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ATP - BIOLUMINESCENCE All images used in this file are reproduced with the permission of the Companies who produce the products illustrated. ATP - BIOLUMINESCENCE • Light produced by reaction of: LUCIFERIN -LUCIFERASE + ATP ATP - BIOLUMINESCENCE • When ATP is present the enzyme - substrate complex is oxidised to an electronically excited state and releases a photon of light which can be measured in a photometer. Some living organisms, such as the firefly can produce light by this activity. • Very sensitive - can detect 10-12 g of ATP (one bacterial cell = 10-15 g ATP) • can detect 102 - 103 cells / g For description of Bioluminescent chemical reaction Refer to Adams and Moss chapter 10 Method MANUALLY • 1 ml of sample in a test-tube. • Add reagents to extract the ATP • Add luciferin-luciferase to start the reaction • Measure the light in a photometer AUTOMATICALLY • reagents in a test-tube (pen) to extract the ATP • Add the sample swab • Push down to start the luciferin-luciferase reaction • Measure the light in a photometer • eg HYLITE 2 by MERCK Sensitivity • Very sensitive - can detect 10-12 g ATP – (1 bacterial cell =10-15 g ATP) – therefore can detect 102-103 cells / ml or 10-100 yeast / ml • Use a calibration curve Uses In Food • -monitoring hygiene in food plants • -starter culture activity • sterility of eg. beer / fruit juice Limitations • ATP comes from all cells(bacteria,yeast, mould) • Sublethally damaged cells have low ATP • Somatic ATP interference (from plant, animals) – requires extraction of somatic ATP with surfactant followed by destruction with ATPase, followed by extraction of microbial ATP Limitations (continued) • Inhibition of luciferin-luciferase by food constituents • Quenching by Cl• ATPase present on some foods or made by some microorganisms • Cleanliness is essential so there is no ATP from finger, glassware etc Advantages • In evaluation of hygiene it is not necessary to distinguish between microbial and nonmicrobial ATP • High levels of ATP, whatever the origin, will tell that the surface is inadequately cleaned and sanitised Advantages (continued) • • • • -portable commercial kits -rapid sensitive indicates presence of viable microorganisms HY-LITE 2 (BY MERCK) FOR ATP MEASUREMENT HY-LITE 2 (BY MERCK) FOR ATP MEASUREMENT Three steps to proper limit setting Step 1 Get an impression about typical limits for HY-LiTE When just starting with HY-LiTE 2, you may use those limits as a very rough guide, which are mentioned in chapter one of the operator manual. Alternatively one may start without setting any limits (or, when using the HACCP plan mode, set PASS = 0 LRU and FAIL = 10000 RLU). As soon as sufficient results have been collected, individual limits should be defined, as mentioned in Step 2 and 3. REPRODUCED WITH PERMISSION FROM MERCK Step 2 Individual limit setting, by using the median as PASS limit As soon as 40 results have been collected, one should define individual PASS / FAIL limits by using the median as PASS limit. To find the median, prepare a list of increasing values out of the 40 RLU readings. Determine the value of the 20th measurement, the "median". This RLU value is your recommended PASS limit, meaning, that 50 % of the results will be PASS. Three times the PASS limit is your FAIL limit. REPRODUCED WITH PERMISSION FROM MERCK Step 3 Use TREND 2, to define the percentage of FAIL results With TREND 2 one may use the expanded statistical capabilities of the software, to choose more detailed criteria for PASS and FAIL limits. TREND 2 can calculate the "quantiles" (or percentile) at any desired level. If for example the customer wants a FAIL results at 15 % of the control points, this percentage can be obtained by defining in TREND 2: FAIL = the upper 15% quantile of the data. For this advanced statistical approach at least 100 results are required, according to the standard rule for „statistical process control“, which says, that you should use at least 5 measurements on each of 20 different occasions. For more information compare the TREND 2 helpfiles and the Tour "Limit setting". REPRODUCED WITH PERMISSION FROM MERCK LUX GENES • Codes for bioluminescence in some bacteria (Vibrio, Photobacterium) Uses of lux gene • • • • To detect a specific pathogen Bacteriophage assessment in a dairy plant Biocide effectiveness Presence of antibiotics