Transcript Physiological characteristics

Physiological characteristics:
Oxidative and fermentation tests
Lab # 8
Medgar Evers College
Prof. Santos
Metabolism
• Metabolism is the sum total of all chemical
reactions that occur in a cell.
• 2 types of metabolic pathways; catabolism
and anabolism.
catabolism
The breakdown of food material either
through respiration or fermentation
Anabolism
• The biosynthesis of materials needed by
the cell such protein production or
photosynthesis
Laboratory Aim today!
• To determine if our unknown sample
carries out respiration (oxidative) or
fermentation (fermentative).
• If our organism is fermentative, which
sugars are specifically fermented?
O/F Glucose test
• This test will be done to determine if our
organism is oxidative or fermentative!
• O/F Glucose is a differential medium that
contains three important ingredients
1- glucose
2- peptone
3- brom-thymol blue pH indicator that turns
yellow at acidic pH
Procedure
1- you will inoculate 2 tubes with your
unknown by stabbing the agar.
2- to one tube, you will aseptically layer with
a couple of drops of mineral oil. This
prevents the diffusion of oxygen and helps
establish anaerobic condition.
• You will repeat the procedure with your
two control organisms; E coli (facultative)
and Pseudomonas (oxidative)
• A color change from green to yellow
means a positive test. Due to pH indicator
changing as a result of slight acid
production due to respiration. Look for
yellow near the top of tube if organism is
aerobic!
results
Aerobic
condition
Anaerobic
condition
interpretati
on
Pseudomo
nas
yellow
green
Strict
aerobe
E. coli
yellow
yellow
facultative
Unknown?
Fermentative tests
1- specific sugar tests such as glucose,
mannitol and lactose.
2- Mixed acid fermentation using MR-VP
medium
3- 2,3- butanediol fermentation
4- citrate fermentation
Specific sugar fermentation
reactions
1- we will use specific sugar broth with a
Durham tube inside.
2-The pH indicator phenol red will be used
to detect the formation of acid that
indicates fermentation.
3- you will inoculate your unknown into a
tube of glucose, lactose and mannitol!
4- the control tube will be E.coli into a
glucose tube.
Mixed acid fermentation
• Some species of bacteria such as
Escherichia, Proteus, Salmonella, and
Aeromonas can ferment glucose and
produce acidic products such a lactic acid,
succinic, acetic, and formic acid.
• In addition they can produce CO2 and H2
gas due to the presence of an enzyme
called formic hydrogenlyase.
• We will use the medium called MR-VP to
inoculate our unknown and your control
which is E. coli.
• MR-VP medium contains glucose,
peptone, and dipotassium phosphate.
• Next week we will use methyl red to detect
the presence of mixed acids!
2,3- Butanediol fermentation
• Some species of bacteria such as
Enterobacter and Serratia can ferment
glucose and produce a neutral end
product such as 2,3-butanediol.
• This is important because usually if a type
of bacteria is + for mixed acid it is – for
neutral fermentation. It allows clinical
microbiologists to distinguish between
important gram – bacteria.
• You will inoculate a tube of MR-VP
medium with your unknown and the
control organism which is Enterobacter
aerogenes.
• Next week, we will discuss how we detect
the neutral end product using Barritt’s
reagent.
Citrate fermentation
• Some bacteria can break down citrate to
oxaloacetate and pyruvate. These
intermediates are then fermented to
produce formate, lactate, acetate, acetoin,
and carbon dioxide.
• We will use Simmons citrate agar. It
contains citrate and ammonium salts to
serve as a source of nitrogen. As the
organisms break down the citrate they
must also break down the ammonium salts
into ammonia. Ammonia raises the pH and
the agar changes color from green to
Prussian blue.
• You will inoculate the Simmons citrate
agar with your unknown and the control
which is Enterobacter aerogenes. First
streak with a needle and then stab it!
Oxidative tests
1- cytochrome oxidase test
2- catalase test
3- nitrate reduction
Cytochrome C oxidase test
• Aerobic organisms such as Pseudomonas
carry out respiration and possess an
enzyme called cytochrome oxidase that
catalyzes the transfer of electrons from
reduced cytochrome c to molecular
oxygen.
• You will streak half a TSA plate with your
unknown and the other half with your
control Pseudomonas aeruginosa.
• Next week, we will use an artificial electron
acceptor to detect the enzyme! The
artificial acceptor is called N,N,N,Ntetramethyl-p-phenylenediamine!
Catalase test
• A dangerous by product of aerobic respiration is
hydrogen peroxide. Aerobes produce an
enzyme called Catalase that breaks down the
hydrogen peroxide into water and oxygen.
• Next week, you will use hydrogen peroxide to
detect the presence of the enzyme by your
unknown. The TSA plate will also be used for
this experiment. The control is a nutrient agar
slant inoculated with S. aureus.
Nitrate reduction
• Some bacteria can use nitrates as the final
electron acceptor instead of oxygen.
• Some bacteria such as Bacillus can
reduce the nitrates to gas end products
such as N2O or N2.
• Others such as E.coli can reduce the
nitrates to nitrites.
• We will assay for the final end product,
gas or nitrite!
• We will use beef extract medium with
potassium nitrate. A Durham tube will be
in place to detect gas formation.
• You will inoculate a tube of beef extract
with your unknown and the control E. coli.
• Next week, we will detect gas formation by
looking at Durham tube and detect nitrite
formation using Nitrate reagent A and B.
These 2 reagents react with nitrite to give
you a deep red color!