(music)
A key objective in microbiology
laboratories is the identification microbes.
You have been utilizing a microscope and
staining techniques to evaluate bacteria
from different genera.
Microscopy and staining however have
their limitations in attempting to
identify bacteria to the level of species.
Let's consider Staphylococcus aureus and
Staphylococcus epidermidis as examples.
If we were to Gram stain these two
species we would find that both
are Gram-positive cocci that grow in clusters. 
If we were then to acid-fast
stain these two organisms we would find
that both are non acid-fast.
So with each of the differential
staining techniques they appear identical
making it difficult to differentiate the
two species.
To aid in identification of bacteria to
the species level
we must employ other tools in the lab.
Biochemical tests, also referred to as
differential test
are important test that allow us to
evaluate differences in metabolic processes
between species .While two species may
look very similar under the microscope they
may be very different metabolically.
We will be evaluating one type of 
biochemical test in the next lab session.
You will be observing a carbohydrate
fermentation test.
Let's explore what carbohydrate
fermentation test
are and what information they provide us
with.
Carbohydrate fermentation in bacterial
cells results in the formation
of an organic acid or in some cases
an organic acid and a gas.
Bacterial cells vary in the types of
carbohydrates they are capable
of fermenting. This is dependent on what
type of enzymes exist in their cells.
So since we know bacterial cells differ
in the carbohydrates they can utilize
we can set up a test in which specific
carbohydrates are present in different
test tubes to determine which carbohydrates are
utilized. In order to determine whether
the carbohydrate is being fermented
we must be able to detect a change in 
the pH of the medium
as the acid by product is produced. So
carbohydrate fermentation media
always contain a pH indicator that
changes color as the pH changes.
The carbohydrate fermentation media we
are using in lab is known as the
phenol red broth. Phenol red broth 
contains
peptones, phenol red, and one carbohydrate.
Peptones serve as a source of amino acids
and the phenol red acts as a pH indicator.
Each test tube will have a different sugar.
A small tube known as a Durham tube
is placed inside the test tube in an
inverted position.
It is placed so that no gas is trapped in
the tube
prior to inoculation with bacteria. 
The Durham tube
serves to trap gases if the bacteria produces
gas as a by-product of fermentation.
The phenol red pH indicator will appear
yellow at a pHs
less than 6.8,  pink to purple at pHs
greater than 7.4
and red in between those two values. The
medium is adjusted to pH 7.3 when the
broth is prepared so that it appears red.
In order to conduct the carbohydrate 
fermentation test the bacteria
of interest are introduced using
aseptic technique
into phenol red broths containing various
sugars.
The broth is then incubated to allow for growth
and evaluated at approximately 18 hours.
Broths older than 24 hours generally will
not produce accurate results.
The reason for this is that a bacterium
capable of fermenting a sugar will first
ferment the sugar as a source of energy.
In doing so the bacteria will produce acids.
that lower the pH causing the
phenol red to turn yellow.
If the bacteria exhaust all the sugar through
fermentation it will then begin to
metabolize the peptones that are present
in the media.
As the bacteria deaminate the amino acids
from the peptones
they produce ammonia, which is basic. As
the production of ammonia increases
the media then becomes alkaline and the
phenol red changes to a pink or purple.
At this point we would not be able to
determine if the bacteria fermented the
sugar and then reverted to amino acids
as a source of energy or if it was 
unable to ferment the sugar and solely
used amino acids as a source of
energy.
Let's consider how one would interpret the results
of the phenol red carbohydrate
fermentation test.
If a bacteria is capable of fermenting
the sugar present in the medium
it will produce acid as a by-product of
the chemical reaction.
In doing so the pH of the medium will be
lowered.
As the pH drops below 6.8 the phenol red
indicator changes to yellow.
After incubation the presence of a yellow
medium indicates a positive reaction
meaning that the bacteria fermented the
sugar that was present.
If the medium remains red, it is a
negative result
indicating that bacteria could not
ferment the provided sugar.
Some bacteria will produce gas in
addition to the acid.
The gas is detected using the inverted
Durham tube. You must look to the top
of the Durham tube to evaluate whether
or not gas was produced.
If gas was produced there will be a
bubble in the top of the Durham tube,
indicating a positive result for gas
production.
If there is no bubble present the
result is negative,
indicating no gas production.
The image seen on the screen shows
potential results from a phenol red broth
carbohydrate fermentation test. Test tube
C is the control,
it has not been inoculated with bacteria.
It shows you what a phenol red broth
looks like when we begin this
differential test.
Test tubes A and B are both positive 
for acid production
as indicated by the change in color of 
the medium from red to yellow.
Test tube A is also positive for gas
production.
The arrow indicates where the bubble
in the Durham tube is located.
Test tube D, has become more
pink in color
indicating that the bacteria have produced
alkaline by-products from peptone break down.
Test tube E has been inoculated
but shows no change.
So let's check your understanding. The
same organism is incubated in
each of the test tubes seen on the screen.
Can you determine what these results mean?
I'll give you a moment.
The results indicate that the test tube containing
glucose shows a positive acid production
with a negative gas production. The tubes
containing maltose and sucrose are negative
for acid production and gas production.
So these results tell us that the
organism can only metabolize glucose
out of  these three sugars and that when it
does so it only produces an organic acid
as its by-product. Please look for the
handout that accompanies this activity
in your pre-lab assignment.
It will be useful to have with you in
the lab session.
(music)
