovac’s reagent. If a red ring forms at
the top of the tube, it is positive for indole, so the organism makes tryptophan
deaminase. Kovac’s reagent 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. (Control =
E. coli). 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.
Motility: This media will show you if your organism is motile.
SIM Media
•
Purpose: To differentiate between bacteria based on three tests: sulfur reduction
(cysteine desulfurase), indole production (tryptophanase), and motility.
A. Indole positive and
hydrogen sulfide
positive
B. Hydrogen sulfide
positive
C. Indole positive and
motility positive (note
fuzzy growth away from
stab line)
D. Negative Control
Decarboxylase Broths
• This tests for the presence of the enzyme
decarboxylase. This test is useful for
differentiating the Enterobacteriaceae. This
enzyme removes and digests the acidic carboxyl
group (COOH) from amino acids, plus cleaves
off NH3, which will raise the pH. The pH indicator
is bromcresal purple. The media is made to
start out slightly acidic (pH 6). Bromcresal
purple is yellow when acids are present and
purple when bases are present.
Decarboxylase Broths
• Three tubes are inoculated. Each tube contains
glucose plus one amino acid; either lysine,
arginine, or ornithine. The carboxylase reaction
requires an anaerobic environment, so each
tube needs to be covered will a layer of sterile
mineral oil to prevent air from reaching the
culture.
• NOTE: PICK THESE TUBES UP FROM THE
RACKS ONE AT A TIME AND LABEL THE
TUBE BEFORE YOU PICK UP THE NEXT
TUBE. THEY ARE ALL THE SAME COLOR
AND YOU MIGHT GET THEM MIXED UP!
Decarboxylase Broths
• Each decarboxylase enzyme produced by an
organism is specific to the amino acid on which it
acts. Therefore, we test the ability of organisms
to produce arginine decarboxylase, lysine
decarboxylase, and ornithine decarboxylase
using three different but very similar media.
• If an organism is able to decarboxylate the
amino acid present in the medium, alkaline
byproducts are then produced and it will turn
purple (positive).
Decarboxylase Broths
ADC, LDC, and ODC
ADC = Arginine decarboxylase
LDC = Lysine decarboxylase
ODC = Ornithine decarboxylase
Media turns red-purple if
it can digest the particular
protein in the tube.
This is a photo of a lysine
decarboxylase broth, so it
only contains the amino
acid, lysine (a protein).
Positive
Negative
Decarboxylase Broths
• ADC: the substrate is arginine. The indicator is bromcresal purple. If
the organism has the enzyme arginine decarboxylase, it can break
down arginine (an amino acid), test is positive (purple). Negative is
yellow. When ADC breaks down argentine, it becomes ornithine,
which is more basic. If the pH goes down, it is acid (yellow), and if
the pH goes up, it is basic (purple). The substrate of the ADC test is
arginine, because that is what is broken down. The product is
ornithine. If the pH becomes alkaline because the organism has
the decarboxylase enzyme, the media will turn purple in 48
hours (pos).
Decarboxylase Broths
•
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). Purple is positive and
yellow is negative. 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 it
turns purple. If you check it in 24 hours, you might see that it is yellow
because it fermented the glucose in the medium, but that does not mean it
is a negative test. You have to check it in 48 hours to allow the
decarboxylase activity to occur. If the pH becomes alkaline because the
organism has the decarboxylase enzyme, the media will turn purple in
48 hours (pos).
Decarboxylase Broths
• ODC: Ornithine decarboxylase. This tests for
decarboxylation of ornithine (an amino acid) into
putricine (putrid smell). Purple is positive and
yellow is negative. Ornithine was the PRODUCT
of the ADC 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. If the pH becomes alkaline
because the organism has the decarboxylase
enzyme, the media will turn purple in 48
hours (pos).
Triple Sugar Iron (TSI) Slants
• 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.
Triple Sugar Iron (TSI) Slants
• 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
Triple Sugar Iron (TSI) Slants
• 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
Triple Sugar Iron (TSI) Slants
A = acid (sugar was fermented)
K = negative result
G = gas formation
H2S = sulfur was used, iron was reduced to black color
K/AG
K/K
K/K
K/A
H2S
A/AG
A/AG
Triple Sugar Iron (TSI) Slants
• 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
Symbols that indicate test
results:
• A: acid has formed. This usually indicates
a sugar nutrient was broken down.
• ALK: means the media is alkaline after the
test. This usually also indicates a negative
test, so we just abbreviate it to “K”.
Therefore:
• K: means the test was negative
• G: means gas formed
• The rest of the following tests we will
not perform, but they are on the exam.
Catalase Test
• Some facultative aerobes have the enzyme called
catalase, which breaks down hydrogen peroxide
(H2O2) into harmless water plus oxygen. Having this
enzyme protects organisms from being destroyed by the
H2O2 in the lysosomes of a white blood cell. Your
instructor will lift the lid on your agar plate next lab
period, and put one drop of H2O2 onto the colony. A
positive test will show the oxygen bubbles rising up from
the plate. That means the organism has the enzyme, so
it is catalase +. NOTE: do not get catalase mixed up with
oxidase. Catalase breaks down into oxygen, but is it not
the oxidase test!
Catalase Test
Oxidase Test
• Some aerobes have the enzyme called cytochrome
oxidase, which is a molecule that is a terminal electron
acceptor in the electron transport chain. On a piece
of paper, place one drop of the oxidase reagent
Dimethyl-p-phenylene diaminic hydrochloride (this
substance is carcinogenic). Then use a toothpick to
obtain the organism from your TSA plate, and scrape the
sample onto the drop of reagent on the paper. The test
should be done in comparison to a positive control,
because time is essential in the development of the test
results. Count the number of seconds it takes to turn
purple and record the time in your journal. If purple is
observed at any time, it is positive for oxidase. If there is
no color change, it is negative.
Oxidase Test
Caseinase Test (Skim Milk Agar)
• Some organisms produce an enzyme called
caseinase (a protease), which breaks down the
protein that makes milk white. It breaks the
protein down into small peptides that can be
absorbed into the cell. Do a heavy streak in the
center. Positive is a clearing (halo) around the
area of growth of the organism because the
milk is broken down and the white color
disappears. Casein is what makes milk look
cheesy when it is left unrefrigerated.
Caseinase Test (Skim Milk Agar)
• A and B are
positive
because of
the clearing
(digestion of
casein)
• C is negative
Litmus milk
• This is done to see if the organism has the enzyme
lactase. If it does, it will ferment lactose, the milk
sugar. The waste product will be an acid, so it will
change the pH indicator from bluish to pink.
• If gas is produced during this process, gas bubble will
also appear. Some organisms ferment lactose and
produce gas, and some ferment lactose without
making gas.
• Since milk also contains a protein called casein, this
test will also determine if the organism has the enzyme
caseinase to break that protein down. If it breaks the
protein down only partially, curds (solid milk) will form.
If it can break the milk protein all the way down to
amino acids, a brown ring appears at the top.
Litmus milk
• 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 has a brown ring in the tube, that is proteolysis,
recorded as “P”.
Litmus Milk Test
P
A
A/C
A/C
G
A
A
K
White (R) means litmus was reduced and became clear.
Pink (A) means it fermented lactose and is acidic.
Bluish (K) means the pH is basic, so lactose was not fermented
Curds formed (C)
Gas in the curd (G)
Brown ring (P) means the casein protein in the milk was broken down (proteolysis)
Litmus Milk
A/C
G
Lipase Test
• The Spirit Blue media has lipids. If the
organism has the enzyme lipase, fatty acids will
be released, and pH will decrease (become
acidic). The indicator dye is called Spirit blue.
When the pH decreases, a positive result is a
dark blue streak in the center of the plate where
you inoculated it. If lipase if produced, the
concentration of the blue will increase where it
was inoculated. Having clearing is NOT a
positive test; it should be darker blue.
Lipase Test (Spirit Blue Agar)
DNAse Test
• This tests for the presence of the enzyme,
DNAse. It contains the indicator dye,
Methyl green complexed with DNA.
Digestion of DNA releases the dye, so in
otherwise green agar, a clear halo
formed around the growth indicates a
positive test.
DNAse Test
• Plate is flooded
with toluidine
blue, positive is
pink area
surrounding
colony.
Nitrate Reduction Test
• Add one drop of Nitrate A and then one
drop of Nitrate B. Red color is positive. If
there is no color change after adding the
two reagents, add zinc powder. If it does
NOT turn red it is positive. If the zinc
powder makes it turn red, it is negative.
Nitrate Reduction Test
• Nitrate is reduced to nitrite, which can be further reduced
to nitrogen. If nitrate (NO3) has an oxygen molecule
removed, it has been reduced. The new molecule is
nitrite (NO2). Nitrite can also be reduced to nitrogen gas
(N2) if it loses oxygen. The reactions look like this:
NO3 (nitrate)  NO2 (nitrite) N2 (nitrogen gas)
• If an organism has the enzyme called nitrate reductase,
it can reduce nitrate like this:
NO3 (nitrate)  NO2 (nitrite)
•
Is this enzyme clinically important? Not really. Some of you have brown eyes and some of you do
not have brown eyes. It serves as a way of classifying organisms on a flow chart.
Nitrate Reduction Test
• After you add the first two reagents, if it turns red, the test is positive
for nitrite, so that means the organism reduced NO3 to NO2. If it
does not turn red, it might be because there is no nitrate present for
the organism to reduce, so we cannot say it is a negative test yet.
• To find out if nitrate is present, add zinc powder. That will reduces
nitrate to nitrite and produce a red color. This time, if it turns red, it
means there was nitrate present during the first part of the test and
the organism did not reduce it, which means the organism is unable
to reduce nitrate. Therefore, the nitrate reduction test is negative. If
there is NO color change after adding zinc powder, the organism
already reduced the nitrate, so the test is positive for nitrate
reduction.
Nitrate Reduction Test
• The nitrate broth we started with contains NO3. If
the organism has nitrate reductase, it will reduce
NO3 to NO2 so there will be no more NO3
present , just NO2. First, we add reagent A to the
tube. Reagent A will bind to NO2 , forming a
complex. However, this complex is clear, so it
does not tell us anything. Then we add Reagent
B, which turns the complex a red color. If you
add reagents A + B and the tube turns red,
the organism has nitrate reductase.
Nitrate Reduction Test
• However, some organisms with that enzyme
reduce NO3 all the way to N2. They take all of the
nitrate and reduce it all the way to nitrogen gas.
In this case, there will be no NO3 or NO2 in the
tube, so there is nothing for reagent A to react
with, and reagent B will not turn the tube red,
even though the organism has the nitrate
reductase enzyme. So, although a red color is a
positive test, a colorless tube is NOT a
negative test.
Nitrate Reduction Test
• When the tube is colorless, there are two possibilities:
• The organism does not have nitrate reductase, and there
is still NO3 in the tube
• The organism has nitrate reductase, and there is no NO3
or NO2 in the tube.
• If a tube is colorless after adding Reagents A + B, we
need to test the tube to see if there is NO3 in the tube.
We do this by adding a little zinc powder by scooping
some on the flat end of a toothpick and adding it to the
tube. Zinc will react with NO3 if it is present (reduces
any residual nitrate to nitrite) and it will turn red. Zinc
is used to confirm a negative test.
Nitrate Reduction Test
Step 1: add naptholamine
The tubes above have been treated with
dimethyl 1-naptholamine and sulfanilic
acid. The tube on the right indicates that
the organism reduced nitrate to nitrite. In
order to further determine
positive/negative nitrate reduction, zinc
powder must be added to detect the
presence of nitrate.
Step 2: add zinc powder
A tube will turn a deep red color on the
addition of zinc if nitrate is
present. The uninoculated control
turned red indicating that nitrate is still
present in the tube, so if it turns red
after adding zinc, it is negative.
Nitrate Reduction Test
API 20E Test Strip
• You could perform each of those tests in
individual test tubes or plates, as shown in
the previous slides.
• Or you could do many of those tests at
once with an API-20E Test Strip.
API 20E Test Strip
All Negative
All Positive
The fast way to run many tests on an organism is to inoculate either an APT 20E test
strip ($12 each) or an Enterotube ($45 each). The organism is placed into each well
and the results are read two days later.
API 20E Test Record Sheets
Enterotube: more expensive than API-20E media
Common Enteric Organisms
•
•
•
•
•
•
•
•
•
Citrobacter freundii
Escherichia coli
Enterobacter aerogenes
Klebsiella pneumoniae
Proteus vulgaris
Providencia stuartii
Salmonella enterica
Shigella somnei
Serratia marcescens
Citrobacter freundii
Citrobacter freundii
• Uses citrate as their sole source of carbon.
• These bacteria can be found almost
everywhere in soil, water, wastewater, etc.
It can also be found in the human
intestine.
• They are rarely the source of illnesses,
except for infections of the urinary tract
and infant meningitis.
Citrobacter freundii
Motility: negative
MR: positive
+
- -
+
-
+
-
Escherichia coli
Non-motile species
Motile species
Escherichia coli
Escherichia coli
• One of the main species of bacteria living in the lower
intestines of mammals, known as gut flora.
• There are about 1 trillion excreted in the feces daily.
• However, the bacteria are not confined to this
environment, and specimens have also been located, for
example, on the edge of hot springs.
• The E. coli strain O157:H7 is one of hundreds of strains
of the bacterium that causes illness in humans.
• E. coli are unable to sporulate. Thus, treatments which
kill all active bacteria, such as pasteurization or simple
boiling, are effective for their eradication, without
requiring the more rigorous sterilization which also
deactivates spores.
Escherichia coli
• E. coli can generally cause several intestinal and extraintestinal infections such as urinary tract infections,
meningitis, peritonitis, septicemia and pneumonia.
• Although it is more common in females due to the
shorter urinary tract, urinary tract infection is seen in both
males and females. Poor toilet habits can predispose to
infection (doctors often advise women to "wipe front to
back, not back to front") but other factors are also
important (pregnancy in women, prostate enlargement in
men).
• If E. coli bacteria escape the intestinal tract through a
perforation (a hole or tear, for example from an ulcer, a
ruptured appendix, or a surgical error) and enter the
abdomen, they usually cause peritonitis that can be fatal
without prompt treatment. However, E. coli are extremely
sensitive to such antibiotics as streptomycin or
gentamycin.
Escherichia coli
• Certain strains of E. coli, such as Escherichia
coli O157:H7, produce a toxin and can cause
food poisoning usually associated with eating
cheese and contaminated meat (contaminated
during or shortly after slaughter or during
storage or display).
• The usual countermeasure is cooking suspect
meat "well done"; the alternative of careful
inspection of slaughtering and butchering
methods (to make sure that the animal's colon is
removed and not punctured).
Escherichia coli
• Escherichia coli O157:H7 is the strain is believed to be
associated with the 2006 United States E. coli outbreak
linked to fresh spinach. Severity of the illness varies
considerably; it can be fatal, particularly to young
children, the elderly or the immunocompromised
• E. coli are susceptable to amoxicillin, cephalosporins,
aminoglycosides, streptomycin, or gentamycin.
• Antibiotic resistance is a growing problem. Some of this
is due to overuse of antibiotics in humans, but some of it
is probably due to the use of antibiotics as growth
promoters in food animals.
Escherichia coli
-
-
Enterobacter aerogenes
Enterobacter aerogenes
• Several strains of the these bacteria are
pathogenic and cause opportunistic infections in
immunocompromised (usually hospitalized)
hosts.
• The urinary and respiratory tract are the most
common sites of infection.
• Enterobacter can be distinguished from other
Gram neg rods by virtue of being a 'fast
fermenter' of lactose (as are E. coli and
Klebsiella).
Enterobacter
Methyl Red: negative
++
Klebsiella pneumoniae
Klebsiella pneumoniae
• This is a nonmotile, encapsulated, lactosefermenting, and facultatively anaerobic
organism.
• It is found in the normal flora of the mouth,
skin, and intestines.
• It naturally occurs in the soil and about
30% of strains can fix nitrogen in
anaerobic conditions.
Klebsiella pneumoniae
• K. pneumoniae can cause bacterial pneumonia, typically
due to aspiration by alcoholics, though it is more
commonly implicated in hospital-acquired urinary tract
and wound infections, particularly in
immunocompromised individuals.
• Klebsiella ranks second to E. coli for urinary tract
infections in older persons.
• Feces are the most significant source of patient infection,
followed by contact with contaminated instruments.
• Klebsiella is resistant to ampicillin but susceptible to
aminoglycosides and cephalosporins.
Klebsiella pneumoniae
Proteus vulgaris
Proteus vulgaris
• Responsible for many human urinary tract
infections.
• Proteus species do not ferment lactose,
are oxidase negative, and urease positive;
some species are motile.
Proteus vulgaris
Providencia stuartii
Providencia stuartii
• This organism is motile.
• They are opportunistic pathogens in
humans and can cause urinary tract
infections, particularly in patients with
long-term indwelling urinary catheters or
extensive severe burns.
• Some strains are sensitive to ampicillin.
Providencia stuartii
IMViC: + + - +
ODC: ONPG: +
H2S: Mannitol: -
Salmonella enterica
Salmonella enterica
Salmonella enterica
• This organism causes typhoid fever and
foodborne illness.
• Salmonella species are motile and
produce hydrogen sulfide.
• They usually do not ferment lactose.
Salmonella enterica
• Food poisoning from Salmonella is usually from
poultry and raw eggs and from food that has
been cooked or frozen, and not eaten straight
away.
• The overuse of antibiotics in both the poultry and
beef industries have created a strain of
salmonella which is potentially resistant to
antibiotics.
• It can also be caught by handling reptiles, such
as iguanas.
Salmonella enterica
• The prevention of Salmonella as a food
illness involves effective sanitizing of food
contact surfaces.
• Alcohol has proven to be an effective
topical sanitizer against Salmonella.
Salmonella enterica
Shigella somnei
Shigella somnei
• This organism is non-motile, non-spore
forming. It is H2S positive.
• It is the causative agent of human
shigellosis.
• Shigella infection is typically via ingestion
(fecal–oral contamination).
• 10 bacterial cells can be enough to cause
an infection.
Shigella somnei
• The stool may contain blood, mucus, or pus (eg
dysentery).
• Shigella cause dysentery that results in the
destruction of the epithelial cells of the intestine.
• Some strains produce an enterotoxin similar to
E. coli O157:H7
• Symptoms usually begin two to four days after
ingestion. Symptoms usually last for several
days, but can last for weeks.
• Severe dysentery can be treated with ampicillin.
Shigella somnei
•
•
•
•
•
•
•
IMViC: - + - - or + + - ODC: + or LDC: Urease: H2S: Sucrose: TSI: K/A
Serratia marcescens
Serratia marcescens
• This organism is involved in nosocomial
infections, particularly urinary tract infections and
wound infections.
• It is motile.
• Due to its preference for damp conditions, it is
commonly found growing in bathrooms
(especially on tile grout), where it manifests as a
pink discoloration.
• Once established, complete eradication of the
organism is often difficult, but can be
accomplished by application of a bleach-based
disinfectant.
Serratia marcescens
• S. marcescens can cause conjunctivitis
and tear duct infections.
• It is common in the respiratory and urinary
tracts of adults and the gastrointestinal
system of children.
Serratia marcescens
•
•
•
•
IMViC: - + + + or - - + +
LDC: +
ADH: ODC: +