Download ATP - BIOLUMINESCENCE

ATP - BIOLUMINESCENCE
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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
•
•
•
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To detect a specific pathogen
Bacteriophage assessment in a dairy plant
Biocide effectiveness
Presence of antibiotics