Transcript Microbial Quality control 4

LAL ATP & DYE
Limulus amoebocyte lysate (LAL)
Gram
negative
bacteria
are
characterized by their production of
endotoxins,
which
consist
of
lipopolysaccharide (LPS) layer (outer
membrane) of the cell envelope. The LPS
is pyrogenic and responsible for some of
the symptoms that accompany infections
caused by gram- negative bacteria.
Horseshoe crab
The Limulus amoebocyte lysate
(LAL) test employs a lysate protein
obtained from the blood cells of the
horseshoe crab (Limulas polyphemous)
the lysate protein is the most sensitive
substrate known for endotoxins.
LAL test
The LAL test is performed by
adding aliquots of food suspensions
or other test materials to small
quantities of a lysate preparation,
followed by incubation at 370C for 1
hour. The presence of endotoxins
causes gel formation of the lysate
material.
Limulus ameboecyte lysate (LAL)
from the blood of horseshoe crabs
Application
Since the normal spoilage of
refrigerated fresh meat is caused by
gram – negative bacteria, the LAL
test is a good, rapid indicator of the
total numbers of gram-negative
bacteria.
Use of LAL test in food!!!
The first food application was the
use of LAL to detect the microbial
spoilage of ground beef. Endotoxins
titers increase in proportion to viable
counts of gram – negative bacteria.
Milk
The method has been found to be
suitable for the rapid evaluation of
the hygienic quality of milk relative
to the detection of coliforms before
and after pasteurization.
Raw fish & Turkey rolls
The method has
been
applied
successfully to monitor
milk,
and
milk
products,
microbial
quality of raw fish, and
cooked turkey rolls.
Japanese traditional dish
Japanese traditional dish
Medical application
The LAL test can now be used to test for
pyrogens. Bacteria often shed little bits of
their outer covering. If these substances,
known as pyrogens, get into the bloodstream,
they raise the body temperature. Even very
tiny levels of pyrogens cause a dangerous
temperature rise, so any liquid going to be
injected or fed into a patient's blood stream
has to be tested for pyrogen contamination.
Previously this was done by injecting the liquid
into a rabbit and monitoring the animal's body
temperature. The new LAL test uses white
blood cells taken from the horseshoe crab
which can detect the pyrogens in a test-tube.
The biomedical industry produces a valuable
substance known as limulus ameboecyte lysate
(LAL) from the blood of horseshoe crabs. This
substance is used to test a variety of biomedical
products and injectable drugs (e.g. vaccines) for
the presence of endotoxins. There are three U.S.
firms that produce most of the LAL in the world.
They generate annual revenues of $60 million.
At the present the U.S. Food and Drug
Administration requires that pharmaceutical and
biomedical manufacturers use LAL to test endproducts for endotoxins before releasing them to the
market.
The LAL test, a widely used
medical tool and a multi-billion
dollar enterprise, arose out of those
early experiments. The test is
routinely used to rapidly and
efficiently detect the presence of
potentially deadly endotoxins in
medicines, blood products, and
medical
devices
such
as
pacemakers and catheters.
Overall, the value of the LAL test
lies in the speed at which results can
be obtained. Foods that have high
LAL titers may be candidates for
further testing by other methods;
those that have low titers may be
placed immediately into categories
of lower risk relative to numbers of
gram – negative bacteria.
ATP assay
Adenosine Triphosphate
The primary source of energy
in all living organisms
ATP is a chemical measure
ATP reacts with chemical found in
fireflies called LUCIFERIN
LUCIFERIN produces light when it is
combined with an enyzme –
LUCIFERASE – and ATP
The light is measured by Luminometer
The entire test is usually
completed with in several minutes
ATP is proportional to
the metabolic activity
High metabolic activity is always
associated with high levels of corrosion,
poor heat transfer, bad practice
This enzyme system has been purified from firefly
tails. The reaction catalyzed by luciferase
converts the chemical energy produced by the
breakdown of ATP into light energy.
Each molecule of ATP consumed in the reaction produces
one photon of light. The relationship between light
production and ATP concentration is linear for many
orders of magnitude. This light is measured by
instruments called luminometers, which essentially
consist of a photo detector, a signal amplifier, and a
signal processor. The enzyme is highly specific for ATP.
When purified enzyme preparations are used in the
assay, false positive reactions are insignificant.
This method is based on the fact that all cells
contain ATP and that the quantity detected in a
certain specimen is referable to a given number
of cells.
It is employed a luciferin-luciferase (EC
1.13.12.7) preparation. During the reaction, ATP is
transformed into adenosine monophosphate
(AMP) and light. The intensity of the light
produced is directly proportional to the cell
concentration of ATP.
A Dream Comes True
Imagine being able to to
immediately determine if a surface is
contaminated by insects, plants,
animals, bacteria, yeast, or fungi.
We can now know when a surface is
truly clean.
It's advantage over conventional
microbiological techniques include:
speed
convenience
assessment of total cleaning
efficiency.
Principle
ATP is an energy molecule found in all
organic substances. The Firefly measures
the amount of light emitted when ATP
and an enzyme known as
lucifern/luciferase (enzyme causing the
glow in fireflies) reacts and releases light.
The light measured gives an indication of
the amount of organism residue on any
given substance.
The ATP method is a well known
method used in the food industry
An assay has been developed whereby it is
possible to assess microbial concentrations in
poultry carcass rinses within 10 minutes. The test is
a modification of one previously developed for
determining raw milk quality. In the latter test,
milk samples were treated with a detergent to
lyse non-microbial cells present in the milk.
Microbial cells were removed by filtration and
the ATP present in the cells extracted and
assayed using the luciferase-luciferin reaction.
The test can be used to make a rapid
assessment of poultry carcass quality, based on
selective cut-offs predetermined by the
processor. If this level of count is exceeded, the
carcass can be subjected to a heat treatment
before sale. The accuracy of the prediction was
high. Work was undertaken to compare
methods used to enumerate bacteria in chicken
carcass rinses.
Therefore, methods for analyzing
food microbial loads within minutes
are needed. A bioluminescence ATP
assay was used to estimate the level
of microorganisms in food before
and after treatments or processing.
Results were obtained within minutes and were
in agreement with conventional plate count
methods which require 2 to 3 days incubation.
The bioluminescence ATP assay may be used by
the produce industry as a quick alternative in
estimating surface microbial load of fruits and
vegetables. This method is fast, sensitive and cost
effective and can be used to help the produce
industries in designing and implementing good
manufacturing practices.
Swab
The ATP assay is a powerful tool
There is no other
rapid test for total
viable biomass as
sensitive, inexpensive,
and as simple as the
ATP assay which
consists of only 2
steps.
The 2 steps
Step 1
The ATP must be
extracted from the
microorganisms
Step 2
Let it mix with
luciferin/luciferase
Then the light is produced and
is measured, compared to a known
standard.
Firefly pocket
Chromogenic and Fluorogenic
substances
Specific detection of microorganisms
Escherichia coli ATCC 25922
Colour change to
blue-green
+
Klebsiella pneumoniae ATCC 13883
+
-
Enterobacter cloacae ATCC 13047
+
-
Citrobacter freundii ATCC 6750
+
-
Citrobacter freundii ATCC 8090
+
-
Shigella flexneri ATCC 12022
Salmonella typhimurium ATCC
14028
-
-
-
-
Test strains
Fluorescence
Indole
+
+
-
Chromogenic and Fluorogenic media help
us detect microorganisms faster due to their
association with media, as selective media.
Principle
Different microorganisms grow in
different kind of media since they
need different nutrients.
The colour helps us differentiate
them faster and precisely.