Download Unit 10: Protein Catabolism - Central New Mexico Community College

Unit 10
Unit 10: Protein Catabolism
By Heather Fitzgerald, Karen Bentz and Patricia G. Wilber.
Copyright Central New Mexico Community College, 2015
Introduction
The thought of rotting meat usually brings up a gagging sensation for most people. Similarly, the smell
of a human cadaver is not associated with pleasant smells. Nonetheless, in part, the smell of the
decaying cadaver comes from the aptly named molecules, cadaverine and putrescine (putrid!).
Cadaverine is produced when microbes break down (catabolize) animal protein to acquire nutrients
and energy for DNA replication and growth. Protein catabolism by microbes is certainly important so
microbes can acquire energy from sources other than carbohydrates and fats, and it is also critical
ecologically. If microbes did not break down animal proteins, the world would be littered with
undegraded carcasses!
Breakdown of specific proteins requires microbes to contain specific genes for specific enzymes
(DNA RNAprotein). While there are many different enzymes involved in converting proteins to
other molecules, we will focus on a few commonly analyzed microbial enzymatic processes.
Proteins consist of amino acids covalently bonded together (See Figure 10-1). There are 21 naturally
occurring (20 are common, one is rare) amino acids (e.g. tryptophan(TRP) and lysine(LYS)). Each amino
acid contains a central carbon atom covalently bonded to both an amino group (NH2) and a carboxylic
acid group (COOH) as well as a side chain that varies in structure (R; R stands for “the Remainder”). The
side chain or R-group, is the unique part of each amino acid and is the unit that gives each of the 21
different amino acids their unique properties
Figure 10-1. Basic structure of proteins (amino acids joined via covalent bond).
Accessed 8/17/2015 from https://commons.wikimedia.org/wiki/File:Protein_primary_structure.svg
Unit 10 Page 1
Unit 10
What part of a protein is targeted for breakdown?
Usually, when enzymes cut the protein chain, they work on a specific amino acid and are named for the
particular bond site that they are able to break. Decarboxylases are enzymes that remove the carboxyl
group from amino acids by breaking the bond between the central carbon and the carboxyl group. (See
Figure 10-2 for examples of general enzyme cut sites.) The specific enzyme lysine decarboxylase,
removes the carboxyl group from the amino acid lysine. Deaminases are enzymes that remove a
molecule’s amine group. Phenylalanine deaminase removes the amino group from the amino acid
phenylalanine. Other enzymes we will study in this lab react with particular atoms in the side chains of
proteins. For instance, the cysteine desulfhydrase enzyme in the SIM test (described below) will
remove the sulfur atom from the R-group in the amino acid cysteine and the tryptophanase enzyme
will remove the indole group and the amino group from the amino acid tryptophan. Finally, we will
study an enzyme that deals with urea. Urea is produced by humans (and other organisms) when
proteins are metabolized for energy and nutrients. The amino group (which contains nitrogen) on the
amino acids in proteins is toxic, so urea is produced and we excrete it in our urine. Some microbes
produce the enzyme urease. The Urease enzyme will convert the urea found in urine to ammonia and
allows the bacteria that produce urease to successfully colonize the urinary tract
While carbohydrate usage generally produces an acid or neutral waste, the break down products of
protein digestion generally increase the pH of the environment by producing basic end products. Just
as in the carbohydrate unit, pH indicators (that change color) are used to visualize the pH change. The
changes in pH in the growth media of a bacterium can provide information about what type of
catabolism has occurred.
Unit 10 Page 2
Unit 10
Figure 10-2. A generalized structure of an amino acid and target sites for enzymes.
Where Decarboxylases cut
Where Deaminases cut
Tryptophanase
works on both the R
group tryptophan and
the amine group
(tryptophan).
Cysteine desulfhydrase
works on the R group of
cysteine
Accessed 8/15-15 from https://commons.wikimedia.org/wiki/File%3AAminoAcidball.svg. By GYassineMrabetTalk This vector image was created with
Inkscape. (Own work). Modifications by H. Fitzgerald.
Specific Protein Catabolism Tests
Lysine Decarboxylase test.
The lysine decarboxylase test shows us if the bacteria produce the enzyme lysine decarboxylase (LDC).
Lysine decarboxylase cleaves the carboxyl group from the amino acid lysine (located on a protein) and
produces the breakdown product cadaverine. Cadaverine has a strong odor that is commonly
associated with rotting animal carcasses (cadavers!).
The differential agent in the LDC test is the amino acid lysine and the pH indicator is Bromocresol
purple. When LDC is present conditions become basic (high pH) and Bromocresol purple becomes
purplish (a positive test result). When LDC is absent, the media turns yellow (pH less than 6.8; low pH).
Uninoculated media is a brownish/purple color. Glucose and peptones are also in the media.
The LDC enzyme requires an acidic environment to become activated, so placing mineral oil over the
broth and capping the tube lids tightly reduces oxygen and encourage fermentation of the glucose to
produce the acidic environment. When this occurs the tube will first turn from brownish/purple to
yellow. If the tubes remains yellow this indicates the bacteria DO NOT contain the LDC enzyme. If the
enzyme is produced by the bacteria being tested, the enzyme will be activated by the low pH. When
LDC cleaves the carboxyl group off the lysine, this generates cadaverine and other basic (high pH) end
products which will raise the pH of the broth back above pH 6.8 and turn the pH indicator
Unit 10 Page 3
Unit 10
(bromocresol purple) back to purple. Therefore a true purple result is a positive reaction indicating the
presence of the LDC, and a yellow or original brownish/purple color is a negative result indicating
growth of the bacteria, but the lack of LDC. See Figure 10-3.
Figure 10-3. Example results of Decarboxylase test. The leftmost tube shows a positive decarboxylase
test, while the two right most tubes show negative results. The browinish tube in the middle shows the
color of an uninoculated tube, while the bright yellow tube indicates a true negative result.
Lysine Decarboxylase
Lysine
Cadaverine
Photo Accessed 8/17/15 from
http://www.microbelibrary.org/library/labor
atory-test/3006-biochemical-test-media-forlab-unknown-identification-part-1.
Phenylalanine Deaminase test.
This test will help distinguish among bacteria by determining whether or not they contain the enzyme
phenylalanine deaminase. In particular, this test helps to distinguish Proteus species (which contain
this enzyme) from bacteria that do not. Bacillus megaterium is also very weakly positive in this test.
This enzyme cleaves off the amino group from the amino acid Phenylalanine. In doing so, the major
products formed are phenylpyruvic acid and ammonia.
This test is performed using an agar slant medium. The differential material is the amino acid
phenylalanine and after incubation a reagent called ferric chloride (FeCl3)(10%) is added to detect if
the phenylpyruvic acid was produced, or not. If phenylpyruvic acid was produced, a light to dark
green, color will appear, and we know the organism produces the enzyme phenylalanine deaminase. .
The medium also contains yeast extract (for carbon and nitrogen. See Figure 10-4 for examples of
positive and negative results after the addition of the ferric chloride solution.
Unit 10 Page 4
Unit 10
Figure 10-4. Examples of positive and negative results in the phenylalanine deaminase test.
Phenylalanine Deaminase
Phenylalanine
Phenylpyruvic acid,
Ammonia
Accessed 8/17/15 from
http://www.microbelibrary.org/library/labor
atory-test/3006-biochemical-test-media-forlab-unknown-identification-part-1.
Urease Test
Ureases are enzymes that will hydrolyze urea to ammonia and carbon dioxide. Urea is a nitrogen
containing waste product found in animal urine. Detection of urease activity (which allows survival of
bacteria in the gastric and/or urinary systems) is a key diagnostic for potential human pathogens. The
ulcer-causing bacteria, Helicobacter pylori uses urease to produce ammonia and help de-acidify its local
stomach environment. Some Proteus species utilize urease to increase the alkalinity of the urine. This
allows the bacteria to survive in the urea-rich environment of the urinary tract. Infection with Proteus
species can lead to inflammation and contribute to urinary tract stone formation. Thus urease
producing bacterial species may be pathogens.
The urease test is done using an agar-based slant medium. The slant contains the differential material
urea, and the pH indicator, phenol red. Phenol red turns yellow when the pH is less than pH 6.8
(acidic), and is a bright pink at a pH ≥ 8.4 (basic/alkaline). Since ammonia is a product of urease
activity and ammonia is basic, a bright pink slant is a positive result, revealing that the bacteria
produces urease. Peptone and other essential growth factors are also in the media.
If at least 80% of the slant and butt, is pink after 24 hours, there is rapid positive urease activity. If
there is pink only along the slant after 24 hours, this is considered weak or delayed activity. These
tubes may become pinker over time. A yellowish colored tube that remains roughly the same color as
uninoculated media indicates a negative urease result. See Figure 10-5 for examples.
Unit 10 Page 5
Unit 10
Figure 10-5. Example results of a Urease test. A rapidly positive urease test by Proteus mirabilis (b) is
indicated by a color change to bright pink (fuchsia) throughout the urea agar slant as compared to the
uninoculated control (a). A delayed positive reaction by Klebsiella pneumoniae is indicated by a color
change only along the slant (c). A negative reaction by Escherichia coli is indicated by the yellow
coloration of the media (d). All samples were incubated at 37oC for 16 hours.
Urease
Urea
Ammonia + CO2
Accessed 8/17/15 from http://www.microbelibrary.org/library/2associated-figure-resource/3301-urease-testlabeled. Cultures and image
description from Benita A. Brink, Adams State College, Alamosa, CO.
Sulfur, Indole and Motility (SIM) test.
The SIM tests analyze three different bacterial characteristics:
(S) The ability of bacteria to produce H2S using cysteine desulfhydrase or thiosulfate reductase (same
as the TSI).
(I) The ability to split the indole group off of the amino acid tryptophan by the enzyme tryptophanase.
(M) The ability of the bacteria to move (be motile) in the media, due to the presence of flagella.
The SIM test is done using a semi-solid agar deep. The semi-solid consistency allows motile bacteria to
move through the media, away from the stab line.
The SIM is a basic T-soy semisolid agar medium made with peptone (a source of cysteine) and beef
extract, thiosulfate, and ferrous sulfate (a source of iron, Fe+2). For examples of expected results for
each of the three components being assayed see Figure 10-6 below.
The (S) or sulfur component of this test identifies the production of hydrogen sulfide (H2S). If a bacteria
produces hydrogen sulfide (H2S), it will react with the iron ions in the ferrous sulfate (an ingredient in
the media) to produce a black color in the bottom of the tube.
Unit 10 Page 6
Unit 10
One means of producing H2S is using the enzyme cysteine desulfhydrase. The cysteine desulfhydrase
enzyme reduces the sulfur in the amino acid cysteine (found in the peptone, an ingredient in the
media) and produces the products of H2S, pyruvate and ammonia.
H2S can also be produced by organisms that produce thiosulfate reductase. Thiosulfate reductase
reduces thiosulfate (an ingredient in the media) to H2S.
SIM media does not allow us to distinguish between these types of sulfide production.
Once H2S is produced by either means, the H2S reacts with the ferrous sulfate in the media to produce
a black color in the butt of the media. The black can obscure the motility results, but fortunately,
organisms that produce a black in the SIM are also motile.
The black in the SIM is produced by the same mechanisms as the black in the TSI.
The black indicates that the organism is a potential pathogen.
The indole (I) component of this test analyzes bacteria for the presence of the enzyme tryptophanase.
Tryptophanse is a deaminase enzyme that breaks down proteins that contain the amino acid
tryptophan (an ingredient in the media) to produce pyruvate, indole and ammonia (amino group).
Some studies suggest that indole is a chemical signaling molecule that helps with bacterial
multiplication and possibly contributes to biofilm formation. Among organisms that infect the
respiratory tract, such as Haemophilus influenzae (the bacteria, not the flu virus), the ability to produce
tryptophanase and break down tryptophan to produce indole is positively correlated with higher
pathogenicity.
The production of indole is observed by the reaction of the indole end product with Kovak’s reagent.
After incubation, Kovac’s is applied to the top of the deep. If a red ring occurs then the bacteria is
positive for the production of indole and thus positive for tryptophanase. If the ring remains clearish or
yellow, the result is negative for indole production and thus negative for tryptophanase.
The (M) test for motility indicates the presence of flagella (see Unit 5) on bacteria. If bacteria have
flagella, then they are able to “swim” away from the inoculation site in a 360 degree manner and
undergo binary fission. This produces a fuzzy haziness extending in all directions (three dimensionally)
from the stab line. If whitish growth is only observed in only two dimensions along the stab line this is a
negative result. Again, all known organisms that can produce H2S in the (S) test are also motile.
Motility is often a sign of pathogenicity. Bacteria that produce flagella can more readily move from the
site of infection in the body to other locations.
Unit 10 Page 7
Unit 10
Figure 10-6. Example SIM test results. Tube A is uninoculated. Note that the stab line is not present. Tube B
has growth from the stab line in a two dimensional pattern but no real change in haziness compared to Tube A,
the uninoculated tube. The lack of haziness and the 2D growth pattern indicates a negative test for
motility. Inoculated tubes should be compared to an uninoculated tube to discriminate between cloudiness
(motility) and lack of motility. Tube B is also negative for indole. Tube C has a red color on the surface of the
medium after addition of Kovac’s reagent. This indicates indole production and the production of
tryptophanase by the bacteria. Tube C also has fuzziness/cloudiness spreading three dimensionally away from
the stab. This indicates motility—the presence of flagella. Tube D is black, which indicates H2S production via
the digestion of cysteine by cysteine desulfhydrase (or reduction of thiosulfate) and therefore is also positive for
motility. D is negative for indole production.
.
Accessed 8/18/15 from http://www.microbelibrary.org/library/2-associated-figure-resource/3645-sim-medium. Image and
results interpretation provided courtesy of Renee Wilkins, University of Mississippi Medical Center, Jackson, MS.
Tryptophanase
Tryptophan
Indole, ammonia
Cysteine Desulfhydrase
Cysteine
H2S, pyruvate, ammonia
Thiosulfate reductase
Thiosulfate
H2S
Unit 10 Page 8
Unit 10
DAY ONE: INOCULATIONS
Video Links:




Lysine Decarboxylase https://www.youtube.com/watch?v=-RtPXdDQVjI
Phenylalanine Deaminase https://www.youtube.com/watch?v=msBI0_Fb_nE
SIM https://www.youtube.com/watch?v=tXNGV6MaNVw
Urease Test https://www.youtube.com/watch?v=1MCWEPhxLZU
Videos created by Corrie Andries.
Materials

Media (per pair of students)
o 2 Lysine broth tubes
o 3 Phenylalanine slants
o 3 SIM deep tubes
o 3 Urea slant tubes

Bacteria Cultures
o Escherichia coli (Ec)
o Proteus vulgaris (Pv)
o Klebsiella pneumoniae (Kp)
o Bacillus megaterium (Bm)
Unit 10 Page 9
Unit 10
Procedure
A. Lysine Decarboxylase Test
Ec or Kp
Bm or Pv
Image created 2015 by H. Fitzgerald and K. Bentz
1. Label your tubes appropriately. Use one tube for each bacterial species, as indicated in the image
above.
2. Use aseptic technique and a cooled, sterile metal loop to acquire a small amount of one bacterial
species.
3. Dip the loop into the broth and swirl the loop around.
4. Resterilize the loop and place it back into the tools container on the lab bench.
5. Add around 2mm (about 5 drops) of mineral oil (make sure NOT to use the microscope objective oil
for this) to the top of the broth.
6. Tightly (!!) cap the tubes and then place into the appropriate inoculation tray.
7. Repeat this procedure for each of the other three bacterial species.
Precautions: In order for the decarboxylases to be well activated the environment must first become
acidic. This will only happen in low oxygen conditions so make sure to add the oil after you inoculate
the tubes, and tighten the caps!!
Unit 10 Page 10
Unit 10
B. Phenylalanine Deaminase Test
Bm
Pv
Choice
1. Label your tubes appropriately. Use one tube for each bacterial species, as indicated in the image
above.
2. Use aseptic technique and a cooled, sterile metal loop to acquire a small amount of one bacterial
species.
3. Create fishtail streak on the surface of the slant.
4. Resterilize the loop and place it back into the tools container on the lab bench.
5. Loosely cap the Phenylalanine tube and place it into the appropriate inoculation tray.
6. Repeat this procedure for each of the other three bacterial species.
7. Remember that on day #2 you will need to add 10% ferric chloride solution to each tube.
Unit 10 Page 11
Unit 10
C. Urease test
Figure 10-8. Inoculation of Urea slants with four different bacteria.
Kp or Bm
Pv
Ec
Image created 2015 by Karen Bentz and modified by H. Fitzgerald
1. Label your tubes appropriately. Use one tube for each bacterial species, as indicated in the image
above.
2. Use aseptic technique and a cooled, sterile metal loop to acquire a small amount of one bacterial
species.
3. Make a fishtail streak on the surface of the slant.
4. Resterilize the loop and place it back into the tools container on the lab bench.
5. Loosely cap the Urea tube and place it into the appropriate inoculation tray.
6. Repeat this procedure for each of the other three bacterial species.
Unit 10 Page 12
Unit 10
D. SIM test
Figure 10-7. Inoculating each of three SIM semi-solid deeps with a different bacteria as shown
Ec
Pv
choice
1. Label your tubes appropriately. Use one tube for each bacterial species, as indicated in the image
above.
2. Use aseptic technique and a cooled, sterile metal needle to acquire a small amount of one bacterial
species.
3. Stab the SIM deep tube about 2/3 of the way into the media. Put the needle in and out as straight as
possible.
4. Resterilize the needle and place it back into the tools container on the lab bench.
5. Loosely cap the SIM deep tube and place into the appropriate inoculation tray.
6. Repeat this procedure for each of the other three bacterial species.
7. Remember that on Day 2, these tubes will to have Kovack’s reagent added to observe the indole
result.
Precautions


If your needle wire is crooked, use a gloved hand to get it as straight as possible
Be careful not to stab all the way to the bottom of the media.
Unit 10 Page 13
Unit 10
DAY TWO: Results and Interpretation
Procedure:
1. Collect the media you inoculated in the previous lab.
2. Add 4 drops of Kovak’s reagent to your SIM tubes.
3. When you are ready to observe your phenylalanine tubes (PA), add 4 drops of FeCl3 reagent to each
of your PA tubes.
4. Observe any color changes, growth patterns or other visible changes to the inoculation tubes. Fill in
your observations in the tables below. Be aware that B. megaterium is likely to VERY WEAKLY green in
the PA tube after addition of the FeCl3 reagent ONLY after about 5-10 minutes.
A. Lysine Decarboxylase Media Results (and a little interpretation)
If the Bacteria Grew, What Color is the Media?
Name of Bacteria
(purple=positive; not purple=negative)
Insert Photos of the Lysine Decarboxylase test results here:
Unit 10 Page 14
Unit 10
B. Phenylalanine Deaminase Media Results (and a little interpretation)
Name of Bacteria
If the bacteria grew, after addition of ferric chloride, what color is the liquid
in the media?
(green=positive; not green, clearish=negative)
Insert photos of your Phenylalanine deaminase test results here:
Unit 10 Page 15
Unit 10
C. Urease Media Results (and a little interpretation)
If the Bacteria Grew, What Color is the Media?
Name of Bacteria
(pink all over = strong positive; pink slant and yellow butt= delayed positive; yellow or original
color all over=negatvie)
Slant color
Insert Photos of the Urease test results here:
Unit 10 Page 16
Butt color
Unit 10
D. SIM Media Results (and a little interpretation)
Sulfur
Indole
(black
ppt.=
positive;
no
(red
ring
at top=positive;
Name of Bacteria
black = negative)
no ring = negative
Insert Photos of the SIM test results here:
Unit 10 Page 17
Motility
(cloudiness or black ppt. away from the
stab line=motility)
Unit 10
Interpretation
Based on all of your protein catabolism results, explain what you know about the enzymes and motility
of each bacterial species you tested. If you didn’t use the test for that species, leave rows blank
Bacterial Species: ___________________________________
Which, if any, Protein Degrading
Enzymes does this Bacteria
Produce?
What evidence from which
media supports your enzyme
choice?
(Cysteine Desulfhydrase, Thiosulfate
reductase, Tryptophanase, Urease,
Phenylalanine Deaminase, Lysine
Decarboxylase)
Enzyme:
Enzyme:
Enzyme:
Enzyme:
Add more rows if you need more room.
Unit 10 Page 18
Does this Bacterial
Species have Flagella?
Evidence?
What is the Evidence (if
any) for this Organism
being a Possible
Pathogen?
Unit 10
Bacterial Species: ___________________________________
Which, if any, Protein Degrading
Enzymes does this Bacteria
Produce?
What evidence from which
media supports your enzyme
choice?
Does this Bacterial
Species have Flagella?
Evidence?
What is the Evidence (if
any) for this Organism
being a Possible
Pathogen?
Does this Bacterial
Species have Flagella?
Evidence?
What is the Evidence (if
any) for this Organism
being a Possible
Pathogen?
(Cysteine Desulfhydrase, Thiosulfate
reductase,Tryptophanase, Urease,
Phenylalanine Deaminase, Lysine
Decarboxylase)
Enzyme:
Enzyme:
Enzyme:
Enzyme:
Add more rows if you need more room.
Bacterial Species: ___________________________________
Which, if any, Protein Degrading
Enzymes does this Bacteria
Produce?
What evidence from which
media supports your enzyme
choice?
(Cysteine Desulfhydrase, Thiosulfate
reductase,Tryptophanase, Urease,
Phenylalanine Deaminase, Lysine
Decarboxylase)
Enzyme:
Enzyme:
Enzyme:
Enzyme:
Add more rows if you need more room.
Unit 10 Page 19
Unit 10
Bacterial Species: ___________________________________
Which, if any, Protein Degrading
Enzymes does this Bacteria
Produce?
What evidence from which
media supports your enzyme
choice?
Does this Bacterial
Species have Flagella?
Evidence?
(Cysteine Desulfhydrase, Thiosulfate
reductase,Tryptophanase, Urease,
Phenylalanine Deaminase, Lysine
Decarboxylase)
Enzyme:
Enzyme:
Enzyme:
Enzyme:
Add more rows if you need more room.
Post-Activity Questions
1. What is the unifying feature of the enzyme tests described in this unit?
Unit 10 Page 20
What is the Evidence (if
any) for this Organism
being a Possible
Pathogen?
Unit 10
2. Indicate with arrows where each of the following enzymes breaks a bond in the amino acid.
Lysine Decarboxylase
Tryptophanase
Cysteine Desulfhydrase
Phenylalanine Deaminase
3. Kovac’s reagent is added to the __________ media and will form a ______ ring at the top of the
media if the result is positive.
4. FeCl3 is added to the _______________________ media and will turn a __________ color if the
result is positive.
5. The pH indicator in the lysine test is _____________ _____________. A positive lysine test will be
a ___________ color.
6. SIM stands for _________, ___________ and __________.
a. Describe what a positive test for sulfur would look like:
b. A positive indole test would have a:
c. If a bacteria is motile, it will have one or more _________ and can move through the media
away from the ______________ _________.
Unit 10 Page 21
Unit 10
7. The pH indicator in the Urease media is _________________ and a positive result will be a
____________ color.
8. List the differential material(s) (if any) for each test.
S
I
M
Phenylalanine Deaminase
Lysine Decarboxylase
Urease
The authors of this lab unit would like to thank Andrea Peterson and Deyanna Decatur for testing new media and organisms, our
associate dean Linda Martin for many kinds of aid, Michael Jillson and Alex Silage for IT support, and our Dean, John Cornish.
Unit 10 Page 22
Unit 10
Table 10-1. Protein Catabolism
Media
Ingredients
SIM deep
T-soy semisolid agar plus:
Inoculate with a
stab into the
media
- beef extract
- peptones (source of
cysteine)
- thiosulfate
- ferrous sulfate as a
source of iron to indicate
H2S production
What Positive/Negative
Results Look Like
1. S A black precipitate in the
butt of the tube is positive
2. I Presence of a red ring at
the top of the media after
Kovac’s reagent is added is
positive
3. M Cloudiness away from the
stab line or BLACK is positive
Interpretation if Results
are Positive
1. S Black. The bacteria digest
cysteine using the enzyme
cysteine desulfhydrase to H2S or
reduces thiosulfate using the
enzyme thiosulfate reductase to
produce H2S. H2S reacts with
ferrous sulfate to produce the
black precipitate.
2. I The bacteria digest
tryptophan to indole, indicating
the presence of tryptophanase
(a deaminase that also works on
the R-group)
Of Special Note
1. S&M If a bacteria
produces a black
precipitate, it also has
flagella. Flagella allow
motility and contribute to
pathogenicity.
2. I Tryptophan is digested
to indole, which reacts with
Kovac’s reagent for form a
red ring. Indole can
contribute to
pathogenicity.
3. M The bacteria produces one
or more flagella
Urease slant
T-soy agar plus:
Use a fishtail
inoculation on
the surface of
the slant
- Urea
- Phenol red pH indicator
Color change to bright pink
(fuchsia) is positive. Fuchsia
color throughout indicates
rapid activity of urease, fuchsia
color only on the top of the
slant indicates weak or delayed
activity of urease.
The bacteria produce the enzyme
urease that converts urea, a
waste product of protein
digestion, to ammonia and CO2.
This raises the pH and the Phenol
red indicator turns a bright pink.
A positive urease result
suggests an organism can
live in the urinary tract and
thus may be pathogenic.
The bacteria produce the enzyme
lysine decarboxylase which
digests lysine to produce
cadaverine. This raises the pH and
the Bromocresol purple turns
purple
Remember to put 5 drops
of mineral oil on the top of
the broth after inoculation
and close the cap of the
tube tightly.
The bacteria produce
phenylalanine deaminase that
digests phenylalanine into
phenylpyruvic acid and ammonia.
The green color will form
almost immediately if the
bacteria is positive for
phenylalanine deaminase.
If the agar remains yellowish,
the test is negative
Lysine
Decarboxylase
broth
Use a loop to
inoculate the
broth
Phenylalanine
Deaminase
Slant
Use a fishtail
inoculation on
the surface of
the slant
Basic nutrient broth plus:
- peptone and glucose
- lysine
- Bromocresol purple pH
indicator
T-soy agar plus:
- yeast extract
- phenylalanine
A color change to purple is a
positive test.
Yellow or any other color is
negative
When 10% ferric chloride is
added to the top of the slant, a
green color is positive.
A yellow color is a negative
result.
Unit 10 Page 23