2. LAL ATP & DYE

3. 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.

4. 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.

5. 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 37 0 C for 1 hour. The presence of endotoxins causes gel formation of the lysate material.

6. Limulus ameboecyte lysate (LAL) from the blood of horseshoe crabs

7. 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.

8. 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.

10. 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.

12. Raw fish & Turkey rolls milk raw fish cooked turkey rolls The method has been applied successfully to monitor milk, and milk products, microbial quality of raw fish, and cooked turkey rolls.

15. Japanese traditional dish

18. 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.

19. At the present the U.S. Food and Drug Administration requires that pharmaceutical and biomedical manufacturers use LAL to test end- products for endotoxins before releasing them to the market. 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. 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.

20. 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.

21. 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.

22. ATP assay Adenosine Triphosphate The primary source of energy in all living organisms

23. 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

24. 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

25. firefly tailsluciferase 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.

26. 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.

28. 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.

29. It's advantage over conventional microbiological techniques include: speed convenience assessment of total cleaning efficiency.

30. Principle lucifern/luciferase 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.

31. 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.

32. rapid assessment of poultry carcass quality 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.

34. bioluminescence 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.

35. Results were obtained within minutes Results were obtained within minutes and were in agreement with conventional plate count methods which require 2 to 3 days incubation. a quick alternative in estimating surface microbial load of fruits and vegetables. 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.

37. Swab

38. 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.

39. 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.

40. Firefly pocket

41. Chromogenic and Fluorogenic substances Specific detection of microorganisms

42. Test strains Colour change to blue-green FluorescenceIndole 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 --

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

45. Principle Different microorganisms grow in different kind of media since they need different nutrients. The colour helps us differentiate them faster and precisely.