LAL ATP & DYELAL ATP & DYE

Limulus amoebocyte lysate (LAL)

Gram negative bacteria are Gram negative bacteria are characterized by their production of characterized by their production of

endotoxins, which consist of endotoxins, which consist of lipopolysaccharide (LPS) layer (outer lipopolysaccharide (LPS) layer (outer membrane) of the cell envelope. The membrane) of the cell envelope. The LPS is pyrogenic and responsible for LPS is pyrogenic and responsible for

some of the symptoms that some of the symptoms that accompany infections caused by accompany infections caused by

gram- negative bacteria.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 testLAL 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 rollsRaw fish & Turkey rolls

The method has been applied successfully to monitor milkmilk, and milk products, microbial quality of raw fishraw fish, and cooked cooked turkey rollsturkey rolls.

Japanese traditional Japanese traditional dishdish

Japanese traditional Japanese traditional dishdish

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.

At the present the U.S. Food and Drug At the present the U.S. Food and Drug Administration requires that pharmaceutical Administration requires that pharmaceutical

and biomedical manufacturers use LAL to test and biomedical manufacturers use LAL to test end-products for endotoxins before releasing end-products for endotoxins before releasing

them to the market.them to the market.

   The biomedical industry produces a valuable The biomedical industry produces a valuable substance known as limulus ameboecyte lysate substance known as limulus ameboecyte lysate (LAL) from the blood of horseshoe crabs. This (LAL) from the blood of horseshoe crabs. This

substance is used to test a variety of biomedical substance is used to test a variety of biomedical products and injectable drugs (e.g. vaccines) for products and injectable drugs (e.g. vaccines) for the presence of endotoxins. There are three U.S. the presence of endotoxins. There are three U.S. firms that produce most of the LAL in the world. firms that produce most of the LAL in the world. They generate annual revenues of $60 million.They generate annual revenues of $60 million.

The LAL test, a widely used The LAL test, a widely used medical tool and a multi-billion medical tool and a multi-billion dollar enterprise, arose out of dollar enterprise, arose out of those early experiments. The those early experiments. The

test is routinely used to rapidly test is routinely used to rapidly and efficiently detect the and efficiently detect the

presence of potentially deadly presence of potentially deadly endotoxins in medicines, blood endotoxins in medicines, blood products, and medical devices products, and medical devices

such as pacemakers and such as pacemakers and catheters.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 TriphosphateThe primary source of energy

in all living organisms

ATP is a chemical measureATP is a chemical measure

ATP reacts with chemical found in ATP reacts with chemical found in fireflies called fireflies called LUCIFERINLUCIFERIN

LUCIFERIN LUCIFERIN produces light when it produces light when it is combined with an enyzme –is combined with an enyzme –

LUCIFERASELUCIFERASE – and ATP – and ATP

The light is measured by The light is measured by LuminometerLuminometer

The entire test is usually The entire test is usually completed with in several minutescompleted with in several minutes

ATP is proportional to ATP is proportional to the metabolic activitythe metabolic activity

High metabolic activity is always High metabolic activity is always associated with high levels of associated with high levels of

corrosion, poor heat transfer, bad corrosion, poor heat transfer, bad practicepractice

This enzyme system has been purified from firefly tailsfirefly tails. The reaction catalyzed by luciferaseluciferase 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 convenienceassessment 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/luciferaselucifern/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 rapid assessment of poultry carcass qualityassessment 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 bioluminescence ATP assay

was used to estimate the level of microorganisms in food

before and after treatments or processing.

Results were obtained within minutesResults 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 a quick

alternative in estimating surface microbial loadalternative in estimating surface microbial load of of fruits and vegetablesfruits 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 stepsThe 2 stepsStep 1Step 1

The ATP The ATP must be must be extracted from extracted from the the microorganisms microorganisms

Step 2Step 2

Let it mix Let it mix with with luciferin/luciferluciferin/luciferase ase

Then the light is produced Then the light is produced and is measured, compared to and is measured, compared to a known standard. a known standard.

Firefly pocket

Chromogenic and Fluorogenic substances

Specific detection of microorganisms

Test strainsColour change to

blue-greenFluorescenc

eIndole

Escherichia coli ATCC 25922 + + +Klebsiella pneumoniae ATCC

13883+ -  

Enterobacter cloacae ATCC 13047

+ - -Citrobacter freundii ATCC

6750+ -  

Citrobacter freundii ATCC 8090

+ -  

Shigella flexneri ATCC 12022 - -  

Salmonella typhimurium ATCC 14028

- -  

Chromogenic and Fluorogenic media Chromogenic and Fluorogenic media help us detect microorganisms faster help us detect microorganisms faster due to their association with media, as due to their association with media, as selective media. selective media.

PrinciplePrinciple

Different microorganisms grow Different microorganisms grow in different kind of media since in different kind of media since they need different nutrients. they need different nutrients.

The colour helps us The colour helps us differentiate them faster and differentiate them faster and precisely.precisely.