Biochemical test set up

An overnight culture is provided

A series of tests are set up to determine the bacterium’s biochemical properties for bacterial
identification

Label all plates tubes and bijoux with your intials and the name of the bacteria you are using before
beginning

Make a single zigzag streak with your bacteria on the phenolphthalein place which is labeled PP

Next, make a single zigzag streak on the 4% tripped on plate which is labeled TRY

For all of the liquid test tubes, add a loophole of culture to each using aseptic technique

For the solid tests gently stab each with a loop full of culture using aseptic technique

Be particularly careful when flaming the loop after inoculation the human lesions test containing oil

Use the part if the flame higher than you normally would use for flaming that is cooler as the oil can
season and spit

For the amino acid decarboxylase test, and a loophole of culture to each of the three B’s use containing
purple liquid

All plates, tubes, and visions??? Will be incubated at 37degress Celsius for 24 hrs.

Biochemical test results

After incubation, the results of biochemical tests for the bacterium under investigation can be
determined.

to determine if the culture can metabolize tryptophan, indole production is measured

to test for indole, place a piece of filter paper in a petri dish and add a few drops from the indole test file

transfer a colony from the 4% tryptone plate to the filter paper and mix using your loop

if a blue green color develops within one minute and the culture is indole positive, no color change will
be seen if the culture is indole negative

to test if the bacterium is positive or negative for the enzyme phosphatase, hold the open place of
colonies grown on the phenolphthalein medium over a bottle of ammonia in a well-ventilated area

If free phenolphthalein has been released by phosphatase action, phosphatase positive colonies will
turn bright pink color.
If the bacteria are negative for the phosphatase, then the colonies will remain colorless.

For the lactose test, the result is determined by observation

The medium has turned yellow, the culture is capable of utilizing lactose

If the media in the tube remains red, the culture is unable to utilize lactose

For the methyl red test, a few drops of methyl red solution must be added to the test tube

If the bacterium is capable of breaking down glucose and an acid has been produced, once the metal red
is added the media will change to a bright red color. This is a positive metyl red test

If the color stays its original yellow color, this indicates a negative metyl red test as no acid has been
produced

For the Vogues-Proskauer test, add six hundred microliters of five percent alpha naphthol solution to
the test tube using a sterile tip

Using a new tip, add 200microliters of 40% Koh to the same test tube

Shake and slope the tube

Examine after 15 minutes and 1 hour

A color change from yellow to a cherry red color indicates a positive votier pro scare result

No color change indicates a negative vor opro test result

The citrate utilization test requires observation only

A green color indicates citrate is not utilized

Blue color indicates citrate has been utilized

The Gelatin hydrolysis test determines whether a bacterial species can breakdown gelatin

This result is visual only

If the media’s solid following incubation, then the bacteria is negative for gelatin breakdown

If the media is partially or totally liquid following incubation run the tube under a cold tap

If the media remains liquid, then the bacteria is positive for gelatin break down

The human lesions test results are observed together

Fermenters produce yellow color in both tubes

Oxidizers show a color change from green to yellow in the tube without oil only

A culture showing no color change in either tube is a non utilizer of glucose

The amino acid decarboxylase test checks for the presence of the enzyme decarboxylase in bacteria

If the bacteria has failed to break down glucose then the media in the control will remain purple

No further analysis is required as the decarboxylation process has failed to begin

If the first stage of the decarboxylation process has begun, then the bacteria will have broken down the
glucose in the media turning the media from its original purple color to yellow

Only if the control has turned yellow do you turn your attention to the other two tubes

In this particular sample, take note of the following:

The control is yellow meaning decarboxylation has begun

The tubes containing the amino acids lysine and ornithine are yellow meaning this particular strain of
bacteria is decarboxylase negative for both lysine and ornithine

In the next example, take note of the ff:

The control is yellow meaning decarboxylation has begun

The tubes containing amino acids lysine and ornithine are purple, meaning this particular strain of
bacteria is decarboxylase positive for lysine and ornithine

welcome to the biochemical virtual

laboratory for microbiology
today we are going to perform several
biochemical tests
the catalase test coagulase test
oxidase test rapid strep a test
latex agglutination test and gonocheck 2
tests we're going to start with the
catalase test
the catalase test is used to
differentiate staphylococci
catalase positive from streptococci
catalase negative the enzyme catalase
is produced by bacteria that respire
using oxygen
and protects them from the toxic
byproducts of oxygen metabolism
[Music]
catalase positive bacteria include
strict aerobes
as well as facultative anaerobes
although they all have the ability to
respire using oxygen as a terminal
electron acceptor
catalase negative bacteria may be
anaerobes
or they may be facultative anaerobes
that only ferment
and do not respire using oxygen as a
terminal electron acceptor
such as straptococci let's start our
experiment
we're going to first click on the
hydrogen peroxide bottle
or h2o2 and drag it to the glass slides
we're going to place one drop of
hydrogen peroxide on each of the slides
now we're going to use a stick and pick
a colony of bacteria
be sure to pick a colony that's off by
itself
now that we have a colony of
streptococcus pyrogenes
we can add it to our slide
[Music]
we see that the test is negative because
there is no bubble formation so
streptococcus pyogenes
must be negative our stick is now
contaminated so we must throw it away in
the biohazardous waste
and choose a new stick let's choose a
colony of staphylococcus aureus for our
next slide
here we see bubbles are produced this
means that staphylococcus aureus
is catalase positive remember to dispose
of your stick properly
contaminated glass slides should go in
the biohazardous sharps container
great we can move on to our next test
our next test
is the coagulase test coagulase is an
enzyme produced by staphylococcus aureus
that converts
soluble fibrinogen in plasma to
insoluble
fibrin other staphylococci do not
produce coagulase
thus this test can be used to
distinguish staphylococcus aureus
from other strains of staphylococci
let's begin our experiment
we will first click on the water bottle
and drag it to the slide and place
one drop of water onto the slide
we'll go ahead and place one drop of
water on the other side as well
to be used later
now we can pick our colonies let's pick
a colony of staphylococcus epidermis
we'll use a colony that's nice and round
off by itself
and let's add the bacteria to our slide
our stick is now contaminated and must
be disposed of
in the biohazardous waste container
now we're going to add rabbit plasma
now we're going to gently mix our slide
to see the results
we see no precipitation so this is
negative
let's dispose of our slide in the
biohazardous sharps container
now let's add staphylococcus aureus to
our second slide
and dispose of our stick
we'll add our rabbit plasma
and gently shake for 15 seconds
here we see a coagulation and
precipitate formation
this means that staphylococcus aureus is
positive
let's dispose of our slide biohazard
sharps waste container
now we can move on to our next test our
next test
is the oxidase test the oxidase test is
used to identify bacteria that produce
cytochrome c oxidase cytochrome c
oxidase is an enzyme of the bacterial
electron transport chain
note all bacteria that are oxidase
positive
are aerobic and can use oxygen as a
terminal electron acceptor
in cellular respiration this does not
mean that they are strict arrows however
bacteria that are oxidase negative may
be anaerobic
aerobic or facultative the oxidase
negative results
only means that these organisms do not
have the cytochrome c
oxidase that oxidizes the test reagent
they may be able to respire using other
oxidases in the electron transport chain
let's begin our experiment we will first
click on the box of sterile sticks and
select a stick
which will become our mouse then we can
choose a colony
of yercinia pseudotuberculosis to place
on the dry slide
we will use the stick with the bacteria
to smear
the bacteria onto one quadrant of the
dry slide
we do not see a color change to purple
so
this bacteria must be negative for
oxidase
let's dispose of our stick and choose a
colony
from pseudomonas originosa be sure to
choose a colony
that is out by itself we'll use our
stick
to smear a small amount of the bacteria
onto
another quadrant of the dry slide
here we see a color change to purple
indicating that this bacteria
is positive for the oxidase test let's
dispose of our stick
in the biohazardous waste
next let's choose a colony from
pseudomonas putina
we'll choose a colony that's out by
itself
and smear a portion of the bacteria onto
one quadrant of the dry slide
again we see the purple has appeared
meaning that this bacteria
is positive for oxidase we'll dispose of
our stick
in the biohazardous waste along with the
dry slide
great now we can move on to our next
test our next test
is the rapid strep a test the quick
view plus strep a test kit is used
to detect streptococcus pyogenes from
a pure culture or directly from a throat
swab
in a lateral flow immunoassay in this
exercise we will test two different swap
samples
let's begin we'll begin by clicking on
the clear tube
and place it in the test cassette to the
tube well
we'll use our reagent a and add it to
the tube in our cassette
we will need to squeeze four drops of
reagent a
into the tube now we will repeat this
using four drops of reagent b
let's test our throat swabs we will
place the swab
in the tube and mix for one minute and
squeeze out the excess liquid
against the side of the tube we'll want
to be sure to dispose of the swab in the
biohazardous waste container
now we can place the cap on the tube
and place two drops of the sample from
the tube
to the round sample well of the test
cassette
then we'll need to dispose of the tube
in the biohazardous waste container now
we can observe the cassette and watch
for the appearance of vertical and
horizontal lines in the three windows
the first or far right is the test
window
a positive result will yield a pink
vertical
line over a blue horizontal line to form
a plus sign
negative would be a blue horizontal line
to only form a negative sign
the second window in the center is the
positive control window
and it will have a pink vertical line
only indicating that the test is working
properly
and will detect a strep a antigen if it
is present in the sample
if no pink line appears in this window a
negative test result would not be valid
the third window at the far left is the
test complete window
and it will have a blue horizontal line
only if no blue line appears
the test is either not complete or not
enough liquid was used in the test
we see our results for our first
bacteria is negative
we'll want to dispose of the cassette in
the biohazardous waste container
now we can repeat the procedure for our
next swab
we'll add four drops of reagent a
four drops of reagent b
and put in the throat swab and mix for
one minute
we'll squeeze out a little bit of the
excess from the cotton swab into the
container
be sure to dispose of it properly
next we can place the lid on the tube
and dispense two drops of the solution
into the testing window
and dispose of our biohazardous waste
now we can incubate for five minutes and
see what our test results are
we see that this test is positive
because we see both a blue line
and a pink line overlapping to make a
positive sign
dispose of your test properly and now we
can move on to our next test
our next test is the latex agglutination
test
the latex agglutination technique is
used to distinguish
different streptococcus species from one
another based on the lancefield
serotype groupings let's begin our
experiment we're going to place
one drop of each of the latex conjugated
antibodies
to strep antigens a b
c f and g on the ovals
labeled one through five on the sample
card
once all five latex reagents are on the
card
we can click on the sample that we wish
to test we can test the control
abc control fg
unknown 1 or unknown 2. let's do our
control
abc we can choose our sample by simply
clicking on it
this will cause a drop of the sample to
be dropped
onto each oval containing the latex
reagents
[Music]
next we'll click on the card that will
enlarge it and bring it into view
in a 50 second timer
we will hold the card and gently rock it
for 15 seconds
let's look at our results the first
three samples
show agglutination or clumping samples 4
and 5 do not
based on these results this is control
abc
because a b and c showed agglutination
[Music]
we will add our reagents again to test
our next control
[Music]
this time let's choose the fg control
we will gently mix for 15 seconds
the first three wells are negative
the wells for f and g are positive
just like we would expect for our
control fg
now that this card is full let's dispose
of it in our biohazardous waste
and set up for our unknown number one
now we're going to add one drop of our
unknown sample number one
to each of these wells
now let's gently rock our slide for 15
seconds
and look at our results
[Music]
here we see that well number three shows
agglutination
this test shows us that we have strep c
in sample one
we have one more sample to try we'll add
our five reagents
to five new wells
and add our sample number two to each of
these wells
now we'll gently rock the slide for 15
seconds
and see our results
here we see that the first well has
agglutination
this means that we have strep group a in
our sample too
we'll be sure to dispose of our cards
properly
now we can go on to our final test the
final test
is the gano check ii test the gonocheck
ii test is used diagnostically to
identify
pathogenic nesseria species in gonorrhea
from in meningitis and samples
distinguishing them
from commensal nasseria and closely
related
moraxella catarrhalis let's begin our
procedure
we will first begin by sterilizing our
inoculation loop by passing it over the
bunsen burner we will place the loop in
the flame
until it is visibly red hot and then
allow a few seconds to cool
now we can drag the loop to one of the
four petri dishes
and pick a colony we will add the
bacteria from the loop
to one of the four test tubes on the
bench
we must decontaminate the loop before
placing it on the binge top
we must do this by flaming the loop once
again
now our tube reappears with a choice of
two caps to place on the tube we are
going to click on the clear cap which
will move to the top of the tube
and the timer will begin to count down
when the timer stops
the sample is yellow due to the
hydrolysis
of glutamal p-nitromanolide by
glutamil aminopeptidase in meningitis
is the only one of these four that
produces this enzyme
let's pick a colony from our next plate
and repeat the procedure
[Music]
we'll want to make sure to decontaminate
our loop before placing it on the bench
we'll incubate our sample for 30 minutes
here we see a color change to blue
indicating that this is in lactamica
blue coloration is due to the hydrolysis
of
5-bromo 4-chloro 3-indole
d-galactopyranocide by galactosidase
an enzyme involved in lactose
fermentation in
lactamica is the only nasseria that
ferments lactose
now we'll choose a colony from our next
plate
and place it into our next tube
and decontaminate our loop
we can place it back on our bench
[Music]
we'll now incubate this sample for 30
minutes
we see no color change so we will select
e to continue with the next test the
clear cap will come off the tube
and you must first discard it in the
biohazardous waste container
the red cap is then placed onto the tube
by clicking onto it
now we can invert and mix the tube
we still observe no color change this
indicates
that this bacteria is more excela
cataracts
let's flame our loop to get our last
sample
now we'll choose a colony from our next
plate
we'll choose our colony and place it
into the last vial
we'll flame the loop once again so we
can place this safely on our bench
now we'll place the white cap and
incubate for 30 minutes
[Music]
we are unable to identify the test so
additional testing will be needed
let's discard the white cap in the
biohazardous waste
and place the red cap onto the tube
we will invert it and mix we observe a
color change to red
indicating that this bacteria is in
gonorrhea