ATP BIOLUMINESCENCE

METHOD IN SURFACE
HYGIENE MONITORING

CREATOR : UTKARSHA KAKATI

BIGTEC LABS INTERN
 ATP BIOLUMINESCENCE ASSAY
• ATP bioluminescence assay cell detection was first developed in the 1950s
by NASA scientists who were interested in finding life (living cells) on other
planets.
• In the food industry, it is a technique used to measure the cleanliness of a
surface.
• ATP bioluminescence detects the amount of ATP, which is an indirect
measurement of the amount of microbial growth on a surface.
• In simple terms, it measures the dirt or filth on a surface indicating the need
for cleaning and sanitizing.
 WHAT IS THE ATP BIOLUMINESCENCE ASSAY
PRINCIPLE?
• All living cells (animals, plants, bacteria, yeast, and mold) contain ATP.
• It is based on the firefly’s ATP luminescent reaction. The firefly has two
chemical compounds, Luciferin and Luciferase, that react with the insect’s
ATP to produce bioluminescence light.
• The ATP collected from a surface reacts with Luciferin/Luciferase
compounds present in the sample swab to create bioluminescence light.
• The amount of bioluminescence light is measured by the Luminometer and is
expressed in Relative Light Units (RLU). RLU numbers are directly proportional
to the amount of ATP.
ATP TEST IN HYGIENE MONITORING IN FOOD
INDUSTRY
• Applied in sectors producing animal products, in particular meat, and in
farms with dairy and slaughter animals as well as in dairy industry .
• Investigating the surfaces of instruments: milking equipment, milk containers,
pipelines, milk receivers etc.
• The highest ATP level (100 000 RLU) was observed on surfaces cleaned with
cool water without any cleaning agents. ₁
• Low ATP levels were found in closed systems, particularly those cleaned
automatically, and in large containers that are easy to clean. The most
discrepant results (ranging from 9 to 38 570 RLU) were obtained in animal
farms in which non-chlorinated water was used for cleaning. ₂
 BIOLUMINESCENCE IN HYGIENE
MONITORING IN HEALTHCARE FACILITIES
• Is not widely acknowledged in the assessment of cleanliness of surfaces in
healthcare institutions. This is because monitoring usually concerns frequently
cleaned and disinfected surfaces, and the expected number of microbes
on these surfaces is low, which, in turn, translates into a weak
bioluminescence signal, and is a limitation of this method.
• To correlate the results of bioluminometric tests and culturebased analyses,
conducted in hospital settings on various surfaces, such as floors, tables,
windows or bathrooms, produced discrepant outcomes.
• Correlation coefficient indicated was very low and equalled 0.078 .
 BIOLUMINESCENCE IN ENVIRONMENTAL
STUDIES
• Checking and validating devices used for environmental measurements.
• Rapid assessment of bioaerosol samples.
• RLU units that corresponded to the number of bacteria was verified by
direct count of acridine orange-stained cells under the fluorescence
microscope.
• Pseudomonas fluorescens and vegetative cells of Bacillus subtilis.
• Bioluminescence method gives better outcomes in assessing efficacy and
bacterial bioaerosol sampling than the microscopy and culture-based
methods since it enables to localise and quantitatively assess losses in
bacterial recovery resulting from their depositing on the sampler’s elements.
 Fig. Bacillus subtilis
Fig. Pseudomonas flourescence
• 10^4 – 10^7 cells suspension - ATP content was higher for B. subtilis than for
P. fluorescens - detect bacterial concentration of 10^5 – 10^9 CFU/ml.
 BIOLUMINESCENCE METHOD IN TEXTILE
HYGIENE MONITORING
• ATP-based method to detect microbial contamination on textiles.
 PERMISSIBLE LIMITS OF MICROBIAL
CONTAMINATION ON DIFFERENT SURFACES
• American Conference of Governmental Industrial Hygienist(ACGIH) :
Bacterial or fungal counts exceeding 500 CFU/m3 in office workplace ~ poor
ventilation or unsanitary condition.
• Medical instrument surfaces & allocated areas surface swabs : Bacterial
count ≥ 5CFU/cm3 ~ high bacterial contamination.
 ATP DETERMINATION KIT (A22066)
• Introduction - Molecular Probesʼ ATP Determination Kit (A22066) offers a
convenient bioluminescence assay for quantitative determination of ATP
with recombinant firefly luciferase and its substrate D-luciferin (Figure 1). The
assay is based on luciferaseʼs requirement for ATP in producing light (emission
maximum ~560 nm at pH 7.8) from the reaction:
3
 STANDARD CURVE
Place an appropriate volume of the standard reaction solution in the
luminometer and measure the background luminescence.

Start the reaction by adding the desired amount of dilute ATP standard solution
and read the luminescence.

Subtract the background luminescence.

Generate a standard curve for a series of ATP concentrations.

 SAMPLE ANALYSIS
Substituting ATP-containing samples for the ATP standard solutions.

Calculate the amount of ATP in the experimental samples from the standard
curve.
 CONCLUSION
• Tool to monitor the efficacy of cleaning and disinfecting practices in both
food , healthcare, textile industries etc.
• Considerable limitations of this method - low sensitivity of commercially
available luminometers used for detection of microbes, poor reproducibility
of results, particularly between surfaces with various properties and purposes,
as well as unfavourable influence of environmental factors on the
measurement outcomes.
 REFERENCES
(1,2)ATP BIOLUMINESCENCE METHOD IN SURFACE HYGIENE MONITORING
~Justyna Syguła-Cholewińska et al, Department of Microbiology, Cracow
University of Economics, Krakow, 31-510, Poland .
3. Molecular Probes Invitrogen Detection Technologies ~ ATP Determination Kit
(A22066)