Lab Exercise: 15
Enzymes:
1. Catalase
2. Proteinase
3. MR-VP
Enzymes

Enzymes are important biosynthetic chemicals found in all
organisms.

The kinds of enzymes produced by an organism dictate the extent of
its biosynthetic abilities.

Enzymes are proteins that act as chemical catalysts (speeding up
chemical reactions).

The ability to produce enzymes is based in the genome (DNA) of the
cell.

If the cell has the appropriate genetic information for the synthesis of
the enzyme then that enzyme is produced and the cell is able to use
the specific reaction.

Properties of enzymes include specificity and reusability.
Enzymes

Microorganisms, specifically bacteria, can be characterized by their
ability to produce various enzymes.

The presence of various enzymes in turn can be determined by the
production of the appropriate end products in the medium (or their
effects on the medium).

Different bacteria produce various complement of enzymes and
hence these can be useful in determining the identity of the
bacterium based on the enzyme profile these organisms present.
Catalase

Bacteria vary in their ability to grow in the presence of atmospheric oxygen.

The reason being that atmospheric oxygen (in the form it is usually found) is
toxic to most cells.

Only cells that have the ability to neutralize the toxic forms of oxygen are
able to thrive in the presence of oxygen.

Aerobes are those organisms that need and thrive in the presence of
oxygen. (Obligate aerobes do not grow in the absence of oxygen)

Anaerobes are those that cannot grow in the presence of oxygen (some
are killed by oxygen)

Facultative anaerobes are those organisms that are able to survive in the
absence of oxygen but grow faster in the presence of oxygen.
Catalase

Catalase is the enzyme that breaks down toxic forms of oxygen (e.g.
peroxides), neutralizing the toxic effect of those compounds.

The presence of catalase indicates the microbe is capable of growing in the
presence of oxygen (aerobe or facultative anaerobe), whereas the absence
of catalase suggests the organism is anaerobic.

The specific reaction catalyzed by catalase is:

H2O2 ----------- H2O + O2 (gas)

Under lab conditions the presence of catalase can be tested by adding
liquid hydrogen peroxide drops directly onto the culture (cells/colony).

If catalase is present in the organism then H2O2 is hydrolyzed and O2
is released as gas bubbles.
Catalase Procedures
• Controls: 1) Divide a DSA plate in half and 2) make a single
streak with one control culture on each side of the plate.
• Unknown: 1) Make a single streak on a DSA plate.
• Incubate at 37° C overnight.
Unknown
DSA
Controls (S.faecealis/ S.aureus)
DSA
Catalase Results:
• Add a few drops of H2O2 over your culture and observe.
Record Results:
Catalase positive (bubbles form)
Catalase negative (no bubbles)
Amylase (Starch Hydrolysis)
• Exoenzymes :enzymes that are produced by the cell
and secreted outside to act on the substrate external to the cell.
The broken down molecules can then be transported easily into the
cell and used as nutrients by the cell.
• Amylase :an exoenzyme that breaks down starch.
• Reaction catalyzed by amylase:
amylase
Starch
amylose + amylopectin
Starch
Amylase Procedures
•Controls: 1) Divide a Starch Agar plate in half and 2) make a
single streak with one control culture on each side of the plate.
• Unknown: 1) Make a single streak on a Starch Agar plate.
• Incubate at 37° C overnight.
Unknown
Controls (E.coli/ B. subtilus)
Starch Agar
Starch Agar
Amylase Results
•In the lab, the reaction can be visualized by addition of a Lugol’s
iodine that will react with starch and produce blue/purple
compound.
• amylase + : a clearing in the blue zone around the growth
indicates starch was broken down (amylase present).
• amylase -: absence of a clearing in the blue indicates that starch
is intact (no amylase).
Controls (E. coli/ S.aureus)
Unknown
_
+
Starch agar
Starch agar
Amylase: Results
Negative for Amylase
Positive for Amylase
Gelatinase

Gelatin is a complex polymeric protein molecule.

Organisms that are able to produce the exoenzyme
gelatinase are able to break down the gelatin structure
and use it as a protein source (nutrient).
Gelatin--polypeptides +free amino acids
gelatinase
Gelatinase

The reaction can be made observable by the addition of chemicals
that will react with the intact gelatin (unused) and form a white
precipitate.

The chemicals are combined in the form of a developer (mercuric
chloride and hydrochloric acid), which precipitate the protein
(gelatin) and form a white color.

Absence of this white color around the area of growth indicate use
of gelatin by the bacterium and hence a positive result for
Gelatinase production by the organism.

Absence of a clearing in the white precipitate formation indicates
presence of intact gelatin (not used by bacterium) and hence a
negative result for the gelatinase production by bacterium.
Gelatinase Procedures
•Controls: 1) Divide a Frazier’s Gelatin Agar plate in half and 2) make
a single streak with one control culture on each side of the plate.
• Unknown: 1) Make a single streak on a Frazier’s Gelatine Agar plate.
• Incubate at 37° C overnight.
Unknown
Frazier’s Gelatine Agar
Controls (E.coli/ B. subtilus)
Frazier’s Gelatine Agar
Gelatinase results
Gelatinase
Negative
Gelatinase
Positive
Methyl Red Voges-Proskauer (MR-VP) test


An important test to differentiate between glucose
fermenting Enterobacteriaceae.
Principle:
 Glucose is fermented to pyruvic acid by one of the
two pathways:
 Fermentation of mixed acids
 Acetoin/ Naphtol+creatine pathway
Methyl Red test

Methyl Red is an acid-sensitive dye that is yellow at a pH above 4.5
and red at a pH below 4.5.

When the dye is added to a culture of organisms growing in glucose
broth, its color indicates whether the glucose has been broken down
completely to highly acidic end products with a pH below 4.5 (methyl
red positive = red), or only partially to less acidic end products with a
pH above 4.5 (methyl red negative = yellow).
Voges-Proskauer test

The Vogues-Proskauer test can be performed on the same glucose
broth culture used for the methyl red test (MRVP broth).

One of the glucose fermentation end products produced by some
organisms is a Naphtol+creatine.

The VP reagents (alphanaphthol and potassium hydroxide solution)
oxidize this compound having a pink to red color.

VP-positive organisms are those reacting in the test to give this pink
color change.
MRVP

Inoculate MRVP broth with your
culture. Incubate at 37oC for 4-5
days.

VP test- Pipette 1.0 ml of MRVP
broth into empty sterile tube. Add
0.6 ml of alphanaphthol and 0.2 ml
of KOH solution. Shake the tube
well and allow it to stand for 10-20
minutes. Observe and record the
color.

Methyl red test - Add 5 drops of
methyl red indicator to the
remaining 5 ml of broth culture.
Observe and record the color of
the dye. (Methyl red test)
Record results of previous lab

Catalase Controls:
S. faecalis, S. aureus
 unknown

Gelatinase Controls:
E.coli, B. subtilis,
 unknown

MRVP E.
coli, Enterobacter aerogenes
 Unknown
MRVP

Inoculate MRVP broth with your culture. Incubate at 37oC
for 4-5 days.

VP test-


Pipette 1.0 ml of MRVP broth into empty sterile tube.

Add 0.6 ml of alphanaphthol and 0.2 ml of KOH
solution.

Shake the tube well and allow it to stand for 10-20
minutes. Observe and record the color.
Methyl red test –


Add 5 drops of methyl red indicator to remaining 5 ml
of broth culture. Observe and record the color of the
dye.
Controls

E.coli:

Enterobacter: MR-, VP+
MR+, VP-
Enzymes II
Lab 16
SIM test- Sulfide, Indole, Motility

Indole is a by-product of the metabolic
breakdown of the amino acid
tryptophan used by some microbes.

The presence of indole in a culture
grown in a medium containing
tryptophan can be readily
demonstrated by adding Kovac's
reagent to the culture.

If indole is present, it combines with
the reagent to produce a brilliant red
color.

If it is not present, there will be no
color except that of the reagent itself.

This test is of great value in the
battery used it identify enteric bacteria.
* RUN THE INDOLE
PART OF THE TEST
FIRST
SIM
Hydrogen sulfide is produced
when amino acids containing
sulfur are metabolized by
microbes. Hydrogen sulfide
formed during growth
combines with the metallic ions
(iron) to form a metal sulfide
that blackens to medium.
SIM- motility

Motility - This is a tubed semisolid
agar that can be used also to
demonstrate motility. It is
inoculated by stabbing the wire
needle straight down the middle of
the agar and withdrawing along
the same path. Motile organisms
will migrate through the agar,
while non-motile microbes will only
grow along the stab line
SIM- Sulfide, Indole, Motility tests
Procedure
Citrate test

The ability of some organisms, such as Enterobacter aerogenes and
Salmonella typhimurium to utilize citrate as a sole source of carbon.

Citrate is a simple carbohydrate and the test is useful differentiation
characteristic in working with intestinal bacteria.

Koser's citrate medium and Simmons citrate agar are two media
used to detect this ability in bacteria. Both of these are synthetic
media in which sodium citrate is the sole source of carbon, and
nitrogen is supplied by ammonium salts instead of amino acids.

Simmons citrate agar contains the indicator bromthymol blue which
changes from green to blue when growth of organisms causes
alkalinity.
Citrate test


Inoculate a slant of Simmons
Citrate agar with your culture, by
streaking the surface. Incubate at
37°C for 1-2 days.

E.coli, and K. pneumonia

Unknown
Examine the slant.

Negative- If the organism did not
grow the slant will remain green.
E. coli

Positive- If the organism did grow,
the slant will be partly or
completely blue in color.
K. pneumonia
Urease reaction

Urea is a protein compound.

Bacteria that are able to break down the molecule have access to
other protein nutrients.

Bacteria that have the enzyme urease are able to use the urea.
Urea------------- CO2 +NH3

The reaction is made observable by including a pH indicator in the
medium.

Phenol red is the indicator (is peach color at neutral pH, turns
yellow below 6.8 and pink above 8.4).
Urease test
Exercise- inoculate


E.coli, Proteus vulgais

unknown
Incubate 48 h at 37 0c
Interpretation:

The release of ammonia by the breakdown
of urea results in the an alkaline pH of the
medium which will turn the medium pink.

Thus pink color indicates production of
urease by the organisms

Absence of pink color indicates a negative
test for urease.
Carbohydrate
utilizationTSI, fermentation tubes
(F-tubes)
Lab 17
Carbohydrate utilization

Carbohydrates are sugars and they serve as energy source for
bacteria

Bacteria use metabolic pathways to degrade sugars and generate
energy (ATP) in aerobic conditions.

In anaerobic conditions some bacteria can switch to fermentation of
sugars with only small gain of ATP.

The ability of cells to catabolize specific substrates is dependent on
their ability to produce appropriate enzymes.
Fermentation

Fermentation is the catabolism of sugars in the absence of
oxygen where the final electron acceptors is an organic
molecule.

Fermentation typically results in the formation of organic acids and
alcohols that will accumulate in the medium. This will result in the
lowering of the pH of the medium which can be visualized by the
addition of an indicator in the media.

Gas production-Some bacteria characteristically produce gases
during the fermentation process, which can be made visible by the
addition of inverted tubes (F-tubes) in the case of liquid media and
cracks in the agar in solid medium.
F-tubes (Fermentation tubes)




The ability to ferment a specific sugars is dependant on the presence of the
enzymes required for the transport and metabolism of that sugar.
Thus fermentation of various sugars can be used to characterize bacteria.
The F-tubes use phenol red in the medium as pH indicator and the use of
inverted tubes to detect production of gases.
Results are recorded as Acid / Acid Gas / Alkaline / No reaction.
KIA – Kligler Iron Agar

KIA is a medium used in the identification of Gram-negative enteric rods.

KIA contains glucose and lactose, but there is 10 times more lactose than
glucose.

KIA contains a pH-sensitive color indicator. Fermentation of these sugars
can be determined by color change of the phenol red indicator, from red to
yellow for acid production.
Principles of the Procedure
KIA contains:





Protein source (beef extract
Two sugars (dextrose 0.1% and lactose 1% )
phenol red for detecting carbohydrate fermentation
ferrous ammonium sulfate for detection of hydrogen sulfide
production (indicated by blackening in the butt of the tube).
The KIA has a two reaction chamber:


Upper/slant part- aerobic –oxidative decarboxylation of amino
acids
Lower/ Butt anaerobic
slant
butt
Reactions tested by KIA test

Fermentation of sugars – acid production lower
the pH (yellow)

Proteins catabolism -broken down resulting in
alkaline products, which increase the pH (pink)

H2S production
Sugar fermentation / protein metabolismas a source of energy- protein sparing.

Cells will prefer to use sugars for energy when they are present,
sparing the proteins; Proteins will be used only after the sugar
source has been depleted.

Protein metabolism results in alkaline end products and an
increased pH of the media.

When a bacterium ferments the sugar and lowers the pH and
depletes all the sugars. The proteins in the medium will then be
broken down resulting in alkaline products, which increase the pH
and reverse the results of sugar fermentation.

Thus fermentation reactions have to be read within 18-24 hours
before sugar reversion can occur.
H2S production

Hydrogen sulfide (H2S) production,
blackens the medium if it occurs. The H2S
reacts with iron in the medium to produce
FeS, which is black.
KIA- procedure

Using a straight inoculating wire, stab the butt and streak
the surface of agar slant. Do not close lid tightly.

Incubate 1-2 days 37oC.

Observe color of butt and slant as described above. Was
there any H2S production? Is the strain motile? This can
be determined by the grow pattern in the stab. Record all
results. Interpretation in following chart:
KIA- Interpretation






butt / slant
Interpretation
yellow / acid yellow glucose and lactose and/or sucrose are
fermented
yellow / acid orange-red glucose only fermented or pink = alkaline
orange-red/ neutral yellow acid glucose only utilized, aerobically
orange-red / neutral orange-red  neutral no fermentation
bubbles cracks - gas production
black - hydrogen sulfide produced
Interpretation
1
2
3
GAS
4
5
KIA test - Results
6
NOTES:
• Red slant = peptone catabolization
•Yellow butt = glucose fermentation only
•Yellow slant = glucose and lactose ferm.
•Black butt = glucose ferm and H2S prod.
C - K/K - control, no inoculation, no change
1- K/K - no sugar fermentation, red slant due to peptone catabolization
2- K/A - glucose only fermentation, facultative anaerobe, no gas
3- K/A/H2S - glucose fermentation, fac. anaerobe, H2S gas production
4- A/AG - glucose and lactose fermentation, gas production
4A- A/A-KG - like #4, but peptone catabolism leadd to pink on slant
5- A/A/H2S - like #4, but the gas produced is H2S
6- A/K - aerobic, capable of glucose and lactose fermentation